The Konnerth group will further improve the multiphoton imaging approach of neuronal networks in the ‘in vivo’ mouse brain. A recent implementation of a newly designed pre-chirping unit helped to increase the depth penetration into the cortical tissue by nearly 100% (from 300 to 600 m). In addition, they will develop an optic fiber-based microendoscopic fluorescence monitoring system that will allow the detection of FRET signals in the intact brain of behaving mice. A prototype of such a device is currently tested in a project investigating the development of the auditory system. A major goal of the lab is the analysis of second messenger dynamics in behaving mice. Therefore, they will establish the Troponin C-based ‘in vivo’ Ca imaging of dendritic function. Further, they began to implement, in collaboration with Dr. Roger Tsien’s group, UC San Diego, USA, a FRET-based cAMP sensor (AKAR 3) for the analysis of brain function in behaving mice. Furthermore, they will develop a targeted electroporation-based method for the delivery of quantum dots to individual neurons. This will give great improvement of the sensitivity, allowing a detailed analysis of the morphological changes of neuronal processes in living brain tissue. The developments will be integrated into the life cell imaging efforts of the groups Bräuchle (area A) and Cremer/Leonhardt (area D).

The Hofmann group will modify the CaM-binding site (IQ motive) at the cytosolic C-terminus of the Cav1.2 protein. The modification of the Cav1.2 gene is associated with embryonic lethality. Therefore, he will introduce a tissue-specific gene modification. He expects reduced spatial learning in the modified animals, if all predictions are correct. As a further and additional mouse line, he is planning to inactivate the putative cAMP kinase site at Ser-1928. These modifications will allow investigating the biological effects of cAMP-dependent phosphorylation of the neuronal L-type Ca2+ channel. The animals will be studied with an array of high-resolution procedures including electrophysiology, learning tests, transcription of Cre dependent genes, real-time detection of phosphorylation of CREB and ERK, and [Ca2+]i (together with the group of Konnerth).

The Haass group is highly interested in a structural analysis of the -secretase complex. This would allow CIPS^M to understand how substrates are selected and where they bind within the complex, how water molecules can penetrate the membrane, and finally which side chains are directly involved in the catalytic activity. In addition important conclusions about the function of Aph-1, Pen-2, and Nicastrin could be made. Such knowledge would dramatically help to understand the normal function of a major disease related enzyme complex. By investigating mutant variants (complexes containing presenilin mutants) insights in the dysfunction will be obtained. This example holds true for all other diseases associated proteins including those associated with PD. Moreover, upon obtaining all this information on protein structure, function, and dysfunction novel domains for therapeutic targets can be identified. An example would be the identification of the binding side of -secretase modifiers and their influence on the overall structural changes of the complex. In addition the Haass Group will conduct a series of projects to understand the in vivo mechanisms leading to neuronal cell death and memory loss in AD.

The Götz group found clear evidence that Pax6 acts as a key neurogenic determinant sufficient to instruct neurogenesis from glial cells in the adult brain after injury. Given this potent function, they now want to understand the molecular basis of Pax6 function in regard to eliciting the neurogenic program. The final aim would be to regulate the neurogenic program elicited by Pax6 by substances easy to apply for therapy (such as small molecules). Towards this aim, they have identified that one of the two DNA-binding domains of Pax6 (the paired domain) exerts all effects in neurogenesis and cell proliferation, while the homeodomain seems not to play any role in this program. However, Pax6 – as most transcription factors – are known to act in a cell-type specific context – interacting with other proteins within a transcriptional network. Indeed, Pax6 function in the retina is rather different where it maintains stem cell identity and inhibits neuronal differentiation. They could already show that the homeodomain plays a crucial role in these processes. Viral constructs containing Pax6 forms with either only the paired or only the homedomain will allow to address the differential function of these domains in different cellular and transcriptional contexts. The function of different Pax6 genes expressed in distinct domains of adult neurogenesis in the zebrafish brain will be elucidated in collaboration with the Bally-Cuif group. Moreover the molecular basis of a novel role of Pax6 in inhibiting neuronal cell death will be examined in collaboration with the Haass lab. Transcriptional networks determining neuronal specificity (GABAergic, glutamatergic, serotonergic, dopaminergic cells) will be studied in collaboration with the Cramer group.

The Biel group is highly interested in understanding the molecular signaling pathways leading to apoptotic cell death in the retina of CNGA3 and CNGB1-deficient mice. To address this problem expression profiling studies complemented with proteomic approaches will be performed in the retina of wild type and knockout mice. These studies will be complemented by rescue experiments aiming at introducing wild type and mutant CNG channels in the murine retina using lentiviral vectors. The studies have potential clinical relevance given that photoreceptor degeneration is a major cause of legal blindness and is a process that so far can be only poorly, if at all, slowed down. To further advance the knowledge of HCN channel function in vivo HCN channels will be mutated using knockin-targeting approaches. An example for these animal models are mouse lines with point mutations in the cAMP binding domain of either the HCN2 or the HCN4 gene. The functional analysis of such mouse lines is expected to clarify the specific contribution of cAMP modulation of HCN channels for the functionality of neural circuits.

Recent work by the Grothe group showed that interaural time differences are encoded in the medial superior olive (MSO) by precisely timed integration of inhibitory and excitatory inputs. Since time differences are within the sub-millisecond time range weak interferences with firing patterns are sought to be sufficient to change MSO output firing. His group will now interfere with the fire properties of the principal neurons in the medial nucleus of the trapezoid body (MNTB) and with the post-synaptic receptor composition and distribution to alter the timing and the efficacy between inhibitory and excitatory inputs to the MSO in a predictable way. One approach will be the manipulation of hyperpolarization activated channels (Ih) in collaboration with the Biel group.

The groups of Klein and Carell (area E) develop new voltage sensitive dyes that are non-toxic and have a large fluorescence wavelength shift of about 20 nm and a signal increase (threefold) upon depolarization. The goal is to use these dyes in order to study depolarization of neurons in whole tissues. The dyes will be optimized so that only certain types of neurons get stained in the tissue.