one hour

Muscarinic receptors are usually involved with in the transduction of cholinergic signals within the central nervous system, smooth muscles, autonomic ganglia and other parasympathetic end organs. Muscarinic receptors belong to the superfamily of G-proteins which are coupled up with receptors, such as the rhodopsin-like receptors. They are related to the ionotropic nicotinic acetylcholine receptors; this is in so far as their physiological acetylcholine is concerned.

Muscarine therefore is an organic molecule which is found in the peripheral nervous system, and does not have any effect central nervous system; this is because it does not get close to the blood brain barrier. Muscarinic receptors (mAChRs) are closely represented in all the organs, cells and tissues. The central mAChRs deal with the regulation of the extraordinary number of behavioral, cognitive, and sensory motor functions (DUJUS, 2008). These neurotransmitter receptors have revolutionized science, because the discovery of increased or reduced signaling through distinct mAChR subtypes has the ability of causing psychophysiology of the major central nervous diseases like schizophrenia, Parkinson’s, Alzheimer’s, epilepsy, and depression. The five subtypes of receptors include M1, M2, M3, M4 and M5. It is important to understand the function of these subtypes so as to be able to develop a drug for the treatment of neurological deficits.

Oxotremorine is a parasympathomimetic medicine which acts as selective muscarinic acetylcholine receptors. Both the immune system and the nervous system communicate bidirectionally, and lymphoid tissues are innervated by the autonomic nervous system. Cells of most immune systems have been found to contain large components of the cholinergic system, and they include choline transporters, choline acetyltransferase, and acetylcholinesterase. The effects of using Oxotremorine were found to activate the antigen-specific antibody cell (AFC). The treatment through using Oxotremorine has improved the response of the AFC of the spleen cells to SRBC (Bering B, 1992). In short, Oxotremorine has the effect of enhancing both the antigen-induced antibody responses and the antigen-mediated T-cell proliferation.

Hyoscyamine is a tropane alkaloid which is a secondary metabolite, it is usually found in plants that belong to the family of Solanaceae (Nightshades). This drug has been used to offer relief to various gastrointestinal disorders. It has also been used to control Parkinson’s disease, heart problem, and respiratory symptoms.

There is a major limitation to the use of drugs that are used to treat muscarinic agents that is attributed to the moderately few selectively for M2/M4 versus M1/M3/M5 receptor subclasses. This is because the subclasses have different proteins, and thus it will be hard to have the drug reacting similarly with the different five proteins. This has therefore brought about different reactions within the body

Drugs that have proven to be effective are those drugs that have less acetylcholine compared to acetylcholinesterase, as the muscarinic has been able to develop resistance towards it. Hyoscyamine has been found to have a lot of antagonist actions through the competitive antagonism, in the sense that they have to bind to muscarinic receptors, and they therefore have no intrinsic activity after this binding which blocks the endogenous ligand (Vrana, 2012). The side effects that can be observed from this drug include nausea and vomiting. When the side effects become more adverse, they can lead to dry mouth, blurred vision (mydriasis), hot and flushed skin, tachycardia, constipation, agitation, delirium, and urinary retention.

Acetylcholine is released by the axonal terminals of the cholinergic receptors, and the cholinergic receptors are found specifically on target neurons. The receptor subtypes can also be expressed through multiple non-neuronal cells, both in the central nervous system and the peripheral nervous system, which includes mesothelial, epithelial, endothelial, and immune cells. The primary role of muscarinic cholinergic receptors is found in the brain; they serve the purpose of ensuring that there is a regulatory role within the brain that is within the venules and the capillaries.

It is important to note that the muscarinic receptors tend to respond more slowly when compared with nicotinic receptors. The effect the muscarinic receptors may be either inhibitory or excitatory. The skeletal muscles are not affected by the muscarinic receptors, but they do have influence over the exocrine glands. The neurons and the skeletal muscles, on the other hand, deal with intrinsic electrical and mechanic rhythmic activities that are modulated, rather than initiated.

