Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle

Abstract

In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α₁-adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α₁-adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

INTRODUCTION

Objectives and Overview

In the present paper, we describe a laboratory practical that demonstrates how the two smooth muscle organs of the lower urinary tract (LUT), the bladder and the urethra, differentially respond to neurotransmitters. The teaching of urinary tract physiology at both the undergraduate and postgraduate level can prove challenging, as students are often confused about the complex nature of LUT innervation. In particular, the observation that, while the urinary bladder and urethra are both smooth muscle organs and are involved in a functionally reciprocal physiological relationship, they are innervated by different neural pathways, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α₁-adrenoceptors. This differential innervation of urinary tract smooth muscle often leads to confusion among students and too often is only partially resolved by repetitive memorization of the innervation pathways.

We have devised a series of simple laboratory experiments as part of a practical class, which will more efficiently impart critical thinking and active student learning on this topic. This is achieved by using classical force measurements of dissected urinary tract smooth muscle tissues to demonstrate the effects of cholinergic and adrenergic agonists and antagonists on the contractions of both the bladder and the urethra.

Background

The mammalian LUT is composed of two organs, the bladder and the urethra (4). Under normal conditions, there is a reciprocal relationship between the two organs: as the bladder fills with urine, it remains relaxed to accommodate increases in volume without a significant increase in intraluminal pressure (4, 14). As this filling stage occurs, the urethra must remain closed to prevent leakage of urine from the bladder to the exterior. At the onset of urination, the bladder contracts to expel urine, while the urethra relaxes to facilitate the passage of urine. Thus contractions of the bladder and urethra are an essential physiological element in maintaining normal urinary continence and voiding.

The bladder is a smooth muscle organ, which possesses a thick smooth muscle coat termed the detrusor (12, 17). These layers are not always observed as distinct components; instead the smooth muscle bundles interlace and appear to form a mesh. This allows the detrusor to act as a syncytial mass that ensures that all dimensions of the bladder change shape uniformly during contraction (13, 18). Internal and external urethral sphincters regulate the flow of urine into and from the urethra, respectively. The external urethral sphincter (also known as the rhabadosphincter) is composed of striated muscle fibers and is contracted by acetylcholine (ACh) on nicotinic receptors via somatic innervation pathways (7). The internal urethral sphincter is composed of urethral smooth muscle (USM) and contributes greatly to urethral contraction during bladder filling (4, 5). Inappropriate or deficient contractions of the bladder or urethral musculature can lead to urinary incontinence (13, 16, 17), and conditions known as overactive bladder or detrusor overactivity (9, 14, 16, 19, 22). Although not life threatening, urinary incontinence and associated pathophysiological conditions can have severe detrimental effects on quality of life, as well as leading to many secondary health problems, such as depression and sleep disturbances (19). Thus it is vital to understand the physiological mechanisms by which the bladder and USM contract to correctly identify therapeutic interventions for alleviating pathophysiological conditions resulting from defects in LUT contractility.

While the bladder detrusor and internal urethral sphincter are both composed of smooth muscle cells and share common fundamental physiological traits [contractions are Ca²⁺ dependent and can be affected by depolarization (10, 11)], they receive differential neural input, which affects contractility. Detrusor smooth muscle (DSM) contraction occurs due to stimulation of the parasympathetic nervous system and subsequent binding of ACh to muscarinic receptors (3, 4, 14). This action causes large contractions of the DSM, resulting in bladder emptying. In contrast, cholinergic innervation is sparse in USM (2, 10), and USM is contracted by stimulation of the sympathetic nervous system via activation of α₁-adrenoceptors (1, 8, 10, 12, 15, 20). In this laboratory practical, students will observe these differential effects on mammalian DSM and USM by performing recordings of contraction of bladder and urethra muscle tissues.

Learning Objectives

The overall learning objective for this practical is to demonstrate to students how the different smooth muscle components of the LUT are affected by sympathetic and parasympathetic neural input.

Specific Learning Outcomes

After completing this activity, the student should be able to:

• • Draw and describe the basic components of the LUT anatomy.
• • Contrast how the sympathetic and parasympathetic nervous systems influence the contractile state of LUT organs.
• • Demonstrate experimentally how cholinergic and adrenergic agonists affect DSM and USM contractions.
• • Predict the effects of cholinergic and adrenergic antagonists on elicited contractions in both DSM and USM.

Activity Level

It should be noted that this practical requires skillful dissection of urinary tract tissues, which require a considerable amount of practice. For this reason, combined with the need for a contractile recording setup, this practical would not be suitable for the majority of second-level education institutions such as high schools. At the undergraduate level, this practical can be incorporated as a laboratory class on LUT/smooth muscle physiology, as part of either an undergraduate course of basic physiology, integrated physiology, human biology, biomedical science, cell biology, or life sciences or as an introductory LUT physiology class to postgraduates in physiology, pharmacology, medicine, or cell biology. Once again, due to the delicate dissections involved and time constraints, either the class instructor or a skilled technician may need to dissect and prepare tissues before the class. However, advanced students may be given the opportunity to perform the dissections themselves. This may be applicable to higher level undergraduate or postgraduate physiology or anatomy courses, where students may be experienced dissectors. If time allows, the instructor or technician may review the anatomy of the LUT as part of the introduction to the class and then demonstrate the dissections to students to make them familiar with the different smooth muscle organs of the LUT.

