MoniTORing neuronal excitability at the synapse

Whether DNA segregates in a biased way has been a subject of intense controversy and debate. Although highly provocative in its biological implications, if true, technical problems have limited researchers from drawing firm conclusions from the data. Elabd et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201307110/DC1) now show a high frequency of nonrandom template segregation during differentiation of embryonic stem cells using rigorous experimentation and implicate the methyltransferase Dnmt3 as a key regulator of this process.Nonrandom template segregation (NRTS) is a phenomenon in which two sister chromatids, supposedly the identical copies of each other produced by a precise DNA replication process, are distinguished and segregated nonrandomly to daughter cells. It has been more than a few decades since the first idea of NRTS was proposed (Cairns, 1975; Potten et al., 1978), and NRTS has been reported in organisms ranging from bacteria to plants (Rando, 2007; Tajbakhsh and Gonzalez, 2009; Lark, 2012; Lopez-Vernaza and Leach, 2013). Yet, speculation on the biological meaning of this phenomenon has remained controversial (Lansdorp, 2007; Rando, 2007; Tajbakhsh and Gonzalez, 2009). A major model, the immortal DNA strand hypothesis, suggested that stem cells or other long-living cells avoid replication-induced mutations by inheriting the old template strands. An alternative model suggested that NRTS carries distinct epigenetic information to daughter cells to allow them to adapt different fates. In addition to these different models in its biological meaning, some studies showed that all the chromosomes follow NRTS, whereas other studies showed that only a subset of the chromosomes follow NRTS. It remains unclear whether these distinct forms of NRTS are mechanistically and/or functionally related. Technical difficulties (described next) have added to the controversy.The scheme of testing NRTS of all of the chromosomes is very simple: cells incorporate BrdU (or other nucleotide analogues) during an S phase. Two divisions later, all BrdU should go to one cell, whereas the other cell becomes completely BrdU negative, if cells follow NRTS (Fig. 1 A). Although addressing NRTS with this scheme seems simple, there are incredible numbers of pitfalls. Did the cells really undergo just one, but not more than one, round of DNA replication with the BrdU? Did they undergo just two rounds of cell division, but not one or more than two, by the time of observation? Did BrdU really label all chromosomes within the cell at a time of incorporation? Furthermore, were the cell pairs used to judge the BrdU inheritance really twin daughters from a single cell division? Do those cells that are observed to segregate BrdU asymmetrically really divide asymmetrically in terms of their fates? What is the frequency of asymmetric cell division of a given cell population, and are those asymmetrically dividing cells really the ones that show NRTS? To really understand this phenomenon, one must consider all of these questions at the same time, and answering all of them conclusively has been quite a mission impossible.Open in a separate windowFigure 1.New evidence for nonrandom template segregation. (A) Assessment of NRTS by pulse–chase BrdU labeling. After BrdU incorporation during an S phase, BrdU will be exclusively segregated into one cell, but not the other, in two cell cycles. (B) NRTS observed during embryonic stem (ES) cell differentiation. The new work by Elabd et al. (2013) suggests that asymmetric BrdU segregation shows striking correlation with the inheritance of Dnmt3a and 3b and expression of differentiation genes, such as Bry and Gata4. Dnmt3a and 3b that are loaded onto the new template-containing sister chromatid (i.e., BrdU⁺) might lead to sister chromatid–specific DNA methylation, leading to lineage commitment.Elabd et al. (in this issue) have now addressed this question using extremely careful approaches. Starting from embryonic stem cell culture, they followed NRTS during embryoid body differentiation. Several lines of technical advancement and the application of rigorous standards by this group led to observations with striking consistency and frequency, thereby providing convincing evidence that NRTS indeed occurs during embryoid body differentiation. First, carefully adjusting the timing of BrdU administration and fixation, aided by live observation, made it possible to exclusively score daughter cell pairs generated by a cell division that incorporated BrdU with the right timing. This yielded a strikingly high frequency (∼50%) of NRTS occurrence, making it unlikely that observed NRTS is caused by experimental artifacts. Furthermore, the inheritance pattern of labeled chromosomes showed tight correlation with the cell fate: BrdU segregation (i.e., newly synthesized DNA) highly correlated with the inheritance of differentiation markers, such as Bry and Gata4, strongly suggesting that it is a biologically relevant, regulated phenomenon (Fig. 1 B).It is of note that the authors found that the “artificial occurrence of NRTS” counts up to ∼5%. Such artificial NRTS occurred when unrelated cells migrate to come in contact, leading to apparent asymmetry in BrdU labeling between two cells. It has been often argued that the probability of labeled strands of all 46 (in humans) or 40 (in mice) chromosomes being segregated to one cell would be extremely low (2⁻⁴⁶ or 2⁻⁴⁰) and that any observation of an asymmetric DNA