Neuronal Plasticity in the Enteric Nervous System Motor

Pathways

Chae Ran Lim

A thesis in fulfilment of the requirement for the degree of

Masters by Research

School of Medical Sciences

Faculty of Medicine

March 2014 Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Originality Statement

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………......

Date ………12th September 2014………………………….

2

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Table of contents

Originality Statement ...... 2

Abstract ...... 6

Acknowledgements ...... 8

List of figures and tables ...... 10

List of abbreviations ...... 11

List of relevant publications or presentations used in thesis ...... 13

Introduction ...... 14

The intrinsic nervous system of the gut ...... 14

The structure of the ENS ...... 15

The myenteric plexus ...... 16

The submucous plexus ...... 16

Morphology of the enteric neurons ...... 18

Neuro-coding concept in the ENS ...... 20

Neurochemistry and neurotransmitters of enteric neurons ...... 20

Electrophysiological characteristics of enteric neurons ...... 21

Functions of the GI tract ...... 22

Control of secretion into the lumen of the GI tract ...... 23

Control of the motor functions of the GI tract ...... 23

Immune functions of the GI tract ...... 26

Inflammatory bowel disease and functional bowel disorders ...... 28

GI tract control of satiety ...... 30

Aims of the study ...... 33

Methods ...... 36

Preparation setup and timeline ...... 36

Threshold tests ...... 39

Electrical stimulation ...... 43 3

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Video recording ...... 44

Drugs and statistical analysis ...... 48

Results ...... 49

Measuring the peristaltic reflex threshold ...... 49

Effects of blockade of fast excitatory neurotransmitter receptors ...... 51

Hexamethonium blocks nicotinic receptors ...... 51

Mecamylamine blocks nicotinic receptors ...... 55

Blockade of 5-HT3 receptors with granisetron ...... 57

Effects of blockade of slow excitatory neurotransmitter receptors ...... 58

Blockade of muscarinic M1 receptors with VU 0255035 ...... 58

Blockade of all muscarinic receptors with hyoscine ...... 61

Effects of blocking both fast and slow excitatory neurotransmitter receptors ...... 61

Effects of other channel blockers and aboral electrical stimulation ...... 63

Blockade of intermediate calcium activated potassium channel (IKCa++) with

Tram-34 ...... 63

++ Blockade of the IKCa and nicotinic receptors ...... 64

Blockade of neuronal conduction with lidocaine ...... 65

Stimulation at the aboral end of the preparation ...... 65

Discussion ...... 67

Electrical stimulation enhanced the peristaltic motor pattern in the presence of

nicotinic receptor blockade...... 68

Blockade of 5-HT3 receptors did not have an effect on recovery ...... 69

Excitation of AH/sensory neurons may enhance the peristaltic reflex ...... 71

An M1 muscarinic receptor antagonist did not have an effect on recovery ...... 71

Overcoming experimental problems and issues ...... 72

Future directions ...... 73

Conclusions ...... 74

4

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

References ...... 75

5

Abstract

Background: Many bowel disorders and diseases are linked to enteric neuronal dysfunction. Details of the mechanisms underlying neurotransmission within in the enteric nervous system (ENS) are not yet known. Acetylcholine acting on nicotinic receptors mediates the majority of fast synaptic transmission between enteric neurons however, recent studies suggest that the peristaltic motor pattern can occur during nicotinic receptor blockade.

Aim: To determine whether the electrical stimulation of the enteric nerves enhances the recovery of the intestinal motility during nicotinic blockade and to determine the mechanisms by which this occurs.

Method: Two segments of proximal ileum measuring 5-7cm were extracted from guinea pigs of either sex (232 to 900g) and positioned in an organ bath. Each segment was cannulated and the peristaltic pressure threshold was determined by step-wise increases of the intraluminal pressure from the oral end while pressure recordings were made from the aboral end. The parameters of the peristaltic motor pattern were compared in electrically stimulated (ES: 1Hz; 1ms; 250 pulses) and non-electrically stimulated (non-ES) preparations using an ANOVA with a repeated measures post-hoc test. Video recordings were made throughout the experiment to detect changes in luminal diameter. Drug containing solutions were added to the serosal side of the intestine.

Results: The peristaltic pressure threshold was taken as the pressure at which four consecutive propulsive contractions were observed. Nicotinic receptor blockade using hexamethonium (300 µM) or mecamylamine (3 µM) inhibited peristalsis. In the presence of hexamethonium, electrical stimulation at the oral end enhanced recovery of peristalsis with 6 out of 20 preparations showing almost full recovery. Peristalsis also Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

showed some recovery when electrical stimuli were applied in the presence of muscarinic blockade (VU 0255035 (150 nM)) or hyoscine (10 µM)); or IKCa2+ blockade (TRAM-34 (100 µM)). A combination of ligand-gated ion channel blockers

(hexamethonium plus RO51 and granisetron) did not prevent the recovery seen following electrical stimulation. When electrical stimulation was applied at the aboral end of the intestinal segment, anterograde propulsive contractions were observed and recovery from nicotinic blockade was enhanced.

Conclusion: The findings of the present study suggest that electrical stimulation improves the recovery of the peristaltic motor pattern in the presence of nicotinic receptor blockade. The mechanism of recovery does not rely on ACh acting at muscarinic receptors, 5-HT or ATP acting at 5-HT3 or P2X receptors (respectively), but may have depended upon an increased excitability of AH neurons. Thus, neuronal plasticity of the ENS is highly adaptable and able to overcome inhibition of multiple receptors in peristaltic motor pathways.

7

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Acknowledgements

It’s been a pretty long time since I’ve first started the Masters degrees and now the end is finally here. I have met a lot of wonderful people who helped me through the process and I want to thank each and every one of them.

Very special thanks to the greatest supervisor Paul, there are so many things I need to thank you for. I enjoyed being your student for the last couple of years!! I had so much fun producing, experiencing, learning and talking about all sorts of interesting things.