Prerequisite Student Knowledge

In order for the practical to be of the most benefit, students should have some appreciation of the differences between the parasympathetic and sympathetic nervous systems and have prior knowledge of the effects of ACh or adrenergic agonists in other systems. Possession of this preknowledge, imparted perhaps in conjunction with a traditional lecture, either before or as an initial part of the practical, will enable students to make predictions about experimental outcomes before they are tested.

Time Required

The bladder and urethral preparations take 30–60 min to set up for force measurement. The experiments will take ~120–150 min to complete.

METHODS

Equipment and Supplies

At the University of Nevada, Reno, our animal model for these experiments is the mouse. It is important to note that, whereas both male and female mice have a functional bladder and urethra, only male mouse urethras have been shown to respond with robust contractions in the presence of adrenergic agonists such as phenylephrine (PE). This is possibly due to a lack of smooth muscle cells or adrenergic receptors in the female mouse urethra (1), and, therefore, only male mice should be used in this experiment. A segment of proximal urethra and the entire bladder are needed from a wild-type mouse to fully complete this practical.

During the course of these experiments, DSM and USM tissues will be incubated in an organ bath. The authors of this study utilized Radnoti organ baths (15 ml volume), each filled with a physiological salt solution, Krebs-Ringer bicarbonate (KRB) solution. The organ baths were kept at physiological temperature during experimentation (37°C) by means of a heated water jacket. Water was heated and circulated through the baths using a water heater circulator (Isotemp 2100 Series Immersion Circulator, Fisher Scientific, Champaign, IL). KRB solution is brought to and maintained at a physiological pH (7.4) by bubbling the solution with a mixture of oxygen and carbon dioxide gas. Oxygenation of the tissues is necessary to keep tissues alive within the organ baths. The oxygen used in the laboratory at the University of Nevada, Reno, is from JW Welding Supply, Fallon, NV, and is 95% O₂/5% CO₂. Oxygen is stored in a tank affixed to a wall adjacent to the contractile setup. Oxygen is then allowed to flow from the tank to the organ baths via plastic tubing, which connects the outflow of the tank to the inflow of the organ bath. The degree of flow of oxygen can be controlled at the level of the tank through adjusting the pressure at the tank regulator or at the level of the organ bath by the use of adjustable plastic or metal clamps on the tubing. Each tissue will require its own bath, so, therefore, a total of six baths are required to perform an experiment with three DSM preparations and three USM preparations.

Once tissues are ready for mounting within the organ baths (each tissue type may be placed in any bath; however it is important to indicate in the recording software which tissue type each bath contains), they are connected on one end to a stable mount and on the other end to a force transducer. The experiment requires a force transducer able to connect to a computer with appropriate data acquisition software. The data acquisition software used should display a graph of force against time and optimally allow for marking drug additions. The authors of this study used AcqKnowledge software (3.9.1; Biopac Systems, Goleta, CA). The force transducer that the authors used in this experiment is a WPI strain gauge. To attach the tissue preparations to the stable mount and force transducer, suture is required, e.g., “Look” 5–0 nonsterile, black braided silk suture (Hospeq, Miami, FL).

A syringe (Becton Dickinson) is required to refill organ baths with oxygenated KRB solution. The authors have used a 60-ml catheter tipped syringe for ease of use. Alternative volume syringes will work, but it should be noted that, for practical use, the syringe volume should be no less than the volume of the bath (in this case, it was 15 ml).

The following drugs (all purchased from Sigma-Aldrich) are used at the given concentrations:

• Phenylephrine (PE) at 0.01 mol/l (10 mM) dissolved in deionized H₂O.

• Carbachol (CCh) at 0.01 mol/l (10 mM) dissolved in deionized H₂O.

• Atropine at 0.01 mol/l (10 mM) dissolved in deionized H₂O.

• Phentolamine at 0.01 mol/l (10 mM) dissolved in deionized H₂O.

Solutions

The physiological solution used in this experiment is KRB solution made up with the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 23.8 NaHCO₃, 1.2 KH₂PO₄, 11 dextrose. The KRB solution should be brought to physiological pH by bubbling with 95% O₂/5% CO₂.

Ethical Approval

All procedures described in this practical were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno. Adopters of this activity are responsible for obtaining permission for human or animal research from their home institution. For a summary of “Guiding Principles for Research Involving Animals and Human Beings,” please see https://www.physiology.org/author-info.animal-and-human-research.

Instructions

Instructions for tissue dissections.

macrodissection.

In our experiments, male mice were anesthetized with isoflurane and then humanely killed by cervical dislocation or decapitation before being used for experimentation. Other methods of animal sacrifice can be used as long as they are within the Institutional Animal Care and Use Committee protocol guidelines and within the regulations of the home institution.

Macrodissection steps are as follows:

• The abdomen of the mouse should then be opened with scissors, e.g., 3 mm Vannas Spring Scissors (Fine Science Tools, Foster City, CA), via one longitudinal incision from the lower abdomen to the sternum of the mouse. During the dissection, vertical cuts should be made from the tail toward the neck to break the pelvic bones. This gives the dissector better access to the LUT and will make LUT removal much easier.