labeling pattern even at a low frequency would support NRTS. However, the new work by Elabd et al. (2013) clearly indicates that additional factors, such as crawling of cells, can cause an artificial NRTS-like outcome at a considerable frequency. Considering this, a new, more rigorous standard must be applied to future studies concerning NRTS.One common feature of controversial fields is often the lack of a strong mechanistic basis from which to test predictions. This has certainly been the case of NRTS. Elabd et al. (2013) provide some of the first mechanistic insights into the process: they showed that the de novo methyltransferases Dnmt3a and 3b are required for efficient NRTS during embryoid body differentiation (Fig. 1 B). It is interesting that de novo DNA methylation, but not maintenance DNA methylation, seems to be required for NRTS. Interestingly, a higher concentration of Dnmt3a and 3b proteins were often cosegregated with BrdU-containing nuclei, suggesting that cells undergoing differentiation inherit the enzyme that allows de novo DNA methylation. This may indicate that cells undergoing differentiation need to methylate DNA to promote differentiation. However, it would not explain why de novo DNA methylation is required to allow the distinction and asymmetric segregation of sister chromatids. A possible unifying theory may be that in a differentiating embryonic stem cell, a newly synthesized strand would be bound by de novo DNA methyltransferases, which may methylate DNA specifically on the newly synthesized strand, before or after the cell division, allowing the cell to molecularly distinguish older versus newer template DNA strands, hence enabling their asymmetric segregation. Upon cell division, DNA methylation in a differentiating cell may initiate the cascade of epigenetic events to program them into a particular cell fate/lineage. If this is the case, this may finally answer the question in epigenetics of whether the epigenome is acquired before fate determination or vice versa: epigenome changes are first triggered in the mother cell—before cell division and fate acquisition—and the daughter cell is born with a suitable epigenome that allows it to adopt a certain cell fate. These data seem to suggest that the meaning of NRTS is the transmission of epigenetic information, instead of protecting the genome stability through avoidance of DNA mutation.Now, if the meaning of the NRTS is transmission of the epigenome, instead of protecting the genome stability through avoidance of DNA mutation, one would wonder whether all the chromosomes should follow NRTS or whether NRTS of only a subset of chromosomes might suffice. It is possible that only a small number of genes must be regulated through NRTS: a few key fate-determining genes, which can trigger a cascade of downstream events, harbor differential epigenetic information between two sister chromatids, and the segregation of these key genes into two daughter cells breaks symmetry and leads to asymmetric cell fates. In particular, once cells settled into a certain lineage (e.g., adult stem cells), the number of fate-determining genes that needs to be regulated through NRTS might be small, leading to chromosome-specific NRTS. It is important to note that in this model, NRTS does not influence all of the genes on a chromosome: not all of the genes on a chromosome are epigenetically marked or have distinct epigenetic marks between two sister chromatids. Therefore, NRTS will influence only a few hypothetical genes whose epigenetic information is distinct between two sister chromatids, leading to asymmetric cell fate. As long as the epigenetic information of key marked genes is segregated nonrandomly, the rest of the genes on the chromosomes, which might be identical genetically and epigenetically to their sisters, do not influence the asymmetric outcome of a cell division. Chromosome-specific NRTS has been proposed previously for chromosome 7 in mice (Armakolas and Klar, 2006), although more recent studies suggested that the finding may be partly caused by the experimental method used in the original study (Falconer et al., 2012; Sauer et al., 2013). A recent observation that Drosophila melanogaster male germline stem cells show NRTS only with X and Y chromosomes, but not autosomes, might be a variation of NRTS, specific to lineage-committed adult stem cells (Yadlapalli and Yamashita, 2013). In contrast, during early stages of development, such as embryonic stem cell differentiation, the number of fate-determining genes that need to be regulated through NRTS might be larger, and those genes may be scattered on many chromosomes, leading to NRTS at the whole genome level. NRTS involving (almost) all of the chromosomes can be easily detected by BrdU pulse–chase experiments. Then, the underlying biological significance of NRTS at the whole genome level and single chromosome level may be the same after all. However, NRTS of a small number of the chromosomes cannot be assessed by pulse–chase labeling with BrdU and requires other methods such as chromosome orientation in situ hybridization at single-chromosome resolution. This method has not been used in many studies yet to address potential NRTS of a small subset of the chromosomes. Therefore, there might be many more cell types that perform NRTS that have yet to be discovered. In summary, the work by Elabd et al. (2013) provides a robust method and model system of NRTS that will allow investigation of its molecular mechanisms and biological meaning.     

