Disruption of the NF-κB/IκBα Autoinhibitory Loop Improves Cognitive Performance and Promotes Hyperexcitability of Hippocampal Neurons
Though originally discovered in the immune system as an important mediator of inflammation, NF-κB has recently been shown to play key roles in the central nervous system, such as synaptogenesis, synaptic plasticity, and cognition. NF-κB activity is normally tightly regulated by its primary inhibitor, IκBα, through a unique autoinhibitory loop. In this study, we tested the hypothesis that the IκBα autoinhibitory loop ensures optimal levels of NF-κB activity to promote proper brain development and function. To do so, we utilized knock-in mice which possess mutations in the IκBα promoter to disrupt the autoinhibitory loop (IκBαM/M KI mice).
Here, we show that these mutations delay IκBα resynthesis and enhance NF-κB activation in neurons following acute activating stimuli. This leads to improved cognitive ability on tests of hippocampal-dependent learning and memory but no change in hippocampal synaptic plasticity. Instead, hippocampal neurons from IκBαM/M KI mice form more excitatory and less inhibitory synapses in dissociated cultures and are hyperexcitable. This leads to increased burst firing of action potentials and the development of abnormal hypersynchronous discharges in vivo.
These results demonstrate that the IκBα autoinhibitory loop is critical for titrating appropriate levels of endogenous NF-κB activity to maintain proper neuronal function.
KeywordsTheta Burst Stimulation Paired Pulse Facilitation Contextual Fear Memory Current Clamp Mode Intrinsic Excitability
The transcription factor NF-κB, originally discovered in B cells of the immune system , has since been shown to be expressed in nearly all cell types. Beyond its role as a critical regulator of the inflammatory response, NF-κB activation can also promote the expression of genes involved in apoptosis and cell survival, thus making it an important mediator of the general stress response . In the central nervous system (CNS), NF-κB signaling regulates neuronal survival following acute pathologic damage such as traumatic brain injury [3, 4, 5] and stroke [6, 7] as well as in chronic neurodegenerative diseases such as Alzheimer's disease [8, 9] and Parkinson's disease [10, 11]. NF-κB also plays an important role in normal development and function of the brain. Recent studies have shown that NF-κB activation is required during long term memory formation [12, 13, 14, 15, 16] as well as during induction of synaptic plasticity [14, 16, 17, 18]. Furthermore, NF-κB plays an important role in neurite outgrowth and synaptogenesis [19, 20, 21]. Indeed, NF-κB is present in post-synaptic compartments and is rapidly transported to the nucleus following stimulation with glutamate or depolarization with KCl .
NF-κB is normally bound by an inhibitory IκB protein and sequestered in the cytoplasm. Activation of NF-κB classically requires the phosphorylation, ubiquitination, and proteasome-mediated degradation of the IκB protein, thereby freeing NF-κB to translocate to the nucleus [22, 23]. NF-κB is quickly silenced by the rapid resynthesis of its principal inhibitor, IκBα . This occurs via direct binding of NF-κB to consensus κB sites in the promoter for IκBα, thus forming a powerful mechanism of feedback inhibition . Complete knockout of IκBα leads to early postnatal lethality in mice [26, 27], highlighting the vital importance of IκBα-mediated inhibition of NF-κB. To investigate the role of the IκBα autoinhibitory loop on NF-κB signaling, we have generated knock-in mice possessing mutations of the κB sites in the IκBα promoter (IκBαM/M KI mice), thus specifically abrogating the NF-κB-mediated feedback arm of this autoinhibitory loop . Here, we show that genetic disruption of this loop leads to enhanced NF-κB activity in neurons. While IκBαM/M KI mice display increased cognitive ability on hippocampal-dependent behavioral tasks, hippocampal plasticity is unchanged. Instead, increased NF-κB signaling alters the balance of excitatory and inhibitory synaptogenesis, leading to hyperexcitability and increased spontaneous burst firing in hippocampal cultures and acute slices. As a consequence, IκBαM/M KI mice display increased seizure-like activity. Together, these data reveal the importance of tight regulation of NF-κB signaling for proper neuronal function.