• The bladder and urethra can then be removed with forceps (e.g., Dumont no. 5 Forceps, Fine Science Tools) and scissors. This is best done by holding the dome of the bladder with the forceps and pulling gently to separate the bladder and urethra from the surrounding adipose tissue. Then the LUT can be removed en bloc by cutting the tissue behind the bladder and below the urethra. The LUT should then be placed in KRB solution to prevent tissue degradation and to preserve physiological conditions.

• The skeletal muscle should be carefully removed from the urethra by sharp dissection. Skeletal muscle, which is striated, will appear as a reddish brown color compared with the translucent smooth muscle, which has no striations. When removing the skeletal muscle from the USM, it is important to cut at an angle away from the urethra to prevent any accidental tears or cuts in the USM.

• The urethra should be measured, and the distal urethra should be cut ~4 mm away from where the ureters enter the bladder. This 4-mm tube of proximal urethra can then be separated from the bladder by cutting where the urethra meets the bladder (Fig. 1A).

Fig. 1.

Preparation of detrusor muscle strips. A: diagrammatic representation of the LUT showing the bladder and urethra. During dissections, the urethra should be removed from the base of the bladder by cutting at the point of the dotted line. B: once the urethra has been removed, the bladder should be opened by making a longitudinal incision along the dotted line shown. C: once opened, the bladder should be pinned out flat on a Sylgard-bottomed dish, and the urothelium layer removed. Strips of DSM should then be taken by cutting the DSM along the dotted lines shown.

dsm strip preparation microdissection.

The steps for DSM strip preparation microdissection are as follows:

• The bladder should be cut longitudinally from the base of the organ to the dome (Fig. 1B). Scissors can be inserted in the opening resulting from the removal of the urethra, and the bladder can be opened with a single longitudinal incision. This results in a rectangle of DSM.

• Using fine dissection pins and a Sylgard-based dish, the bladder should be pinned out to hold the rectangular shape in place (Fig. 1C). A Sylgard 184 Silicone Elastomer Kit (Dow Corning, Midland, MI) is necessary to make the Sylgard-bottomed dish if none are premade and available. To make the Sylgard-bottomed dish, add one part Sylgard curing agent to nine parts Sylgard 184 silicone elastomer. This mixture should be mixed thoroughly and poured into a glass or plastic dish of desired diameter, taking precautions to allow air bubbles to rise to the surface of the dish. The Sylgard should be of sufficient depth to allow for the insertion of dissection pins. The dish should then be left to cure and dry until no longer tacky, i.e., ~12–24 h. The elastomer and curing agent have a dynamic curing range from 77°F to 302°F. Dishes can be placed beneath a heating lamp overnight to aid in the curing and drying process. The dish can be used for dissection once the Sylgard has cured and is no longer tacky.

• The innermost layer of bladder tissue, the urothelium, should be removed by holding the urothelium with one set of forceps and the underlying bladder smooth muscle tissue with another set of forceps. Ideally, the urothelium will peel away from the smooth muscle with ease. If the urothelium does not easily separate from the smooth muscle, small scissors should be used to gently cut away the urothelium. It is imperative to be patient and cautious while cutting the urothelium away, as a cut into the smooth muscle could destroy the tissue. Cuts made with scissors should be parallel to the DSM sheet to prevent any accidental cuts or tears in the smooth muscle.

• DSM strips of ~8 mm in length and ~2 mm in width can be cut from the bladder with the urothelium removed. Separate sutures should then be tied to both ends of the DSM strip. An initial double knot should be tied on one side of the bladder strip, and tying a second double knot with the loose end of the suture thread should create a loop. The loops should be secure on the tissue (helped by the use of double knots), as they will be hooked onto the stable mount and the transducer. As such, the loops should be an appropriate size to ensure the entire tissue is submerged in KRB solution in the bath when attached to the transducer and stable mount. If loops are too large, the tissue will not be fully submerged, which will degrade tissue health and prevent exposure to drug application. Knots should be tightened on the tissue and the loops, as loose knots will prevent accurate transducer readings and could result in unsuccessful experimentation.

usm ring preparation microdissection.

The steps for USM ring preparation microdissection are as follows:

• The urethra should be isolated from the bladder as described above to make a ring preparation from the remaining urethral tube (Fig. 2A).

• Sutures should be passed through the urethra using a sewing needle (Fig. 2B). Once the suture has been passed through the urethral ring, the looped end of the suture should be cut, revealing two threads (Fig. 2C). In the absence of suture thread to create sutures, wires may also be used to attach the USM ring to the contractile mounts. In our laboratory, we prefer the use of suture thread, as it is more malleable for dissectors and can be adjusted for tissues of different sizes.

• The ends of the threads should then be tied with a tight double knot (Fig. 2D). The double knot should be tight, as a loose knot will mask contractions and could result in an unsuccessful experiment. The result should be an intact urethral tube with two tied loops on either side. This urethral preparation will measure contractions in the circular direction as the tube constricts.

Fig. 2.

Preparation of the urethra tube. A: diagrammatic representation of the urethra tube after removal from the bladder. B: a suture should be passed through the opening in the urethral tube with the aid of a sewing needle. C: scissors should be used to cut the suture once it has passed through the urethral tube, resulting in two separate strings. D: these strings should be tied to create a loop above and below the urethral tube. The tissue is now ready for setup on the force transducer.

