From art to engineering? The rise of in vivo mammalian electrophysiology via genetically targeted labeling and nonlinear imaging

Abstract

A convergence of technical advancements in neuroscience has begun to transform mammalian electrophysiology from an art into a precise practice.

Figure 1. Schematic for the Production of a Modified BAC for the Targeted Expression of an XFP or an XFP-Based Reporter in Mice

(A) A library of suitable BAC clones is scanned using bioinformatics and an appropriate clone, encoding a suitable cell-type-specific transcriptional unit with ample flanking regions, is selected. Note that only a few of the many possible enhancer and silencer regions are drawn. An exon that lies downstream of the ATC start sequence is selected to be replaced by the XFP/reporter sequence by homologous recombination (exon 2 in this example), and a shuttle vector that codes for the label together with flanking regions around the exon (“a” and “b”) is constructed. The enzyme RecA is used to interchange the sequence for the exon and the label to form a modified BAC clone that codes for the label. The modified clone is injected into a mouse oocyte, where the dominant incorporation into the host DNA occurs through nonhomologous recombination. (B) Many factors influence the phenotype of a given transgenic mouse, and thus the same clone may result in a number of lines with slightly different properties. The insert shows the XFP expression pattern for a line based on a BAC clone that contains the transcriptional unit of a glycine transporter. (Image: Jean-Marc Fritschy and Hanns-Ulrich Zeilhofer)

Figure 2. Targeted Electrical Recording of Transgenically Labeled Inhibitory Interneurons in Mouse Cortex

(A) The two-photon laser scanning microscope is shown schematically. The critical features are the use of separate fluorophores, one for the label (GFP in this example) and another to mark the intracellular fluid of electrode (Alexa in this example) that have overlapping excitation spectra and different emission spectra (see [B]). The intracellular voltage shows a trace obtained under whole-cell patch of the response to vibrissa stimulation. Alexa, Alexa 594 dye; fs laser, titanium:sapphire mode-locked laser with 100 fs output pulse width; GFP, green fluorescent protein; PMT, photomultiplier tube. (B and C) Emission spectra and fluorescent images from the GFP and Alexa channels. Confirmation of whole-cell patch is achieved by injecting Alexa into the GFP-filled cell, as illustrated in the overlay. (Images: Troy Margrie)

Figure 3. Prospects for Recording from Awake but Head-Restrained Animals

(A) Photograph of a trained rat that is awake and head-restrained, ready for imaging of organic [Ca²⁺]-sensitive dyes. All aspects of the recording procedure demonstrated in primates are expected hold for mice as well. (Image: Jack Waters) (B) Photograph and set-up of visual virtual reality for rodents. In this example, the rat is body-fixed, and can rotate on an axis, but is not head-fixed. The visual world of the animal is controlled by projected images, and reward is administered through a food tube. (Images: Hansjuergen Dahmen)