Brain and Gut Dissection in Surface Fish and Cavefish Populations of Astyanax mexicanus

Summary

Here, we present a protocol for dissecting and extracting whole brain and gut components of surface and cave-adapted morphotypes of Astyanax mexicanus. This protocol can be adapted for use in downstream applications, including transcriptomics, metabolomics, and gut microbiota analysis.

Abstract

Teleost fish are used to answer a wide range of research questions spanning from development to genetics and neuroscience, and more recently, to understand the complexities of the gut-brain axis, and even in a wider context, the gut-brain-liver axis. The Mexican tetra, Astyanax mexicanus, has been increasingly used in comparative and developmental evolution frameworks due to the availability of morphologically and physiologically distinct morphotypes, including river-dwelling surface fish and cave-adapted, blind cavefish. Adaptation to cave habitats is associated with changes in morphological traits, behavioral traits, and gut microbiota diversity that are also found in laboratory conditions. This has opened up the possibility to ask questions about the interactions between the microbiome, metabolic activity, neural adaptation, and complex behavioral outputs. These interactions can now be addressed through whole-brain mapping studies with cleared brains and gut microbiota sequencing to detect bacterial diversity associated with surface and cave-adapted phenotypes. However, most of these studies would benefit from intact, carefully dissected, and preserved whole organs and tissues, which allow for post-processing in downstream applications. This protocol describes a step-by-step method for brain and gut dissection, where the whole brain and abdominal organs are removed and isolated for both surface fish and cavefish morphotypes.

Introduction

Animal models, including vertebrate fish, are critical tools for modeling biological phenomena and complex pathologies in relatively simpler, accessible systems. For instance, the zebrafish, Danio rerio, is one of the most widely used teleost fish models, with a plethora of methods applied to study their development, genetics, physiology, and behavior, with significant impact in the biomedical and neurobehavioral sciences^{1,2,3,4,5,6,7<....}

Protocol

This protocol is conducted in accordance with IACUC-approved protocols 2024-0004 and A790125 at the Molecular Sciences Center and Medical Sciences Campus of the University of Puerto Rico.

1. Pre-dissection set-up

1. If using a Sylgard Petri (SP) dish to dissect, make sure they are prepared prior to the procedure, taking into account that it may take a few days to set.
2. To prepare a SP dish, use Sylgard 184 and Elastomer 184 in a 10:1 ratio. Mix 200 g of Sylgard 184 base with 20 g of elastomer in a disposable cup. Pour into Petri dishes to about one-third to one-half depth. Tap the dish to remove bubbles an....

Results

Following this protocol, brain and gut contents were extracted, and measurements of the length of the complete gut, telencephalon, and optic tectum were taken and compared between surface fish and Pachón cavefish morphotypes (Figure 15). We used independent-sample Welch's t-tests for statistical comparisons, with a sample size of n = 6 surface fish and n = 6 Pachón cavefish. Surface fish had significantly larger optic tectum dimensions than pachón cavefish, in line with previously p.......

Discussion

The increasing relevance of A. mexicanus as a research model in a number of fields, ranging from evolution to biomedical neuroscience and microbiology, calls for a standardized organ dissection and collection method. While isolated structures have been studied and documented previously, including the optic tectum^16,38, adult heart¹¹, and others^11,44, a systematic guideline.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.0 Liter Zebrafish Breeding Tank	Tecniplast	https://shop.tecniplastusa.com/products/1-0-liter-zebrafish-breeding-tank	To prepare the euthanasia ice slurry. The tank is half-filled with ice and then the perforated internal tank is placed on top. Then it is filled with water to the top.
4L Polyurethane Ice Pan	USA Scientific	Item #2328-7242
Biohazard Waste Bags	Fisherbrand	14-828-244
Calbiochem OmniPur PBS 10x Liquid Concentrate, Molecular Biology Grade, Sterile	MilliporeSigma	65074L
Dissecting Microscope Olympus SZ51	Olympus	OLSZ51NPS-LED/R
Dissecting Tissue Forceps	Fisherbrand	08-953G
Dissection Pins, 100 pack	Home Science tools	https://www.homesciencetools.com/product/dissection-pins-100-pack/?srsltid=AfmBOoofk8r6_hd4nx_7bjjP2 GzkNBlnOJ3vUKor5 rFCJvVxPN9xRlpc
Forceps, Delicate, Extra Fine Sharp Tips	MOPEC	AB132
Forceps, Tissue, Delicate Super Fine Tip	MOPEC	AB047
Hemostat, 12.5 cm, straight	World Precision instruments	https://wpiinc.com/products/var-15920-halsted-mosquito-hemostatic-forceps
Jewelers forceps, Dumont No. 5	MilliporeSigma	https://www.sigmaaldrich.com/US/en/product/sigma/f6521
Kimwipes EX-L	Kimberly-Clark	34155
Liquid Nitrogen			Provided by Institution
Noyes Micro Scissors, 12 cm	World Precision instruments	500228
Paper Towels			Provided by Institution
Petri Dishes with Clear Lid, 95 mm, 15 mm, Round	Fisherbrand	FB0875714G
Scalpel Blades, #22, 10 pack	Home Science Tools	https://www.homesciencetools.com/product/scalpel-blades-22-10-pack/
Scalpel handle #4	Surtex	https://surtex-instruments.com/product/scalpel-handle-no-4/
Scissors, Iris Dissection	MOPEC	AA146
Scissors, Superfine Micro	MOPEC	AA152
Standard Disposable Transfer Pipettes	Thermo Scientific	13-711-9D
Sterile Eppendorf Microcentrifuge 1.5 µL tubes	Eppendorf	05-402-25
Sylgard 184	Electron Microscopy Sciences	24236-10
Thermo-Flask	Lab-Line Instruments Inc.	2119
Ultra Fine Scissors	NEWMED Instruments	NM-DI-206023
Water suitable for electrophoresis, PCR	Sigma-Aldrich	W4502-1L