Recording software setup.

The data acquisition software should be set up and calibrated to an appropriate weight (e.g., 1 g). Data should be collected at an acquisition rate of 20.0 samples/s for the entirety of the experiment. The acquisition rate of 20.0 samples/s ensures enough data points are collected for accurate analysis without creating an excessively large file. The time and amplitude scale should be adjusted to an appropriate level to easily view contractions. Once the tissue preparations are ready to be attached to the transducer, the recording should be started. It is suggested to keep recording data throughout the entire experiment, as this will allow easy comparison and constant monitoring of the tissue responses. The USM ring preparation and the DSM strip preparation should then be attached to the transducer and initially stretched to ~0.25 g of tension. To attach the tissues to the force transducer, one of the tied loops should be put around the thin wire recording contractions, and the other should be attached to the stable mount (Fig. 3). Once on the force transducer, tissues need to be equilibrated for ~1 h in oxygenated, warmed KRB solution (KRB solution is maintained at 37°C by heating jackets surrounding the organ bath, as described above). Tissue equilibration is necessary to restore ionic gradients that were altered while the tissue was immersed in cold KRB solution during dissection and to improve the reliability of consistent responses to any applied drugs. KRB solution is maintained at 37°C by storing KRB solution inside a glass beaker, which is kept in a heated water bath maintained at 37°C. This KRB solution should be brought to physiological pH by bubbling with 95% O₂/5% CO₂. Similar to oxygenating the organ baths, this beaker of KRB solution is oxygenated via plastic tubing, which connects the outflow of the tank to a piece of plastic tubing that ends inside the KRB solution. Tissues should be washed by replacing the KRB solution every 15 min. Washing the tissues is important to prevent the build up of toxins and possible debris, which can be detrimental to tissue health. Moreover, the washing process is essential to remove drugs from the baths and to keep tissues alive and healthy. To wash the tissues, the KRB solution that is in the bath should be drained, and new KRB solution should be added. Our laboratory uses a vacuum system to drain the KRB solution from the tissue baths; this KRB solution is subsequently diverted to a waste storage container. If a vacuum system is not available for use, gravity can be used to drain KRB solution from the organ baths. To use gravity, the baths should be above the waste storage container with tubing connecting the tissue baths to the waste storage container. Washing should be repeated a minimum of three times in quick succession to fully wash out drugs from the bath (each wash will reduce the concentration of drug in the bath ~10-fold). It is important to avoid disturbing the tissues when reintroducing KRB solution, and this can be accomplished by inserting the fresh solution down the walls of the bath in a calm and controlled manner. Disruption to tissue during washing can cause several problems, including the possibility of tearing tissue, changing the tension applied to the tissue, and creating artifacts on the data acquisition software. Finally, it is important for students to practice good organization by properly marking all events in the recording software and in a laboratory notebook. The time of event, concentration, name of drug applied, and the cause of an apparent artifact (e.g., increased bubbling, application of tension, or accidental disruption of the wire during washes, etc.) should be noted.

Fig. 3.

Organ bath used in experimentation. Diagrammatic representation is shown of the organ bath setup used in performing force measurements with the urethral tube set up to the force transducer, as detailed in the text. The looped sutures of the ring preparation and the strip preparation should be placed on the wires of the force transducer and the stable mount (A). The inflow of oxygen is indicated (B). Oxygen bubbles should be present in the bath in a manner that does not interfere with the tissue preparations.

Due to the delicate nature of the dissection and the time required for tissues to equilibrate, students should not attempt to perform the dissection and mounting to the force transducer if there is a time constraint. As mentioned above, in most classes, either the instructor or a technician will need to dissect and prepare tissues ahead of time, as the dissections are intricate and require a great deal of experience to perform quickly. However, advanced students with dissecting skills, perhaps in anatomy or physiology undergraduate/postgraduate laboratories, may be allowed to perform dissections if time and tissue availability permits.

Experiments

Experiment 1: Effect of CCh on DSM and USM contractions.

CCh is a cholinergic agonist that activates muscarinic cholinergic receptors, i.e., the main excitatory pathway in DSM. Begin the experiment by recording ~5 min of baseline activity. Using a pipette, draw 1.5 µl of 10 mM CCh stock solution and expel into the baths containing the DSM and USM preparations. (Note that this volume is for a 15-ml bath. If a different volume bath is being used, the volume of stock added will need to be recalculated to ensure a final concentration of 1 µM CCh.) The bubbling O₂ should mix the drug into solution to a 1 µM final concentration. Indicate the addition of 1 µM CCh (final concentration) in the acquisition software and observe the resulting contractions for 5 min. After 5 min have transpired, drain the KRB solution from the bath and quickly add fresh, warm KRB solution. Any contraction should cease after washing the tissue with fresh KRB solution. If not, students should repeat the washing process. It is important to wash out completely so the tissue does not become desensitized. To test the validity of the results observed, students should repeat the addition of CCh to the baths to see if the effect is the same. A second contraction should be similar in amplitude to the first contraction. Allow 15 min after washout for tissues to reequilibrate before eliciting a second control response. These reproducible controls are vital to validate any inhibition responses seen later in any other experiments. An example of a reproducible contraction to CCh in the DSM is seen in Fig. 4A. It is important to note that the two contractions have similar amplitudes. After 5 min, wash the bath and quickly replace with warm, oxygenated KRB solution. Allow tissue to equilibrate for 15 min after washout before proceeding with the next experiment.