You have always been so patient with me. Without your patience, I wouldn’t have been able to learn so many things (there are just so many things, I can’t list them all!!). Also,

I am very grateful for all the opportunities you created for me. I know I will never get this amount of support and care from anywhere else!!

Thank you to Lulu who took on the role as my supervisor when Paul had to go to

Melbourne. You have provided and shared so many things- like helping me out with the

Annual review and letting us use your room during building renovation!! I will never forget your kind smiles!!

Kate! Thank you so much for being there whenever I needed you and for being a friend.

Your kind assistance and opinions throughout these years were amazingly supportive!!

Thank you Hin and Mel for being great colleagues! Thank you Jeremy for helping me with picking out guinea pigs!! Thank you Joel for producing Scribble- it was easy and useful for data analysis!!

Lastly, a huge gratitude goes to my wonderful parents!! I don’t know how to thank you for all the hard work you have done for me. Without your support and sacrifice over the last 25years of my life, I wouldn’t be able to be the person I am today. I hope this accomplishment will make you happy and proud! I love you both very much!! Thank 8

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

you to my baby brother for being my baby brother- you know I rely on you very much.

Thank you JK for being “you” and for everything you have said and done for me over the last precious 7 years!

I wouldn’t have made it without the encouragements and love from everyone!! Thank you all once again!!

9

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

List of figures and tables

Figure 1. Schematic diagram of the muscle and nerve plexuses of the Enteric Nervous

System

Figure 2. Predicted mechanisms by which peristalsis could recover during nicotinic receptor blockade

Figure 3. Schematic side view of the experimental setup

Figure 4. Timeline for experiment

Figure 5. Types of peristaltic contractions

Figure 6. Example of edge detection software used to find the upper and lower bounds of the ileal segment

Figure 7. Example of a spatial temporal map generated from diameter

Figure 8. Example of how pressure waves were analysed

Figure 9. Illustration of recovery status in presence of hexamethonium

Figure 10. Intestinal segments in the absence or presence of hexamethonium

Figure 11. Graph of partial and full recovery of peristaltic reflexes

Table 1. Drugs and drug combinations used

10

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

List of abbreviations

5-HT Serotonin

ACh Acetylcholine

AER Ascending enteric reflex

AIHW Australian Institute of Health and Welfare

ATP Adenosine triphosphate

CCK Cholecystokinin

CD4+ Immune cells expressing the Cluster of Differentiation 4 glycoprotein

such as helper T cells cGMP Cyclic guanosine monophosphate

CNS Central nervous system

CRF Corticotrophin releasing factor

DMSO Dimethyl sulfoxide

ENS Enteric nervous system

EPSP Excitatory postsynaptic potential

ES Electrically stimulated

FT Flow through

GALT Gut-associated lymphoid tissue

GI tract Gastrointestinal tract

GLP-1 Glucagon-like peptide-1

GLP-1R Glucagon-like peptide-1 receptor

IBD Inflammatory bowel disease

IBS Irritable bowel syndrome

ICV Intracerebroventricular

IgA Immunoglobulin A

11

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

IgG Immunoglobulin G

IPANs Intrinsic primary afferent neurons

LPDCs Lamina propria dendritic cells

MMC migrating motor (or myoelectric) complex nNOS Nitric oxide synthase

NHMRC National Health and Medical Research Council

NO Nitric oxide

Non-ES Not electrically stimulated

NPY Neuropeptide Y

PACAP Pituitary adenylyl cycles activating peptide

PKG1 cGMP dependent protein kinase type 1

PYY Peptide YY

PT Peristaltic Threshold

RO water Reverse osmosis water

SEM Standard error of mean

SSPE Sustained slow postsynaptic excitation t80 Time to 80% return to baseline (secs)

TLCR5 Toll-like cell receptor 5

VIP Vasoactive intestinal peptide

YLD Years lived with disability

12

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

List of relevant publications or presentations used in thesis

Lim CR, Tay P, Bertrand RL & Bertrand PP. (2012). The persistence of peristalsis and why electrical stimulation gets things moving. In The Enteric Nervous System

Workshop #4. Adelaide, SA. Oral presentation (PPB).

Lim CR, Tay P, Bertrand RL & Bertrand PP. (2013). Electrical stimulation enhances recovery of the peristaltic reflex during nicotinic blockade in guinea pig ileum. In

Australian Neuroscience Society. Melbourne, Australia. Poster presentation (CRL).

13

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Introduction

The significance of the intrinsic innervation of the gastrointestinal (GI) tract, the enteric nervous system (ENS), is often underestimated as many consider the central nervous system to be the chief mechanism by which the human body controls its organ systems. However in recent years, there has been a rising awareness that many diseases or disorders of the GI tract are related to a dysfunction of the enteric nervous system.

Starting with pioneers like Langley, Trendelenburg and Starling, the connections and neurotransmitters utilised by the enteric nervous system have been studied widely under diverse experimental conditions. The following literature review aims to provide background on the functions of the gastrointestinal tract with a focus on the role of the enteric nervous system in controlling these functions.

The intrinsic nervous system of the gut

The enteric nervous system (ENS) is a part of the autonomic nervous system, and is primarily located in the wall of gastrointestinal (GI) tract (i.e., stomach, small intestine and colon), pancreas and gall bladder (Furness, 1987). The ENS is responsible for the moment-to-moment control of the GI tract including motility, secretion, absorption and immune functions. The neurons of the ENS can be placed into three major functional classes: motor neurons, interneurons and sensory neurons. These form a complex network of connections and circuits which are responsible for enteric reflexes and motor patterns.

