Generation of mouse mutants
Generation of mouse mutants via gene manipulation in embryonic stem cells for the regulated expression of sequence-altered subunits for ionotropic glutamate receptors of the AMPA, NMDA and kainate subtypes. Sequence changes comprise functional ablation, green fluorescent protein (GFP)-fusion, mutations in the inner channel lining, mutations affecting gating kinetics and desensitization, C-terminal alterations affecting trafficking to synapses and interaction with components of intracellular signaling cascades. Expression regulation is attempted by the use of newly engineered versions of the Cre system and of tetracycline-mediated transcriptional activation/repression. Aims are to study molecular mechanisms at the postsynaptic side underlying fast excitatory synaptic transmission and activity-modulated synaptic strength.
Studies of the physiological relevance of RNA editing by adenosine deamination
These studies include the conditional ablation in mice of the three known mammalian RNA dependent adenosine deaminases, ADAR1-3, which are currently the only candidate enzymes for RNA editing by adenosine deamination. ADAR2 edits in vitro the Q/R site of GluR-B pre-mRNA and thus ensures the low permeability to calcium of hetero-oligomeric AMPA receptor channels configured with this edited subunit. Interference with Q/R site editing of GluR-B, either by an appropriate sequence change in the GluR-B gene or by ADAR2 ablation leads in the mouse to a prematurely lethal, seizure-prone phenotype. No obvious phenotypical consequences arise from ADAR2 ablation if within the GluR-B gene the edited codon is exchanged for the unedited one. This demonstrates that of all the possible sites edited by ADAR2 (the enzyme is found in many tissues), the Q/R site in the GluR-B transcript is physiologically the most important one.
Gene expression in, and pulsatile activity of, hypothalamic gonadotrophin releasing hormone (GnRH) producing neurons in transgenic mice expressing GFP in these cells
GnRH cells project their axons to the median eminence where they release GnRH synchronously every 20 minutes (rodent) into the hypothalamic-hypophyseal portal blood circulation. Origin and mechanism of the pulse generator for the pulsatile activity of this small (800 in the rodent) and scattered population of neurosecretory cells are unknown and constitute the focus of this project.
Function and physiology of cells expressing GnRHII
Function and physiology of cells expressing GnRHII, a close relative of GnRH, but sequence-conserved across vertebrate evolution.
The molecular mechanism for the activity dependent generation of the immediate early gene homer1a
The homer 1 gene products serve to link (constitutive products), or sever (immediate early form) the link, between components mediating calcium-induced calcium release in neurons. The constitutively expressed homer forms and the activity-dependent homer version originate from the same gene, but are probably not generated by alternative transcript splicing.