Objective Enduring, abnormal expression and function of the ion channel hyperpolarization-activated cyclic-AMP gated channel type 1 (HCN1) occurs in temporal lobe epilepsy (TLE). We examined the underlying mechanisms, and queried if interfering with these mechanisms could modify disease course. Methods Experimental TLE was provoked by kainic acid-induced status epilepticus (SE), HCN1 channel repression was examined at mRNA, protein and functional levels. Chromatin immunoprecipitation was employed to identify the transcriptional mechanism of repressed hcn1 expression, and the basis for their endurance. Physical interaction of the repressor, NRSF, was abolished using decoy oligodeoxynucleotides (ODNs). Video-EEG recordings were performed to assess the onset and initial pattern of spontaneous seizures. Results Levels of NRSF and its physical binding to the hcn1 gene were augmented after SE, resulting in repression of hcn1 expression and HCN1-mediated currents (Ih), and reduced Ih-dependent resonance in hippocampal CA1 pyramidal cell dendrites. Chromatin changes typical of enduring, epigenetic gene repression were apparent at the hcn1 gene within a week after SE. Administration of decoy ODNs comprising the NRSF DNA-binding sequence (NRSE) in vitro and in vivo, reduced NRSF binding to hcn1, prevented its repression and restored Ih function. In vivo, decoy NRSE-ODN treatment restored theta rhythm and altered the initial pattern of spontaneous seizures. Interpretation Acquired HCN1 channelopathy derives from NRSF-mediated transcriptional repression that endures via chromatin modification and may provide insight into the mechanisms of a number of channelopathies that co-exist with, and may contribute to, the conversion of a normal brain into an epileptic one.

Increased sensory input from maternal care attenuates neuroendocrine and behavioral responses to stress long term and results in a lifelong phenotype of resilience to depression and improved cognitive function. Whereas the mechanisms of this clinically important effect remain unclear, the early, persistent suppression of the expression of the stress neurohormone corticotropin-releasing hormone (CRH) in hypothalamic neurons has been implicated as a key aspect of this experience-induced neuroplasticity. Here, we tested whether the innervation of hypothalamic CRH neurons of rat pups that received augmented maternal care was altered in a manner that might promote the suppression of CRH expression and studied the cellular mechanisms underlying this suppression. We found that the number of excitatory synapses and the frequency of miniature excitatory synaptic currents onto CRH neurons were reduced in "care-augmented" rats compared with controls, as were the levels of the glutamate vesicular transporter vGlut2. In contrast, analogous parameters of inhibitory synapses were unchanged. Levels of the transcriptional repressor neuron-restrictive silencer factor (NRSF), which negatively regulates Crh gene transcription, were markedly elevated in care-augmented rats, and chromatin immunoprecipitation demonstrated that this repressor was bound to a cognate element (neuron-restrictive silencing element) on the Crh gene. Whereas the reduced excitatory innervation of CRH-expressing neurons dissipated by adulthood, increased NRSF levels and repression of CRH expression persisted, suggesting that augmented early-life experience reprograms Crh gene expression via mechanisms involving transcriptional repression by NRSF.

The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (∼10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts.DOI: http://dx.doi.org/10.7554/eLife.01267.001

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

Epigenetic mechanisms are involved in programming gene expression throughout development. In addition, they are key contributors to the processes by which early-life experience fine-tunes the expression levels of key neuronal genes, governing learning and memory throughout life. Here we describe the long-lasting, bi-directional effects of early-life experience on learning and memory. We discuss how enriched postnatal experience enduringly augments spatial learning, and how chronic early-life stress results in persistent and progressive deficits in the structure and function of hippocampal neurons. The existing and emerging roles of epigenetic mechanisms in these fundamental neuroplasticity phenomena are illustrated. KeywordsCRH; epigenetic; hippocampus; neonatal; early-life experience; maternal care; learning and memory; stress; corticotropin releasing hormone; glucocorticoids; GR; neuroplasticity; programming; resilience The clinical problem: association of early-life experience with learning and memoryNumerous clinical reports demonstrate a strong association between early-life experience and subsequent cognitive functions. Chronic childhood stress (such as extreme poverty, loss of parent, social deprivation or abuse) correlates with learning and memory impairments later in life (Kaplan et al., 2001;Nelson et al., 2007;Wilson et al., 2007). As examples, lower socioeconomic level early in life correlates with cognitive function in adulthood, and post-institutionalized orphans have abnormal neuronal function in limbic areas including the hippocampus, as shown by functional MRI studies (Chugani et al., 2001), and by worse cognitive performance when compared to never-institutionalized children (Nelson et al., 2007). Improving the experience of these institutionalized infants by placing them in families significantly improves learning and memory long-term. Remarkably, the timing of the placement into foster care is crucial, and placement before the age of two years is Corresponding author: Tallie Z. Baram, MD, PhD, tallie@uci.edu, Tel.: +1-949-824-1063; fax: +1-949-824-1106;, Address: Med Sci I; ZOT 4475, University of California at Irvine, Irvine, California 92697-4475, USA. * Equal contributorsPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeurobiol Learn Mem. Author manuscript; available in PMC 2012 July 1. (Bos, Zeanah, Smyke, Fox, & Nelson, 2010). These data suggest that critical developmental periods exist for the processes by which early-life experience shapes cognitive function throughout life.The impact of early-life expe...

The dorso-ventral and developmental gradients of entorhinal layer II cell grid properties correlate with their resonance properties and with their hyperpolarization-activated cyclic nucleotide-gated ion channel current (HCN, Ih) characteristics. We investigated whether such correlation existed in rat hippocampal CA1 pyramidal cells, where place fields also show spatial and temporal gradients. Resonance was absent during the first postnatal week, and emerged during the second week. Resonance was stronger in dorsal than ventral cells, in accord with HCN current properties. Resonance responded to cAMP in ventral but not in dorsal cells. The dorso-ventral distribution of HCN1 and HCN2 subunits and of the auxiliary protein tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) could account for these differences between dorsal and ventral cells. The analogous distribution of the intrinsic properties of entorhinal stellate and hippocampal cells suggests the existence of general rules of organization among structures that process complementary features of the environment.

Background:Information is differentially processed in the dorsal versus ventral hippocampus. Results: Expression levels of Kv4.2 and HCN1 varied and conferred distinct integrative properties to CA1 pyramidal cell dendrites in dorsal and ventral hippocampus. Conclusion: Molecular and physiological differences provide the basis for the specific properties of dorsal and ventral CA1 pyramidal cells. Significance: Channel expression and function enable specific processing roles.

Epidemiological and recent prospective analyses of long febrile seizures (FS) and febrile status epilepticus (FSE) support the idea that in some children, such seizures can provoke temporal lobe epilepsy (TLE). Because of the high prevalence of these seizures, if epilepsy was to arise as their direct consequence, this would constitute a significant clinical problem. Here we discuss these issues, and describe the use of animal models of prolonged FS and of FSE to address the following questions: Are long FS epileptogenic? What governs this epileptogenesis? What are the mechanisms? Are there any predictive biomarkers of the epileptogenic process, and can these be utilized, together with information about the mechanisms of epileptogenesis, for eventual prevention of the TLE that results from long FS and FSE.