The autonomic nervous system was originally split in to three sub-divisions: sympathetic, parasympathetic and enteric (Langley, 1921). In the GI tract, both the sympathetic and parasympathetic pathways have extensive innervation including many connections to the ENS. The post-ganglionic sympathetic neurons that innervate the GI

14

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

tract have cell bodies in the thoracic and lumbar regions of the spinal cord (Furness,

2006). Parasympathetic pathways arise from the brainstem and the sacral spinal cord to innervate the upper and lower parts of the GI tract, respectively. The various activities carried out by the GI tract can be modified by sympathetic or parasympathetic nerves via changes to enteric neuron activity (J.C. Bornstein, 2002). In addition to the autonomic input, the gut is innervated by sensory neurons from the nodose ganglia (via the vagus) and dorsal root ganglia (via spinal nerves) which can also modulate ENS function.

The enteric neurons also connect with the post-ganglionic neurons in autonomic ganglia via a small number of intestinofugal neurons. This allows the gut to send information from one region of the GI tract via the prevertebral sympathetic ganglia to other regions of the GI tract (Furness, 1987).

Because sympathetic, parasympathetic and afferent neurons have cell bodies which lie outside the gut, they are called “extrinsic neurons” while the neurons of the

ENS are termed “intrinsic neurons” in relationship to the GI tract (Furness, 1987).

The structure of the ENS

The ENS is typically arranged into two main plexuses, or layers of nerves: the myenteric (Auerbach’s) and submucosal (Meissner’s). A myenteric plexus is found in the esophogus, stomach, small intestine and large intestine (Horina et al., 1992; Costa et al., 1996). In contrast, the submucosal plexus is absent from esophogus and there are only a small number of ganglia in the stomach (Furness, 1987). The submucous plexus has been found to contain neurons responsible for secretion and vasodilation, and the myenteric plexus has neurons for controlling the smooth muscle as well as many

15

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

interneurons. Both of these plexuses have been found to contain intrinsic sensory

(afferent) neurons.

The myenteric plexus

To look in more detail, the myenteric plexus consists of large ganglia with on average 100 neuronal cell bodies per ganglion that are connected by nerve strands all of which are situated between the external longitudinal and the inner circular smooth muscle layers (Furness, 1987). The myenteric plexus contains more functional sub-types of neurons than does the submucous plexus (Costa et al., 1996). The myenteric plexus is primarily involved in processing of information via interneurons and control of GI tract motility via the inhibitory and excitatory motor neurons.

The submucous plexus

As for the submucous plexus, it consists of smaller ganglia containing on average 7 neuronal cell bodies per ganglion of a small number of neuronal types with many interconnecting nerve strands (Furness, 1987; Costa et al., 1996). Despite the smaller ganglia size the submucous plexus contains as many neurons as does the myenteric plexus. Each ganglia was estimated to contain from a single neuron up to 30 neurons in a study conducted in guinea pig (Furness, 1987). As mentioned above, the submucous plexus is mainly involved in mucosal function (secretion via secretomotor neurons) and blood flow (vasodilatation via vasodilator neurons).

16

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 1. Schematic diagram of the muscle and nerve plexuses of the Enteric Nervous

System. The ENS has a myenteric and submucosal plexus that are composed of nerve fibres that connect the cell body-containing ganglia together. The myenteric plexus is located between the circular muscle and longitudinal muscle and is involved in processing information through interneurons and control of GI tract motility. The submucosal plexus is located between the circular muscle and muscularis mucosae layer and is involved in mucosal function (secretion and dilatation). (Furness, 2012)

17

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Morphology of the enteric neurons

Enteric neurons can be classified as motor neurons, interneurons or sensory neurons (or intrinsic primary afferent neurons; IPANs) (Furness, 2000) based mainly on the structures they innervate (i.e., their targets). Some classes may play more than one role such as the IPANs also acting as interneurons. These classes of neurons can be further sub-divided based on neurochemistry (i.e., the types of neurotransmitters or other markers contained within the neuron) and on their projection pattern (i.e., anal, oral or local innervations).

In 1899 Dogiel differentiated and categorised the neurons of the ENS according to morphological features such as the number of projections and shape of dendrites.

Today, the morphology of enteric neurons is identified according to a simplified nomenclature as Dogiel type I, II, III and filamentous neurons.

Dogiel Type I

Dogiel type I morphology has been found to describe mainly the motor neurons of the ENS (Dogiel, 1899; Brookes et al., 1992). Dogiel (1899) stated that these cells were star-shaped, flat with round nucleus. Some of the other features included short and thin fibres extending from the flat lamellar dendrites with axons that projected from the ganglion. The neurons of type I are unipolar and are smaller than that of the other three types measuring 13 to 34 µm in diameter. Type I cells are mostly found in the submucosal (Meissner’s) plexus of humans as well as animals such as guinea pigs, rabbits and dogs (Dogiel, 1899). In more recent studies by Messenger et al. (1994), histochemical methods were used to show that neurons with Dogiel type I morphologies mostly innervate the circular muscle of the small intestine and were electrophysiologically identified as S neurons.

18

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Dogiel Type II

Dogiel type II cells are star-shaped, angular and spindle shaped (Dogiel, 1899).

Type II cells are measured differently from type I cells as the measurements are made according to the short and long axis. The nucleus of this type is large and round, it contains one to two nucleoli and there are from 3 to 10 axons/dendrites. Cells of type II have been mostly identified as sensory neurons/IPANs (Dogiel, 1899; Furness et al.,

1998). According to Costa et al (1996), approximately 30% of myenteric neurons are

Dogiel type II cells. These cells are also prominent in submucous plexus (Wattchow et al., 1995).

Dogiel Type III

Type III cells are not discussed in modern morphological identification (Furness,

1987). This is included to acknowledge the morphological system created by Dogiel in

1899. Type III cells share morphological similarities with type II cells. Dogiel (1899) stated that the type III cells are mostly found embedded in the centre of the ganglia.

Also the branches of the dendrites are short.

Filamentous neurons

The filamentous dendrites of these neurons are relatively short (less than 50 µm) with branches and single long process found in myenteric ganglia (Furness et al., 1988;

Clerc et al., 1998). Filamentous neurons have also been further divided in to type I to VI neurons (Brehmer & Stach, 1998) though this nomenclature is not commonly used.

