Mice lacking the transcriptional regulator Bhlhe40 have enhanced neuronal excitability and impaired synaptic plasticity in the hippocampus

<div><p>Bhlhe40 is a transcription factor that is highly expressed in the hippocampus; however, its role in neuronal function is not well understood. Here, we used <i>Bhlhe40</i> null mice on a congenic C57Bl6/J background (<i>Bhlhe40</i> KO) to investigate the impact of Bhlhe40 on neuronal excitability and synaptic plasticity in the hippocampus. <i>Bhlhe40</i> KO CA1 neurons had increased miniature excitatory post-synaptic current amplitude and decreased inhibitory post-synaptic current amplitude, indicating CA1 neuronal hyperexcitability. Increased CA1 neuronal excitability was not associated with increased seizure severity as <i>Bhlhe40</i> KO relative to +/+ (WT) control mice injected with the convulsant kainic acid. However, significant reductions in long term potentiation and long term depression at CA1 synapses were observed in <i>Bhlhe40</i> KO mice, indicating impaired hippocampal synaptic plasticity. Behavioral testing for spatial learning and memory on the Morris Water Maze (MWM) revealed that while <i>Bhlhe40</i> KO mice performed similarly to WT controls initially, when the hidden platform was moved to the opposite quadrant <i>Bhlhe40</i> KO mice showed impairments in relearning, consistent with decreased hippocampal synaptic plasticity. To investigate possible mechanisms for increased neuronal excitability and decreased synaptic plasticity, a whole genome mRNA expression profile of <i>Bhlhe40</i> KO hippocampus was performed followed by a chromatin immunoprecipitation sequencing (ChIP-Seq) screen of the validated candidate genes for Bhlhe40 protein-DNA interactions consistent with transcriptional regulation. Of the validated genes identified from mRNA expression analysis, insulin degrading enzyme (<i>Ide</i>) had the most significantly altered expression in hippocampus and was significantly downregulated on the RNA and protein levels; although Bhlhe40 did not occupy the <i>Ide</i> gene by ChIP-Seq. Together, these findings support a role for Bhlhe40 in regulating neuronal excitability and synaptic plasticity in the hippocampus and that indirect regulation of <i>Ide</i> transcription may be involved in these phenotypes.</p></div

<i>Bhlhe40</i> KO mice have increased neuronal excitability.

<p>(A) <i>Bhlhe40</i> KO mice have a 14.4% decrease in IPSC amplitude (<i>Bhlhe40</i> KO: 85.6 ± 12.2, WT: 100 ± 11.6); n = 4 mice for each <i>Bhlhe40</i> KO (5 cells) and WT (6 cells). (B) Left: <i>Bhlhe40</i> KO mice have a 40% increase in mEPSC amplitude, relative to WT levels (<i>Bhlhe40</i> KO: 18.99 ± 0.69 pA, WT: 13.6 ± 0.88 pA). Right: Example traces of mEPSCs from WT and <i>Bhlhe40</i> KO slices. Error bars are standard error of the mean; unpaired t-tests were used for both mEPSC and IPSC amplitude comparisons <i>Bhlhe40</i> KO hippocampal slices compared to WT slices; * = p<0.05.</p

Insulin levels trended to an increase but are not changed in the hippocampus of <i>Bhlhe40</i> KO mice.

<p>(A) Protein (Western) Blots were used to determine expression levels of proteins for candidate genes identified from the mRNA validation experiments. Single hippocampi from six WT and <i>Bhlhe40</i> KO mice were used for protein-level analysis by western blotting. Representative autoradiograms from WT (+) and <i>Bhlhe40</i> KO (-) samples are shown for IDE (120kDa), Bhlhe41(50kDa), and Kctd12 (36kDa). (B) Protein quantification by western blot was determined via densitometry using Fiji (Image J). In <i>Bhlhe40</i> KO hippocampus, IDE was 1.6-fold downregulated (p<0.0001), Kctd12 was 30% upregulated (p<0.05), and Bhlhe41 was 18% upregulated (p<0.05) relative to WT levels. For Bhlhe41, a triplet band was observed, likely due to the two possible post translation sumoylations. The relative expression for Bhlhe41 represents a combination of all three bands. Unpaired t-tests were used to test for significance changes for protein levels. Values shown are <i>Bhlhe40</i> KO protein level relative to WT levels by arbitrary units (AUs); * = p<0.05 **** = p<0.0001. Additionally, Clock, Camk1d, Gabbr1, GluR1, Scn1a, and Chl1 were tested and found to be not significantly changed in <i>Bhlhe40</i> KO hippocampus. (C) Single hippocampi from six WT and <i>Bhlhe40</i> KO mice were used for BDNF quantification by ELISA. Mature BDNF (top) and Pro BDNF (bottom) were tested on independent ELISAs. BDNF levels are expressed in ng of BDNF relative to mg of total protein. Top) pro BDNF (unpaired t-test p>0.05); Bottom) mature BDNF (unpaired t-test p>0.05). (D) Single hippocampi from seven WT and <i>Bhlhe40</i> KO mice were used for insulin quantification by ELISA. Insulin levels are expressed in ng of insulin relative to mg of total protein (insulin ELISA students t-test p = 0.07). All error bars are standard error of the mean.</p

Quantitative RT-PCR Primers.