Control of neuronal excitability by GSK-3beta: Epilepsy and beyond

Glycogen synthase kinase 3beta (GSK-3β) is an enzyme with a variety of cellular functions in addition to the regulation of glycogen metabolism. In the central nervous system, different intracellular signaling pathways converge on GSK-3β through a cascade of phosphorylation events that ultimately control a broad range of neuronal functions in the development and adulthood. In mice, genetically removing or increasing GSK-3β cause distinct functional and structural neuronal phenotypes and consequently affect cognition. Precise control of GSK-3β activity is important for such processes as neuronal migration, development of neuronal morphology, synaptic plasticity, excitability, and gene expression. Altered GSK-3β activity contributes to aberrant plasticity within neuronal circuits leading to neurological, psychiatric disorders, and neurodegenerative diseases. Therapeutically targeting GSK-3β can restore the aberrant plasticity of neuronal networks at least in animal models of these diseases. Although the complete repertoire of GSK-3β neuronal substrates has not been defined, emerging evidence shows that different ion channels and their accessory proteins controlling excitability, neurotransmitter release, and synaptic transmission are regulated by GSK-3β, thereby supporting mechanisms of synaptic plasticity in cognition. Dysregulation of ion channel function by defective GSK-3β activity sustains abnormal excitability in the development of epilepsy and other GSK-3β-linked human diseases.     

Memory beyond the synapse

Based on studies of the molecular and cellular cascades that occur during memory consolidation for a one-trial passive-avoidance learning task in the young chick, I review the evidence that memory is encoded in permanent changes in synaptic connectivity ina specific brain region, the Hebb hypothesis. I conclude that despite the fact that such a cascade occurs, culminating in the synthesis of cell-adhesion molecules that are involved in synaptic remodelling, synaptic events are not in themselves sufficient to account for the phenomena of memory. Both whole brain (neuromodulator) and whole body (hormonal) processes are engaged.Memories are labile, disarticulated and stored in a distributed manner; how the mind/brain recreates coherent memories from this pattern is a mystery.     

Plasticity of dendritic excitability

Dendrites are equipped with a plethora of voltage-gated ion channels that greatly enrich the computational and storage capacity of neurons. The excitability of dendrites and dendritic function display plasticity under diverse circumstances such as neuromodulation, adaptation, learning and memory, trauma, or disorders. This adaptability arises from alterations in the biophysical properties or the expression levels of voltage-gated ion channels-induced by the activity of neurotransmitters, neuromodulators, and second-messenger cascades. In this review we discuss how this plasticity of dendritic excitability could alter information transfer and processing within dendrites, neurons, and neural networks under physiological and pathological conditions.     

Experience-driven brain plasticity: beyond the synapse

The brain is remarkably responsive to its interactions with the environment, and its morphology is altered by experience in measurable ways. Histological examination of the brains of animals exposed to either a complex ('enriched') environment or learning paradigm, compared with appropriate controls, has illuminated the nature of experience-induced morphological plasticity in the brain. For example, this research reveals that changes in synapse number and morphology are associated with learning and are stable, in that they persist well beyond the period of exposure to the learning experience. In addition, other components of the nervous system also respond to experience: oligodendrocytes and axonal myelination might also be permanently altered, whereas changes in astrocytes and cerebrovasculature are more transient and appear to be activity- rather than learning-driven. Thus, experience induces multiple forms of plasticity in the brain that are apparently regulated, at least in part, by independent mechanisms.     

Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron‐specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high‐content microscopy, and live‐imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP‐dependent protein kinase A (PKA)‐mediated phosphorylation of a synapse‐confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA‐R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA‐dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR‐dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.     

Calretinin: Modulator of neuronal excitability

Calretinin is a member of the calcium-binding protein EF-hand family first identified in the retina. As with the other 200-plus calcium-binding proteins, calretinin serves a range of cellular functions including intracellular calcium buffering, messenger targeting, and is involved in processes such as cell cycle arrest, and apoptosis. Calcium-binding proteins including calretinin are expressed differentially in neuronal subpopulations throughout the vertebrate and invertebrate nervous system and their expression has been used to selectively target specific cell types and isolate neuronal networks. More recent experiments have revealed that calretinin plays a crucial role in the modulation of intrinsic neuronal excitability and the induction of long-term potentiation (LTP). Furthermore, selective knockout of calretinin in mice produces disturbances of motor coordination and suggests a putative role for calretinin in the maintenance of calcium dynamics underlying motor adaptation.     

Nanoscale Remodeling of Functional Synaptic Vesicle Pools in Hebbian Plasticity

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Plasticity of neuronal receptors

This article describes ways in which receptors, key components of signal propagation through a synapse, can mediate changes in that propagation. Changes occur at four levels: in the signal-transducing capability of a single receptor molecule, in the number of receptors per cell, in the subcellular placement of receptor molecules, and in the cytoarchitecture of receptor-rich regions. The ability of receptors to shift between different desired states is called plasticity, and such shifts can be long-lived as well as transient. In this article we focus on neuronal receptors, although key findings from a variety of cell systems are reported. Neuronal receptor plasticity may have a special role in the assembly as well as the adaptability of the nervous system.     

Cyclin beyond the cell cycle: new partners at the synapse

In this issue of Developmental Cell, Odajima, Wills, and colleagues (2011) demonstrate that the cell-cycle regulator, cyclin E, sequesters Cdk5, a key regulator of neuronal development and synaptic plasticity. This cell-cycle-independent function of cyclin E reveals an exciting mode of Cdk5 regulation in postmitotic neurons and offers a window into evolutionary parsimony.     

Hebbian Plasticity Guides Maturation of Glutamate Receptor Fields In Vivo

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Outside and in: development of neuronal excitability

Investigation of the development of excitability has revealed that cells are often specialized at early stages to generate Ca(2+) transients. Studies of excitability have converged on the central role of Ca(2+) and K(+) channels in the plasmalemma that regulate Ca(2+) influx and have identified critical functions for receptor-activated channels in the endoplasmic reticulum that allow efflux of Ca(2+) from intracellular stores. The parallels between excitability in these two locations motivate future work, because comparison of their properties identifies shared attributes.     

Mathematical properties of neuronal TD-rules and differential Hebbian learning: a comparison

A confusingly wide variety of temporally asymmetric learning rules exists related to reinforcement learning and/or to spike-timing dependent plasticity, many of which look exceedingly similar, while displaying strongly different behavior. These rules often find their use in control tasks, for example in robotics and for this rigorous convergence and numerical stability is required. The goal of this article is to review these rules and compare them to provide a better overview over their different properties. Two main classes will be discussed: temporal difference (TD) rules and correlation based (differential hebbian) rules and some transition cases. In general we will focus on neuronal implementations with changeable synaptic weights and a time-continuous representation of activity. In a machine learning (non-neuronal) context, for TD-learning a solid mathematical theory has existed since several years. This can partly be transfered to a neuronal framework, too. On the other hand, only now a more complete theory has also emerged for differential Hebb rules. In general rules differ by their convergence conditions and their numerical stability, which can lead to very undesirable behavior, when wanting to apply them. For TD, convergence can be enforced with a certain output condition assuring that the δ-error drops on average to zero (output control). Correlation based rules, on the other hand, converge when one input drops to zero (input control). Temporally asymmetric learning rules treat situations where incoming stimuli follow each other in time. Thus, it is necessary to remember the first stimulus to be able to relate it to the later occurring second one. To this end different types of so-called eligibility traces are being used by these two different types of rules. This aspect leads again to different properties of TD and differential Hebbian learning as discussed here. Thus, this paper, while also presenting several novel mathematical results, is mainly meant to provide a road map through the different neuronally emulated temporal asymmetrical learning rules and their behavior to provide some guidance for possible applications.     

