Determination Of The Physiological Effects Of Vagus Nerve Biology Essay

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The autonomic nervous system plays an important role in the maintenance of homeostatic processes in the body. The cardiovascular system (CVS) which includes the heart and the circulatory system is regulated by the ANS. Any changes in heart rate (HR) and blood pressure (BP) are homeostatically adjusted by the ANS via sympathetic or parasympathetic nervous system.

The primary cardiac control centre (cardioacceleratory, cardioinhibitory) and vasomotor centres (large group of neurons for vasoconstriction and small group for vasodilation) are located in the medulla oblongata. The cardioacceleratory centre controls the sympathetic nervous system which responds by increasing HR and force of contraction. The cardioinhibitory centre controls the heart via parasympathetic neurons which in turn reduces HR. Vasoconstriction is brought about by the SNS and vasodilation by PNS. These centres are influenced by reflex pathways and via input from higher centres especially from the cerebral cortex, bulbar reticular area, upper spinal cord and hypothalamus (Williams et al, 1981). Ex: Reduction in partial pressure of oxygen (POâ‚‚) in the blood is detected by chemoreceptors which stimulate the cardioacceleratory centre to increase cardiac activity and stimulate vasodilation.

The sympathetic (SNS) and parasympathetic (PNS) nervous system controls the heart by means of the cardiac plexus and both innervate the Sinoatrial Node (SAN), Atrioventricular Node (AVN) and the myocardium. The ventricular myocardium predominantly has SNS fibres compared to PNS fibres (Martini and Nath, 2008).

The SNS preganglionic fibres synapse with postganglionic fibres in the cervical and superior thoracic ganglia (t1-t4) and the postganglionic fibres innervate the heart. Increased SNS activity releases

Noradrenaline (NA) which interacts with β receptors in the nodes causing rapid depolarisation which in turn results in increased HR. It also interacts with α

receptors in the blood vessels promoting vasoconstriction. The end result is increase in blood pressure. SNS also has a positive ionotropic effect on the heart, thereby increasing Stroke Volume (SV). Increase in SV and HR contributes to increase Cardiac Output (CO) (Williams et al, 1981).

It is essential for the heart to receive this constant autonomic tone. This is to prevent SNS from dramatically elevating HR and PNS reducing HR. During the 'rest and relaxation' phase the PNS dominates maintaining HR between 70-80 beats per minute and in the case 'flight or fight response' it is the SNS. If SNS activity falls below normal it results in reduced HR and BP in major arteries contributing to complications such as ischemia. In the case of blood vessels the sympathetic vasoconstrictor nerves are constantly active keeping arterioles partially constricted (Martini and Nath, 2008)

The vagus nerve is the primary parasympathetic nerve fibre which controls the heart. It releases Acetycholine (Ach) which interacts with Muscarinic receptors (M2) in the heart, diminishing rate of depolarisation of the nodes and thereby reducing HR. PNS does not have any direct effect on blood vessel diameter (Martini and Nath, 2008). The PNS reduces force of contraction of the atria but has little effect on the ventricular myocardium. Therefore PNS reduces HR, CO and BP.

The main factors influencing the cardiac centre and vasomotor center include chemoreceptors and baroreceptors which are innervated by the glossopharyngeal and vagus nerves. Baroreceptors are primarily located in the carotid artery, carotid sinus, and aortic arch. They monitor acute changes in BP via measuring the degree of distention within walls of carotid artery, sinus and aortic arch. The threshold for stimulation of receptors in carotid artery and sinus is 60mmHg with peak activity at 175-200 mmHg. Aortic receptors have a higher threshold (Williams et al, 1981). Alterations in BP affect the rate of firing of these receptors and variation in oxygen or carbon dioxide levels in the plasma alters firing rate of chemoreceptors. When BP or plasma POâ‚‚ increases or plasma PCOâ‚‚ decreases, the receptors inhibit vasomotor and cardioaccelerator tone and increase activity in cardioinhibitory area, resulting in reduced HR and BP. If BP or plasma PCOâ‚‚ increase or plasma POâ‚‚ decrease then the reverse is true.

This experiment was carried out to determine the effects of NA on the CVS and establish whether atropine affects this response. The effect of histamine on the CVS was also investigated. Finally the effect of an unknown drug on the heart was examined and thereby established its identity.

Method

Participants were told to access a program called 'The virtual cat v2.5.6' which is a computer simulation program of an anesthetised cat preparation, using the computers available in the physiology suite. It was accessed using the following pathway:

Access My Computer displayed as 45000000(student number) on SCI 000000 (PC number). Then use the following pathway

G:\Strathclyde University\Anaesthetized Cat

They were initially told to access and read the 'contents' section using the 'Help' tab. Then they were told to click the 'Options' tab and uncheck nictitaing membrane and skeletal muscle so that only heart rate (HR) and blood pressure (BP) showed up on screen. They were also told to uncheck the 3 boxes at the bottom of the screen which included 'stimulate vagus nerve', 'stimulate spinal cord ganglion' and 'stimulate skeletal muscle'.

