Mar 13, 2024
Extracellular fluid extraction in zebrafish brain tissue and samples
- 1Universidade Federal do Sul e Sudeste do Pará
Protocol Citation: Caio Maximino 2024. Extracellular fluid extraction in zebrafish brain tissue and samples. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3p1zl5d/v1
Manuscript citation:
Maximino C, Puty B, Benzecry R, Araújo J, Lima MG, Batista EJO, Oliveira KRM, Crespo-Lopez ME, Herculano AM (2013). Role of serotonin in zebrafish (Danio rerio) anxiety: Relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models. Neuropharmacology 71: 83-97. https://doi.org/10.1016/j.neuropharm.2013.03.006
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 12, 2024
Last Modified: March 13, 2024
Protocol Integer ID: 96603
Keywords: Zebrafish, Neurochemistry, Extracelllular fluid, Cerebrospinal fluid, Neurotransmitter release, Neurotransmitter transort
Abstract
This protocol is used to extract the contents of the extracellular fluid of the adult zebrafish brain, allowing quantification of analytes such as neurotransmitter (e.g., Maximino et al., 2013) or proteins (e.g., Pradel et al., 1999).
Protocol materials
Tris HClP212121
Step 1.1
Glutathione
Step 1.4
GlucoseP212121Catalog #Glucose
Step 2.1
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Step 2.1
Potassium chlorideP212121
Step 2.1
Potassium phosphate (monobasic)P212121
Step 2.1
Tris BaseFisher ScientificCatalog #BP152-1
Step 1.1
Sodium chlorideP212121
In 2 steps
Calcium Chloride
In 2 steps
Magnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
Step 2.1
Reagent preparation
Reagent preparation
Prepare the extraction fluid. The recipe below is good for 1 L.
CITATION
To make 1 L Tris buffer
- Add 4.44 g Tris HClP212121 to 1 L double distilled water
- Add 2.65 gTris BaseFisher ScientificCatalog #BP152-1
- Check if 8.0 . If not, adjust with acid or base
To the Tris buffer, add 5.2596 g Sodium chlorideP212121
Add 0.27745 g Calcium ChlorideContributed by users
Add 0.30733 gGlutathioneContributed by users
Adjust pH to 7.4
Prepare dissection solution (artificial cerebrospinal fluid). The recipe below is good for 1 L.
CITATION
- Add 7.65564 g Sodium chlorideP212121 to 1 L double distilled water
- Add 0.1491026 g Potassium chlorideP212121
- Add 0.1701 g Potassium phosphate (monobasic)P212121
- Add 0.240732 gMagnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
- Add 1.80156 g GlucoseP212121Catalog #Glucose
- Add 0.27745 g Calcium ChlorideContributed by users
- Add 1.68014 g Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Keep dissection solution on the fridge (4 ºC) for a maximum of 30 days before use.
Euthanasia and dissection
Euthanasia and dissection
Sacrifice animals in ice-cold water.
CITATION
Add ice to a beaker filled with at least 1 L system water. Measure temperature so that it falls between 0 ºC and 4 ºC.4 °C
Add a tea strainer above the ice layer, where individual animals will be placed. The use of the strainer allows cooling the water while avoiding direct contact of the fish body with the ice.
Transfer animals individually to the strainer. Maintain animals for at least 5 min in contact with the cold water. If animals show any sign of activity, allow for more time in the ice.
Decapitate and dissect the animal.
Transfer the animal to a Petri dish filled with dissection fluid.
Equipment
Cell Culture/Petri Dishes or equivalent
NAME
10X35 mm
TYPE
Nunc™
BRAND
150318
SKU
LINK
Decapitate the animal by cutting cleanly through the pectoral girdle with dissection scissors. The cut
should be made immediately anterior to the articulation of the pectoral fin with the girdle, severing the heart.
Equipment
Fisherbrand™ Dissecting Scissors
NAME
Scissors
TYPE
Fisherbrand
BRAND
15277168
SKU
LINK
Using the dissecting scissors, remove the skin and bones from the head, exposing the brain. To avoid damaging the forebrain, start dissection at the level of the junction between medulla and spinal cord. Gently raise the medulla with an insulin needle and cut the ventral roots of the cranial nerves using microdissection pincers.
The tissue sample can be assessed as a whole, or microdissected into forebrain, midbrain, and hindbrain.
ECF extraction
ECF extraction
In a 1.5 mL microtube filled with extraction fluid, add one brain (or fraction). Keep the microtube on ice or on the fridge, maintaining the temperature at 4 ºC. Incubate for 30 min.
4 °C
00:30:00
30m
Quantification
Quantification
Samples can be analyzed using sensitive techniques, such as HPLC, to assay neurotransmitter content.
Expected result
Since the tissue is not used during the analysis, most of the neurotransmitter content is expected to represent extracellular levels (i.e., released or not transported.
The remaining tissue can be used to assay other analytes, such as second messenger levels.
Protocol references
Pradel G, Schachner M, Schmidt R (1999). Inhibition of memory consolidation by antibodies against cell adhesion molecules after active avoidance conditioning in zebrafish. Journal of Neurobiology 39: 197-206. https://doi.org/10.1002/(SICI)1097-4695(199905)39:2%3C197::AID-NEU4%3E3.0.CO;2-9
Maximino C, Puty B, Benzecry R, Araújo J, Lima MG, Batista EJO, Oliveira KRM, Crespo-Lopez ME, Herculano AM (2013). Role of serotonin in zebrafish (Danio rerio) anxiety: Relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models. Neuropharmacology 71: 83-97. https://doi.org/10.1016/j.neuropharm.2013.03.006
Citations
Step 1
Maximino C, Puty B, Benzecry R, Araújo J, Lima MG, de Jesus Oliveira Batista E, Renata de Matos Oliveira K, Crespo-Lopez ME, Herculano AM. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.
https://doi.org/10.1016/j.neuropharm.2013.03.006Step 2
Vargas R, Jóhannesdóttir IT, Sigurgeirsson B, Thorsteinsson H, Karlsson KA. The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity.
https://doi.org/10.1152/advan.00099.2010Step 3
Wallace CK, Bright LA, Marx JO, Andersen RP, Mullins MC, Carty AJ. Effectiveness of Rapid Cooling as a Method of Euthanasia for Young Zebrafish (Danio rerio).
https://www.ingentaconnect.com/content/aalas/jaalas/2018/00000057/00000001/art00009