Combined membrane potential imaging and connectome of behavioral circuits in an annelid worm

In most animal phyla, nerve cells form highly specific connections with each other. The resulting intricate networks determine what activity patterns a nervous system can sustain, and hence what behaviors an animal can exhibit. Accordingly, understanding the relationship between connectivity and activity is a major goal of neuroscience. However, despite major recent advances, no current technology can comprehensively record activity in large nervous systems such as the human or even the mouse brain, nor can connectivity be reconstructed at synaptic level in such systems. Doing both at once in the same specimen remains a distant goal. However, smaller nervous systems offer exciting opportunities today, and here we report for the first time obtaining joint functional and anatomical data from a major functional unit of the nervous system of the medicinal leech, by combining voltage-sensitive dye (VSD) imaging with serial blockface electron microscopy (SBEM). We simultaneously recorded from the majority of the neurons in a segmental ganglion during several motor behaviors with a VSD sufficiently sensitive to record subthreshold neuronal activity. We then anatomically imaged the entire ganglion with SBEM. As a proof of concept, we have manually traced a critical motor neuron and identified all of its numerous presynaptic partners. The thorough reconstruction of individual synapses in combination with functional data enabled a quantitative analysis that revealed spatial clustering of synapses from functionally synchronized cell assemblies. All of our data is publicly available.