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Spatial chromatin organization is crucial for transcriptional regulation and might therefore be particularly dynamic in neurons since these terminally differentiated cells dramatically change their transcriptome in response to external stimuli. Here, we show that stimulation of neurons causes condensation of large chromatin domains. We find that this phenomenon is not only induced in rat hippocampal neurons cultured in vitro, but is also present in vivo in amygdala neurons of rats subjected to fear conditioning, and hippocampal neurons of animals subjected to kainate evoked seizures or High-Frequency Stimulation (HFS). The activity-induced chromatin condensation is an active, very rapid, and reversible process, that is independent of transcription and precedes the expression of Immediate Early Genes (IEG). It is accompanied by the redistribution of posttranslational modifications of histones, and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that neurons depleted of the histone deacetylase HDAC1 fail to condense chromatin upon stimulation, a phenomenon that can be fully reversed by the introduction of human HDAC1. Taken together, our results suggest that the HDAC1-dependent chromatin reorganization might constitute an important level of fine-tuning of transcriptional regulation in stimulated neurons.

Mammalian excitatory synapses express diverse types of synaptic plasticity. A major challenge in neuroscience is to understand how a neuron utilizes different types of plasticity to sculpt brain development, function, and behavior. Neuronal activity-induced expression of the immediate early protein, Arc, is critical for long-term potentiation and depression of synaptic transmission, homeostatic synaptic scaling, and adaptive functions such as long-term memory formation. However, the molecular basis of Arc protein function as a regulator of synaptic plasticity and cognition remains a puzzle. Recent work on the biophysical and structural properties of Arc, its protein-protein interactions and post-translational modifications have shed light on the issue. Here, we present Arc protein as a flexible, multifunctional and interactive hub. Arc interacts with specific effector proteins in neuronal compartments (dendritic spines, nuclear domains) to bidirectionally regulate synaptic strength by distinct molecular mechanisms. Arc stability, subcellular localization, and interactions are dictated by synaptic activity and post-translational modification of Arc. This functional versatility and context-dependent signaling supports a view of Arc as a highly specialized master organizer of long-term synaptic plasticity, critical for information storage and cognition.

Activity-regulated cytoskeletal-associated protein (Arc) is implicated as a master regulator of long-term synaptic plasticity and memory formation in mammalian brain. Arc acts at synapses and within the nucleus, but the mechanisms controlling Arc localization and function are little known. As Arc transcription and translation are regulated by extracellularsignal-regulated kinase (ERK) signaling, we asked whether Arc protein itself is phosphorylated by ERK. GST-fused Arc of rat origin was able to pull down endogenous ERK2 from rat hippocampal lysates. Using a peptide array, we show that ERK binds a non-canonical docking (D) motif in the C-terminal domain of Arc, and this interaction is abolished by phosphorylation of Tyr309. Activated ERK2 phosphorylated bacterially expressed Arc in vitro at all five predicted sites, as confirmed by phospho-specific protein staining and LC-MS/MS analysis. In neuroblastoma cells expressing epitope tagged-Arc, we demonstrate ERK-dependent phosphorylation of Arc in response to activation of muscarinic cholinergic receptors with carbachol. Using phosphosite-specific antibodies, this stimulus-evoked phosphorylation was shown to occur on Ser206 located within the central hinge region of Arc. In cultured hippocampal neurons expressing phosphomutant Arc under control of the activity-dependent promoter, we show that Ser206 phosphorylation regulates the nuclear:cytosolic localization of Arc. Thus, the neuronal activity-induced phosphomimic exhibits enhanced cytosolic localization relative to phosphodeficient and wild-type Arc. Furthermore, enhanced Ser206 phosphorylation of endogenous Arc was detected in the dentate gyrus cytoskeletal fraction after induction of long-term potentiation (LTP) in live rats. Taken together, this work demonstrates stimulus-evoked ERK-dependent phosphorylation and regulation of Arc protein.

