Homer1a knockout mice exhibit a deficit in Pavlovian Cued Fear Conditioning
KO mice were crossed to generate wildtype and KO littermates and genotyped as previously described (Hu et al., 2010
; ). 6–10 week old male and female Homer1a
wildtype and KO littermates were then fear conditioned and tested to determine if Homer1a was required for normal fear learning. When we examined shock reactivity, we found that there was no difference in activity response to a 0.6mA footshock, suggesting that both mice of both genetic backgrounds have similar pain sensitivity and unconditioned footshock responses. Following a single session of 5 trials of 0.6mA footshock paired with 6kHz tone, with a pre-training saline injection stress, mice were tested for fear expression to the tone 24 and 48 hours later. During fear testing, we found a significant decrease (F(1,16)=4.96, P<.05, N=9/group) in the expression of cued fear across both testing sessions (). These data suggest that Homer1a expression, even in the presence of normal Homer1b,c, 2, and 3, is required for normal cued fear learning.
Pavlovian Fear conditioning results in an increase in Homer1a mRNA
Homer1a was dynamically regulated during consolidation of Pavlovian fear conditioning. Homer1a contains a unique stop site at the end of exon 5 that makes its sequence unique to the longer gene variants of homer such as homer1c. Primers for RTPCR were designed based on this sequence to differentiate between homer1a and homer1c expression (). For all of the experiments described amygdala and hippocampal tissue was extracted during consolidation of fear, 2 hours after training (). As illustrated in , all animals were measured for baseline freezing to presentation of a tone in one context (Context A). One day later, animals were presented with 5 tone-shock pairings (30 sec 6kHz tone co-terminating with 0.5sec, 1mA shock) or 5 tones without any shock in a novel context (Context B). On the third day, animals were tested in Context B without any tones or shocks for 3 minutes as an assessment of contextual fear conditioning and then immediately placed into context A where freezing in response to 5 tone-alone trials is assessed. Using this procedure, we found that one training session achieved both retention of contextual and cued fear conditioning () (b: T (18) = 4.20; p < 0.05, n = 10/group; c: T(18)= 2.69, p < 0.05, n = 10). In a separate cohort of animals quantitative PCR demonstrated an increase in homer1a mRNA in the hippocampus () (T(18) = 3.35, p < 0.05, n = 10) and in the amygdala () (T(18) = 2.39, p < 0.05, n = 10) 2 hours after fear conditioning. RNA for homer1c (a longer gene variant of the Homer1 gene family) was not increased during Pavlovian fear conditioning in either brain region () (hippocampus; amygdala). No changes in homer1a mRNA levels were seen in the striatum.
Pavlovian Cued and Contextual fear conditioning
Expressional analysis of homer1a and homer1c after Pavlovian fear conditioning in the hippocampus and amygdala
BDNF causes an upregulation of homer 1a in primary amygdala and hippocampal cultures, which is transcription dependent, MEK dependent and ERK dependent
To assess differential regulation of Homer1a through TrkB signaling, primary hippocampal and amygdala cell cultures were used. Much like in fear conditioning, BDNF-induced plasticity increased homer1a mRNA levels in both hippocampal and amygdala cell culture () (hippocampus T (10) = 3.25, p < 0.05, n = 6/group; amygdala T(10) = 2.67, p < 0.05, n = 6) but not homer1c levels. The trkB-specific agonist (7, 8-DHF) upregulated homer1a in cell culture (hippocampus T(10) = 2.25, p < 0.05, n = 6/group, amygdala T(10)= 4.86, p < 0.05, n = 6). are representative pictures of immunostainting for CamKII in both hippocampal (b) and amygdala (e) primary neuronal cultures. are representative pictures of immunostaining for parvalbumin in both hippocampal (c) and amgydala (f) primary neuronal cell cultures.