Mice were housed 2-5 per cage with ad libitum access to food and water in a room with a 12 h light/dark cycle in a sterile pathogen-free mouse facility. All procedures were performed in accordance with NIH guidelines and with the approval of the Baylor College of Medicine Institutional Animal Care and Use Committee. Initial IκBαM/M KI mice were generated as described previously . For the current studies, mice were further backcrossed onto a pure C57BL/6 background for a minimum of five generations. Heterozygous mice were then intercrossed to obtain littermate IκBα+/+ (WT) mice or IκBαM/M KI mice. For our behavioral experiments, we subsequently set up WT × WT and KI × KI breeding cages to obtain larger cohorts of age-matched male mice. Genotyping was performed by PCR of tail DNA at time of weaning.
Neonatal pups were collected from heterozygous breeding cages and genotyped. For molecular experiments, whole brains were removed and pooled by genotype into ice cold dissection buffer (Hanks Buffered Saline Solution supplemented with 10 mM HEPES, pH 7.5, 0.6% glucose, 20 U/ml penicillin, 20 μg/ml streptomycin). Using a dissecting microscope, cortices were isolated and meninges removed. The tissue was then cut into small pieces and transferred to 10 ml of dissection buffer. Following addition of 500 μl trypsin (2.5%), the tissue was incubated at 37°C for 12 min. 400 μl soybean trypsin inhibitor (1 mg/ml) and 100 μl DNase I (1%) were then added and the tissue collected by centrifugation for 5 min at 1200 rpm. The supernatant was decanted off and replaced with 2 ml of culture media (Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, 40 U/ml penicillin, 40 μg/ml streptomycin) and 20 μl DNase I (1%). The tissue was then gently triturated with a cut P1000 pipette tip 8-10 times. After allowing the remaining pieces to settle, the supernatant was collected into a fresh tube and the remaining tissue pieces were again triturated in 2 ml fresh culture media with an uncut P1000 pipette tip. This was repeated once more, after which no tissue pieces were visible. The collected supernatants were then centrifuged for 5 min at 1200 rpm and the cell pellet resuspended in 5 ml culture media. This was repeated once more before the cells were counted and plated onto poly-D-lysine coated 6 cm dishes at a density of 2.5-3 × 106 cells/dish in a volume of 5 ml culture media. For electrophysiology and immunostaining experiments, only hippocampi were isolated and plated onto poly-D-lysine coated glass coverslips in 24-well plates at a density of 1 × 105 cells/well. To improve local density, cells were initially applied to the center of the coverslip in a 40 μl bubble and allowed to settle before filling the well with 500 μl culture media. All cultures were grown in tissue culture incubators at 37°C, 5% CO2, 95% humidity.
Protein isolation and Western blot
To measure NF-κB kinetics, cortical cultures were stimulated with 10 ng/ml recombinant TNFα (Chemicon, GF023) or IL-1β (Calbiochem, 407617) at 10 DIV by replacing half the culture media with fresh culture media containing 2x drug for the indicated duration. The media was then completely removed and the neurons were scraped into 100 μl Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 500 μM DTT) supplemented with complete protease and phosphatase inhibitor cocktails. The lysate was incubated on ice for 1 hr followed by addition of 5 μl 10% NP-40 and then vortexed for 10 sec. The lysate was then centrifuged for 20 sec at 14,000 rpm and the supernatant containing cytoplasmic proteins was collected and stored at -80°C for future use. The pellet was then incubated in 50 μl Buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 500 μM DTT) supplemented with complete protease and phosphatase inhibitor cocktails and vortexed for 10 sec every 10-15 min for a total of 1 hr. The resulting lysate was then spun at 4°C for 5 min at 14,000 rpm and the supernatant containing nuclear proteins was collected and stored at -80°C for future use. For Western blot analysis, the cytoplasmic fractions were quantified using a DC colorimetric protein assay (Bio-Rad) and boiled at 95°C for 7 min in sample buffer. 15 μg of protein samples were then loaded onto 12% SDS-polyacrylamide gels, run at 100 mV for 2 hr, transferred onto nitrocellulose membranes (Bio-Rad) at 90 V for 1.5 hr at 4°C in transfer buffer (50 mM Tris, 40 mM glycine, 20% methanol, 0.01% SDS), and then blocked with 5% milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were then probed with primary antibody (Rabbit anti-IκBα, Santa Cruz, sc-371, 1:1000; Mouse anti-α-tubulin, Sigma, 1:20,000) diluted in blocking solution overnight at 4°C. Membranes were washed 4 × 10 min in TBST and then blotted with secondary antibody (Horse anti-Rabbit-HRP, Vector Labs, 1:5000; Horse anti-Mouse-HRP, Vector Labs, 1:5000) for 2 hr at room temperature. The membranes were again washed 4 × 10 min in TBST, incubated in ECL solution (GE Healthcare Life Sciences), and exposed to film. After developing, the films were digitized on a flatbed scanner and band intensities quantified using ImageJ software (NIH). Levels of α-tubulin served as control for loading.
