This document outlines the procedure for performing an assay to determine the concentration of serotonin (5-HT) using rat stomach fundus tissue. Key steps include: 1) Preparing and cleaning the rat stomach fundus, making cuts to isolate the longitudinal muscle; 2) Attaching the muscle to an organ bath for relaxation and experimentation; 3) Exposing the tissue to standard and test doses of serotonin and measuring the response to determine the unknown concentration. The rat stomach fundus is a sensitive tissue for bioassays of serotonin and other drugs like acetylcholine due to the presence of receptors, though its preparation requires longitudinal cuts to study circular muscle contraction.

1. Assay of Serotonin
Presenter:
Dr Arun Singh
MBBS, MD

2. • Aim: To determine unknown concentration of
serotonin (5-HT) using rat stomach (fundus).

3. Background
• Stomach fundus is useful in the
bioassay of 5-HT which is
considered to be most sensitive
tissue among the three parts of
stomach namely, fundus, corpus
and pylorus.
• Fundus is identified by its gray
color and situated above the pink
thick pyloric region.

4. Drugs like histamine are little or insensitive to he rat fundus due to the lack of H1
receptors in the stomach muscle.
The other drugs which have sensitivity against the fundus are ACh, bradykinin
and prostaglandin (PGE2).
The muscle preparation is the important step in the bioassay using stomach
fundus.
Both longitudinal and circular muscle may be used in the experiment which
depends on the transverse cut made to prepare the tissue (Figs 16.1A to E).

6. Materials and
Method
Animal/tissue : Rat/Stomach fundus
PSS : Tyrode/deJalon/Krebs
Lever : Frontal writing/ simple lever
Magnification : 7-15x
Tension : 1-4g
Air : O2/Carbogen
Temperature : 35-37°C
Drug : Serotonin (5-HT) [M.wt: 176. 218]
Acetylcholine chloride (ACh) [M. wt: 181.68]

7. Precautions
before
Experimentation
Clean the organ bath before starting the experiment
specially inner organ bath (chances of presence of
previous drug used or to avoid any contamination)
Clean
Balance the simple writing lever, tangential to the
rotating drum
Balance
Prepare PSS for the experiment, while taking exact
quantity of chemicals (1% variability is acceptable)
Prepare

8. Add the calcium chloride at the end of PSS preparation
(to avoid any precipitation: PSS should be clear)
Try to minimize the handling of tissue (especially at
the middle part)
Maintain the dose cycle properly (tissue sensitivity
depend on this cycle)
Animal should be fasted properly (any food present in
the stomach may interfere with the assay)
Carefully clean the fundus from the adjacent tissues.

9. Method
Step III Rat is fixed on the DB by tying it legs with the
help of thread.
Step II
Sacrifice the rat by stunning (a strong blow) on
the head, and then keep on the dissecting
board (DB).
Step I Keep the animal (rat) fasting for at least 24hr,
water is given ad libitum.

10. Step VI
Cut and separate the stomach attachment at
the end of esophagus and down at the
duodenum.
Step V Identify the stomach (just down the line of
peritoneal cavity touches the liver and spleen.
Step IV Cut open the abdomen of rat by a vertical long
cut, and then expose the abdominal viscera.

11. Step IX Then, cut the fundus at the midline into two
equal parts.
Step VIII
Wash the stomach contents properly with the
PSS and then cut open the fundus by lesser
curvature.
Step VII
Identify the fundus (grey part) which
discriminates from the pyloric region that is
pink in appearance.

12. Step XII Select the experimental design and get the response with
standard and test drug.
Step XI Then, attach into the organ bath and leave for relaxation
at least 30-45 min.
Step X
For preserving the longitudinal muscle the alternate
opposite vertical cut should be given (as shown in the
figure) and then tie the thread on the either end of the
muscle and stretch out the muscle.

13. Calculation and Result
• The calculation and graph depend on the
method / design of experimentation adopted
either 3- point, 4-point or matching, etc. (see
the bioassay section for calculation).
• Inference Write the findings of the study.

14. Discussion
Fundus contains the swallowed
air and has functions mainly
concerned with pressure
changes.
Its preparation is somewhat
tedious than the other tissue
preparations because it require
alternate longitudinal cutting of
tissue in which contraction of a
circular muscle is measured in a
longitudinal fashion.

15. It is a slow-contracting muscle hence, sometimes it needs a
stretching weight to bring it to the baseline.
Bradykinin produces slow contraction of stomach fundus
whereas it is a strong stimulator of isolated intestinal tissue.
This tissue well responds to tryptamine and prostacyclin
(PGE2).