Talley J. Lambert

Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo.

Activity‐dependent changes in gene‐expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB‐regulated microRNA miR‐132. To determine if production of miR‐132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT‐PCR (RT‐qPCR). Pilocarpine‐induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR‐132 (pri‐miR‐132) followed by a subsequent rise in mature microRNA (miR‐132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor‐exposure, and cocaine‐injection, respectively, also increased pri‐miR‐132. Induction kinetics of pri‐miR‐132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature‐miR‐132 increased significantly between postnatal Days 10 and 24. We conclude that miR‐132 is an activity‐dependent microRNA in vivo, and may contribute to the long‐lasting proteomic changes required for experience‐dependent neuronal plasticity.

Different types of environmental enrichment have discrepant effects on spatial memory and synaptophysin levels in female mice

Environmental enrichment paradigms that incorporate cognitive stimulation, exercise, and motor learning benefit memory and synaptic plasticity across the rodent lifespan. However, the contribution each individual element of the enriched environment makes to enhancing memory and synaptic plasticity has yet to be delineated. Therefore, the current study tested the effects of three of these elements on memory and synaptic protein levels. Young female C57BL/6 mice were given 3h of daily exposure to either rodent toys (cognitive stimulation) or running wheels (exercise), or daily acrobatic training for 6 weeks prior to and throughout behavioral testing. Controls were group housed, but did not receive enrichment. Spatial working and reference memory were tested in a water-escape motivated radial arm maze. Levels of the presynaptic protein synaptophysin were then measured in frontoparietal cortex, hippocampus, striatum, and cerebellum. Exercise, but not cognitive stimulation or acrobat training, improved spatial working memory relative to controls, despite the fact that both exercise and cognitive stimulation increased synaptophysin levels in the neocortex and hippocampus. These data suggest that exercise alone is sufficient to improve working memory, and that enrichment-induced increases in synaptophysin levels may not be sufficient to improve working memory in young females. Spatial reference memory was unaffected by enrichment. Acrobat training had no effect on memory or synaptophysin levels, suggesting a minimal contribution of motor learning to the mnemonic and neuronal benefits of enrichment. These results provide the first evidence that different elements of the enriched environment have markedly distinct effects on spatial memory and synaptic alterations.

Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms

Overexpression of the amyloid precursor protein (APP) in hippocampal neurons leads to elevated β-amyloid peptide (Aβ) production and consequent depression of excitatory transmission. The precise mechanisms underlying APP-induced synaptic depression are poorly understood. Uncovering these mechanisms could provide insight into how neuronal function is compromised before cell death during the early stages of Alzheimers disease. Here we verify that APP up-regulation leads to depression of transmission in cultured hippocampal autapses; and we perform whole-cell recording, FM imaging, and immunocytochemistry to identify the specific mechanisms accounting for this depression. We find that APP overexpression leads to postsynaptic silencing through a selective reduction of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents. This effect is likely mediated by Aβ because expression of mutant APP incapable of producing Aβ did not depress transmission. In addition, although we eliminate presynaptic silencing as a mechanism underlying APP-mediated inhibition of transmission, we did observe an Aβ-induced presynaptic deficit in vesicle recycling with sustained stimulation. These findings demonstrate that APP elevation disrupts both presynaptic and postsynaptic compartments.

Age-dependent effects of environmental enrichment on spatial reference memory in male mice

Although environmental enrichment has been shown to improve various types of memory in young and aging mice, no study has directly compared the degree to which enrichment improves memory at different ages throughout the lifespan in male mice. Therefore, the present study investigated the effects of long-term continuous enrichment in young (3 months), middle-aged (15 months), and aged (21 months) male C57BL/6 mice. Spatial reference memory was tested in the Morris water maze. Results demonstrate that 24h/day environmental enrichment for approximately 6 weeks significantly improved spatial memory in the Morris water maze in aged males, but not in young or middle-aged males. These data also indicate that 24h exposure to complex enriched housing conditions increases the magnitude of enrichment-induced improvements in memory among aged mice relative to those previously reported by this lab and others.

MicroRNA132 Modulates Short-Term Synaptic Plasticity but Not Basal Release Probability in Hippocampal Neurons

MicroRNAs play important regulatory roles in a broad range of cellular processes including neuronal morphology and long-term synaptic plasticity. MicroRNA-132 (miR132) is a CREB-regulated miRNA that is induced by neuronal activity and neurotrophins, and plays a role in regulating neuronal morphology and cellular excitability. Little is known about the effects of miR132 expression on synaptic function. Here we show that overexpression of miR132 increases the paired-pulse ratio and decreases synaptic depression in cultured mouse hippocampal neurons without affecting the initial probability of neurotransmitter release, the calcium sensitivity of release, the amplitude of excitatory postsynaptic currents or the size of the readily releasable pool of synaptic vesicles. These findings are the first to demonstrate that microRNAs can regulate short-term plasticity in neurons.

A Genome-Wide Screen of CREB Occupancy Identifies the RhoA Inhibitors Par6C and Rnd3 as Regulators of BDNF-Induced Synaptogenesis

Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis.

Leptin Induces Hippocampal Synaptogenesis via CREB-Regulated MicroRNA-132 Suppression of p250GAP

Leptin acts in the hippocampus to enhance cognition and reduce depression and anxiety. Cognitive and emotional disorders are associated with abnormal hippocampal dendritic spine formation and synaptogenesis. Although leptin has been shown to induce synaptogenesis in the hypothalamus, its effects on hippocampal synaptogenesis and the mechanism(s) involved are not well understood. Here we show that leptin receptors (LepRs) are critical for hippocampal dendritic spine formation in vivo because db/db mice lacking the long form of the leptin receptor (LepRb) have reduced spine density on CA1 and CA3 neurons. Leptin promotes the formation of mature spines and functional glutamate synapses on hippocampal pyramidal neurons in both dissociated and slice cultures. These effects are blocked by short hairpin RNAs specifically targeting the LepRb and are absent in cultures from db/db mice. Activation of the LepR leads to cAMP response element-binding protein (CREB) phosphorylation and initiation of CREB-dependent transcription via the MAPK kinase/Erk pathway. Furthermore, both Mek/Erk and CREB activation are required for leptin-induced synaptogenesis. Leptin also increases expression of microRNA-132 (miR132), a well-known CREB target, which is also required for leptin-induced synaptogenesis. Last, leptin suppresses the expression of p250GAP, a miR132 target, and this suppression is obligatory for leptins effects as is the downstream target of p250GAP, Rac1. LepRs appear to be critical in vivo as db/db mice have lowered hippocampal miR132 levels and elevated p250GAP expression. In conclusion, we identify a novel signaling pathway by which leptin increases synaptogenesis through inducing CREB transcription and increasing microRNA-mediated suppression of p250GAP activity, thus removing a known inhibitor of Rac1-stimulated synaptogenesis.

Leptin-Induced Spine Formation Requires TrpC Channels and the CaM Kinase Cascade in the Hippocampus

Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and β-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, β-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptins induction of dendritic morphological changes that initiate spine and excitatory synapse formation.

Navigating challenges in the application of superresolution microscopy

In this review, Lambert and Waters focus on the current practical limitations of superresolution microscopy (SRM) and provide information and resources to help biologists navigate through common pitfalls when designing an SRM experiment.

Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop.

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation. DOI: http://dx.doi.org/10.7554/eLife.23437.001