EMSA and NF-κB p65 ELISA
For EMSA experiments, NF-κB consensus probes (Santa Cruz, sc-2505) were end-labeled with 32P using T4 polynucleotide kinase (New England Biolabs) and purified using nucleotide purification columns (Qiagen). 1 μl of labeled probe was then mixed with 1 μl poly dI:dC, 3 μl 5x binding buffer (75 mM Tris, pH 7.5, 375 mM NaCl, 7.5 mM EDTA, 25% glycerol, 100 μg/ml BSA), and 3-5 μg of nuclear protein in a final volume of 15 μl. For cold competition, 1 μl of unlabeled consensus or mutant (Santa Cruz, sc-2511) probe was added at 10-fold excess. For supershift, 1 μl of anti-p50 (Santa Cruz, sc-1190X), or anti-p65 (Santa Cruz, sc-372X) antibody was added to the mixture. After a 30 min incubation at 4°C, the samples were loaded onto a 6% non-denaturing polyacrylamide gel and run in 0.5x TBE at 4°C for 30 min at 200 V, then 2 hr at 250 V. The gel was then dried down onto filter paper using a slab gel dryer and then exposed by autoradiography to Kodak-MR film at -80°C. For quantification of p65, nuclear samples were analyzed with the ELISA-based TransAM NFκB p65 kit (Active Motif) according to the manufacturer's instructions.
For quantitative real-time PCR experiments, total RNA was isolated from cortical neuronal cultures and analyzed as described previously . The primer sequences are as follows: 5'-TCGCTCTTGTTGAAATGTGG-3' (IκBα-Fwd), 5'-TCATAGGGCAGCTCATCCTC-3' (IκBα-Rev), 5'-AATGTGTCCGTCGTGGATCTGA-3' (GAPDH-Fwd), and 5'-GATGCCTGCTTCACCACCTTCT-3' (GAPDH-Rev).
All mouse behavior experiments were performed in the Mouse Neurobehavior Core facility with age-matched cohorts of 2-4 mo old male mice from homozygous WT or KI breeding. The initial test battery consisted of, in order, open field, light-dark, rotarod, prepulse inhibition, conditioned fear, and hotplate. There was a minimum separation of one day between tests, which has been shown previously not to have significant carryover effect using the same test protocols and equipment [30, 31]. Additional cohorts of mice were later tested with elevated plus maze or Morris water maze as described previously .
For extracellular field potential recordings, brains were isolated from 3-5 mo old mice and cut into 400 μM horizontal or transverse slices using a vibratome sectioning system (PELCO) in ice cold cutting artificial cerebral spinal fluid (cutting ACSF: 110 mM sucrose, 60 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 28 mM NaHCO3, 7 mM MgCl2, 0.5 mM CaCl2, 5 mM glucose, and 0.6 mM ascorbate, saturated with 95% O2 and 5% CO2). Hippocampal slices were then transferred to a heated recording chamber filled with recording ACSF (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, and 10 mM glucose, saturated with 95% O2 and 5% CO2) maintained at 32°C. Stimulation of Schaffer collaterals from the CA3 region was performed with bipolar electrodes, while borosilicate glass capillary pipettes filled with recording ACSF (resistances of 1 to 1.5 MΩ) were used to record field excitatory postsynaptic potentials (fEPSPs) in the CA1 region. Signals were amplified using a MultiClamp 700 B amplifier (Axon), digitized using a Digidata 1322A (Axon) with a 3 kHz low pass filter and a 0.1 Hz high pass filter, and then captured and stored using Clampex 9 software (Axon) for offline data analysis. Before each experiment, input-output recordings were made by stimulating for 0.1 ms at intensities ranging from 50 to 500 μA and calculating the slope of the fEPSP responses. A stimulation intensity corresponding to 30% of the maximal fEPSP slope was then chosen. For paired-pulse experiments, pairs of stimuli were delivered with interpulse intervals ranging from 10 to 200 ms and the fEPSP amplitude from the second stimulus (P2) was divided by the fEPSP amplitude from the first stimulus (P1) to calculate paired pulse facilitation (P2/P1). For LTP measurement, baseline transmission was measured every 20 sec for 10 min. A weak LTP induction protocol was then applied using one train of theta burst stimulation (1 × TBS) consisting of ten 5 Hz clusters of four 