Fig. 4.

Effect of CCh on DSM and USM contractions. A: representative contractile trace showing the effect of 1 µM CCh on DSM contractions. B: representative contractile trace showing the effect of 1 µM CCh on USM contractions. *Artifact resulting from washing of the tissue within the organ bath.

Experiment 2: Effect of PE on DSM and USM contractions.

The next experiment students will perform is observing the effect of PE on the DSM and the USM. PE is a selective α₁-adrenergic agonist; α₁-adrenoceptors are the main excitatory pathway in USM. To begin the experiment, stable baseline activity should be recorded before addition of any drugs. Using a pipette, draw 15 µl of the 10 mM PE stock solution, ensuring it contains no bubbles. (Note that this volume is for a 15-ml bath. If a different volume bath is being used, the volume of stock added will need to be recalculated to ensure a final concentration of 10 µM PE.) Insert the pipette tip into the bath containing the USM and cautiously eject the PE into the bath in a slow and uniform manner. Be cautious to avoid touching the tissue with the pipette tip and be careful that the force of PE ejection does not disrupt the tissue. Repeat this process for the bath containing the bladder. The bubbling O₂ should mix the drug into solution to a 10 µM final concentration. Indicate the time of drug addition in the acquisition software. Observe effects of PE for ~5 min. After 5 min have transpired, drain the KRB solution from the bath and quickly add fresh, warm KRB solution. Any contraction should cease after washing the tissue with fresh KRB solution. If not, students should repeat the washing process. It is important to wash out completely so the tissue does not become desensitized. To test the validity of the results observed, students should repeat the addition of PE to the bath to see if the effect is the same. A second contraction should be similar in amplitude to the first contraction. Allow 15 min after washout for tissues to reequilibrate before eliciting a second control response. These reproducible controls are vital to validate any inhibition responses seen later in any other experiments. An example of a reproducible contraction to PE in the USM is seen in Fig. 5B.

Fig. 5.

Effect of PE on DSM and USM contractions. A: representative contractile trace showing the effect of 10 µM PE on DSM contractions. B: representative contractile trace showing the effect of 10 µM PE on USM contractions. *Artifact resulting from washing of the tissue within the organ bath.

Experiment 3: Effect of phentolamine on CCh-induced DSM contractions.

To begin this experiment, obtain control responses to CCh in the DSM preparations as described in experiment 1. Then wash tissues with regular KRB solution and record a baseline for ~15 min. Add 1.5 µl of 10 mM phentolamine stock to the bath for a final concentration of 1 µM phentolamine using a pipette, being cautious not to disrupt tissue with the pipette. Allow the DSM to be exposed to phentolamine for 20 min. The KRB solution in the baths should be oxygenated during this time. After the 20-min incubation period, introduce 1 µM CCh into the bath containing the DSM. Observe and record effects of phentolamine on tissue contractions for 5 min or longer until tissue contractions have plateaued and stabilized and then wash the tissues. Phentolamine will require multiple washouts to reverse effects.

Experiment 4: Effect of phentolamine on PE-induced USM contractions.

To begin this experiment, obtain control responses to PE in the USM preparations as described in experiment 2. Then, wash tissues with regular KRB solution and record a baseline for ~15 min. Add 1.5 µl of 10 mM phentolamine stock to the bath for a final concentration of 1 µM phentolamine using a pipette, being cautious to not disrupt tissue with the pipette. Allow the USM to be exposed to phentolamine for 20 min. The KRB solution in the baths should be oxygenated during this time. After the 20-min incubation period, introduce 10 µM PE into the bath containing the USM. Observe and record effects of phentolamine on tissue contractions for 5 min or longer until tissue contractions have plateaued and stabilized then wash the tissues. Phentolamine will require multiple washouts to reverse effects.

Experiment 5: Effect of atropine on CCh-induced DSM contractions.

After phentolamine has been washed out, students will experiment with the effects of blocking cholinergic receptors with the muscarinic cholinergic antagonist atropine. To begin this experiment, obtain control responses to CCh in the DSM preparations as described in experiment 1. Then record ~15 min of baseline before adding 1.5 µl of 10 mM atropine stock to the DSM baths. This will give a final atropine concentration of 1 µM in a 15-ml bath. Allow tissues to incubate in atropine for 20 min. After the 20-min incubation period has concluded, add 1 µM CCh to the bath containing the DSM. Allow tissues to be exposed to CCh for 5 min. Observe effects of atropine on CCh-induced contractions. After 5 min, wash tissues.

Experiment 6: Effect of atropine on PE-induced USM contractions.

To begin this experiment, obtain control responses to PE in the USM preparations as described in experiment 2. To begin the experiment, record ~15 min of baseline before adding 1.5 µl of 10 mM atropine stock to the USM baths. This will result in a final atropine concentration of 1 µM in a 15-ml bath. Allow tissues to incubate in atropine for 20 min. After the 20-min incubation period has concluded, add 10 µM PE to the bath containing the USM. Allow tissues to be exposed to PE for 5 min. Observe effects of atropine on PE-induced contractions. After 5 min, wash tissues.

Troubleshooting

There are three key points during the experiment to be aware of: the handling and dissection of USM and DSM preparations, setup and maintenance of the contraction apparatus, and drug administration.

Preparation handling.

It is of utmost importance to keep tissues in physiological solution at all times to prevent tissue degradation. The KRB solution should be replaced regularly (every 15 min), and baths should be monitored to ensure tissues are well oxygenated. One must be extremely cautious when dissecting to avoid any tissue damage, e.g., when removing skeletal muscle from USM or the urothelium from DSM, as any unwanted tears or cuts in the smooth muscle will result in nonviable tissues. When tying the knots on the tissues, it is important to tie sturdy double knots that will hold. The double knots should be rechecked before being put on the contraction apparatus. In the USM preparation, the two looped threads should not cross each other at any point in the urethral tube, as this will obscure any contractions and confound results.

Contraction apparatus.

It should be taken into consideration that constant oxygenation of the KRB solution in the organ baths (to maintain pH and oxygenate tissues), application of appropriate tension, and the maintenance of KRB solution at physiological temperature in the baths (maintained by the heating jackets in the baths) are required. The level of oxygen flow into the baths should never be so high that it creates artifacts in the recording; however, constant oxygenation of the KRB solution is needed to ensure mixing of any drugs applied as well as to prevent tissue degradation. Thus appropriate oxygenation should be monitored throughout the experiment. The difference between a contraction and an artifact from high oxygen flow will be apparent as contractions are consistent, whereas artifacts will be variable and disordered. If students are unsure, the oxygen flow into the organ bath may be stopped completely for a short time; if the “contractions” cease immediately, then the students were observing artifacts resulting from high oxygen flow to the organ baths. The USM and DSM preparations should be stretched to an appropriate tension to allow for accurate contractile recordings. The USM and DSM tissue preparations should be stretched to 0.25 g at the start of the experiment. The tissues will likely relax to an ideal range of 0.1–0.2 g, which will allow for accurate readings. If too much tension is applied, it is possible that the tissues will tear. On the contrary, the acquisition system will not be able to accurately record contractions in a tissue that is understretched. The temperature in the baths should always be kept at physiological temperature throughout the entire experiment, a constant 37°C. Physiological temperature is required to keep tissues healthy and to best represent tissue responses under accurate physiological conditions. If abnormal contraction responses are observed in the experiments, students and instructors should first check the above items, oxygen flow, temperature, and appropriate tension on tissue preparation, as disruptions in any of these can cause tissues to rundown prematurely.

Students should be aware that the time scale and force scale on the acquisition software should be set to an appropriate range. USM contractions can be viewed at a force scale of 0.25 g, with the midpoint at 0.00 g. DSM contractions can be viewed at a force scale of 0.4 g, with the midpoint at 0.00 g. However, these are only guidelines, and each tissue varies to a certain extent, so students should be aware that they might need to adjust the force scale to view tissue contractions on their computer. Both USM and DSM contractions can be seen in real time when the time scale is set to 5 min.

It is also important for students to check that the wires connecting the preparation to the force transducer are not touching the bath, the tissue, or any other wires. If these wires touch, any muscle contraction will be masked, and inaccurate results will be obtained.

Drugs.

Addition of correct concentrations of drugs is imperative to a successful experiment. Students should take the bath volume into consideration when calculating the volume of drug needed. The authors used 15-ml baths, but bath volume may vary between different setups, so students should be aware that the values presented in this paper are only guidelines and should be adjusted for the bath volume that they are using.

Appropriate concentration of drugs should be applied accurately and consistently in the same location within the bath during each addition. Finally, it is important to emphasize to students the necessity of keeping records of the drugs and their relevant concentrations that they are adding, by noting it both in the acquisition software (if it allows) and in their laboratory notebook.

There are also several general points to consider while experimenting. Foremost is the importance of generating reproducible, similar control contractions to PE in the USM and CCh in the DSM at the start of the experiment. Good control contractions are vital to accurately evaluate the effect of atropine and phentolamine. If the control contractions are dissimilar in amplitude, it will be necessary to contract the tissue a third time to generate an appropriate control. Second, it is important to follow the guidelines for washout times. The effect of phentolamine can be reversed, but it will take several washouts to remove the drug from the tissues. The effect of atropine is difficult to reverse and should thus be the last drug used in the experiment.

RESULTS

Expected Results

Experiment 1: Effect of CCh on DSM and USM contractions.

Students should expect to see a robust DSM contraction in response to 1 µM CCh (Fig. 4A), but should see no contraction to CCh in the USM preparation (Fig. 4B). CCh is a cholinergic agonist that mimics ACh, an activator of muscarinic cholinergic receptors that results in DSM contraction. In the DSM, cholinergic receptors are the main excitatory pathway, but in the USM the main excitatory pathway is activation of α₁-adrenergic receptors.

Experiment 2: Effect of PE on DSM and USM contractions.