Summary Levels of adult neurogenesis in the dentate gyrus (DG) of the hippocampus are correlated with unique cognitive functions. However, the molecular pathways controlling it are poorly understood. Here, we found that the known physiological ways to enhance neurogenesis converged on the eEF2/eEF2K pathway via AMPK in the DG. Enhancing the elongation phase of mRNA translation in eEF2K-knockout (eEF2K-KO) mice induced the expression of neurogenesis-related proteins in the hippocampus. We thus tested the hypothesis that inducing eEF2K-KO in mature neurons of the DG controls neurogenesis. Indeed, both general eEF2K-KO and targeted KO in DG excitatory mature neurons resulted in enhanced neurogenesis levels and upregulation of neurogenesis-related proteins. Increased neurogenesis was correlated with enhanced performance in DG-dependent learning. Moreover, general and local eEF2K-KO in old mice rejuvenated the DG, paving the way for better mechanistic understanding of how neurogenesis is controlled in the mature DG and possible treatments for incurable aging-associated diseases.

Background Primary neuronal cultures are widely used to elucidate fundamental aspects of neuronal anatomy, physiology, cell biology, and neuronal dysfunction in animal models of disease. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it is often difficult to produce high-quality cultures consistently in a single laboratory, and to compare results between laboratories. To overcome these issues, cryopreservation can be used to obtain more standardized, high-quality banks of neuronal cultures. New Method In this study, we present a simplified cryopreservation method for rodent primary hippocampal and cortical neurons from embryonic day 18.5 fetuses, using DMSO-containing traditional cell freezing medium. Results Cryopreserved neurons stored for more than 1 year in liquid nitrogen were assessed by cell imaging, as well as biochemical signaling transduction and gene expression in response to pharmacological treatments. Cryopreserved neuronal cultures were comparable to freshly prepared cultures in terms of: (1) neuronal viability, (2) neuronal morphology and maturation, (3) functional synapse formation, (4) stimulus responsiveness. These results indicate that DMSO-cryopreserved neurons are equivalent to freshly prepared neurons both developmentally and functionally. Comparison with Existing Methods Our method is simple and does not require special reagents or equipment. Conclusions Introduction of the cryopreserved neurons as a standard laboratory practice has the potential to increase the robustness and reproducibility of findings between laboratories and reduce the number of animals used in research.

The Kavli Prize in Neuroscience was awarded for the third time in September 2012, by the Norwegian Academy of Science and Letters in Oslo. The accompanying Kavli Prize Symposium on Neuroscience, held in Bergen and Trondheim, was a showcase of excellence in neuroscience research. The common theme of the Symposium presentations was the mechanisms by which animals adapt to their environment. The symposium speakers--Michael Greenberg, Erin Schuman, Chiara Cirelli, Michael Meaney, Catherine Dulac, Hopi Hoekstra, and Stanislas Dehaene--covered topics ranging from the molecular and cellular levels to the systems level and behavior. Thus a single amino acid change in a transcriptional repressor can disrupt gene regulation through neural activity (Greenberg). Deep sequencing analysis of the neuropil transcriptome indicates that a large fraction of the synaptic proteome is synthesized in situ in axons and dendrites, permitting local regulation (Schuman). The nature of the 'reset' function that makes animals dependent of sleep is being revealed (Cirelli). Maternal behavior can cause changes in gene expression that stably modify behavior in the offspring (Meaney). Removal of a single sensory channel protein in the vomero-nasal organ can switch off male-specific and switch on female-specific innate behavior of mice in response to environmental stimulation (Dulac). Innate behaviors can be stably transmitted from parent to offspring through generations even when those behaviors cannot be expressed, as illustrated by the elaborate burrowing behavior in a rodent species, in which independent genetic regions regulate distinct modules of the burrowing pattern (Hoekstra). Finally, at the other extreme of the nature-nurture scale, functional magnetic resonance imaging (fMRI) analysis in children and adults identified a brain area specifically involved in reading (Dehaene). As the area must originally have developed for a purpose other than reading, such as shape recognition, this illustrates the use of a previously formed neural structure to tackle a new challenge.

Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP3-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