100 Hz pulses. fEPSP traces were again recorded for 20 min before a standard LTP induction protocol was applied using three trains of TBS (3 × TBS) spaced 20 sec apart, followed by 60 min of fEPSP recording. For data analysis, every 3 consecutive fEPSP traces were averaged together and the initial slope of the fEPSP measured. For spontaneous burst activity, the recording electrode was placed in either CA3 or CA1 and the perfusion buffer changed to modified ACSF containing 8.5 mM K+ and 0.5 mM Mg2+ as described previously . Five consecutive traces of 50 sec each were recorded and the number of interictal bursts counted manually. For whole cell patch clamp experiments, hippocampal cultures were transferred to a recording chamber perfused with oxygenated Tyrode solution (25 mM HEPES, 129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, 10 μM glycine, and 50 μM picrotoxin). Neurons were recorded under whole cell patch using electrodes filled with recording solution (40 mM HEPES, pH 7.2, 110 mM K-Gluconic acid, 10 mM phosphocreatine, 10 mM EGTA, 2 mM MgATP, 2 mM Na2ATP, 0.3 mM Na2GTP) with a resistance of 3-8 MΩ. For spontaneous firing, neurons were held in current clamp mode without any current injection and recorded for 100 sec. For evoked firing, neurons were held in current clamp mode and steps of current ranging from 0 to 200 pA (in 20 pA increments) were injected for 100 ms and the number of action potentials elicited were counted.
To quantify synaptic puncta formation, 14-15 DIV hippocampal neuronal cultures were washed with phosphate buffered saline (PBS) 2 × 5 min and then fixed in 4% PFA overnight at 4°C. Coverslips were again washed with PBS 3 × 5 min before permeabalization in PBS with 0.1% Triton X-100 (PBST) for 15 min at room temperature. Neurons were then blocked with 3% goat serum in PBST for 1 hr and then stained with primary antibody (Rabbit anti-VGLUT1, Synaptic Systems, 1:2000; Mouse anti-MAP2, Chemicon, 1:2000; Mouse anti-VGAT, Synaptic Systems, 1:1000; Rabbit anti-MAP2, Chemicon, 1:1000; Mouse anti-PSD-95, Chemicon, 1:500; Rabbit anti-GAD65, Chemicon, 1:500; Mouse anti-Gephyrin, Synaptic Systems, 1:500) diluted in blocking solution overnight at 4°C. Coverslips were then washed in PBST 5 × 3 min before incubation with secondary antibody (Goat anti-Rabbit-Alexa555, Invitrogen, 1:2000; Goat anti-Mouse-Alexa488, Invitrogen, 1:2000) diluted in blocking solution for 2 hr at room temperature. Coverslips were again washed in PBST 5 × 3 min and then mounted onto glass slides with Prolong Gold AntiFade Reagent with DAPI (Invitrogen). The coverslips were sealed with clear nail polish and stored at 4°C. Images were taken using a 63x oil objective on a Nikon epifluorescent microscope using Metamorph software. Image analysis was performed using ImageJ software. For colocalization experiments, images were split into red and green channels and a threshold was applied. Presynaptic puncta were automatically outlined and the percent area which also stained positive for each postsynaptic marker was calculated. Puncta which contained at least 25% overlap were considered positive.
For analysis of seizure-like activity, mice were anaesthetized and cranial burr holes drilled to allow placement of electrodes. Teflon-coated silver wire electrodes (0.005 inch diameter) were implanted in the subdural space overlying temporal and parietal cortex bilaterally. The electrodes were then connected to a microminiature connector (Omnetics). After a 24 hr recovery period, simultaneous video and EEG recordings were captured in freely behaving mice for multiple 2-4 hr intervals over a one week period with a computer system running Harmonie software (Stellate Systems).
All data is presented as mean ± SEM. Outliers were identified using Grubbs' method with α = 0.05. Pairwise comparisons were analyzed using a two-tailed Student's t-test, while a two-way ANOVA followed by Bonferroni post-hoc analysis was used for multiple comparisons. P values less than or equal to 0.05 were considered statistically significant.
