Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy

Cell and Tissue Research
K A MesceT C Brelje

Abstract

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the moth Manduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10,000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMRFamide and SCPB, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.

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Citations

Jan 14, 1999·The Journal of Comparative Neurology·T R Bhatt, F M Horodyski
Jul 25, 2000·The Journal of Comparative Neurology·H K LehmanK A Mesce
Jan 1, 1995·Archives of Insect Biochemistry and Physiology·M K Choi, S E Fahrbach
May 29, 2003·Journal of Insect Physiology·J Clark, A B. Lange
Oct 21, 1996·Journal of Neuroscience Methods·L P TolbertJ G Hildebrand
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May 14, 1999·Trends in Neurosciences·F C Rind, P J Simmons
Jul 22, 1994·The Journal of Comparative Neurology·L D AcevedoB Mulloney
Apr 30, 2003·Journal of Neurobiology·Marla SpivakKaren A Mesce
Apr 13, 2002·Frontiers in Neuroendocrinology·Karen A Mesce, Susan E Fahrbach
Nov 26, 2003·International Immunopharmacology·Zeno Földes-PappGernot P Tilz
Apr 16, 2002·The Journal of Comparative Neurology·Dan LuJane L Witten
Dec 26, 2001·The Journal of Comparative Neurology·Kevin M CrispKaren A Mesce
May 22, 1998·The Journal of Comparative Neurology·F C Rind, P J Simmons
Dec 24, 1997·Journal of Neurobiology·D Hess, A Büschges
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Feb 13, 2001·The Journal of Comparative Neurology·K A MesceK A Klukas
Oct 27, 1997·Journal of Neurophysiology·L S Gilchrist, K A Mesce

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