Background Patients with schizophrenia often suffer from cognitive dysfunction, including impaired learning and memory. We recently demonstrated that long-term potentiation in rat hippocampus, a mechanistic model of learning and memory, is linked to gene expression changes in immunity-related processes involved in complement activity and antigen presentation. We therefore aimed to examine whether key regulators of these processes are genetic susceptibility factors in schizophrenia. Methods Analysis of genetic association was based on data mining of genotypes from a German genome-wide association study and a multiplex GoldenGate tag single nucleotide polymorphism (SNP)-based assay of Norwegian and Danish case–control samples (Scandinavian Collaboration on Psychiatric Etiology), including 1133 patients with schizophrenia and 2444 healthy control subjects. Results Allelic associations were found across all three samples for eight common SNPs in the complement control-related gene CSMD2 ( CUB and Sushi Multiple Domains 2 ) on chromosome 1p35.1-34.3, of which rs911213 reached a statistical significance comparable to that of a genome wide threshold ( p value=4.0 × 10 −8 ; odd ratio=.73, 95% confidence interval=.65–.82). The second most significant gene was CSMD1 on chromosome 8p23.2, a homologue to CSMD2 . In addition, we observed replicated associations in the complement surface receptor CD46 as well as the major histocompatibility complex genes HLA-DMB and HLA-DOA . Conclusions These data demonstrate a significant role of complement control-related genes in the etiology of schizophrenia and support disease mechanisms that involve the activity of immunity-related pathways in the brain.

Summary The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2 −/− neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Regulation of translation factor activity plays a major role in protein synthesis-dependent forms of synaptic plasticity. We examined translational control across the critical period of Arc synthesis underlying consolidation of long term potentiation (LTP) in the dentate gyrus of intact, anesthetized rats. LTP induction by high frequency stimulation (HFS) evoked phosphorylation of the cap-binding protein eukaryotic initiation factor 4E (eIF4E) and dephosphorylation of eIF2alpha on a protracted time course matching the time-window of Arc translation. Local infusion of the ERK inhibitor U0126 inhibited LTP maintenance and Arc protein expression, blocked changes in eIF4E and eIF2alpha phosphorylation state, and prevented initiation complex (eIF4F) formation. Surprisingly, inhibition of the mTOR protein complex 1 (mTORC1) with rapamycin did not impair LTP maintenance or Arc synthesis nor did it inhibit eIF4F formation or phosphorylation of eIF4E. Rapamycin nonetheless blocked mTOR signaling to p70 S6 kinase and ribosomal protein S6 and inhibited synthesis of components of the translational machinery. Using immunohistochemistry and in situ hybridization, we show that Arc protein expression depends on dual, ERK-dependent transcription and translation. Arc translation is selectively blocked by pharmacological inhibition of mitogen-activated protein kinase-interacting kinase (MNK), the kinase coupling ERK to eIF4E phosphorylation. Furthermore, MNK signaling was required for eIF4F formation. These results support a dominant role for ERK-MNK signaling in control of translational initiation and Arc synthesis during LTP consolidation in the dentate gyrus. In contrast, mTORC1 signaling is activated but nonessential for Arc synthesis and LTP. The work, thus, identifies translational control mechanisms uniquely tuned to Arc-dependent LTP consolidation in live rats.

Long term facilitation (LTF) of C-fiber-evoked firing of wide dynamic range neurons in the spinal dorsal horn in response to conditioning stimulation (CS) of afferent fibers is a widely studied cellular model of spinal nociceptive sensitization. Although 100 Hz CS of primary afferent fibers is commonly used to induce spinal cord LTF, this frequency exceeds the physiological firing range. Here, we examined the effects of electrical stimulation of the sciatic nerve within the physiological frequency range on the magnitude and stability of the C-fiber-evoked responses of wide dynamic range neurons and the expression of immediate early genes (c-fos, zif268, and Arc) in anesthetized rats. Stimulation frequencies of 3, 30 and 100 Hz all induced facilitation of similar magnitude as recorded at 1 h post-CS. Strikingly, however, 3 Hz-induced potentiation of the C-fiber responses was decremental, whereas both 30 and 100 Hz stimulation resulted in stable, non-decremental facilitation over 3 h of recording. The number of dorsal horn neurons expressing c-fos, but not zif268 or Arc, was significantly elevated after 3 Hz CS and increased proportionally with stimulation rate. In contrast, a stable LTF of C-fiber responses was obtained at 30 and 100 Hz CS, and at these frequencies there was a sharp increase in zif268 expression and appearance of Arc-positive neurons. The results show that response facilitation can be induced by stimulation frequencies in the physiological range (3 and 30 Hz). Three hertz stimulation induced the early phase of LTF, but the responses were decremental. Arc and zif268, two genes previously coupled to LTP of synaptic transmission in the adult brain, are upregulated at the same frequencies that give stable LTF (30 and 100 Hz). This frequency-dependence is important for understanding how the afferent firing pattern affects neuronal plasticity and nociception in the spinal dorsal horn.