Mutation of the IκBα promoter delays IκBα resynthesis and enhances NF-κB activity in neurons
Hippocampal-dependent learning and memory is enhanced in IκBαM/MKI mice
Synaptic plasticity is unchanged in IκBαM/MKI hippocampal slices
Enhanced NF-κB activity alters the balance of excitatory to inhibitory synapse density
Hippocampal neurons from IκBαM/MKI mice exhibit spontaneous burst firing and hyperexcitability
IκBαM/M KI mice exhibit increased interictal epileptiform activity in vivo
We employed a mouse knock-in model in which we genetically disrupted the IκBα autoinhibitory loop by mutating the κB binding sites in the IκBα promoter, which abolishes NF-κB binding and promotion of IκBα expression, leading to delayed resynthesis of IκBα and enhanced NF-κB activity in multiple peripheral organs . Here, we demonstrate that this IκBα autoinhibitory loop operates in the central nervous system (CNS) to tightly regulate NF-κB activity and is required to maintain normal levels of excitability. Specifically, we found that the normal NF-κB directed resynthesis of IκBα is delayed in neurons from IκBαM/M KI mice. It is important to note that mutation of the κB sites does not completely abolish IκBα expression, as this would lead to an early postnatal lethality [26, 27]. Rather, we see almost normal expression of IκBα at baseline in IκBαM/M KI neurons. Instead, the NF-κB mediated rapid resynthesis of IκBα is blocked, with IκBα gradually reappearing after prolonged TNFα stimulation. This lagging IκBα resynthesis is possibly mediated by the activity of other transcription factors at the IκBα promoter, such as SP1, CREB, or AP-1 . As a consequence, we believe that NF-κB is not constitutively active within IκBαM/M KI neurons, but rather undergoes stronger and prolonged activation in response to each acute stimulation.
There are over 150 stimuli known to activate NF-κB, ranging from infectious pathogens and inflammatory cytokines to growth factors and hormones . In this study, we utilized in vitro stimulation with TNFα and IL-1β to directly activate NF-κB in neuronal cultures. Within the CNS, these cytokines are secreted not just in response to injury, but also during normal brain function, specifically following changes in neuronal activity [42, 43] or during cognitive tasks . Furthermore, application of glutamate or KCl activated NF-κB in cultured neurons [13, 45] indicating that activation of glutamate receptors or neuronal depolarization both can lead to NF-κB activation. As these occur spontaneously within neural networks, NF-κB activity can be detected under baseline conditions in vitro and in vivo [13, 46]. Thus, we believe that IκBαM/M KI mice represent a unique model of increased NF-κB activity following endogenous activation and allow the study of NF-κB under physiologic conditions.
One consequence of enhanced NF-κB activation is an improvement in both contextual and spatial learning and memory in IκBαM/M KI mice. Numerous studies have been published implicating the role of NF-κB in learning and memory. Knockout of the p65 subunit of NF-κB (balanced with knockout of TNFR to bypass embryonic lethality) leads to a deficit in spatial memory formation , while knockout of the p50 subunit leads to an impairment in active avoidance memory . Similarly, inhibition of NF-κB activation via transgenic overexpression of an IκBα super-repressor in the forebrain leads to impaired spatial memory formation . A bioinformatics analysis demonstrated that many of the genes whose expression was altered by conditioned fear training were regulated by the NF-κB subunit c-Rel , and that c-Rel-/- mice demonstrate a deficit in contextual memory formation . These studies provide strong evidence that loss of NF-κB function leads to learning and memory deficits. Importantly, our studies demonstrate that the converse is also true, that boosting NF-κB signaling in vivo can lead to enhanced learning and memory.
In addition to improved cognitive performance, enhanced NF-κB activity also promotes neuronal hyperexcitability and the spontaneous firing of bursts of action potentials. In vivo, IκBαM/M KI mice display elevated seizure-like activity, evidenced by increased epileptiform activity in hippocampal slices and increased interictal spike (IIS) frequency on cortical EEG. While it is not clear whether IIS events drives epileptogenesis or are an adaptive mechanism to prevent ictal events , IIS typically occur between seizure episodes and suggest a pro-epileptic phenotype in IκBαM/M KI mice. At the neuronal level, IIS are caused by a slow depolarization current known as a paroxysmal depolarizing shift (PDS) and may reflect an increase in excitatory or a decrease in inhibitory connectivity within the network . Our immunostaining experiments demonstrate that both of these changes in synaptic density are present in IκBαM/M KI hippocampal neuronal cultures, and our patch-clamp recordings reveal characteristic PDS currents, suggesting that this imbalance forms the basis for IIS activity in IκBαM/M KI mice. Alternatively, increased NF-κB activity may be directly enhancing the intrinsic excitability of neurons, which can in turn drive changes in synaptic connectivity . This hypothesis is supported by the increased action potential firing in response to current injections in IκBαM/M KI neurons, however very little is known about the role of NF-κB in regulating voltage-gated ion channels or leak channels to alter membrane excitability. In all likelihood, both synaptogenesis and intrinsic excitability are affected in IκBαM/M KI mice and together contribute to the improved cognition and increased seizure-like activity.
