Evaluation of Voltage-Sensitive Fluorescence Dyes for Monitoring Neuronal Activity in the Embryonic Central Nervous System

Abstract

Using an optical imaging technique with voltage-sensitive dyes (VSDs), we have been investigating the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity, and small dye bleaching. In the present study, we evaluated the suitability of voltage-sensitive fluorescence dyes for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem-spinal cord preparations from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion, and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N, but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS, and di-3-ANEPPDHQ, but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.

A simultaneous optical and electrical in-vitro neuronal recording system to evaluate microelectrode performance

“…This was still within the manufacturers recommended loading concentration of 0.2—2 μ M. Increases in the excitation intensity or exposure time to improve signal quality were avoided to reduce the occurrence of phototoxicity and maintain temporal resolution, respectively. It should also be noted that while this study employed Di-4-ANEPPS, newer generation VSDs, such as di-2-ANEPEQ, di-3-ANEPPDHQ and di-4-AN(F)EPPTEA may have resulted in improved imaging [ 34 ]. Camera acquisition settings were adjusted to maximise the quality of digitised fluorescent signals.…”

Optical Imaging-Based Guidance of Viral Microinjections and Insertion of a Laminar Electrophysiology Probe Into a Predetermined Barrel in Mouse Area S1BF

“…Membrane-bound dyes such as voltage-sensitive dyes (VSD) used in vivo report voltage changes in neurons at an excellent temporal and spatial resolution ( Grinvald et al, 1988 , 1999 ; Grinvald and Hildesheim, 2004 ; Devonshire et al, 2013 ). The VSD pharmacological and cytotoxic side effects have recently been alleviated to near-negligible levels, using newer generations of blue dyes and lower dye concentrations ( Ross et al, 1977 ; Grinvald et al, 1988 ; Shoham et al, 1999 ; Grinvald and Hildesheim, 2004 ; Lippert et al, 2007 ; Grandy et al, 2012 ; Devonshire et al, 2013 ; Habib et al, 2013 ; Carter and Shieh, 2015 ). Compared to in vivo VSD imaging, OI-IS is an indirect indicator of neural activity.…”

Altered NMDAR signaling underlies autistic-like features in mouse models of CDKL5 deficiency disorder

“…Following recovery, each slice was bulk-loaded with 100 μL of a solution containing the voltage-sensitive dye di-2-ANEPEQ 49,50 (JPW1114, Molecular Probes) diluted at 0.05 mg/mL in aCSF. Each slice was stained for 14 min, washed with aCSF, and imaged in an interface chamber using an 80 x 80 CCD camera recording with a 1 kHz frame rate (NeuroCCD; RedShirt Imaging).…”