Abstract
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.
Highlights
The receptive field properties of visual cortical neurons are governed by long-range synaptic connections formed across brain areas and local inputs formed between cells within a cortical area
In this study, we examined the applicability of two-photon optogenetics to a probe a diverse array of potential synaptic connections by measuring the reliability and specificity of two-photon optogenetic stimulation using 7 different Cre lines with either transgenic or adeno-associated virus (AAV)-mediated expression of the opsin ChrimsonR (Klapoetke et al, 2014)
To what extent might the relative locations of pre- and postsynaptic cells influence connection strength? We examined synaptic connection strength as a function of the horizontal distance between connected cells and the distance of the presynaptic neuron from the pia (Figure 7D-F). Within Cre lines, we observed little to no correlation between postsynaptic potentials 116 (PSPs) amplitude and the horizontal distance between cells (Table 3)
Summary
The receptive field properties of visual cortical neurons are governed by long-range synaptic connections formed across brain areas and local inputs formed between cells within a cortical area. Discovered and engineered opsin variants were subsequently combined with rapid scanning and patterned illumination methods to demonstrate feasibility of two-photon optogenetic stimulation in acute brain slices and in vivo (Andrasfalvy et al, 2010; Papagiakoumou et al, 2010; Prakash et al, 2012; Packer et al, 2012; Packer et al, 2015; Chaigneau et al, 2016; Ronzitti et al, 2017; Pégard et al, 2017; Forli et al, 2018; Mardinly et al, 2018; Yang et al, 2018; Marshel et al, 2019) In recent years, these methods have been combined with electrophysiological recordings to measure synaptic connectivity, with individual studies focused on recurrent excitatory connectivity within cortical layers (Izquierdo-Serra et al, 2018; Seeman et al, 2018) or distinct connectivity of subtypes of Sst interneurons (Naka et al, 2019). Differences in the strength, kinetics, and short-term dynamics of synaptic responses were found across presynaptic and postsynaptic subclasses of cells; the amplitudes of less common synaptic connections were often similar to the more canonical connection paths when probed at the single cell level
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