There was a selection of 'unknown drugs' ranging from A to Q and 'standard drugs' which include, Tubocurarine, NA, Acetylcholine, Neostigimine, Carbachol, Atropine, Histamine, Mepyramine, Hexamethonium, Gallamine, Verapamil, Morphin, Naloxone, and Adenosing 8-SPT.

In the initial part of the experiment participants were told obtain a sample control data by simply clicking the 'Start' button. After approximately 1 minute they were told to check the 'stimulate vagus nerve' option and observe and record any changes in HR or mean BP for about 1 minute after which they stopped the trace. Average HR and BP were determined by moving the cursor over the recording.

Afterwards, it was told to plot a dose-response curve for NA by injecting into a vein a starting dose of 0.1µg/kg and then doubling each subsequent dose reaching to a maximum of 100µg/kg. For the response only maximal HR and BP following administration was recorded. Then standard dose 10mg/kg of Atropine was administered which was followed by administration of increasing doses of NA. Changes in HR and BP were recorded.

On the 3rd phase of the experiment they were told to plot a dose-response curve for Histamine. The dosage was similar as NA. They were then told to identify a method that can be used to investigate how histamine administration altered HR and BP.

Finally from the list of unknown drugs, Drug A was chosen and participants were told to determine its effect on the sample preparation and in doing so identify what type of drug it is.

Results

Dose-Response curves were plotted using % of maximal response (HR or BP) as the dependant variable and Log₁₀ values of the respective drug concentrations as the independent variable

% max. response x 100

Blood pressure is given as 'mean values' and not in the form of systolic/diastolic.

Normal heart rate was 108 beats per minute (bpm) and mean blood pressure was 94.2 mmHg (133/75 mmHg).

Frequency of vagal stimulation 0.03 stimulations/second (3 stimulations per 100 seconds). Immediately after each stimulation HR reduced to 61 bpm and mean BP was reduced to 65 mmHg and after 19 seconds both HR and BP returned to normal.

Table 1. Comparison between the Effect of NA (NA - µg/kg) and the Effect of

10mg/kg dose of Atropine on Heart Rate (beats per minute)

Dosage (µg/kg)

Heart Rate with NA (bpm)

Heart Rate with NA + Atropine (bpm)

0.1

106.5

121.7

0.2

107.6

128.3

0.5

114.4

130.0

1

117.4

130.0

2

126.1

131.0

5

134.8

132.0

10

144.6

142.4

20

159.8

153.3

50

138.0

156.5

100

121.7

167.4

Graph 1. Dose Response Curve for the effect of NA (Log₁₀ [NA]) and 10mg/kg dose of Atropine on Heart Rate (% maximal HR)

Dosage (µg/kg)

Mean BP with NA (mmHg)

Mean BP with NA + Atropine (mmHg)

0.1

92.0

105.1

0.2

94.2

108.0

0.5

97.8

108.3

1

105.8

115.2

2

113.0

123.2

5

129.0

131.0

10

137.0

160.1

20

162.3

182.6

50

187.7

200.2

100

197.8

220.6

Table 2. Comparison between the Effect of NA (NA - µg/kg) and the Effect

of 10mg/kg dose of Atropine on Mean Blood Pressure (BP - mmHg)

Graph 2. Dose Response Curve for the effect of NA (Log₁₀ [NA]) and 10mg/kg dose of Atropine on Mean Blood Pressure (% maximal BP)

Table 3. Changes in Heart Rate (beats per minute) and Mean Blood Pressure (mmHg) in response to Histamine (mg/kg)

Histamine Dosage

(mg/kg)

Heart Rate

(bpm)

Mean Blood Pressure (mmHg)

0.1

108.0

82.6

0.2

108.0

68.8

0.5

107.0

57.2

1

107.0

47.1

2

108.0

38.4

5

107.0

34.1

10

108.0

30.4

20

107.0

29.7

50

108.0

29.0

100

108.0

28.5

Graph 3. Dose Response Curve for the effect of Histamine (Log₁₀ [Histamine]) on Heart Rate (HR) and Blood Pressure (BP) - in the form of % of maximal response

Table 4. Changes in Heart Rate (beats per minute) and Mean Blood Pressure (mmHg) in response to Drug A (mg/kg)

Drug A Dosage (mg/kg)

Heart Rate (bpm)

Mean Blood Pressure (mmHg)

0.1

112.0

95.0

0.2

113.0

96.0

0.5

114.0

97.0

1

117.0

98.0

2

121.0

99.0

5

124.0

105.0

10

126.0

105.0

20

126.0

105.0

50

128.0

105.0

100

128.0

105.0

Graph 4. Dose Response Curve for the effect of Drug A (Log₁₀ [Drug A]) on Heart Rate (HR) and Blood Pressure (BP) - in the form of % of maximal response

Discussion

It was found that stimulation of the vagus nerve (VN) reduced HR by 42.6% and BP by 30.9%.

