Dopaminergic stimulation of local protein synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal neurons.

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.

Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.

In vivo imaging has revolutionized our understanding of biological processes in brain physiology and pathology. However, breathing-induced movement artifacts have impeded the application of this powerful tool in studies of the living spinal cord. Here we describe in detail a method to image stably and repetitively, using two-photon microscopy, the living spinal tissue in mice with dense fluorescent cells or axons, without the need for animal intubation or image post-processing. This simplified technique can greatly expand the application of in vivo imaging to study spinal cord injury, regeneration, physiology and disease.

Measuring dendritic distribution of membrane proteins.

Neurons perform much of their integrative work in the dendritic tree, and spinal motoneurons have the largest tree of any cell. Electrical excitability is strongly influenced by dendrite membrane properties, which are difficult to measure directly. We describe a method to measure the distribution of ion channel membrane densities along dendritic trajectories. The method combines standard immunohistochemistry with reconstruction procedures for both large-scale and small-scale optical microscopy. Software written for Matlab then extracts the colocalization of the target ion channel with the target dye injected cell, and calculates the relative channel density per square micron of cell surface area, as a function of distance from the cell body. The technique can be used to quantify the localization and distribution of any immunoreactive moiety, and the software provides a flexible vehicle for sensitivity analysis, to validate heuristics for selecting thresholds.

Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy.

Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of alpha-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis.