Kontakt

Dr.
Sidney Cambridge

Group Leader


Anatomie und Zellbiologie
Im Neuenheimer Feld 307
69120 Heidelberg
Tel. 06221 – 54 8671
Fax 06221 – 54 4952
Email


Gruppe Cambridge

Research Summary

Cellular Neurobiochemistry & Optogenetics

There are two main research areas in the group. First, in a very interdisciplinary approach we developed a new method called ‘photoactivated gene expression’ that allows high resolution induction of transgene expression upon irradiation with one or two-photon light. Based on the inducible Tet system and a caged, i.e. light-sensitive, analog of doxycycline we can induce transgene expression in any cell at any time. We are beginning to use this method in vivo to silence neuronal activity in single cells and to study its effects on network plasticity and maintenance. The second research area is based on quantitative mass spectrometry to characterize synaptic proteomes from different tissues, stages, following induction of plasticity, or to analyze synaptic protein turnover. Interesting candidate proteins are being biologically validated by GFP-tagging and live imaging, siRNA knock-downs, and electrophysiological recordings.


Group Leader

Dr. Sidney Cambridge

Group Members

Terzi, Firat (Ph.D. student)

Eric Brandhorst (M.D. student)

Krämer, Gabriele (Technical Assistant)



Current Projects

Photoactivated Gene Expression

Regulated neuronal network activities are fundamental to proper brain function. To maintain network functionality at steady state and under acute changes of synaptic input, there must be exquisite homeostatic mechanisms at the level of single cells, small groups of cells, and large neuronal ensembles. To induce and then characterize physiological changes upon genetic manipulation of neurons, we developed a method that allows targeted transgene expression in single cells by irradiation with UV or 2-photon light so that the manipulated neuron can be analyzed before, during, and after gene expression. This photoactivated gene expression method is based on the inducible Tetracycline system and the reversible inhibition of tetracycline analogs with photosensitive protection compounds (“caging”) (Cambridge et al 2006, Angewandte Chemie). The feasibility of this approach was demonstrated in several different eukaryotic systems including plant leafs, brain slices, and living Xenopus tadpoles (Cambridge et al. 2009, Nature Methods). High-resolution 2-photon mediated photoactivated transgene expression was also possible. Being able to control gene expression with light is a key missing dimension in the field of neuroscience as researchers are using light to study neuronal morphology and to regulate neuronal activity. We are now in the process of establishing in vivo photoactivated gene expression to allow the targeted manipulation of neurons in defined neuronal networks.

Quantitative mass spectrometry of synaptic proteomes


The remarkable adaptive and plastic capabilities of the human brain are important for higher cognitive functions such as learning and memory. The neuronal changes that accompany synaptic plasticity and long-term potentiation (LTP), a process that is thought to form the basis of learning and memory, are often electrophysiologically well described. There is, however, surprisingly little knowledge about the underlying synaptic molecular events. One major problem is that synapses are comprised of at least two thousand proteins while being highly dynamic, highly diverse, and highly interconnected. We used in-depth, high-resolution mass spectrometry to quantitatively compare synaptic proteomes in more detail. By applying a stringent biochemical purification protocol for synapses, we routinely obtained high accuracy proteomes of about 2000 proteins.  Advanced mass spectrometry tools allowed quantitative comparison of protein abundance from two different synaptic preparations and thus identified these proteins that changed upon stimulation, between different tissues, or under various conditions. This unprecedented pool of differentially abundant synaptic proteins is now the foundation of our continuing research efforts as we try to identify and validate protein candidates that are key mediators of synaptic function, signaling, and plasticity. We are using standard procedures to analyze promising candidates further including the use of GFP-tagged versions for live imaging, siRNA knock-downs, and electrophysiological recordings to monitor any changes in synaptic currents.

Masters Project

 

 

 

 

 

 

Verantwortlich: E-Mail,   Letzte Änderung: Thu, 12.05.2016
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