The 10th cranial nerve, which is part of the parasympathetic nervous system, reduced the force of contraction of the myocardium with a characteristic reduction in number of action potentials. The left vagus nerve innervates the AVN and the right vagus nerve innervates the SAN. Ach released from VN interacted with M2 receptors in the nodes which increased conductance of K+ and inhibited Ca²+ channels by increasing K+ permeability and reducing Ca²+ permeability in pacemaker cells thereby hyperpolarised the sinoatrial cells. This extended period of repolarisation and reduced spontaneous depolarisation and thereby reduced the overall time it took for the SAN to reach threshold potential and finally resulted in reduced HR (Martini and Nath, 2008). The Ach released by the vagus nerve interacted with M3 receptors in the blood vessels which resulted in the release of nitric oxide (NO) from endothelial cells. NO simulated vasodilation which resulted in reduced BP (Patel et al, 2006).

Vagal tone can be inhibited by a muscarinic antagonist. Ex: Atropine - used in the treatment of Sinus bradycardia and AVN block. Atropine (competitive muscarinic antagonist) prevents binding of Ach to M2 receptors in the heart and M3 receptors in blood vessels (Walch et al, 2001) thereby preventing effect of vagal stimulation resulting in tachycardia (Rang and Dale, 2007) and vasoconstriction. However atropine's effect on blood pressure is not strikingly significant (Rees et al, 1989).

NA elevated HR by 51.8 bpm and BP by 103.6 mmHg. HR reached a maximum of 159.8 bpm for a dose of 20 µg/kg and BP was elevated to 197 mmHg for a dose of 100 µg/kg. This confirms that NA released by the SNS, is highly effective during a 'flight-or-fight' response and preparing the body for vigorous activity by elevating HR and promoting vasoconstriction (Craig and Stitzel, 2003).

When dose was increased to 50 and 100 µg/kg HR reduced to 138 and 121.7 bpm respectively. This reduction in HR was due to reflex bradycardia which was achieved via stimulation of baroreceptors in response to elevated levels of BP.

Bradycardia was achieved by increased vagal stimulation and this restored homeostasis.

When atropine was added it resulted in tachycardia reaching a maximum of 167.4 bpm for a dose 100 µg/kg NA. Atropine blocks the effect of bradycardia reflex by inhibiting vagal stimulation. Since it is a muscariinc antagonist it does not affect responses of NA (Craig and Stitzel, 2003). Atropine did not have a significant effect on blood pressure which supports the findings of Rees et al (1989)

The main adrenoreceptors that mediate the effect of NA in cardiac and vascular smooth muscle include; α1, α2, β1, and β2. All these receptors involve second messenger systems. The characteristics of these receptors are discussed in detail in table 5.

Histamine had no significant effect on HR but reduced blood pressure by 65.7 mmHg reaching a minimum of 28.5 mmHg for a dose of 100 mg/kg. Therefore histamine was effective as a vasodilator and the degree of vasodilation depends on the concentration of histamine and the degree of baroreceptor reflex compensation (Craig and Spitzel, 2003).

The main receptors involved here are H1 (rapid short lived response) and H2 (slow and sustained response) receptors. Activation of H1 (heart and endothelium) lead to activation of Gq₁₁ protein leading to activation of Phospholipase C resulting in elevation of IP3 and Ca²+ levels. H2 (heart) activation in turn activated Gs protein leading to increased cAMP levels thereby increasing intracellular Ca²+ levels. To a lesser degree H3 (in some endothelia) receptors are too involved (Craig and Stitzel, 2003). They act by inhibiting release of NA from SNS thereby preventing vasoconstriction. Once H3 is activated this in turn activates Gi/o protein resulting in reduced Ca²+ influx via G protein coupled N-type Ca²+ channels.

