It is remarkable that experience can modify neuronal function in a manner that is rapid and which can last for an entire lifetime as long-term memory. It is now well established that particular patterns of synaptic activity can give rise to alterations in synaptic strength, and these alterations (called LTP and LTD) are believed to underlie both memory storage and the activity-dependent fine-tuning of brain development. Both LTP and LTD have been shown to have late phases that require the synthesis of new proteins. Thus, synaptically-driven gene transcription is likely to be a key event in laying down long-term memories. There is general agreement that this process requires postsynaptic Ca influx. However, the details of how Ca influx is coupled to transcriptional events remain poorly understood. What are the spatial and temporal requirements for Ca signals to trigger transcription in neurons? Attempts to address this question have almost exclusively involved bath application of glutamate or high K to dissociated neuronal cultures. Given the nonphysiological nature of these stimuli, it is not surprising that conflicting results have emerged, with some investigators claiming a requirement for a Ca transient in the nucleus while others have reported that a Ca transient restricted to dendrites is sufficient. We will address this issue using a preparation that more closely resembles the intact brain. Here, we propose to stimulate glutamatergic synapses impinging upon dendrites of neurons in brain slices while measuring Ca concentration throughout the neuron at high resolution using multiphoton microscopy and simultaneously measuring both somatic membrane potential and transcription factor activity. The latter will involve both dynamic measurements using a CREB/CBP FRET system and posthoc analyses using high resolution in situ hybridization (CATFISH) and immunohistochemistry with phosphorylation-state specific antibodies. This analysis will be performed in two types of CNS neurons with different dendritic morphologies and firing properties. What are the critical nuclear targets for Ca triggered transcriptional events in neurons? Much attention has been directed towards the transcription factor CREB, to the exclusion of other potentially important targets. Cell culture experiments have indicated that the transcription factor SRF (Serum Response Factor) is robustly activated by Ca signaling in neurons. Moreover, many if not all activity-dependent IEGs contain binding sites for SRF and its associated factors. Thus we hypothesize that SRF is a key target of synaptically-driven Ca signals that supports initiation of new gene transcription. We propose to use novel spatial and temporal gene ablation techniques to address the necessity of SRF for the activation of a panel of neuronal immediate-early genes and in several forms of activity-dependent plasticity known to have transcription-dependent late phases (including hippocampal LTP and cerebellar LTD). Moreover, a novel small molecule SRF activator will be used to acutely induce SRF-dependent transcription in the absence of synaptic stimulation. The latter approach will be used to address the sufficiency of SRF-dependent transcription for long-term changes in synaptic efficacy and IEG transcription.
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