Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons <em>In vivo</em>

Abstract

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular 1 , juxtacellular 2 or loose-patch 3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visuallyguided single-cell electrophysiology [4][5][6] . However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons 7 , but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult [8][9][10][11] . The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals 12 . We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes 13 . However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods 11,14 . Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation. Video LinkThe video component of this article can be found at https://www.jove.com/video/3921/ Protocol 1. Prepare Dye-coated Needles 1. Electrolyt...

Detection of submillisecond spike timing differences based on delay-line anticoincidence detection

“…We obtained extracellular single-unit recordings from 76 retrogradely labeled small cell axons in 37 fish as described in detail in a previous methods article ( Lyons-Warren et al 2013). Individuals ranged from 5.5 to 8.6 cm in standard length.…”

Short-Term Depression, Temporal Summation, and Onset Inhibition Shape Interval Tuning in Midbrain Neurons

“…Passive electrical properties owing to morphology, as well as active dendritic conductances, can influence linear and nonlinear synaptic integration along dendrites (Magee, 2000;Gulledge et al, 2005;London and Häusser, 2005). We have not yet looked in detail for morphological differences between ELp neurons of different tuning types, but previous anatomical studies have not revealed any obvious morphological classes of ELp neurons (George et al, 2011;.…”

“…Differences in the degrees of depression of excitation and inhibition also contributed to interval tuning. In addition to GABAergic inhibition from local interneurons (George et al, 2011), ELp neurons receive excitation from ELa small cells (Friedman and Hopkins, 1998;Lyons-Warren et al, 2013a) and from other ELp neurons Fig. 10A).…”

“…For transverse stimulation to the fish, stimuli were delivered between three vertically oriented electrodes on one side of the chamber and three vertically oriented electrodes on the other side of the chamber. For longitudinal stimulation to the fish, stimuli were delivered between two vertically oriented electrodes on the front of the chamber and two vertically oriented electrodes on the back of the chamber (Lyons-Warren et al, 2013b). Stimuli consisted of monophasic square electric pulses.…”

“…The surgical procedure has been described previously Lyons-Warren et al, 2013b). Briefly, fish were anesthetized in a solution of 300 mg/L tricaine methanesulfonate (MS-222) and paralyzed with an intramuscular injection of 100 l of 3 mg/ml gallamine triethiodide (Flaxedil).…”