The purpose of this experiment was to understand and quantify the effects of drugs on biological systems, using in vivo pharmacology methods. Five compounds were administered one apiece to five different mice. Mouse #32, with the red/blue marked tail, was the control, and injected with a dose of 0.9% saline. Mouse #29, with the red marked tail, was injected with a low dose (0.01 mg/kg) of Oxotremorine. Mouse #30, with a blue marked tail, was injected with a high dose (0.1 mg/kg) of Oxotremorine. Mouse #31, with the black marked tail, was injected with a single dose (1.0 mg/kg) of Hyoscyamine. Mouse #28, with the unmarked tail, was injected with a dose of Hyoscyamine, followed thirty minutes later by a high dose of Oxotremorine. Mouse #37, from a separate pen, also with a blue and red marked tail, was also injected with a single dose (1.0 mg/kg) of Hyoscyamine. The parameters of interest included the parasympathetic nervous system, the sympathetic nervous system, and the central nervous system. The key observations fell within the categories of the mice's typical/atypical behavior, CNS excitation/depression, reflexes, ocular responses, and dermal anomalies. Afterwards, an assessment of potential adverse effects was performed.

The monitored observations included behavior, such as aggressiveness, fearfulness, passiveness, the presence/absence of grooming, and vocalization. CNS excitation, such as increased motor activity, tremors, fasciculations, clonic convulsions, tonic convulsions, increased respiratory rate, lack of respiratory depth, and straub tail were recorded if present. CNS depression, such as a lack of motor activity, ataxia, analgesia, catatonia, a lessened respiratory rate, and an increased respiratory depth were also watched conditions. The mice's reflexes, such as their corneal, pinnal, grasping, and righting reflexes were monitored and recorded every fifteen minutes as well. Ocular effects, such as enopthalmos, exopthalmos, lacrimation, chromodacryorrhea, and ptosis were all watched for, despite not being expected adverse effects from either drug used in the experiment. Dermal observations, including blanching, hyperemia, and cyanosis were noted if they occurred Some general observations, including muscular weakness, salivation, piloerections, micturition, diarrhea, writhing, body posturing, and rectal temperatures were observed and recorded as necessary.

Before any drugs were given, the baseline for each mouse was observed and noted. If the behavior or effect was present (1), it was noted, and the absences (0) were noted as well. After the mice were injected, the above mentioned observations were monitored every fifteen minutes for a period of ninety minutes. At the thirty minute mark, sixty minute mark, and at the end of the ninety minute period, the observations were tallied, averaged, and then plotted via Excel spreadsheet.

Methods

The mice used should be 3-4 week old and weigh between 29 and 32g. Before injecting the animals, observe the baseline behavior and check the baseline temperature. Each mouse will get the following injections:

1 Control (0.9% Saline). Inject 100 to 200 ml/animal.

2 Low dose of Oxotremorine (0.01mg/kg).

3 High dose of Oxotremorine (0.1mg/kg).

4 Hyoscyamine (1mg/kg).

5 Hyoscyamine (1mg/kg) and high dose of Oxotremorine (0.1mg/kg). Inject with Hyoscyamine first and observe the animal for 30 min. Then, inject the animal with the high dose of Oxotremorine.

Volume may need to be adjusted based on the weight of the animal. Inject the animals and observe the effects. The effects need to be checked every 10 minutes, up to 90 minutes. Temperature will be checked at the end of 30 min, 60 min and 90 min. The calculation for saline is as follows:

1mg/kg = 0.001mg/g

Saline: 100 - 200ul/animal

Treatment: 100 - 200ul/animal

The calculation for each drug dose is listed below. The doses below were calculated based on a weight average of 31 g per mouse. If the mice weight more or less, re-calculate the volumes needed for the injections.

1- Oxotremorine (0.01mg/kg) Low Dose (0.01mg/kg)

0.00001mg/g x 31g mouse = 0.00031mg (=0.31 mg) Oxotremorine is required for a 31g mouse. 0.31 mg Oxotremorine is present in 100 ml Stock Oxotremorine solution.

2- Oxotremorine (0.1mg/kg) High Dose (0.1mg/kg)

0.0001mg/g x 31g mouse = 0.0031mg (=3.1 mg) Oxotremorine is required for a 31 g mouse. 3.1 ug Oxotremorine is present in 100 ml of Stock Oxotremorine solution (labeled OxoHigh).

