The very detailed behavioral and physiological analyses of our mice with altered GluA1 expression revealed, for the first time, that AMPA receptor plasticity is also critically involved in hippocampus-based learning and memory, thus illuminating the endogenous regulation of AMPA receptors. Several studies from us and our collaborators showed, with no doubt, that AMPA receptors with the GluA1 subunit are not essential for spatial reference memory - the gold standard for spatial learning -, but are critically involved in behavioral tasks for the spatial working memory; a learning deficiency which we could correlate with lack of some forms of long-term potentiation at CA3-to-CA1 hippocampal connections (for review see: Sanderson et al., 2007).
Besides the deficiency in spatial working memory, we could show that also in some other learning paradigms relying on amygdala and thalamus the GluA1 subunit is needed for proper operation of the respective brain regions (Humeau et al., 2007, Engblom et al., 2008). As shown in collaboration with Dr. Rohini Kuner, GluA1 receptor-mediated plasticity modulates inflammatory pain as well (Hartmann et al 2004). Currently and in the next few years this traditional research program is continued, as can be noticed from the different funding resources of my group.
However, in order to shift the analysis of all our different mouse models with altered AMPA receptor expression to a new level, we need to establish and develop technologies which permit (a) a fast and reversible gene regulation and (b) an optical analysis of neuronal activity in the behaving mouse with long-term recording of population activity in the brain of mice or rats. First, the reversible gene regulation should allow us to monitor the function of the controlled gene in learning behavior when the gene is in the "ON" or "OFF" state in the very same mouse. Second, the simultaneous optical recording of neurons will reveal the ongoing changes in neuronal activity during and after learning in our different mouse mutants.
Both goals are tremendously challenging. But our experience over the last years and the infrastructure of the Max Planck Institute of Medical Research will support this endeavor.
The successful achievement of both goals is absolutely necessary for the molecular understanding of specific neuronal circuits in complex brain functions.
There are a few pioneering studies from us an others, demonstrating that the doxycycline-controlled reversible gene regulation can be used in behavioral research (Mansuy et al., 1998, Reijmers et al., 2007, Mack et al., 2001). Yet, the epigenetic silencing of the doxycycline-controlled gene was a major difficulty with the current technology (Zhu et al., 2007). Recently, we showed a faithful, efficient co-expression of several proteins in neurons (Tang et al., 2009).
This possibility of efficient, multiple gene transfer opens attractive options to get around the silencing problem of transcriptionally inactive genes and permits the combination of several regulatory genes for fast reversible gene expression. The co-expression of counteractive doxycycline-regulated activators and silencers will be, therefore, investigated in great detail.
For the fast analysis of the different approaches, the rAAV technology is the best tool, since several transduced gene cassettes can be tested side by side in primary cultures, organotypic cultures and, finally, in the mouse (for review see Osten et al., 2007).
Monitoring the neuronal activity
In collaboration with Haydon-Wallace et al., 2008, using one of the many and novel fluorescent Ca2+ indicator proteins.from (see report by Dr. Mazahir Hasan) we have access to the world’s leading technology of optogentic tools, and we feel confident that optical analysis in behaving mice will be possible in the near future, as indicated by the studies of
AMPA receptor regulators
In addition to our conditional mouse models for AMPA and NMDA receptor 'knockouts' we will investigate a series of new endogenous proteins regulating AMPA receptor transmission. Besides the TARPs, new proteins with a strong effect on the function of the AMPA receptor complex appear on the horizon. In particular, the reversible over-expression or inactivation of these AMPAR regulating proteins during learning tasks will be investigated together with changes of neuronal activity and learning behavoiur.
We hope that our future work will provide novel insights into the function of AMPA receptors in specific neuronal circuits during learning together with solid, new methodology useful for the analysis of genes in neuronal circuit activity during higher brain functions. Needless to say that our knowledge of anatomical and physiological studies will assists our efforts, and that the contribution of motivated students of the Universities of Heidelberg and Mannheim play a active part in this adventure.
If sufficient resources are provided by the Max Planck Institute for Medical Research, we will include gene manipulation in rat ES cells and new approaches for gene targeting, using Zink-finger proteins, in our research projects.