Students should observe a robust contraction of USM in response to 10 µM PE (Fig. 5B). However, no DSM contraction should be observed in response to PE (Fig. 5A). PE is an α₁-adrenergic agonist. Adrenergic receptors are the main excitatory pathway in the USM. Thus PE introduced to USM will act on the adrenergic receptors and cause a contraction. However, PE will have no effect in the DSM because the main excitatory pathway in the DSM is through cholinergic receptors.

Experiments 3 and 4: Effect of phentolamine on CCh-induced DSM contractions and PE-induced USM contractions.

Students should expect to see an abolition of PE-induced contraction in the USM, but no effect on CCh-induced contraction in the DSM. Phentolamine, a potent α-adrenergic antagonist, blocks the adrenergic receptors to which PE binds to cause contraction of USM. With these receptors blocked, PE cannot induce the expected contractile response, and no contraction would be expected in the USM. Conversely, phentolamine will have no effect on CCh-induced contractions in DSM, because CCh acts on muscarinic cholinergic receptors and not adrenergic receptors (see Figs. 6 and 7).

Fig. 6.

Effect of phentolamine on DSM contractions induced by CCh. Representative contractile trace shows the effect of 1 µM phentolamine on DSM contractions induced by CCh. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Fig. 7.

Effect of phentolamine on USM contractions induced by PE. Representative contractile trace shows the effect of 1 µM phentolamine on USM contractions induced by PE. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Experiments 5 and 6: Effect of atropine on CCh-induced DSM contraction and PE-induced USM contraction.

Students should expect atropine to inhibit contractions induced by CCh in the DSM, but atropine should have no effect on PE-induced contractions in the DSM. Atropine, a powerful anticholinergic drug, blocks the muscarinic receptors that CCh activates. When these receptors are blocked, CCh cannot bind and initiate contraction; therefore, the CCh-induced contraction in DSM will be inhibited by 1 µM atropine (Fig. 8). However, since PE acts on adrenergic receptors and not cholinergic receptors, atropine is expected to have no effect on PE-induced contractions in the USM (Fig. 9).

Fig. 8.

Effect of atropine on DSM contractions induced by CCh. Representative contractile trace shows the effect of 1 µM atropine on DSM contractions induced by CCh. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Fig. 9.

Effect of atropine on USM contractions induced by PE. Representative contractile trace shows the effect of 1 µM atropine on USM contractions induced by PE. *Artifact resulting from washing of the tissue within the organ bath. Double slash marks denote time period of 15 min.

Evaluation of Student Work

The purpose of this laboratory practical is to educate students on how the organs of the LUT are innervated by different pathways and thus have differences in their responses to neurotransmitters. Students should complete a laboratory report summarizing the practical. This should contain at a minimum: 1) a detailed labeled drawing of the LUT before dissection, highlighting the location of the bladder and urethra; 2) a description of the experimental protocol and the predicted results of each experiment (with justification for this rationale); and 3) a description of results obtained and interpretation. As well as this laboratory report, which may constitute a summative assessment assignment, student learning and understanding of the concepts involved should also be assessed in a formative manner.

Student understanding may be best evaluated with a classroom assessment technique (CAT). Three different appropriate CATs for this purpose are described below. After all CATs, the instructor can quickly review the answers given by students to ascertain what has been understood and what may be unclear and warrants further revision, either directly in that class (this may be a reason to have a supportive lecture after the practical rather than before) or in a future tutorial. These CATs can be used for nongraded, formative assessment to allow the instructor to correct any misalignments of student understanding and stated learning objectives and could also be used as a base for creating summative assessment exam questions and exercises.

• A 5-min three-point paper: At the start of the next class following the practical, students are given 5 min to quickly write down three important points taken from the practical. The three main points should be some variation of the following: 1) the bladder is contracted by cholinergic/parasympathetic input/the bladder is contracted by ACh; 2) the urethra is contracted by adrenergic/sympathetic input/the urethra is contracted by PE; and 3) the bladder and urethra are involved in a reciprocal relationship, and maintenance of normal LUT function relies on different innervation pathways acting on each organ.

• Empty-outline CAT: At the start of the next class following the practical, students are given a blank diagram of the LUT with missing anatomical and innervation information. Students are given 5 min to use their understanding of the laboratory practical content to fill in the empty outlines.

• Critical thinking quiz: Students are presented with a series of questions designed to test their critical thinking and not merely recall abilities. Answering the questions correctly involves systematically thinking about the experiments performed in the practical and the results achieved. Examples of questions (Q) to use are shown below; correct answers (A) are included. These questions may be best given after the completion of a lecture that compliments the practical.

  • Overactive bladder is a condition in which the bladder smooth muscle is too excitable and overcontracts when the bladder is not yet full. This can manifest as an increased voiding frequency and sudden urge to urinate. Would anticholinergic or antiadrenergic drugs be more appropriate to use in this condition and why?

  • Anticholinergic drugs are more appropriate as CCh contrasts the bladder smooth muscle, an effect blocked by atropine. Antiadrenergic drugs have no effect on bladder contractions as seen in the laboratory practical.

  • Describe what symptoms might be expected in the bladder if ACh release is increased.

  • Overactive bladder/detrusor overactivity. As the increased ACh will lead to increased contractions of bladder smooth muscle, this could manifest as increased voiding frequency or increasing urge to urinate.