3- Hyoscyamine (1mg/kg)

0.001mg/g x 31g mouse = 0.031mg (=31 mg) Hyoscyamine is required for a 31g mouse.

31 mg of Hyoscyamine is present in 100 ml of Stock Hyoscyamine solution.

Some examples of calculating the correct volumes are as follows, with a 38g mouse and Hyoscyamine (1mg/kg).

First, calculate the amount of Hyoscyamine required for the given mouse.

Animal weight x dosage concentration = Concentration administered to animal

38g x 1mg/1000g = 0.038mg, (38 mg) is required per mouse.

Next, calculate the volume of stock solution that contains 0.038mg (=38 mg) of Hyoscyamine.

Drug/Stock concentration x Concentration administered to animal = volume to deliver to animal

38 mg x 1 ml/0.31mg = 0.123ml = 123 ml

Then, withdrawal 123 ml from the given stock solution and administer to the mouse.

Results

Table 1: Control Mouse #32, 0.9% Saline

Graph 1: Control Mouse #32, 0.9% Saline

Table 1 and Graph 1 show the results of the control mouse that was dosed with saline. The mouse (#32) weighed 27.9g was injected with a dose of 0.9% saline based on its weight, using the aforementioned calculation. Its baseline showed normal grooming behavior and normal reflexes. Its rectal temperature was 38.5 degrees Celsius. After thirty minutes it became passive, but continued its grooming behaviors and normal reflexes. Its rectal temperature was 37.7 degrees Celsius. After sixty minutes, its grooming behaviors and reflexes remained normal, while it became slightly less passive. Its rectal temperature was 38.0 degrees Celsius. After ninety minutes, its grooming and reflexing was still normal, and its rectal temperature was 37.8 degrees Celsius.

Table 2: Low Oxotremorine (0.01 mg/kg) Mouse #29

Graph 2: Low Oxotremorine (0.01 mg/kg) Mouse #29

Table 2 and Graph 2 show the results of the low dose of Oxotremorine. The mouse (#29) weighed 38.1g was injected with a low dose (0.01 mg/kg) of Oxotremorine (112.96ul). Its baseline showed normal grooming behavior and normal reflexes. Its rectal temperature was 38.0 degrees Celsius. After thirty minutes, it still showed normal grooming and reflexes. Its rectal temperature dropped to 37.7 degrees Celsius. After sixty minutes, mouse #29 still displayed normal reflexes, but its grooming decreased. It also started showing the CNS depressive effects of decreased motor activity, slowed respiratory rate, and increased respiratory depth. Its rectal temperature was 37.8 degrees Celsius. After ninety minutes, it started grooming slightly more but not as much as its baseline, and its motor activity stayed depressed and decreased. However, when handled its reflexes remained the same as its baseline. Its rectal temperature was 37.5 degrees Celsius.

Table 3: High Oxotremorine (0.1 mg/kg) Mouse #30

Graph 3: High Oxotremorine (0.1 mg/kg) Mouse #30

Table 3 and Graph 3 show the results of the high dose (0.1 mg/kg) of Oxotremorine. The mouse (#30) weighed 34.3g and was injected with 110.6ul of Oxotremorine. The mouse’s baseline was fairly typical, with normal grooming behaviors and normal reflexes. Its rectal temperature was 38.7 degrees Celsius. Thirty minutes after the injection was given, the mouse began to show signs of becoming passive, as well as the CNS depression effects of lowered respiratory rate, increased respiratory depth, some analgesia, and decreased motor activity. Its reflexes remained the same except for a slight decline in grasping, and it started having an odd body posture. Its rectal temperature was 34.1 degrees Celsius. After sixty minutes the mouse remained passive, stopped grooming as much, and continued to show the exact same CNS depression effects and odd body posture. All reflexes returned to baseline levels, however. Its rectal temperature was 35.5 degrees Celsius. After ninety minutes, the mouse became slightly less passive and stopped grooming altogether. CNS depression effects were still observed, but lessened; motor activity had increased slightly, but respiratory rate stayed the same. Its reflexes stayed at their baseline values, while the strange body posture lessened while not vanishing completely. Its rectal temperature was 36.8 degrees Celsius.

