10:15am - 10:30am
Ablation of the RyR2-Ser2030 Phosphorylation Site Limits Changes in RyR2 Sensitivity During ß-Adrenergic Stimulation
Department of Physiology, University of Bern, Switzerland
During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (AR) activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2. Hyperphosphorylation by PKA at the RyR2-S2808 site has been proposed as a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Recently, a new PKA phosphorylation site has been identified and proposed as a mediator of the adrenergic response, S2030. We examined the contribution of RyR2-S2030 to the excitation contraction (EC)-coupling mechanism using experimental approaches on cellular and subcellular levels and a transgenic mouse with ablated RyR2-S2030 phosphorylation site (RyR2-S2030A). EC-coupling gain was assessed with the whole-cell patch-clamp technique and confocal Ca2+ imaging while the ß-ARs were stimulated with Isoprotenerol (Iso). At matched Ca2+ loading of the sarcoplasmic reticulum (SR), the EC-coupling gain in Iso was diminished in mutant compared to WT cardiomyocytes. In addition, Ca2+ waves elicited by SR Ca2+ overloading showed no acceleration during Iso treatment, unlike WT cells. Recent studies have suggested that Ca2+ spark refractoriness depends on the sensitivity of the ryanodine receptors. Moreover, it was demonstrated that adrenergic stimulation shortens RyRs refractoriness. We measured Ca2+ spark restitution using the “low-dose ryanodine method” in which repetitive sparks originating from the same cluster of RyRs are recorded. We found that mutant RyRs sensitivity was not enhanced by Iso application, contrary to WT cells. Together, our results suggest that ablation of the RyR2-S2030 site may result in a blunted increase of RyR2 Ca2+ sensitivity upon ß-adrenergic stimulation, and that the site represents a link between the adrenergic pathway and modulation of RyR2 channel activity.
10:30am - 10:45am
Culture of Lgr5-Positive Cells from the Early Postnatal Murine Retina
1Department of Ophthalmology, Inselspital, Bern University Hospital, Switzerland; 2Department for BioMedical Research, University of Bern, Switzerland; 3Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland; 4Inner Ear Research Laboratory, Department of Otorhinolaryngology, Head & Neck Surgery, Inselspital, Bern University Hospital, Switzerland
Recently it has been shown that the retina harbors a cell population which expresses the Leucine-rich repeat containing G-protein receptor 5 (Lgr5). This marker was identified as an adult stem cells marker in the intestine and was found to be expressed in several other adult SC populations of epithelial tissues. In the retina Lgr5 is expressed in glycinergic amacrine interneurons. Even though Lgr5-postive amacrine cells demonstrate properties of differentiated interneurons they also contribute to the generation of new retinal cells in adult animals. Therefore, we would like to investigate if Lgr5-postive cells can be cultured and expanded in vitro and whether they can differentiate into different retinal cell types.
For the experiments Lgr5EGFP-Ires-CreERT2 knock-in mice were used. In a first step whole retinae from P1 and P5 animals were cultured as spheres in low adherence culture plates. In the neonatal mouse retina Lgr5 can be detected from P4. In accordance with this finding no GFP-positive cells were initially found in P1 cultures. Yet after 8 days in culture GFP expression could be detected. Immunohistochemistry performed on 3rd generation spheres showed that not all Lgr5-positive cells expressed Syntaxin, a marker of mature amacrine cells. Furthermore, Lgr5-positive cells from P5 retinae were sorted by FACS and cultured as spheres. However, the cells did not proliferate under the chosen culture conditions.
These preliminary results demonstrate that Lgr5-positive cells can be cultured in vitro, albeit no proliferation was seen. Therefore, in a next step different culture conditions will be assessed. Furthermore, Syntaxin-negative Lgr5-cells will be further investigated to analyze whether these cells correspond to a progenitor-like cell type.
10:45am - 11:00am
Predictive Plasticity in Dendrites: From a Computational Principle to Experimental Data
1Department of Physiology, University of Bern, Switzerland; 2Computational Neuroscience Laboratory, Imperial College London, UK
Plasticity of excitatory cortical synapses is thought to be the main mediator of mammalian learning.
Due to the selective advantage that the ability to adapt to a changing environment grants, it is reasonable to assume that the processes governing these changes in connection strength are in some sense optimal. Yet, this has been difficult to reconcile with an «embarrassment of riches» of the LTP and LTD phenomenology. Here, we present an attempt at bridging this gap by showing that a mathematically derived model can exhibit some of these experimentally observed effects, while still retaining functional capabilities under diverse learning paradigms.
Our work is based on a published plasticity model that postulates that learning is driven by an intraneuronal prediction error where the weights of «student inputs» onto a dendritic compartment change in order to reproduce voltage changes imposed on a somatic compartment by «teacher inputs».
We show here that this two-compartment model of a pyramidal neuron can be extended in some simple ways, such as using conductance-based instead of current-based inputs, bringing it closer to the biophysics of pyramidal neurons. This allows us to reproduce a diverse set of experimental observations on cortical plasticity, such as different characteristics of the spike-timing dependence of plasticity.
Additionally, we show within a simple setup of a pattern recognition task that the extended model, while being less analytically tractable, can still perform well under unsupervised and reinforcement learning paradigms. Therefore, a single learning rule derived from the optimization of a well-defined cost function can be brought into correspondence with a large body of experimental evidence on synaptic plasticity, while still providing a diverse set of relevant functionality.
11:00am - 11:15am
Value Learning in Drosophila Explain Odor Conditioning
1Department of Physiology, University of Bern, Switzerland; 2Zoology Department, University of Fribourg, Switzerland
Associative learning in the fruit fly olfactory system has been studied in many aspects from molecular level to behavior level. Fruit flies are able to associate conditional stimulus such as odor with unconditional aversive stimulus such as electrical shock, or appetitive stimulus such as sugar or water. Here, we propose a model with value learning rule to explain the mechanism underlying associative learning. In our model, the unconditional stimulus acts as a teaching signal, synaptic weight is updated to represent the predict value of the conditional stimulus.
With this model, we can reproduce the data from different experiment protocols using the same set of parameters. The next question is, if the fruit fly do can learn values, where that could happen in its brain. Previous studies suggest that the mushroom body is crucial for associative learning, the synaptic connections between Kenyon cells and mushroom body output neurons are considered to be where the learning is taken place. Thus, we look closely into the anatomy of the mushroom body, and try to justify that our model can physically be imbedded in the connections between the different types of neurons in the mushroom body.