LSPS/Optogenetics to Improve Synaptic Connectivity Mapping: Unmasking the Role of Basket Cell Mediated Feed-Forward Inhibition

Abstract

Neocortical pyramidal cells (PYRs) receive synaptic inputs from many types of GABAergic interneurons. Connections between parvalbumin-positive, fast-spiking interneurons (“PV-cells”) and PYRs are characterized by perisomatic synapses and high-amplitude, short latency IPSCs. Here, we present novel methods to study the functional influence of PV-cells on layer 5 PYRs using optogenetics combined with laser-scanning photostimulation (LSPS). First, we examined the strength and spatial distribution of PV-to-PYR inputs. To that end, the fast channelrhodopsin variant ChETA was expressed in PV-cells in somatosensory cortex of mice using an AAV-based viral construct. Focal blue illumination (100-150 µm halfwidth) was directed through the microscope objective to excite PV-cells along a spatial grid covering layers 2-6, while IPSCs were recorded in layer 5 PYRs. The resulting optogenetic input maps showed evoked PV-cell-inputs originating from a ∼500 um diameter area surrounding the recorded PYR. Evoked IPSCs had short latency/high amplitude characteristic of PV-cell inputs. Secondly, we investigated how PV-cell activity modulates PYR output in response to synaptic excitation. We expressed halorhodopsin (eNpHR3.0) in PV-cells using the same strategy as for ChETA. Yellow illumination hyperpolarized eNpHR3.0-expressing PV-cells, effectively preventing action potential generation and thus decreasing inhibition of downstream targets. Synaptic input maps onto layer 5 PYRs were acquired using standard glutamate-photolysis LSPS either with or without full-field yellow illumination to silence PV-cells. The resulting IPSC input maps selectively lacked short-latency perisomatic inputs, while EPSC input maps showed increased connectivity, particularly from upper layers. This indicates that glutamate uncaging LSPS-based excitatory synaptic maps will consistently underestimate connectivity.

Significance Statement: Neural computations depend on the interplay of synaptic and intrinsic neuronal properties within complex networks of interconnected neurons. It is of particular interest how individual GABAergic interneuron types modulate network dynamics. Here, we outline novel methods to study connectivity between PV-cells, a prominent type of neocortical GABAergic interneuron, and PYRs, the main principal neurons of the neocortex. Using optogenetic methods and LSPS, we map the spatial extent and synaptic characteristics of PV-to-PYR connections, and demonstrate a substantial role of PV-cells in spatially specific feed-forward inhibition of PYRs. These methods can be used to better quantify the role of interneuron types in modulating input to and output of PYRs.