During the first reporting period, we have established the methods to obtain the data that we proposed to obtain. Since most of these methods are non-standard, this involved development of custom equipment, which by itself warrants methods publications. Thus far, we have built and tested the behaviour setups, the data acquisition equipment, and the two-photon microscope. Beside establishing all the methods, we have obtained a significant amount of data towards fulfilling our objectives. These data are currently being analysed. During the first reporting period, 5 our of 6 aims and sub-aims were launched. Note that because all aims and sub-aims span 3 years, these also span two reporting periods, so none of these objectives are yet finished.
Aim1-Question1. Are distinct 5HT3a cells also functionally diverse?
Towards this aim, we have build the behavioral apparatus, the electrophysiology and a two-photon imaging microscope. All these are now functional. With electrophysiological methods and two photon microscopy we have already obtained the first data sets of 5HT3a neurons. Interestingly during the mouse whisker behavior, 5HT3a neurons were clearly diverse. We currently are investigating whether cellular responses can be clustered in sub-groups using statistical clustering methods or whether the diversity of 5HT3a neurons lays on a continuum.
Aim1-Question2 How does the activity of 5HT3a cells correlate with the activity of pyramidal cell dendrites?
In a subset of experiments, we also have imaged the Ca2+ changes in the dendrites of pyramidal neurons (the tuft dendrites). Some of these were clearly, co-modulated with the activity of 5HT3a neurons and VIP cells specifically. The analysis of these data is currently ongoing.
Aim2-Question1. How do single 5HT3a cells affect dendritic activity in pyramidal neurons, and behavior?
While we originally suggested to use single-neuron juxtacellular recordings to manipulate the activity of single neurons, we have instead used single-neuron electroporation of plasmids that encode an opsin, thus making single neurons sensitive to light. Currently this setup is built and we are performing the first light-manipulation experiments to see how the activity of these cells affects the dendritic integration in the tuft dendrites of pyramidal neurons.
Aim2-Question2. How does silencing or activation of only VIP- versus all 5HT3a cells affect network activity and behavior?
So far we have built the setup for doing these experiments and we will soon begin to perform these experiments.
Aim2-Question3. How do layer-1 5HT3a neurons affect behavior?
As in the previous aim and sub-question, here we have so far only built the apparatus for doing the experiments but the data is incomplete.
We expect that the work carried out so far will result in at least 3 publications. Two papers are currently being written up in a manuscript. The first, is a methods paper about the microscope that we developed. The second, is also a methods paper, outlining a new method to measure sub-microm movements of electrodes. This work is a corollary of the methods that we had to develop. The third publication will take longer but will concern the diversity of 5HT3a neurons in the somatosensory cortex.