Table 4: Hyoscyamine Mouse #31

Graph 4: Hyoscyamine Mouse #31

Table 4 and Graph 4 show the results of the dose of Hyoscyamine. Mouse #31 weighed 33.1g and was injected with a dose (1.0 mg/kg) of Hyoscyamine (106.8ul). Its baseline showed passivity, normal grooming, normal reflexes, and its rectal temperature was 38.6 degrees Celsius. After thirty minutes it showed the same passivity, grooming, and reflexes, along with the CNS excitation effects of increased respiratory rate and increase motor activity. Its rectal temperature was 38.1 degrees Celsius. After sixty minutes the mouse did not groom quite as much, but retained the same passivity, reflexes, increased motor activity, and increased respiratory rate. Its rectal temperature was 38.6 degrees Celsius. After ninety minutes, the mouse showed the same passivity, grooming, and reflexes, along with its increased respiratory rate and increased motor activity. Its rectal temperature was 38.6 degrees Celsius.

Table 5: Hyoscyamine Mouse #37

Graph 5: Hyoscyamine Mouse #37

Table 5 and Graph 5 also show the results of another dose (1.0 mg/kg) of Hyoscyamine. Mouse #37 weighed 29.7g and was injected with roughly 100ul of Hyoscyamine. Its baseline showed fearfulness and normal grooming, and its rectal temperature was 38.3 degrees Celsius. After thirty minutes it showed increased fearfulness, passivity, and decreased grooming. Its body and extremities showed signs of tremors, and normal grasping reflexes. It also showed signs of ptosis, increased respiratory rate, straub tail, and decreased motor activity. Its rectal temperature was 38.3 degrees Celsius. After sixty minutes, the mouse was still fearful, stopped grooming, and still showed signs of straub tail, tremors, ptosis, and yet also increased motor activity. Its grasping reflex remained normal, and its rectal temperature remained 38.3 degrees Celsius. After ninety minutes, mouse #37 started grooming again, and had even higher degrees of increased motor activity. Its rectal temperature was 38.4 degrees Celsius.

Table 6: (1.0 mg/kg) of Hyoscyamine followed by high dose (0.1 mg/kg) of Oxotremorine, Mouse #28

Graph 6: (1.0 mg/kg) of Hyoscyamine followed by high dose (0.1 mg/kg) of Oxotremorine, Mouse #28

Table 6 and Graph 6 show the results of the dose (1.0 mg/kg) of Hyoscyamine followed by the high dose (0.1 mg/kg) of Oxotremorine. The mouse (#28) weighed 28.5g and was first injected with a dose of Hyoscyamine based on its weight, using the aforementioned calculation. Its baseline showed normal reflexes, normal grooming habits, and the CNS effect of excitatory motor activity. Its rectal temperature was 38.8 degrees Celsius. After thirty minutes, mouse #28 was injected with a high dose of Oxotremorine based on its weight. It continued to show normal grooming habits, normal reflexes, and the CNS effect of excitatory motor activity. Its rectal temperature was 38.1 degrees Celsius. After sixty minutes, mouse #28 continued to show normal grooming behavior, some vocalization, normal reflexes, and the CNS effect of excitatory motor activity. Its rectal temperature was 38.2 degrees Celsius. After ninety minutes, mouse #28 stopped grooming and vocalizing, started having an odd body posture, and had much less CNS excitatory motor activity. However, its reflexes remained normal, while its rectal temperature dropped to 28.6 degrees Celsius.

Discussion/summary

Reference

Bering B, M. H. (1992). Muscarinic Cholinergic Receptors on Intact Human Lymphocytes. Properties and Subclass Characterization. Biol. Psychiatry. PubMed, 1987;22:1451–1458.

DUJUS. (2008). Muscarinic Acetylcholine Receptors. Dartmouth Undergraduate Journal of Science.

Vrana, M. K. (2012). Automatic Nervous System. Elsevier's Integrated Review Pharmacology (Second Edition).