Dendritic spines on pyramidal neurons receive the vast majority of excitatory input and are considered electrobiochemical processing units, integrating and compartmentalizing synaptic input. Following synaptic plasticity, spines can undergo morphological plasticity, which possibly forms the structural basis for long-term changes in neuronal circuitry. Here, we demonstrate that spines on CA1 pyramidal neurons from organotypic slice cultures show bidirectional activity-dependent morphological plasticity. Using two-photon time-lapse microscopy, we observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, whereas theta burst stimulation led to the formation of new spines. Moreover, without stimulation the number of spine retractions was on the same order of magnitude as the stimulus-induced spine gain or loss. Finally, we found that the ability of neurons to eliminate spines in an activity-dependent manner decreased with developmental age. Taken together, our data show that hippocampal neurons can undergo bidirectional morphological plasticity; spines are formed and eliminated in an activity-dependent way.

The turnover of each protein in the mammalian proteome is a functionally important characteristic. Here, we employed high-resolution mass spectrometry to quantify protein dynamics in nondividing mammalian cells. The ratio of externally supplied versus endogenous amino acids to de novo protein synthesis was about 17:1. Using subsaturating SILAC labeling, we obtained accurate turnover rates of 4106 proteins in HeLa and 3528 proteins in C2C12 cells. Comparison of these human and mouse cell lines revealed a highly significant turnover correlation of protein orthologs and thus high species conservation. Functionally, we observed statistically significant trends for the turnover of phosphoproteins and gene ontology categories that showed extensive covariation between mouse and human. Likewise, the members of some protein complexes, such as the proteasome, have highly similar turnover rates. The high species conservation and the low complex variances thus imply great regulatory fine-tuning of protein turnover.

Neuronal processing is classically conceptualized as dendritic input, somatic integration, and axonal output. The axon initial segment, the proposed site of action potential generation, usually emanates directly from the soma. However, we found that axons of hippocampal pyramidal cells frequently derive from a basal dendrite rather than from the soma. This morphology is particularly enriched in central CA1, the principal hippocampal output area. Multiphoton glutamate uncaging revealed that input onto the axon-carrying dendrites (AcDs) was more efficient in eliciting action potential output than input onto regular basal dendrites. First, synaptic input onto AcDs generates action potentials with lower activation thresholds compared with regular dendrites. Second, AcDs are intrinsically more excitable, generating dendritic spikes with higher probability and greater strength. Thus, axon-carrying dendrites constitute a privileged channel for excitatory synaptic input in a subset of cortical pyramidal cells.

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility.