The cytochrome c gene proximal enhancer drives activity-dependent reporter gene expression in hippocampal neurons

The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP+ neurons, while the frequency of mEPSCs was not significantly different. Increasing neural activity by applying a GABAA receptor antagonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity

Rapid homeostatic downregulation of LTP by extrasynaptic GluN2B receptors

Although the activation of extrasynaptic GluN2B-containing N-methyl-D-aspartate (NMDA) receptors has been implicated in neurodegenerative diseases, such as Alzheimer's and Huntington's disease, their physiological function remains unknown. In this study, we found that extrasynaptic GluN2B receptors play a homeostatic role by antagonizing long-term potentiation (LTP) induction under conditions of prolonged synaptic stimulation. In particular, we have previously found that brief theta-pulse stimulation (5 Hz for 30 s) triggers robust LTP, whereas longer stimulation times (5 Hz for 3 min) have no effect on basal synaptic transmission in the hippocampal CA1 region. Here, we show that prolonged stimulation blocked LTP by activating extrasynaptic GluN2B receptors via glutamate spillover. In addition, we found that this homeostatic mechanism was absent in slices from the SAP102 knockout, providing evidence for a functional coupling between extrasynaptic GluN2B and the SAP102 scaffold protein. In conclusion, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction via the activation of extrasynaptic GluN2B-containing NMDA receptors.NEW & NOTEWORTHY Although long-term potentiation (LTP) is an attractive model for memory storage, it tends to destabilize neuronal circuits because it drives synapses toward a maximum value. Unless opposed by homeostatic mechanisms operating through negative feedback rules, cumulative LTP could render synapses unable to encode additional information. In this study, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction under conditions of prolonged synaptic stimulation via the activation of an extrasynaptic GluN2B-SAP102 complex

Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies

Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system

The Homeobox Transcription Factor Barx2 Regulates Plasticity of Young Primary Myofibers

Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation

A Revised View on the Role of Surface AMPAR Mobility in Tuning Synaptic Transmission: Limitations, Tools, and Alternative Views

Calcium dynamics in presynaptic terminals regulate the response dynamics of most central excitatory synapses. However, this dogma has been challenged by the hypothesis that mobility of the postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPAR) plays a role in tuning fast excitatory synaptic transmission. In this review, we reevaluate the factors regulating postsynaptic AMPAR mobility, reassess the modeling parameters, analyze the experimental tools, and end by providing alternative ideas stemming from recent results. In particular, newer methods of labeling AMPARs with small fluorophores in live neurons, combined with super-resolution microscopy and sub-second dynamics, lends support to the idea that AMPARs are primarily within the synapse, are greatly constrained, and have much slower mobility than previously thought. We discuss new experiments which may be necessary to readdress the role of postsynaptic AMPAR mobility in tuning fast excitatory synaptic transmission

Differentiating myoblasts and maturating myotubes have different levels of Barx2 expression.

<p><b>A.</b> Cultured primary myoblasts were induced to differentiate by serum withdrawal and changes in cell shape, actin remodeling and myogenin expression were monitored over the first 6 hours. <b>B–D.</b> Myotube maturation was examined between 24 and 72 hours post serum-withdrawal. At 24 hours myotubes were thin and appeared immature (<b>B</b>). Between 48 and 72 hours, thicker myotubes appeared often with local aggregations of nuclei (<b>C</b> and <b>D</b>) and frequent strong contractions. <b>E.</b> Observations of myotube maturation in culture presented schematically. <b>F.</b> Barx2 expression was measured by RT-PCR at different stages of differentiation. Scale bars represent (A–C) - 20 µm, D–50 µm.</p