Homer1a is upregulated through the BDNF-trkB signaling pathway in cultured amygdala and hippocampal neurons
Blocking transcription with Actinomysin D (ActD) inhibited BDNF-induced upregulation of homer1a in both hippocampus and amygdala cell cultures () (a: F(3,20) = 27.34, p < 0.05, n = 6/group; d: F(3,20)=258.90, p < 0.05, n = 6/group). In addition MEK inhibition by U0126 blocked BDNF induced increases in homer1a in both hippocampus and amygdala cells () (b: F(3,20)= 7.039, p < 0.05, n = 6/group, e: F(3,20) = 14.57, p < 0.05, n = 6/group). We next utilized primary cell culture from floxed-ERK knockout mice, in which we transfected cells with a Cre Recombinase expressing lentivirus to delete the ERK gene. We found that genetically deleting ERK impaired BDNF induced upregulation of homer1a in both hippocampus and amygdala cells as well () (c: F(3,20) = 23.42, p < 0.01, n = 6/group, f: F(3,20) = 89.61, p < 0.01, n = 6/group). Thus, BDNF appears to upregulate homer1a in a transcriptionally dependent manner, and through MEK and ERK signaling mechanisms. None of these manipulations had any effect on homer1c mRNA levels (). Genetic deletion of ERK was demonstrated through QT-PCR in amygdala (T(10) = 2.28, p < 0.05, n = 6/group) and hippocampal cells (T(10) = 2.37; p < 0.05, n = 6).
Homer1a upregulation by BDNF is transcription dependent, MEK dependent, and ERK dependent
Homer1cvalues during BDNF induced plasticity
BDNF application onto amygdala cells results in histone modifications along the Homer1 promoter
In order to determine the epigenetic role of BDNF signaling on homer1a expression we examined histone modifications around the homer1a promoter region after BDNF induced plasticity. Chromatin immunoprecipitation (ChIP) assays were performed to measure the levels of several histone modifications around the Homer1 promoter after BDNF-induced plasticity. Levels of promoter enrichment were quantified by QT-PCR. We found that BDNF application had distinct effects in hippocampal and amygdala primary cell culture. In the hippocampal cell cultures, there was a significant increase in H3 acetylation (T(10) = 6.80, p < 0.05, n = 6/group) following BDNF application, but no changes were apparent in H4 acetylation, H3K9 methylation or H3K27 methylation (). In amygdala cell cultures, however, there appears to be a decrease in H3K9 methylation (T(10) = 2.44, p < 0.05, n = 6/group) following BDNF application, but no changes in H3 acetylation, H4 acetylation of H3K27 methylation (). Significant changes in acetylation or methylation were not detected at the GAPDH promoter region.
BDNF-induced and fear conditioning-induced histone modifications around the Homer1 promoter
Pavlovian fear conditioning induces epigenetic modifications of histones along the Homer1 promoter
We next examined the effect of Pavlovian fear conditioning on histone modifications around the Homer1 promoter region. In the hippocampus there was a significant increase in H3 acetylation (T(18) = 2.37, p < 0.05, n = 10/group), but no difference in H4 acetylation, H3K9 methylation or H3K27 methylation (). In the amygdala, however, there was a significant decrease in H3K9 methylation (T(18) = 3.14, p < 0.05, n = 10/group), but no changes in H3 acetylation, H4 acetylation, or H3K27 methylation (). Significant changes in acetylation or methylation were not detected at the GAPDH promoter region. Notably, these in vivo results parallel the histone modification-specific findings seen in amygdala and hippocampal primary cell culture.
HDAC inhibition enhances fear conditioning, Homer1a expression, and modifications of the Homer1 promoter
HDAC inhibitors have been shown to enhance contextual fear conditioning(Levenson et al., 2004
). In this experiment, we examined the effect of the HDAC inhibitor, sodium butyrate (NaB) on fear conditioning. Note that the overall level of fear retrieval in controls () was less than in our initial study (), which is likely due to injection stress. We showed that IP administration of NaB can induce increases in contextual fear memories (T(18) = 2.10, p < 0.05, n = 10/group) but did not appear to cause an increase in cued fear conditioning (). NaB also appeared to enhance hippocampal homer1a
mRNA expression (; F(3,36) = 5.01, p < 0.05, n = 10/group), but seemed to reverse the mRNA increase in amygdala tissue (, F(3,36) = 5.45, p < 0.05, n = 10/group). Sodium butyrate enhanced H3 acetylation in hippocampal tissue (, F(3,36) = 9.54, p < 0.01, n = 10/group) but reversed fear conditioned induced decreases in H3K9 methylation in amygdala tissue (, F(3,36) = 4.58, p < 0.05, n = 10/group).
Effects of histone deacetylase inhibition on fear conditioning, homer1a mRNA levels, and histone modifications around the Homer1 promoter