Clearance of glutamate inside the synapse and beyond.

The heated debate over the level of postsynaptic receptor occupancy by transmitter has not been extinguished - indeed, new evidence is fanning the flames. Recent experiments using two-photon microscopy suggest that the concentration of glutamate in the synaptic cleft does not attain levels previously suggested. In contrast, recordings from glial cells and studies of extrasynaptic receptor activation indicate that significant quantities of glutamate escape from the cleft following exocytosis. Determining the amount of glutamate efflux from the synaptic cleft and the distance it diffuses is critical to issues of synaptic specificity and the induction of synaptic plasticity.     

Amnesia and neglect: beyond the Delay-Brion system and the Hebb synapse.

Hippocampal damage in people causes impairments of episodic memory, but in rats it causes impairments of spatial learning. Experiments in macaque monkeys show that these two kinds of impairment are functionally similar to each other. After any lesion that interrupts the Delay-Brion system (hippocampus, fornix, mamillary bodies and anterior thalamus) monkeys are impaired in scene-specific memory, where an event takes place against a background that is specific to that event. Scene-specific memory in the monkey corresponds to human episodic memory, which is the memory of a unique event set in a particular scene, as opposed to scene-independent human knowledge, which is abstracted from many different scenes. However, interruption of the Delay-Brion system is not sufficient to explain all of the memory impairments that are seen in amnesic patients. To explain amnesia the specialized function of the hippocampus in scene memory needs to be considered alongside the other, qualitatively different functional specializations of other memory systems of the temporal lobe, including the perirhinal cortex and the amygdala. In all these specialized areas, however, including the hippocampus, there is no fundamental distinction between memory systems and perceptual systems. In explaining memory disorders in amnesia it is also important to consider them alongside the memory disorders of neglect patients. Neglect patients fail to represent in memory the side of the world that is contralateral to the current fixation point, in both short- and long-term memory retrieval. Neglect was produced experimentally by unilateral visual disconnection in the monkey, confirming the idea that visual memory retrieval is retinotopically organized; patients with unilateral medial temporal-lobe removals showed lateralized memory impairments for half-scenes in the visual hemifield contralateral to the removal. Thus, in scene-memory retrieval the Delay-Brion system contributes to the retrieval of visual memories into the retinotopically organized visual cortex. This scene memory interpretation of hippocampal function needs to be contrasted with the cognitive-map hypothesis. The cognitive-map model of hippocampal function shares some common assumptions with the Hebb-synapse model of association formation, and the Hebb-synapse model can be rejected on the basis of recent evidence that monkeys can form direct associations in memory between temporally discontiguous events. Our general conclusion is that the primate brain encompasses widespread and powerful memory mechanisms which will continue to be poorly understood if theory and experimentation continue to concentrate too much, as they have in the past, on the hippocampus and the Hebb synapse.     

Degeneracy in the nervous system: from neuronal excitability to neural coding

Degeneracy is ubiquitous across biological systems where structurally different elements can yield a similar outcome. Degeneracy is of particular interest in neuroscience too. On the one hand, degeneracy confers robustness to the nervous system and facilitates evolvability: Different elements provide a backup plan for the system in response to any perturbation or disturbance. On the other, a difficulty in the treatment of some neurological disorders such as chronic pain is explained in light of different elements all of which contribute to the pathological behavior of the system. Under these circumstances, targeting a specific element is ineffective because other elements can compensate for this modulation. Understanding degeneracy in the physiological context explains its beneficial role in the robustness of neural circuits. Likewise, understanding degeneracy in the pathological context opens new avenues of discovery to find more effective therapies.