Synaptic connectivity to L2/3 of primary visual cortex measured by two-photon optogenetic stimulation

Understanding cortical microcircuits requires thorough measurement of physiological properties of synaptic connections formed within and between diverse subclasses of neurons. Towards this goal, we combined spatially precise optogenetic stimulation with multicellular recording to deeply characterize intralaminar and translaminar monosynaptic connections to supragranular (L2/3) neurons in the mouse visual cortex. The reliability and specificity of multiphoton optogenetic stimulation were measured across multiple Cre lines, and measurements of connectivity were verified by comparison to paired recordings and targeted patching of optically identified presynaptic cells. With a focus on translaminar pathways, excitatory and inhibitory synaptic connections from genetically defined presynaptic populations were characterized by their relative abundance, spatial profiles, strength, and short-term dynamics. Consistent with the canonical cortical microcircuit, layer 4 excitatory neurons and interneurons within L2/3 represented the most common sources of input to L2/3 pyramidal cells. More surprisingly, we also observed strong excitatory connections from layer 5 intratelencephalic neurons and potent translaminar inhibition from multiple interneuron subclasses. The hybrid approach revealed convergence to and divergence from excitatory and inhibitory neurons within and across cortical layers. Divergent excitatory connections often spanned hundreds of microns of horizontal space. In contrast, divergent inhibitory connections were more frequently measured from postsynaptic targets near each other.

Automated summary

Understanding cortical microcircuits requires thorough measurement of physiological properties of synaptic connections formed within and between diverse subclasses of neurons. In this study, the researchers combined optogenetic stimulation and multicellular recording to deeply characterize intralaminar and translaminar monosynaptic connections in the mouse visual cortex. They measured the reliability and specificity of the optogenetic stimulation and verified the connectivity measurements using paired recordings and targeted patching. The study revealed the abundance, spatial profiles, strength, and short-term dynamics of excitatory and inhibitory synaptic connections between genetically defined presynaptic populations. They found that layer 4 excitatory neurons and interneurons within L2/3 were the most common sources of input to L2/3 pyramidal cells, but also observed excitatory connections from layer 5 intratelencephalic neurons and translaminar inhibition from multiple interneuron subclasses.