In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Abstract

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices. ABSTRACT: Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain. Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

Projection-specific integration of convergent thalamic and retrosplenial signals in the presubicular head direction cortex

“…Viral injections were performed unilaterally (Mathon et al, 2015; Richevaux et al, 2019) at the coordinates −0.82 AP, 0.75 ML and −3.2 mm DV for the ADN, and at −2.1 to −2.15 AP, 0.65 ML and −0.65 mm DV for the RSC. Volumes of 200 to 250 nl were injected with a 10 µL Hamilton syringe equipped with 33ga needle over a time of 10 min.…”

“…Fluorescent beads for retrograde tracing were injected (300-500 nl) into the PrS at the coordinates: -4.06 antero-posterior (AP), 2.00 medio-lateral (ML) and -2.15 mm dorso-ventral (DV) and into the LMN (-2.8 AP, 0.75 ML, -5.35 DV) with respect to the bregma. Viral injections were performed unilaterally (Mathon et al, 2015;Richevaux et al, 2019) at the coordinates -0.82 AP, 0.75 ML and -3.2 mm DV for the ADN, and at -2.1 to -2.15 AP, 0.65 ML and -0.65 mm DV for the RSC. Volumes of 200 to 250 nl were injected with a 10 µL Hamilton syringe equipped with 33ga needle over a time of 10 min.…”