Often the filamentous neurons are found to be descending interneurons. In a study by

Clerc et al (1998), the identification of filamentous descending interneurons was done by the projections and similarity to other neurons in the guinea pig ileum. Furthermore, the neurons were found to be small (approximately 26 µm diameter) with an axonal

19

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

process that was mainly innervating the circular muscle or longitudinal muscle of guinea pig ileum (Clerc et al., 1998; Furness et al., 1998).

Neuro-coding concept in the ENS

Neurochemical markers can be combined in diverse ways to identify unique classes of neurons in the ENS. Then, the morphology, functions and properties of a neuron may be inferred from this unique mixture of markers. As mentioned earlier, from the results provided by Costa et al (1996) it can be seen that vasoactive intestinal peptide (VIP), enkephalin, serotonin (5-HT), somatostatin, calretinin, substance P and calbindin can be used together to form a code. A neuron does not contain all of these markers at once, but the immunoreactivity to one or the other markers can be used to correlate the type of neuron.

Neurochemistry and neurotransmitters of enteric neurons

Various types of neurons of the ENS are recognized by particular neurochemical factors. In order to start the process of identifying these factors, it is important to classify the histochemical markers. Costa et al (1996) were the first to describe various classes of enteric neurons by using six different histochemical markers present in the nerve cell body. The six markers were calbindin, calretinin, VIP, 5-HT, somatostatin and substance P. Therefore, immunoreactive neurons for one or more of the listed histochemical markers can be cross-referenced with Dogiel morphological types. The study conducted by Costa et al (1996) showed that immunoreactivity for calbindin was expressed in Dogiel type II neurons. Additionally VIP and somatostatin were both expressed in Dogiel type III, and 5-HT and substance P were present in Dogiel type I enteric neurons. For Dogiel type II neurons, substance P was also found but with weak

20

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

immunoreactivity. Each histochemical marker identified neurons with different projections and locations.

Neurotransmitters allow communication amongst various neurons. In the ENS, there are numerous types of synapses which may be activated by situations such as, stretch of the gut wall, compression of the mucosa and chemicals applied to the mucosa

(Monro et al., 2002). More generally, the neurotransmitters are released when an action potential invades the nerve terminal. Synaptic transmission in the ENS includes fast ligand-gated ion channels and slow G-protein-coupled receptors (Ren & Bertrand,

2008). Neurons that normally utilize ATP or adenosine are known as purinergic neurons.

Ren et al in 2008 stated that the purinergic receptors involved are P2X (involved in fast synaptic transmission), P2Y (involved in slow synaptic transmission) and adenosine

(involved in presynaptic inhibition) receptors. Furthermore, these purinergic receptors are localised throughout the ENS and influence various reflexes and motor patterns

(Bornstein, 2008). For excitatory motor neurons of the ENS, acetylcholine (ACh) is the main neurotransmitter (Furness, 2000). VIP and NO (nitric oxide) are neurotransmitters used by descending inhibitory longitudinal muscle motor neurons (Costa et al., 2000).

Tachykinins are contained in excitatory motor neurons which in particular are associated with the ascending enteric reflex (AER) in the circular muscle of the ENS

(Holzer et al., 1987). For fast EPSPs (excitatory post-synaptic potentials) in the myenteric plexus, ACh, ATP and 5-HT are all involved (Galligan, 2002); ACh, histamine and or/NO may be co-transmitters with ATP (Ren & Bertrand, 2008) while

5-HT and ACh are co-localised.

Electrophysiological characteristics of enteric neurons

In the myenteric plexus, electrophysiologically characterised neurons classed as

AH or S neurons exist (Hirst et al., 1974). AH neurons have a large Ca2+ component in 21

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

their action potential and also have a large afterhyperpolarisation (i.e., an AH) following the action potential - AH neurons are mainly sensory neurons. As for the S neurons, they are mainly interneurons and motor neurons. Fast EPSPs observed in S neurons are larger and much more frequently observed than in AH neurons (Furness, 2000; Monro et al., 2002). There are sensory neurons in both plexuses which typically have AH type electrophysiological properties and Dogiel type II morphology. Neurons with electrophysiological properties of S type show Dogiel type I morphology. AH and S type neurons vary in relation to characteristic features such as, membrane potential, chemical and morphological properties (Messenger et al., 1994). Neurons immunoreactive for markers such as VIP, dynorphin and enkephalin were found to be S type (Messenger et al., 1994; Costa et al., 1996). A study conducted by Messenger et al.

(1994) found that with internodal strand (i.e., nerve tracts between enteric ganglia) stimulation, AH neurons showed a delayed afterhyperpolarization following the action potential and S neurons showed fast EPSPs and slow EPSPs. Though these experiments were performed in guinea pig colon; many of the features were similar to the ileum.

Intracellular recordings with electrical stimulation of the neurons give various measurements. As mentioned earlier, AH and S types have dissimilar action potential properties. In addition, AH neurons have a greater amplitude of action potential and a membrane potential that is more negative than in S neurons (Messenger et al., 1994).

Functions of the GI tract

The gastrointestinal (GI) tract has various functions which aid in breaking down and digesting food into a size suitable for rapid and efficient absorption. This is possible due to the ENS which plays a critical role in controlling motility, secretion and contributing to the immune system. Secretion assists in mixing food in the lumen and

22

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

releases digestive enzymes and hormones such as 5-HT. Peristalsis and segmentation are the primary kinds of motility in the GI tract. Finally, the GI tract is an important piece of the immune system. There are two types of immunity seen in the GI tract, both innate and adaptive. The immune system is vital as a defence against pathogenic material which is commonly found in the lumen of the GI tract. Finally, the control of satiety is via signalling through extrinsic sensory (afferent) neurons from the GI tract to the central nervous system (CNS).