We sought to determine whether the fragile X mental retardation gene fmr1 is regulated in long-term potentiation (LTP) and electroconvulsive shock (ECS). In situ hybridization of fmr1 mRNA in hippocampus of rats given LTP in vivo showed no change in fmr1 mRNA levels relative to control. However, ECS induced a selective increase in fmr1 mRNA expression in the dentate gyrus (DG) granule cell layer at 6 h post-ECS. The ECS paradigm may unmask relevant activity-dependent regulatory mechanisms that modulate fmr1 gene transcription in vivo.

Major learning events are typically followed by a period during which the number and/or duration of rapid-eye movement sleep episodes is increased. Processes critical to memory formation are thought to take place during this interval of ‘enhanced’ rapid-eye movement sleep. We therefore compared the capacity for long-term potentiation during rapid-eye movement sleep and alert wakefulness after learning. Rats were chronically implanted with electrodes for stimulation of the perforant path and recording of evoked potentials and EEG in the dentate gyrus. After obtaining baseline recordings, rats were trained on a 40-trial two-way active avoidance task. Conditioned rats exhibited a two-fold increase in the mean duration of rapid-eye movement sleep episodes, as reflected by a prolongation of the hippocampal theta rhythm. There was no change in the sleep pattern of pseudoconditioned controls, which received explicitly unpaired tones and foot shocks in a yoked design. High-frequency stimulation was applied during the second, third, and fourth major rapid-eye movement sleep episodes after active avoidance training. Another group was tetanized at matching time points during alert wakefulness. After pseudoconditioning, tetanus applied during wakefulness or rapid-eye movement sleep readily induced long-term potentiation, and there was no difference between groups in the magnitude of increase for the population excitatory postsynaptic potential slope or the population spike height as measured 1 h, 24 h, and 5 days post-tetanus. By contrast, in conditioned rats, tetanus applied during wakefulness failed to elicit long-term potentiation of the excitatory postsynaptic potential slope (7.6% increase 1 h posttetanus), while the group stimulated during ‘enhanced’ rapid-eye movement sleep exhibited a 32% potentiation, equivalent to that obtained after pseudoconditioning. Analysis of individual experiments revealed a marked reduction in the probability of inducing long-term potentiation during post-learning wakefulness. We conclude that avoidance learning affects the induction of long-term potentiation in the dentate gyrus, suppressing induction during alert wakefulness while releasing the potential for synaptic modification during episodes of rapid-eye movement sleep. The effect is compatible with the hypothesis that memory consolidation involves a dynamic regulation of events such as long-term potentiation during sleep and wakefulness.

A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.

The possible importance of opioid peptides in the induction of long-term potentiation (LTP) was investigated in the perforant pathgranule cell system. A high-frequency train (400 Hz) was delivered to the lateral or medial perforant path, during push-pull perfusion of the dentate molecular layer with artificial cerebrospinal fluid (CSF) alone, or with CSF containing naloxone (10 −4 M). Naloxone effectively blocked the induction, but not the maintenance of LTP in the lateral perforant path, a putative proenkephalin system. Naloxone did not affect the production of LTP in the medial pathway. These findings suggest that activation of naloxone-sensitive receptors is necessary for the full expression of LTP in the lateral perforant pathway.

We investigated the possible importance of stimulus train frequency for the induction and magnitude of long-term synaptic plasticity in the perforant path-granule cell pathway. Under the same experimental conditions, low- (15 Hz) or high-frequency (400 Hz) stimulation could elicit a profound long-term depression (LTD), or typical long-term potentiation (LTP), of the population spike amplitude, excitatory postsynaptic potential (EPSP) amplitude and spike onset latency. In addition, changes in the relationship between the EPSP and population spike amplitude indicated that granule cell excitability was enhanced during LTP and reduced during LTD. LTD occured primarily after low-frequency stimulation (5 of 6 cases), and was always accompanied by striking changes in the EEG, most notably a biphasic slow potential. While the EEG changes were confined to the first 5 min after the tetanus, LTD lasted from 1 to 4 h. The nature of the EEG events is still unclear, it is suggested that they may represent a spreading depression-like episode. Finally, we found that LTP evoked by high-frequency stimulation was larger and generally reached peak magnitude faster than when it followed low-frequency stimulation. A possible mechanism and role for hippocampal LTD is proposed.