From the list of standard drugs Mepyramine which is a first generation antihistamine (h1 receptor antagonist), can be used to investigate the effect of histamine by administering the drug to a sample preparation with histamine and constructing a dose response curve. When comparing this (histamine + mepryamine) to a dose response curve of histamine it should show a parallel shift to the right. This finding is supported by Cardell and Edvinson (1994)

Receptor Type

Location with regards to the Cardiovascular system

2nd Messenger

Effect on Cardiovascular System

Agonist

Antagonist

α1

Blood vessels (arteries and veins)

Activates G protein (Gq) which in turn activates Phospholipase C resulting in the production of 2nd messengers inositol triphosphate (IP3), Ca²+ and Diacylglycerol

Ca²+ will bind to calmodulin which in turn activates various proteins

Vasoconstriction

Methoxamine - used in the prevention and treatment of acute hypotension occurring with spinal anesthesia

Prazosin - used in treatment of hypertension and heart failure (Wishart, 2009)

α2

Blood vessels (arteries and veins)

Activates Gi protein which inhibits adenyl cyclase contributing to reduction in the formation of cyclic AMP (cAMP) from ATP resulting in protein kinase A (PKA) being unable to phosphorylate other proteins.

There is also inhibition of Ca²+ channels and stimulation of K+ channels.

Vasoconstriction (via post junctional α2a receptor) of arteries and veins

Vasodilation (via pre-junctional and endothelial α2b receptor) of arteries (Craig and Stitzel, 2003)

Clonidine - used in the treatment of hypertension and alcohol withdrawal.

Yohimbine - used in treatment of erectile dysfunction (Wishart, 2009).

β1

Heart (SAN, AVN, atrial and ventricular myocardium)

Activates Gs protein which in turn elevates levels of cAMP and increased activity of PKA

Increased HR, BP, CO and force of contraction

Dobuatmine - used in treatment of heart failure and cardiogenic shock.

Metoprolol - used for treating hypertension and angina (Wishart, 2009).

β2

Blood vessel (artery - to skeletal muscle) and Heart (Rang and Dale, 2003)

Same as β1

Vasodilation, minor increase in HR and force of contraction (Rang and Dale, 2003).

Formoteroll - used in management of asthma and chronic obstructive pulmonary disease

There is no clinical use for antagonists. Ex: Butoxamine (Wishart, 2009).

Table 5. Characteristics of Adrenoreceptors involved in the Cardiovascular System

2-(3-trifluoromethyl) phenyl histamine is a H1 receptor agonist and its use is still being researched

(Craig and Stitzel, 2003). Diphenhydramine (antihistamine) is a H1 antagonist used as an antiemetic, sedative and provides temporary relief of allergic symptoms ex: rhinitis. Antihistamines are usually classified as first generation or second generation substances. Second generation is less potent than first generation and is less able to cross the blood brain barrier thereby producing less sedation (Craig and Stitzel, 2003).

Betazole is H2 receptor agonist used to test for gastric secretory function. Cimetidine is a H2 anatagonist used in treatment of heart burn and peptic ulcers. Imetit is a H3 agonist used for research purposes and Ciproxifan is a potent H3 antagonist (Wishart, 2009).

Drug A elevated HR and BP by 20 bpm and 10.8 mmHg reaching a maximum of 128 bpm and 105 mmHg respectively after which BP remained stagnant. The elevated HR, the superficial effect on BP and the absence of bradycardia reflex indicate that Drug A was a muscarinic antagonist, namely Atropine. Also it only caused slight tachycardia and vasoconstriction compared to NA (Valentin, 1985).

Limitations

One important limitation is that there was no direct interaction with living tissue and the program used prefixed doses - so there is no variation in results and it was difficult to study any unique observations since the computer simulated program (CSP) often produced the same results (responses) every time. Some expertise was required to handle the software; for an observer it is more likely for him/her to forget the methods used and there was lack of knowledge of how to actually carry out a real life experiment. (Baby, 2009)

Advantages

The main benefit was that there was no need to use actual animals. This saved time required to carry out the experiment; minimised difficulties in handling animals (living or dead) which is labour-intensive and costly; eliminated problems with availability of animals and alleviated any emotional problems that might affect the observer. It also eliminates possible ethical issues (baby, 2009)

With the use of CSP many students can carry out their own experiment and observe results and there are fewer chances of any experimental errors occurring and the experiment can be visualised clearly.

In a 'real life' experiment a spinalized cat is used. The cat is initially anesthetized and a column is inserted into the spinal cord. It therefore destroys CNS control of HR and BP, which in turn damages the blood pressure reflex and baroreceptor reflex originating from carotid artery, carotid sinus and aortic arch (Kitagawa and Walland, 1982) thereby affecting results (bradycardia reflex). However, use of spinalized cats is effective in evaluating effect of drugs on spinal reflexes and it is possible to observe only the drug's effects on the body without any interference from the body's natural biological responses (Kehne, 1985).

Conclusion

The parasympathetic nervous system reduced HR and BP via stimulation of vagus nerve and it was found that the muscarinic antagonist, Atropine abolishes this response. However its effect on BP was not profound. This was confirmed when tested with NA where it only caused profound tachycardia. Histamine had no significant effect on HR but was responsible for vasodilation resulting in significant reduction in BP. Drug A was identified as atropine.

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