Control of secretion into the lumen of the GI tract

A large quantity of fluid is produced and secreted by the human GI tract to optimize the environment for digestion and uptake of nutrients. The fluid consists of digestive enzymes, ions (i.e., H+, K+, Na+ and Cl-), bile and mucus with water following down the osmotic gradient. Although food intake may seem like a simple process, the system itself is very complicated. Some of the most significant organs which secrete the above elements include the salivary gland, pancreas, liver and epithelial cells of the gastrointestinal tract. Secretion of these products is most significant during the digestion of food. The excessive secretion by the gut is a pathological occurrence which can be caused by bacterial infection (e.g., cholera) or from increased interstitial hydrostatic pressure (Barbezat & Grossman, 1971).

Control of the motor functions of the GI tract Since the 19th century, from Bayliss and Starling, and Trendelenburg to modern day research, gut motility is an area of great interest to many researchers. Motility occurs throughout the GI tract and there are three main types of motor patterns seen, each made up of several simpler reflexes. Motility of the intestine is composed of the peristaltic motor pattern (the main propulsive pattern), segmentation (the main mixing pattern) and the migrating motor (or myoelectric) complex (MMC) (the main 23

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

housekeeping pattern). The contribution of the three types of motility determines the rate of content transit along the GI tract (Bornstein, 2008). The peristaltic motor pattern consists of circular and longitudinal muscle layers contracting and relaxing in synchrony (Bayliss & Starling, 1899). These muscle layers are innervated by the neurons from the myenteric plexus. As described, the neural pathways consist of sensory neurons, interneurons, and excitatory and inhibitory motor neurons (Monro et al., 2002).

Peristaltic 'reflexes' Bayliss and Starling were the first to observe polarised reflexes of the gastrointestinal tract in 1899. They noted that the contractions of the circular muscle of the small intestine reduced the luminal diameter and in some instances the gut increased its length. This comprises the essential contractile pattern for peristalsis and segmentation. In addition, they noticed that the contraction of the longitudinal muscle shortened the gut which increased its transverse diameter but did not cause any propulsive effects. (Bayliss & Starling, 1899; Hansen, 2003). These observations led to a conclusion which is now well known as the “law of the intestine” which stated that

"Local stimulation of the gut produces excitation above and inhibition below the excited spot". This was used to explain the onward movement of the bolus within the intestine.

Excitation of the intestine is seen in the presence of a bolus which affects the wall of the intestine (i.e., either by stretch or mucosal stimulation).

More recently, the basis of peristalsis has been explained as the actions of three simpler reflexes: ascending and descending excitation, and descending inhibition

(Bayliss & Starling, 1899; Bornstein et al., 2004). These reflexes combine to form the most important type of motility in the intestine: the peristaltic motor pattern. Peristalsis involves oral contractions and aboral relaxation which causes a propulsive movement 24

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

which pushes a bolus forward (Olsson & Holmgren, 2001). Peristaltic motor patterns are initiated as the intestinal wall is very sensitive to changes such as, stretch, mucosal deformation, luminal contents and local blood flow (Bayliss & Starling, 1899). Stretch of the intestinal wall probably activates both vagal afferents present in the intestinal wall that are “load sensitive” (Schwartz & Moran, 1998) and enteric intrinsic sensory neurons that respond to stretch (Kunze et al., 1998).

The neurons underlying the simpler reflexes have been identified in the guinea pig small intestine. The excitatory motor neurons extend orally for 6 to 12mm and the inhibitory motor neurons extend 3 to 25mm in the aboral direction (Bornstein et al.,

2004). These neurons utilize numerous transmitters such as, ACh, ATP, tachykinins, VIP,

NO, pituitary adenylyl cycles activating peptide (PACAP) and 5-HT and play a role in the peristaltic motor pattern (Bennett & Whitney, 1966; Bornstein et al., 2004). Despite the number of transmitters, the most critical factor in control of the gut motility is ACh.

ACh is able to stimulate contraction of the smooth muscle and cause depolarisation (an excitatory junction potential) when excitatory motor neurons innervating the circular muscle are activated (Bornstein et al., 2004). The major inhibitory neuromuscular transmitter of the GI tract is nitric oxide. NO is a physiological mediator of the relaxation of the smooth muscle of the GI tract (Groneberg et al., 2011). Groneberg et al., (2011) found that mice lacking in neuronal nitric oxide synthase (nNOS), nitric oxide-guanosine 3’,5’-cyclic monophosphate (cGMP)-dependent protein kinase type 1

(PKG1) had delayed emptying of the stomach but a normal motility rate. Furthermore, the NO/cGMP cascade was found to have impact on the motility regulation in the knockout mice. In addition to the listed transmitters, corticotrophin releasing-factor

(CRF) which is mainly associated with activities such as endocrine and visceral responses to stress is another factor that changes gut motility along the GI tract (Tache 25

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

& Perdue, 2004). A study conducted by Tache and Perdue in 2004 showed that when

CRF was injected intravenously into conscious rats, the gut motility pattern changed from fasted to fed pattern and the antral motility of the stomach was reduced. This indicated that CRF inhibited gut motility and delayed transit along the small intestine.

Segmentation and the MMC Segmentation is the main mixing pattern of the GI tract. Throughout the process of digestion and absorption of nutrient, 90% of the contractile activity is made up of segmentation (Bornstein et al., 2004). Segmentation occurs when circular muscle displays rhythmic constrictions alternating with relaxations which act to divide the contents (Olsson & Holmgren, 2001; Bornstein et al., 2004). This action helps the intestinal contents to be absorbed after intestinal contents are mixed with factors such as, enzymes, bile, mucin and bicarbonate (Bornstein, 2004).

The main housekeeping reflex is known as the migrating motor complex (MMC).

