Optical Imaging of Neuronal Activity and Visualization of Fine Neural Structures in Non-Desheathed Nervous Systems

Abstract

Locating circuit neurons and recording from them with single-cell resolution is a prerequisite for studying neural circuits. Determining neuron location can be challenging even in small nervous systems because neurons are densely packed, found in different layers, and are often covered by ganglion and nerve sheaths that impede access for recording electrodes and neuronal markers. We revisited the voltage-sensitive dye RH795 for its ability to stain and record neurons through the ganglion sheath. Bath-application of RH795 stained neuronal membranes in cricket, earthworm and crab ganglia without removing the ganglion sheath, revealing neuron cell body locations in different ganglion layers. Using the pyloric and gastric mill central pattern generating neurons in the stomatogastric ganglion (STG) of the crab, Cancer borealis, we found that RH795 permeated the ganglion without major residue in the sheath and brightly stained somatic, axonal and dendritic membranes. Visibility improved significantly in comparison to unstained ganglia, allowing the identification of somata location and number of most STG neurons. RH795 also stained axons and varicosities in non-desheathed nerves, and it revealed the location of sensory cell bodies in peripheral nerves. Importantly, the spike activity of the sensory neuron AGR, which influences the STG motor patterns, remained unaffected by RH795, while desheathing caused significant changes in AGR activity. With respect to recording neural activity, RH795 allowed us to optically record membrane potential changes of sub-sheath neuronal membranes without impairing sensory activity. The signal-to-noise ratio was comparable with that previously observed in desheathed preparations and sufficiently high to identify neurons in single-sweep recordings and synaptic events after spike-triggered averaging. In conclusion, RH795 enabled staining and optical recording of neurons through the ganglion sheath and is therefore both a good anatomical marker for living neural tissue and a promising tool for studying neural activity of an entire network with single-cell resolution.

Spatial distribution of intermingling pools of projection neurons with distinct targets: A 3D analysis of the commissural ganglia in Cancer borealis

“…Di-4-ANEPPDHQ is a lipophilic fluorescent dye that preferably stains neuronal membranes in the stomatogastric nervous system (Goldsmith, Städele, & Stein, 2014) and highlights cell somata. Di-4-ANEPPDHQ is a lipophilic fluorescent dye that preferably stains neuronal membranes in the stomatogastric nervous system (Goldsmith, Städele, & Stein, 2014) and highlights cell somata.…”

“…Nonneuronal dye affinity was low enough to visibly exclude any sheath staining from subsequent analyses (Goldsmith et al, 2014;Preuss & Stein, 2013). For long incubation periods (>2 days) we supplemented the saline with penicillin-streptomycin (Sigma) at 10 mg/L to prevent bacterial contamination.…”

“…The voltage-sensitive dye Di-4-ANEPPDHQ was used to indiscriminately stain neural membranes. Di-4-ANEPPDHQ is a lipophilic fluorescent dye that preferably stains neuronal membranes in the stomatogastric nervous system (Goldsmith, Städele, & Stein, 2014) and highlights cell somata. A 5-mM stock solution was prepared by diluting 1 mg dye in 300 ml of dimethyl sulfoxide (DMSO), aliquotting for singlestaining use, and kept in darkness at -20 8C.…”

A robust data-driven approach to the decoding of pyloric neuron activity

“…Traditionally, methods developed to identify pattern generating neurons routinely require the laborious task of analysing the related neuron/network activities that are recorded intracellularly and extracellularly [1]- [4]. More importantly, many of these methods have relied on and been limited by the necessarily subjective judgement of the neuron location even in such a small nervous system, as the densely packed neurons that can exist in different layers are often covered by ganglion and nerve sheaths which impede access for recording electrodes and neuronal markers [5]. Voltage sensitive dye imaging (VSDi), an optical recording technique which can in principle report the membrane potential of multiple neurons, offers a promising alternative.…”

Automated detection and analysis of depolarization events in human cardiomyocytes using MaDEC

“…This allows for non-invasive, non-destructive, and longitudinal assessment of hiPS-CM electrophysiology [11, 29]. VSDs have previously been used in neuronal [8, 21] and cardiac [7, 10, 15] cells and tissues. A wide range of VSD compounds and properties have been synthesized [29].…”