Pharmacology of the Autonomic Nervous System Utilizing the Isolated Ileum Model System I Introduction Mammalian smooth muscle typically occurs as sheets of contractile fibers surrounding the intestinal tract uterus spleen and blood vessels It is both anatomically and physiologically different from skeletal muscle The fibers are short narrow spindle shaped cells with little sarcoplasmic reticulum The highly ordered structure of skeletal muscle is absent and the contractile properties reflect the differences in structure Smooth muscle contraction is slow sustained and spontaneous If smooth muscle tissue is stretched it will first relax to this new length and then begin to resume spontaneous activity Therefore the muscle can work over a wide range of lengths a definite advantage for a hollow organ e g intestine uterus or blood vessel subject to distention due to filling Smooth muscle contractions occur in the absence of any innervation unlike skeletal muscle contractions However smooth muscle activity is mediated by the two divisions of the autonomic nervous system sympathetic and parasympathetic The sympathetic innervation is inhibitory while the parasympathetic is excitatory In the intestinal smooth muscle preparation utilized in these experiments the parasympathetic ganglia and the post synaptic receptors are contained in the tissue However the sympathetic ganglia are absent Why is this Review the structure and pharmacology of the autonomic nervous system to familiarize yourself with these details II Summary of Experimental Procedures You will isolate a segment of rat ileum and suspend it in an isotonic buffered saline solution Tyrode s One end of the muscle is fixed while the other end is attached to a force displacement transducer for recording muscle contractions You will use this preparation to demonstrate and examine 1 The principles of a bioassay and Dose Response curves 2 The effects of sympathetic and parasympathetic agonists and antagonists 3 The use of this preparation in order to determine the site of action of an unknown agent All of the data will be recorded on disk using the WINDAQ 200 program and then analyzed using the WINDAQ playback feature see the sections in this manual on the use of these programs III METHODS 164 Data Recording While one members of the group prepares the tissue another member can set up the Transbridge with a FORT 10 force displacement transducer and ready the WINDAQ program for recording on one channel Since you will be examining contractions that invoke an upward deflection of the computer screen trace set the baseline 0 tension near the bottom of the screen The resulting changes in muscle tension are slow to occur but last up to 4 or 5 minutes Select a slow sampling rate e g 10 50 sec Do not tweak flick or bend the transducer It is a fragile instrument Dissection Techniques Your TA will mercifully dispatch the rat with CO2 narcosis After the animal stops breathing make an abdominal midline incision to expose the viscera Take care to avoid cutting any visceral organs Expose the viscera and cut the diaphragm at the top of the abdominal cavity Note any spontaneous movements of the intestine in situ Carefully lift out the intestine without stretching it and lay it in a Petri dish containing Tyrode s solution warmed to room temperature Locate a segment of the small intestine as close to the ileo cecal junction as possible and cut away the mesentery beginning at this point and proceeding for 10 15 cm toward the stomach Handle the intestine gently and keep it moist Feel free to perform a dissection of the rat Your TA will point out the major organs Sever the ileum as close to the colon as possible and again about 12 15 cm toward the mouth Place this isolated section into a separate Petri dish containing fresh Tyrode s solution Flush and rinse out the lumen by gently forcing Tyrode s through it with a 5 ml syringe Place this rinsed section of intestine into another Petri dish containing fresh Tyrode s solution Do not let it touch or sit in fluid containing the intestinal contents Attach surgical thread to one end of the ileum One end of this thread should be left about 10 12 inches long see Figs 1 2 Be sure to tie square knots no granny knots please A second thread should be tied about 2 0 3 0 cm down the ileum from the first thread The ends of this second thread should be formed into a small loop with another square knot Figs 1 2 Carefully isolate this segment of ileum by cutting it outside of the attached ligatures Repeat this procedure on the remaining length of ileum until one segment has been prepared for each lab group Figure 1 illustrates a resulting section of ileum 165 LOOP LIGATURES tied with square knots 6 8 LIGATURE Ileum 2 3 cm FIGURE 1 Attach one end of your segment to the hook in the bottom of the tissue bath see Fig 2 3 using the small loop in the thread Attach the long thread attached to the other end of the muscle to the lever of the transducer Tie the ileum FIRST Then move the transducer up in order to place a very small amount of tension on the ileum segment Watch the screen in order to determine when you start to place tension on the transducer Be careful not to damage the transducer Fill the bath with a Tyrode s solution by clamping the tubing at the base of the tissue bath and adding Tyrode s to the top 166 167 Check the setup to make certain that the suture is as vertical as possible and is not touching the side of the tissue bath and the muscle has a slight tension on it Very gently start aerating the solution using the attached air pump and record the contractions using the WINDAQ data acquisition program The sampling rate should be set at a low frequency After suspending the muscle but before adding any drugs calibrate the transducer with a 10 g weight You will probably also need to turn the gain down on the transbridge amplifier probably to x100 or even x10 in order to observe full contraction of the ileum segment IV EXPERIMENTS A Bioassay for Acetylcholine ACh ACh interacts with post synaptic cholinergic receptors on the ileum to initiate and potentiate contractions Remember that the most peripheral receptors are affected first So are these nicotinic or muscarinic The magnitude of the resulting contraction is proportional to the dose of ACh applied within a restricted range of ACh concentrations which varies from muscle to muscle You will take advantage of this to construct a Dose Response curve and then use this