<p>Quantitative RT-PCR Primers.</p

<i>Bhlhe40</i> KO mice have unique changes in gene expression in the central nervous system.

<p>Unilateral Hippocampus (HIPP), Cortex (CTX), and Cerebellum (CER) were isolated from naïve <i>Bhlhe40</i> KO and WT animals (n = 4). MRNA was extracted and run on an Illumina whole genome expression array. (A) <i>Bhlhe40</i> KO and WT hippocampal gene expression had non-overlapping separation. One WT CER sample was removed as an outlier and one <i>Bhlhe40</i> KO CTX clustered with the WT samples but was included in the analysis. (B) Venn Diagram of significant genes changed (p<0.05, FDR<0.3) in <i>Bhlhe40</i> KO Cerebellum, Cortex, and Hippocampus, relative to WT). (C) Pathway analysis of a whole genome expression array on hippocampal genes identified several highly upregulated pathways in energy metabolism and downregulated pathways involved in synaptic activity in <i>Bhlhe40</i> KO mice compared to WT mice. The top 20 upregulated and top 20 downregulated pathways are shown, organized by Z score; n = 4. R = Reactome; K = Kegg; B = Biocarta.</p

<i>Bhlhe40</i> KO mice do not have more-severe behavioral seizures.

<p>Animals were infused with 227ngs of KA and monitored for six hours. (A) Latency from infusion to first seizure, unpaired t-test p>0.05. (B) Total time seizing over the first 15 minutes, first 30 minutes, and full 6 hours, unpaired t-test p>0.05. (C) Latency from last seizure to the end of the six-hour monitoring period, unpaired t-test for each p>0.05. (D) Seizure scale for maximum seizure response for the first 15 minutes, first 30 minutes, and full 6 hours, unpaired t-test for each p>0.05. n = 6 for WT mice and n = 5 for <i>Bhlhe40</i> KO mice (Figs 2B and 2C), n = 6 for <i>Bhlhe40</i> KO mice (Figs 2A and 2D; including an animal that died during the monitoring period). Error bars are standard error of the mean.</p

<i>Bhlhe40</i> KO mice have reduced hippocampal synaptic plasticity.

<p>(A) EPSP slope input/output curves were significantly different (p<0.001; n = 9). (B) Paired pulse facilitation is not significantly impaired (p = 0.063; n = 5). (C) There was a 60% reduction in LTP measured by EPSP slope 1hr following 1s of 100Hz stimulation (<i>Bhlhe40</i> KO: 120 ± 20.7%, WT: 150.5 ± 21%; p<0.001; n = 5 mice, 6 slices each). (D) There was a 52% reduction in LTD measured by EPSP slope 1hr following 15min of 1Hz stimulation (<i>Bhlhe40</i> KO: 85 ± 12.8%, WT: 68.5 ± 19.5%; p<0.001; n = 5 mice, 6 slices each). Data from Figs 3A, 3B, 3C, and 3D were p<0.05 on Shapiro Wilk normality test and tested for significance by Mann-Whitney Rank Sum Test. Error bars are standard error of the mean.</p

<i>Bhlhe40</i> KO mice have impaired relearning on the MWM.

<p>(A) There was no change in escape latency during initial learning and (B) initial probes revealed no cognitive deficits of <i>Bhlhe40</i> KO animals as indicated by latency to platform. (C)or time spent in the goal (NE) quadrant. (D) <i>Bhlhe40</i> KO mice had impaired relearning of the new platform location (SW quadrant) (linear regression line elevation difference p<0.01) (two-way RM-ANOVA p = 0.067). (E) Similarly, <i>Bhlhe40</i> KO animals were impaired in the latency to the new platform area on the reversal probes (linear regression line elevation difference p<0.05 t-test) (two-way RM-ANVOA p = 0.059). (F) On the 4hr reversal probe <i>Bhlhe40</i> KO showed no preference for the new platform location (SW) quadrant compared to the initial platform location (NE) quadrant, whereas WT animals spent more time in the NE compared to SW quadrants (p<0.05). (G) There was no significant difference in time spent in the SW quadrant compared to the NE quadrant for either <i>Bhlhe40</i> KO or WT animals on the 24hr and 48hr reversal probes (p>0.05), although there was a near-significant trend for WT animals on the 48hr probe (p = 0.0531). Across all conditions there was no effect on swim speed (p>0.05) WT n = 12, <i>Bhlhe40</i> KO n = 12. Initial 24hr probe measurements included only n = 11 for <i>Bhlhe40</i> KO mice due to a software error in recording one animal. One <i>Bhlhe40</i> KO animal was removed from the dataset due to repeated thigmotaxis-like behavior. Error bars are standard error of the mean. Statistical significance (*p<0.05) was tested by two-way RM-ANOVA (Figs 4A, 4B, 4C, 4D, 4E, and 4F), comparison of linear regression lines (Figs 4A, 4B, 4D, and 4E) and Mann Whitney U test (Figs 4E, 4F, and 4G).</p