The MMC is highly variable and is initiated in the gastroduodenal area during the interdigestive (fasting) period (Dooley et al., 1992; Olsson & Holmgren, 2001). Motilin, another gut hormone, is the main driving factor of the MMC that leads to a burst of regular contractions that depend on smooth muscle slow waves (Vantrappen et al.,

1979). According to Bornstein (2004), there are 3 distinct phases of MMC activity - phase 1: motor silence without contractile activity; phase 2: irregular contractions; and phase 3: regular contractions - whereas older literature by Vantrappen et al., (1979) suggests there are 4 phases of the MMC (Bell, 2013). This difference may be due to the animal models used.

Immune functions of the GI tract The functions of the GI tract include breaking down and digesting food for fast and efficient absorption. The ENS is the main controller for the various behaviours 26

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

observed in the GI tract. The ENS not only manages motility and secretion, but also modulates the immune system of the GI tract. The regulation of the immune system is important as excessive stimulation can trigger disorders such as inflammatory bowel disease or functional bowel disorders (which will be discussed later in the thesis).

Immunity exists as two types: innate immunity and adaptive immunity. Innate immunity is the first line of defence and it is non-specific. Innate immunity is initiated once a pathogen is detected however there is no long-lasting protection. In contrast, adaptive immunity is exceedingly specialized as it can eliminate or prevent current or future pathogenic growth. Adaptive immunity is the second line of defence which commences after innate immunity. The GI tract is a critical part of the overall immune system. Some of the most obvious and simple lines of defence include, the low pH (1 to

4) of the stomach, enzymes in saliva and bile with destroy bacteria. In addition, though many different cell types in the intestinal immune system are responsible for defence against countless pathogens are not specifically characterized (Uematsu & Akira, 2008).

Evolutionary pressures on the mucosal immune system of GI tract ultimately led to the development of two adaptive types of defence (Brandtzaeg, 1998). One involves secretory antibodies, known as immunoglobulin A (IgA) and IgM. This type of defence helps with the inhibition of the colonization and invasion of various pathogenic microorganisms and luminal antigens (Brandtzaeg, 1998). Lamina propria dendritic cells (LPDCs) express Toll-like cell receptor 5 (TLCR5) in the small intestine. TLCR5 is crucial in the differentiation of naïve B cells in to IgA, producing plasma cells by activating gut-associated lymphoid tissue (Uematsu & Akira, 2008). B cells are important in the production of secretory antibodies of the lymphoepithelial tissues. The second type of defence is oral tolerance which activates helper T cells (CD4+); maturity

27

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

of oral tolerance occurs with the introduction of the dietary antigens via consumption of various foods. (Brandtzaeg, 1998).

Peyer’s patches are mucosal structures found in distal ileum (i.e., lower part of the small intestine). It consists of a group of aggregated lymphoid nodules and IgA- positive plasma cells for immune surveillance. Peyer’s patches are a type of gut- associated lymphoid tissue (GALT) (Brandtzaeg, 1998) that may be required for initiation of mucosal IgA antibody responses in the GI tract (Yamamoto et al., 2000).

Inflammatory bowel disease and functional bowel disorders

As mentioned, an overactive or defective immune system can have drastic consequences for the function of the GI tract and can cause inflammatory bowel disease or post-infectious IBS (a functional bowel disorder). Inflammatory bowel diseases (e.g.,

Crohn’s disease and ulcerative colitis) and the functional bowel disorders are some of the most concerning issues in our society. Although the mortality rate is low, the quality of one’s life may be jeopardised. In Australia, statistics taken from 2002 to 2003 have shown that gastroscopy and colonoscopy had accounted for 152,000 admissions to public hospitals (Australian government department of health and ageing). This was one of the top five reasons for medical admissions. According to AIHW (Australian Institute of Health and Welfare), a total of 35,719 persons suffered from diseases of the digestive system in the 1999. Of that, 16,729 persons were recorded to have an inflammatory bowel disease of some sort. Furthermore, statistics obtained by AIHW for 1999 from international and Australian studies showed that Crohn’s disease was more common in females and the incidence of ulcerative colitis was more prevalent in males. Years lived with disability (YLD) was recorded for all imflammatory bowel diseases and showed that the total number of female patients with of Crohn’s disease had 5511.4 YLD and

28

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

while males had a reduced 3868.9 YLD. For ulcerative colitis, males and females had roughly the same YLD with 2959.3 YLD for males and 2548.6 YLD for females.

Due to the growing problems relating to digestive diseases, the National Health and Medical Research Council (NHMRC) of Australia has increased support and funding for research into bowel disease and disorders. Funding in 2000 totalled $1,812,

219, whereas in 2010 the amount was dramatically higher at $7,122,514. More specifically, the government support of inflammatory bowel disease research increased from $729,485 to $2,838,412 over 10 years (2000-10). These facts underline the importance of digestive system diseases.

Inflammatory bowel disease and functional bowel disorders (such as irritable bowel syndrome) may share some symptoms, but their underlying cause is different. A functional bowel disorder is an idiopathic disorder which can be associated with three factors; functional abdominal bloating, functional constipation or functional diarrhoea

(Thompson et al., 1999). Furthermore, disorders may be categorised by epidemiology, symptoms, histological appearance and pathogenic response to various treatments

(Lennard-Jones, 1989).

Inflammatory bowel disease may be caused by numerous factors including infection, ischemia, immunological disruption or physical damage (corresponding to the location of the impairment) (Lennard-Jones, 1989). Some limitations in social/recreational interaction, home arrangements and restriction in lifestyle (i.e., food and immediate toilet access) are some factors that define the severity of the IBD

(Drossman et al., 2007). Ulcerative colitis and Crohn’s disease are two very well- known types of inflammatory bowel disease. As the name suggests, ulcerative colitis is most common in the colon and rectum. Histologically, it shows muscular thickening in

29

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

the mucosa and glandular damage. As for Crohn’s disease, the region of damage may be anywhere from mouth to anus. According to Fiocci (1998), ulcerative colitis is an autoimmune disease. In the mucosa, a prominent infiltration of neutrophils was noted with normal to hyporeactive T cells. Also, cytokine production increased in the mucosa.