  • Bladder sphincter dyssnergia occurs after injuries to the spinal cord, and in this condition the bladder and urethra contract simultaneously during voiding. Why is this undesirable?

  • Normally there is a synergistic relationship between the bladder and urethra. When the bladder contracts, the urethra relaxes, allowing it to open and thus allowing urine to pass through. If the urethra remained contracted during urination, voiding could not occur, and urine could not pass through the constricted sphincter.

Additionally, using a variation of the empty outline CAT in a follow-up question, which could be used in a summative examination as well as a CAT, students can be shown an example trace of one of the contractile experimental protocols (e.g., CCh on DSM) with the name of the tissue absent (or the tissue named but drug name absent). Then students could be asked to explain which tissue (or agonist) is presented in the trace and why. This will evaluate how well students have grasped the concept of differential urinary tract innervation.

In more clinically applicable programs, such as medicine or clinical pharmacology, students could be tested on their understanding of drug actions and uses pertaining to urinary tract function. To do this, students should be presented with a list of commercially available pharmacological treatments for urinary tract dysfunction, including anticholinergic (e.g., oxybutynin) and antiadrenergic (e.g., doxazosin) drugs. Using this list and drawing on observations from the laboratory practical, students could be asked to determine on what urinary tract tissue this drug will have an effect. Alternatively, a clinical case (e.g., overactive bladder, urethral sphincter obstruction) could be presented and students asked to prescribe an appropriate pharmacological treatment.

As mentioned previously, a traditional lecture that takes place in conjunction with the practical would help to reinforce the desired learning outcomes and, if taking place after the practical, will allow opportunities to evaluate student understanding through some of the CATs described above. Thus student understanding of the practical can be assessed and any issues addressed in the lecture.

A summary of how these assessment techniques are constructively aligned with the desired learning outcomes and planned teaching strategies for this practical are shown in Table 1.

Table 1.

Constructive alignment of learning outcomes, teaching strategies, and assessment

Learning Outcomes	Teaching Strategies	Assessment
Draw and describe the basic components of the LUT anatomy.	Demonstration/review of dissection.	Laboratory report.
	Related class lecture.	Empty outline CAT.
	Initial practical introduction.
Contrast how the sympathetic and parasympathetic nervous systems influence the contractile state of LUT organs.	Students test agonists on LUT organs during practical.	Monitored during practical.
	Related class lecture.	5-min three-point paper.
Demonstrate experimentally how cholinergic and adrenergic agonists affect DSM and USM contractions.	Students test agonists on LUT organs during practical.	Monitored during practical.
Predict the effects of cholinergic and adrenergic antagonists on elicited contractions in both DSM and USM.	Ask students to make these predictions during the practical, in their laboratory reports and again as a class wide Q&A session in the related lecture.	Monitored during practical.
		Laboratory report.
		Empty outline CAT.
		Critical thinking quiz.

Limitations/Adaptations

This laboratory practical has been designed for the use of mouse tissues. However, in the absence of mice, other mammalian tissues, such as those from rabbit, guinea pig, or rat, would be expected to yield similar results. It should be noted that the female mouse urethra does not yield a robust reproducible response to PE, possibly due to a lack of smooth muscle cells or adrenergic receptors in the female mouse urethra (1); thus male mice should be utilized. While large amounts of DSM strips can be harvested from a single mouse bladder, the availability of sufficient urethral tissue can be problematic in the mouse. Due to the limited size of the male mouse urethra, a single animal will be needed for a single organ bath, as the urethra will be attached to the transducer as an intact ring preparation. We have found that attempting to use dissected strips of mouse USM are unviable due to the small size and delicate nature of the tissue. However, the use of larger animals, such as rabbit, guinea pig, or rat, might facilitate the use of urethral strips (dissected in a similar manner to DSM strips) instead of ring preparations, thus allowing multiple urethral strip preparations to be harvested from a single animal. To reduce the number of animals used per experiment, a limited number of urethra preparations should be used and perhaps even a single representative preparation could be used, depending on class size and number of animals available. Instructors should be aware that, while the current paper utilized CCh and PE to contract DSM and USM, any agonist of M₃ receptors or α₁-receptors, respectively, would suffice.

If time or tissue availability is a constraint, either the bladder or urethra experiments on their own may be performed as a stand-alone practical. The bladder may be preferable in such a situation, as the DSM dissection is more straightforward and can be more easily handled by students of varying skill levels. As noted above, multiple DSM strips may be harvested from a single animal, thus making a bladder-only practical preferable if tissue availability is limited.

Additional Resources

For additional information on this topic, please see Refs. 6, 8, 9, 11, 13–15, 21, 22.

GRANTS

This project was supported by exploratory funding from the University of Nevada, Reno. B. E. Rembetski and B. T. Drumm received salary support from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant R01-DK-091336. C. A. Cobine received salary support from NIDDK Grant R01-DK-078736.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.E.R. performed experiments; B.E.R. analyzed data; B.E.R. and C.A.C. prepared figures; B.E.R., C.A.C., and B.T.D. edited and revised manuscript; B.E.R., C.A.C., and B.T.D. approved final version of manuscript; B.T.D. conceived and designed research; B.T.D. interpreted results of experiments; B.T.D. drafted manuscript.