Shedding light on hub cell function in vivo – HOLOHUB

We will address the following specific objectives:
(1) We will develop an optical system for in-vivo light-activation of a single cell or a subpopulation of cells deep inside scattering tissue while reading out network dynamics through calcium imaging. The microscope will combine two-photon (2P) imaging with 2P holographic patterned photoactivation and temporal focusing. Different combinations of opsins and calcium indicators will be tested to individuate the optimal candidate for combined photostimulation and functional imaging.
(2) We will interrogate hub function in vivo in the healthy and epileptic hippocampus based on the a priori assumption that early born GABA neurons are potential hub cells.
(3) We will identify candidate hub cells in vivo by looking for neurons which activation has a significant impact on the activity of many others without any a priori assumption on a possible subtype but only based on the analysis of effective connectivity maps.
This project breaks scientific barriers:
(1) it will demonstrate for the first time the use of patterned 2P optogenetics in vivo with single cell precision.
(2) it will address the novel theory that adult cells are functionally different based on their temporal developmental origin.
(3) it will define the role of developmentally defined GABAergic subpopulations in specific hippocampal physio-pathological functions.
(4) it will address the idea that specific neuronal subsets fulfill a “hub-like” function that endows them with the ability to strongly affect the activity of hundreds of other cells.

- We built up an optical system enabling in vivo simultaneous 2P imaging and 2P holographic photostimulation using a amplified fiber laser. The system has been also equipped with electrophysiology.
- We started to discuss the optimal design to duplicate the microscope in Cossart Laboratory. To increase the illumination field in the system for Cossart Laboratory, we tested a new model of SLM (having 4 times more pixels).
-We tested and characterized the biophysical properties of different opsins under 2P illuminations. These include Chronos, ReachR and CoChR.
-We used GCaMP transgenic mice and wild type mice bulk loaded with OGB to test the damage threshold under 2P illumination in vivo using the amplified fiber laser.
-We optimized the injection protocol in the hippocampus for Chr2-mcherry, Chronos TdTomato and GCaMP6.
- We found an efficient protocol to express Chr2-mcherry and GCamP6s in CA1 hippocampus area in order to stimulate and record at the same time the activity of CA1 microcircuits. Precisely we optimized the co-localisation of GCamP6s and ChR2 (quantified using immunohistochemical approaches) and tested their functionality using electrophysiology and 2P imaging in brain slices.
The described experiments in hippocampal preparations have been performed by Thomas Tressard, a Phd student from the Cossart lab (co-directed by R. Cossart and V. Emiliani), who has moved starting from January 2016 to the Emiliani lab for 6 months.

- Test experiments of photostimulation and Ca imaging in vivo at shallow depths (L2/3 in visual cortex)(Emiliani lab)
- Test of combined in vivo photostimulation and Ca imaging in the hippocampus (Emiliani lab)
- Selective expression of opsins in hub neurons identified by their early birthdate.
- Duplication of the optical set up in Marseille

1) E. Ronzitti1, R. Conti, E. Papagiakoumou, D. Tanese, V. Zampini, E. Chaigneau, A.J. Foust, N. Klapoetke, E.S. Boyden and V. Emiliani
Sub-millisecond optogenetic control of neuronal firing with two-photon holographic photoactivation of Chronos, submitted
Here we characterized the biophysical properties of the opsin Chronos under 2P holographic illumination
2) R. Conti1, O. Assayag, V. de Sars, M. Guillon and V. Emiliani
submitted

The coordinated activation of neuronal microcircuits is proposed to support the ongoing computations of the cerebral cortex in health and disease. A common approach to reduce network complexity is to outline microcircuits and infer their functional role by selectively modulating them. Targeted cell modulation has proven successful in vitro as it enabled the finding of a major functional subtype of cells, i.e. hub neurons that function in synchronizing the developing hippocampus through a dense and extended axonal arborisation (Bonifazi et al. Science 2009). If hub neurons are critically involved in hippocampal development, it remains unknown whether the same or functionally similar hub cells orchestrate physiological or pathological network oscillations occurring in the adult brain in specific behavioural contexts. We therefore aim at exploring hub function in vivo in the adult hippocampus. Our ultimate goal is twofold. From the perspective of optics development, we want to provide an experimental approach for a dynamic 3D description of hippocampal effective connectivity in awake mice with single-cell resolution. From the side of neurobiology, we work at linking single or assemblies of hub cells with behaviour and at containing pathological dynamics by modulating the activity of sparse microcircuits. To this aim, we will follow 3 objectives. The first will consist in the development of an optical system for in-vivo light-activation of a single cell or a subpopulation of cells deep inside scattering tissue while reading out network dynamics through calcium imaging. The microscope will combine two-photon (2P) imaging with 2P holographic patterned photoactivation and temporal focusing, a new photoactivation technique recently developed in the Emiliani lab (Lutz et al. Nature Methods 2008, Anselmi et al. PNAS 2011, Papagiakoumou et al. Nature Methods 2010, Nature photonics 2013). The next two objectives will be run in parallel and rely on precise multiple-cell stimulation. One will interrogate hub function in vivo in physiological and epileptic conditions based on the a priori assumption that early born GABA neurons are potential hub cells, as recently proposed by the Cossart lab (Picardo et al. Neuron 2011). The second will find hub cells based on effective connectivity maps obtained in vivo through patterned light activation and study their function by mimicking spontaneous activity. Altogether, this proposal will bridge the gap between the cellular and behavioural levels of cortical network description by providing precisely spatially patterned neuronal stimulation in vivo based on the combined efforts of a strong multidisciplinary task force covering a broad range of complementary expertise going from non linear optics, and wave front shaping to neurobiology and genetics.