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Understanding the neural basis of behavior requires studying the activity of neural networks. Within a neural network, single neurons can have different firing properties, different neural codes and different synaptic counterparts. Therefore, it will be useful to readout from the brain and control it at a single-cell resolution. However, until recently, single cell readout and control in the brain were not feasible. The first scientific problem we addressed, is this regard, was the low spatial resolution of light based neural activation. Opsins are genetically encoded light switches for neurons that cause neural firing, or inhibition, when illuminated (and are therefore called “opto-genetic” molecules). However, optogenetic experiments are biased by ‘crosstalk’: the accidental stimulation of dozens of cells other than the cell of interest during neuron photostimulation. This is caused by expression of optogenetic molecules through the entirety of the cells, from the round cell body (“soma”) to the elongated neural processes. Our solution was molecular-focusing: by limiting the powerful opsin CoChR to the cell body of the neuron, we discovered that we could excite the cell body of interest alone. This molecule, termed “somatic-CoChR” was stimulated with state of the art holographic stimulation to enable millisecond temporal control which can emulate actual brain activity. Thus, we achieved for the first time single cell optogenteic stimulation at sub millisecond temporal precision. A second challenge was imaging the activity of multiple cells at a single cell resolution. The most popular neural activity indicator is the genetically encoded calcium sensor GCaMP, due to its optical brightness and high sensitivity. However, the fluorescent signal originating from a cell body is contaminated with multiple other fluorescent signals that originate from neurites of neighboring cells. This leads to a variety of artifacts including non-physiological correlation between cells and an impaired ability to distinguish between signals coming from different cells. To solve this, we made a cell body-targeted GCaMP. We screened over 30 different targeting motifs for somatic localization of GCaMP, and termed the best one, in terms of somatic localization, “SomaGCaMP”. This molecule was tested in live mice and zebrafish and can report the activity of thousands of neurons at a single cell resolution. A third challenge was voltage imaging in the brain, since genetically encoded indicators still suffered from either low sensitivity, or from low brightness. To record voltage, we used nitrogen vacancy nanodiamonds, known to be both very bright and sensitive to electric fields. Our aim was to bring the nanodiamonds to the membrane so the large electric field created by the action potential could impinge upon them and change their fluorescence. By making the nanodiamonds hydrophobic through surface chemistry modification, and inserting them into micelles, we labeled neural membranes with monodisperse diamonds for hours. We are now in the process of assessing the sensitivity of the nanodiamonds to the membrane voltage.
Altogether, thinking backwards from fundamental limitations in neuroscience is instrumental in deriving strategies to fix these limitations and study the brain. In the future, we will use similar approaches to study and heal brain disease, at single-cell and subcellular resolutions.