Given its known role as a transcription factor, enhanced NF-κB activity must be exerting its effect on cognitive performance and network excitability through altered gene transcription. However, given the extremely large and diverse set of target genes thought to be regulated by NF-κB , the precise molecular mechanism has proven difficult to define. We measured the mRNA and protein levels of various suspected target genes which play important roles in neuronal function, including the AMPA-type glutamate receptor subunit GluR1  and the NMDA-type glutamate receptor subunit NR1 , but were unable to find consistent changes in brain tissue or neuronal cultures. We believe this may be a consequence of the subtle nature of our genetic model, and that the phenotypes evident in IκBαM/M KI mice may be a result of the cumulative effect of small changes in many genes. Additionally, gene transcription in IκBαM/M KI mice may be most altered during specific contexts. Recent detailed work from the Meffert lab demonstrated that NF-κB directly regulated excitatory synaptogenesis, but only during developmental or plasticity-induced periods of rapid synapse development, and was not active during maintenance of synapses in mature neurons . Interestingly, this effect was due to NF-κB-dependent transcription of the scaffolding protein PSD-95, an important regulator of excitatory synapses  which, through interaction with the cell adhesion molecule neuroligin, can also modulate the balance of excitatory and inhibitory synapses . It will be interesting to see whether expression of PSD-95 is enhanced in IκBαM/M KI mice during contextual or spatial memory consolidation.
While the focus of our experiments was on neuronal function, IκBαM/M KI mice possess mutations of the IκBα promoter in all cell types. Enhanced NF-κB activity in resident glial cells might lead to increased levels of inflammatory cytokines in the brain and is thought to drive the neurotoxicity seen in chronic neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease . We see no evidence of neuronal loss in IκBαM/M KI mice, perhaps due to a protective effect of increased NF-κB in neurons, or because the enhancement of NF-κB is too mild to trigger toxicity. Cytokine secretion has also been shown to modulate neuronal function and excitability [54, 55]. Specifically, secretion of TNFα from glial cells can increase neuronal expression of AMPA-type glutamate receptors [42, 43], and infusion of low levels of IL-1β into the brain can improve contextual learning and memory . Furthermore, intra-hippocampal injections of IL-1β can lead to an increase in IIS frequency and sensitivity to kainate-induced seizures . While we cannot exclude the possibility of contribution from these other cell types to the improved cognitive performance and hyperexcitability in IκBαM/M KI mice, our experiments using primary neuronal cultures strongly suggest a direct effect in neurons.
Overall, the results of our experiments confirm a role for NF-κB in regulating the formation of synaptic connections and encoding of long term memory, and demonstrate the importance of the IκBα autoinhibitory loop in properly regulating endogenous NF-κB activity in neurons to ensure healthy physiologic levels. On the one hand, we have shown that disruption of this loop can lead to enhanced learning and memory. Indeed, these results suggest that pharmacologic activation of NF-κB might be a viable therapeutic option to improve cognitive function in various forms of dementia, such as Alzheimer's disease, or following traumatic or ischemic brain injury. However as with any strategy that impairs synaptic inhibition, this must be carefully titrated so that NF-κB activity remains within a physiologic range to prevent an imbalance in synaptic strength and the potential promotion of seizure activity.
We are grateful to N. Aithmitti and X. Chen for expert technical support, Z. Wang and B. Peng for assistance with initial breeding and characterization of the mice, and N. Justice and members of the Zheng laboratory for constructive discussions and help with the manuscript. We thank C. Spencer and the Baylor College of Medicine IDDRC Administrative, Mouse Neurobehavior, and Mouse Physiology cores (HD24064) for their assistance. This work was supported by grants from NIH (AG20670, AG32051, and AG33467 to HZ; NS614283 to DS).
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