Crohn’s disease is dissimilar to ulcerative colitis in that there is moderate antibody secretion and a lack of autoimmunity. In addition, hyper-reactive T cells exist with prominent T cell infiltration in the mucosa (Fiocchi, 1998). In Crohn’s disease, cytokine production also increased but the mucosa did not have a great impact in its production.

Although there is not definitive evidence, the number of IgG1 appears elevated in ulcerative colitis whereas in Crohn’s disease, IgG2 level is increased (Fiocchi, 1998).

In functional bowel disorders, studies done by Sanger in 1996 showed that 5- hydroxytryptamine is involved. A 5HT4 receptor antagonist is mainly useful in cases of

IBS and functional diarrhoea. The excessive release of serotonin also leads to pain and discomfort. Mast cells are also important in IBS as a study conducted by Barbara et al.

(2006) indicated that with growing stress levels, mast cell numbers increase. The activation of mast cells and their proximity to nerve fibres correlate with the severity of abdominal pain perceptions. Pharmacologically, drugs that can control the activities of mast cells such as anti-IgE antibodies, intracellular protein tyrosine kinase inhibitors or anti-histamines are very helpful in relieving the distress of functional bowel disorders

(Barbara et al., 2006).

GI tract control of satiety

The GI tract initiates motility and secretion upon food intake. As mentioned earlier, the gut responds to distension and ultimately sends this information to the central nervous system (Schwartz & Moran, 1998). The control of satiety is via afferent

30

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

signalling from the GI tract to the CNS (Gutzwiller et al., 2004). This signal is from three different sources: gastrointestinal distension, release of gastrointestinal hormones and luminal nutrients. According to Gutzwiller et al., when this appetite regulation is kept tightly under the “normal” condition (i.e., when the study subject is seen without anorexia or obesity), it is thought that the nutrient balance or homeostatic model is at the optimal level (Gutzwiller et al., 2004).

There are several gastrointestinal peptides that are released both from small intestine in presence of nutrients (i.e., carbohydrates); these include cholecystokinin

(CCK), ghrelin and peptide YY (PYY) which suppresses excessive eating (Thomas et al., 1979). Studies conducted from the 1970's (Barbezat & Grossman, 1971) until very recently show that glucagon-like peptide-1 (GLP-1) is the most extensively investigated gastrointestinal peptide (Gutzwiller et al., 2004; Steinert et al., 2012). Furthermore, in the study conducted by Steinert et al, the stomach was identified as the chief component in short-term control of appetite and satiation was augmented with the release of GLP-1 and PYY. GLP-1 is known as the derivative of the proglucagon gene that is classified as an incretin hormone. Nutrients stimulate the secretion of GLP-1 by the enteroendocrine

L cell which then binds to the GLP-1 receptor (GLP-1R) (Jang et al., 2007). Some of the main functions of GLP-1 involve gut motility, regulating blood glucose level (by glucose-dependent insulin secretion and inhibiting glucagon secretion) and influences to insulin transcription (Jang et al., 2007; Puddu et al., 2010). GLP-1R is located in the brain stem, hypothalamic arcuate nucleus as well as periphery (such as pancreas, lung, kidney, GI tract and heart) (Gutzwiller et al., 2004; Puddu et al., 2010).

According to Turton et al (1996), neuropeptide Y (NPY) is the most important factor for controlling stimulation of feeding. Also, throughout the conducted experiments, it was found that GLP-1 functioned as an inhibitor as well. Turton (1996)

31

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

found that the administration of intracerebroventricular (ICV) GLP-1 before the stimulation of NPY prominently reduced food intake. Furthermore, it is important to note that gustducin-coupled sweet taste receptors (otherwise known as T1R2+T1R3) on the epithelium of the intestine are a crucial part of the release of GLP-1 as T1R2+T1R3 acts as a sensor for the presence of nutrients in the GI tract (Jang et al., 2007).

The feedback system which is induced upon food intake serves various functions such as secretion; activation of the controller in the CNS (as mentioned earlier), nutrient intake, digestion, absorption, storage and metabolism, and feedback on the current state of control system, and lastly efferent control (primarily for food intake and energy use)

(Gutzwiller et al., 2004).

32

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Aims of the study

The neurotransmitters involved in synaptic transmission in the ENS are not known precisely. This study is designed to determine whether electrical stimulation enhances motor pathways in the guinea pig ileum. Inhibition of nicotinic receptors will be used as a tool to reveal non-nicotinic neurotransmission. Electrical stimulation will be used to help restore the neurally mediated peristaltic motor pattern. Other drugs or drug combinations will be used to inhibit or excite specific motor pathways to help dissect out the transmitter systems involved in the control of intestinal motility. The effect of electrical stimulation on the recovery of peristalsis in presence of these drugs has led to the three specific hypotheses and aims below.

33

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 2. Predicted mechanisms by which peristalsis could recover during nicotinic

receptor blockade. Schematic of synaptic potentials in enteric neurons. Red arrows indicate

when electrical stimulation was used. Electrical stimulation was applied to evoke neuronal

plasticity in guinea pig ileum segments. Left, control fast EPSP (red) is large enough to

evoke action potentials while during nicotinic blockade (Nic-, blue) the fast EPSP is

substantially reduced and does not trigger action potentials. A. Following electrical

stimulation, new transmitters/receptors may be recruited resulting in a bigger fast EPSP

which is mediated by 5-HT or ATP. B. Electrical stimulation may make it more likely that

ACh evokes muscarinic (or other) slow EPSPs which, when combined with a small fast

EPSP can evoke action potentials. Slow EPSPs may be mediated by muscarinic,

tachykinergic, serotonergic or purinergic receptors (or others). C. Electrical stimulation may

enhance slow EPSPs or evoke an SSPE which can by itself evoke action potentials.

34

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Hypothesis 1: Non-nicotinic fast EPSPs are responsible for recovery of peristalsis.

