Final Report Summary - RAPHE (Cell-type specific features of serotonergic neurons in the raphe nuclei) The focus of the proposal was on identifying the cell-type specific features of serotonergic neurons in the brainstem raphé nuclei (RN) using channelrhodopsin (ChR2)-mediated photostimulations (PS) of 5-HT neurons in behaving rats. 8 months after the start of the Intra-European (IEF) Fellowship I decided to switch to another postdoctoral fellowship, therefore the results of the final report will be similar to those in the midterm report. After the initial success with viral delivery of channelrhodopsin (ChR2) to the raphe nucleus 5-HT neurons in rats using a serotonergic cell specific promoter (Pet1) we realised, that the expression levels are not high enough to induce action potentials with photostimulation in a reliable way. We performed extensive extracellular unit recordings in rats previously infected with ChR2 and the number of cells responding to photostimulation was very low (2 neurons out of 109, 1.8 %). We then quantified the intensity of light needed for action potential generation in brain slices in vitro and found that the overall thresholds for action potential generation are high and variable (mean=43.77±37.98 range: 2.7-97.0 mW / mm2, n=20 neurons). Our explanation for the observed results is that the ChR2 expression driven by the Pet1 promoter is not strong enough to lead to reliable action potential generation following low intensity photostimulation in 5-HT neurons. To overcome these limitations we genetically targeted raphe nucleus 5-HT neurons by stereotaxically injecting a Cre-recombinase-dependent adeno-associated virus (rAAV)2.1 into knockin mice that selectively expressed Cre in serotonin transporter-expressing neurons. Our results show that the expression is more robust in SERT-CRE mice compared to rats. The efficiency of the PS was verified using electrophysiological recordings. Recordings were performed in acute slices, where pulses of light were able to elicit spiking in ChR2-YFP positive cells and intensity of light required for action potential generation in raphe slices in vitro is more than an order of magnitude lower than in rats and very consistent throughout multiple cells from different mice (mean=2.3±1.4 range: 1.1-6.0 mW / mm2, n=17 neurons). In anesthetised mice we were able to induce action potentials with short light pulses in mice previously infected with DIO-ChR2 in about 30 % (13 out of 40 neurons, 32 %) of the cells recorded, consistent with the percentage of 5-HT neurons in the raphe nucleus. Thus, it is possible to identify ChR2 expressing 5-HT neurons during extracellular recordings in vivo by their positive responses to brief pulses of light. Behavioural effects of 5-HT PS: SERT-Cre mice previously infected with ChR2 in DRN 5-HT neurons were implanted with a fibreoptic cannula for PS and electroencephalography (EEG) electrodes. 5-HT PS changed the behavioural state of 3/3 animals tested in the following way: when the animals were in slow-wave sleep (SWS, evident from the EEG) a short PS (5-15 s train of 10 ms long light pulses of 5mW power at 30 Hz) was sufficient to awaken the animals. This was evident at both behavioural and electrographic levels: animals started walking in the cage while the EEG slow waves (characteristic of SWS) were substituted by faster and lower amplitude waves characteristic of wakefulness.
