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Imaging Interneurons in Epilepsy

Final Report Summary - IMAGINE (Imaging Interneurons in Epilepsy)

The research project revolves around the three-dimensional imaging of interneurons in the hippocampus under different conditions. I will summarize the work which already been published in three publications in prestigious journals (Chiovini et al, 2014 Neuron; Williamson, Kaszas et al., 2015 Advanced Materials, Deneux et al., 2016 Nature Communications).
As a beginning, I have been working on different types of interneurons, including fast-spiking parvalbumin-positive (FS-PV) and somatostatin-positive (SOM) interneurons. The work has been started on FS-PV interneurons, and many of the Objectives have been fulfilled concerning their dendritic properties.
Objective 1 & 2 deals with the characterization of the active properties of dendrites and the action potentials (AP) in interneurons, and we have shown how the AP-evoked Calcium influx propagates all through the dendritic arbour of the neuron. The propagation has been observed under silent conditions of the surrounding neuronal network, as described in Objective 1. Furthermore, these neurons have been observed in active neuronal networks, also. When examining interneurons in complete hippocampal preparations, the neuronal network was showing rhythmic activity that shows many similarities to sharp-wave ripples (SPW-R). I have taken part in the testing of a novel organic electrochemical transistor (OECT) device that I was using to stimulate activity in the imaged neuronal population. The same device is also capable of recording, which was useful as a second verification of the oscillations besides imaging, by electrophysiology.
Our results showed that oscillations have a major impact on the dendritic arbor. In particular, as stated in Objective 2, we have defined the active properties of dendrites using 3D calcium imaging, and determined that even when the neuron is not firing action potentials, activity hot spots appear on the dendrite in correlation with the SPW-Rs. To answer the questions posed in Objective 3 regarding synaptic integration during physiological oscillations, we have also observed the same dendrites during action potentials, and saw that the Calcium responses are summating supralinearly when the neuron fires an AP synchronously to the SPW-Rs.
Following these single cell patch-clamp recordings, in Objectives 3 & 4 the aim was posed to study network oscillations. As such, I have recorded the activity of many cells in the hippocampus in 3D, using multicell bulk loading with a calcium-sensitive dye. As stated in the grant proposal, I have taken part in the development of a novel algorithm that aims to clarify a major disadvantage of calcium imaging methods. Namely, though two-photon calcium imaging shows the activity of neurons, it does not show the exact activity level that can be stated as the exact number of action potentials. To circumvent this issue, our novel algorithm is able to screen and filter the calcium traces acquired from the somata of the neurons, and give a high-accuracy estimate (>90%) of the corresponding number of action potentials (Deneux et al, 2016 Nature Communications). When combined with the previously described OECT device, the two provide a powerful tool to convert the so far ambiguous calcium responses to exact firing patterns. I have used similar methods to show correlated Calcium imaging through transparent, organic cortical surface probes with simultaneously recorded multi-channel electrophysiological activity (Donahue, Kaszas et al., 2017 submitted).
In conclusions, my most significant results are the following:
1. I have succeeded in imaging the complete dendritic tree in its active and inactive states (Chiovini et al. 2014 Neuron).
2. When active, the interneuron dendrites showed hot spots of activity in correlation with the network events (Chiovini et al. 2014 Neuron).
3. When single neuronal suprathreshold activity synchronized with the SPW-Rs, the responses showed supralinear characteristics (Chiovini et al. 2014 Neuron)
4. I have conducted synchronous 3D two-photon calcium imaging of neuronal populations and electrophysiological recording and stimulation with novel organic transistor devices (Williamson, Kaszas et al., 2015 Advanced Materials)
5. Could record the populational activity of the neuronal network in 3D (Deneux et al, 2016 Nature Communications)
6. Using our novel algorithm, I can define exact firing patterns for a 3D neuronal network based on the the 3D two-photon calcium imaging