Aim 1: Determine if blockade of nicotinic fast EPSPs results in the up regulation of other non-nicotinic fast EPSPs (Figure 2A) which can then be blocked with selective receptor antagonists.

Hypothesis 2: The muscarinic slow EPSPs are responsible for the recovery of peristalsis.

Aim 2: Determine the effects of muscarinic blockade on the recovery of peristalsis from nicotinic blockade (Figure 2B).

Hypothesis 3: The excitation of AH/sensory neuron networks helps to recover peristalsis.

++ Aim 3: Determine if electrical stimulation and IKCa blockade improve recovery from nicotinic blockade to a similar extent (Figure 2C).

35

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Methods

Preparation setup and timeline

Tissue was obtained from guinea pigs of either sex, in the weight range of 232 to

900g (n = 63). The guinea pigs were sacrificed by stunning on the head followed by severing the carotid arteries and the spinal cord. All animals were humanly killed in accordance with the guidelines provided by University of New South Wales, Animal

Care and Ethics Committee. Two segments of proximal ileum measuring approximately

5-7 cm were removed. The oral end of the tissue was removed from approximately 10 cm from the pyloric sphincter. The luminal contents of both tissue segments were cleaned out with oxygenated physiological saline (NaCl 118 mM, NaHCO3 25 mM, D- glucose 11 mM, KCl 4.8 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaH2PO4 1.0 mM). 95%

O2/5% CO2 was bubbled through the physiological saline.

The intestinal segments were cannulated in an organ bath designed to hold two pieces of tissue and perfused with physiological saline at 36 – 37 °C. A continuous flow of warm oxygenated physiological saline was delivered to the organ bath to superfuse the tissue. The physiological saline was connected to the main reservoir and flow at a rate of 8 mL per minute (refer to Figure 3). This was to ensure the distribution of sufficient nutrients to the tissue as well as to maintain the bath temperature and oxygen levels. To detect the variations in intraluminal pressure, the oral end of the intestine was attached to an adjustable pressure reservoir while a pressure transducer (DTX/Plus,

Viggo-Spectramed, Singapore) was attached to the aboral end of the preparation.

Furthermore, a draining line with two-way stopcock was connected to the aboral end of expel any excess contents and to release pressure of the lumen. The pressure reservoir was also filled with physiological saline and was used to induce a peristaltic reflex. The

36

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

intraluminal pressure signals were recorded by an in-house amplifier (gain: x1000) and

National Instruments A/D Converter (United States) to a PC. An in-house computer program (University of New South Wales, Sydney, Australia) was used to capture and modify analogue signals to digital signals. A video camera was used to record the intestinal pattern and diameter of the intestinal segments (see below ‘Video Recording).

Silver/silver chloride electrodes were placed on either side of the tissue in preparation for electrical stimulation during the experiment. After cannulation and the set up of experimental materials, the intestinal segments were left for 30 minutes at 0 mmH2O for recovery and equilibration.

Figure 3. Schematic side view of the experimental setup. Left, (oral) a 50mL syringe with

stopper and glass tube contained physiological saline to raise intraluminal pressure of the

guinea pig intestinal segments. A video camera was suspended above the organ bath by a

retort stand. Silver electrodes were placed on one of the intestinal segments at the start of the

experiment. Although not illustrated, the main reservoir was connected directly to the organ

bath via a heat exchange system. (Modified from Paul P. Bertrand, unpublished).

37

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 4. Timeline for experiment. The first 30 minutes of the experiment was the equilibration period, where the two segments of ileum are unstimulated (either by pressure

or electrical stimulation). Drug incubation was normally carried out for 30 minutes, but if

needed incubation was extended to 1 hour where previous experiments have shown that the drug takes longer to equilibrate with the tissue. The rest of the protocol after the drug

incubation was kept the same. Two separate electrical stimuli sessions were included in the

experimental protocol. Electrical stimulation (ES) session was to find the threshold for the electrical training (ET) session which required voltage 150% above the threshold. A 30

minute washout period was used to allow the drug to leave the organ bath as well as the

intestinal segments.

38

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Threshold tests

After the 30 minute equilibration period, the pressure reservoir connected to the oral end of the preparation was raised to cause an increase in the intraluminal pressure.

The pressure reservoir was increased and held in 10 mmH2O increments to a maximum of 60 mmH2O or until a peristaltic reflex was induced. A successful peristaltic reflex was taken as 4 consecutive propulsive contractions, from the oral to the aboral end of the preparation. An illustration of a peristaltic reflex can be seen in the pressure traces in

Figure 5. During each 10 mmH2O increase, the tissue was observed for 30 seconds for any signs of contraction. Once the peristaltic threshold was reached, the pressure reservoir was lowered back to 0 mmH2O. In order to ensure that the tissue would not tire, 5 minutes of rest was given before beginning the next set of threshold testing. This process was repeated on both intestinal segments 3 times to establish an average peristaltic threshold (PT). The repetition of the protocol was to ensure consistency during the control conditions as well as in post-stimulation conditions. The pressure waves associated with the contractions were recorded for analysis of characteristics such as frequency, amplitude and time. From these data, comparisons of control, stimulation period and post-stimulation period were established.

During the stimulation period of the experiment, various drugs were first introduced via the main reservoir along with the oxygenated physiological saline. In drug conditions, intestinal segments were left to incubate for 30 minutes (sometimes longer depending on the properties of the drug). Unlike the control experiment, the threshold tests were repeated only 2 times with the same 5 minute resting periods.

Numerous drugs and combination of drugs were used to block specific sets of receptor hypothesised to contribute to the electrically stimulated recover in these experiments. As drugs were administered via the main reservoir, they were applied to the serosal side of

39

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

the intestinal segments where they have rapid access to the myenteric plexus and the motor pathways contained therein. Drug combinations can be seen in Table 1.

40

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 5. Types of peristaltic pressure waves. Three pressure traces from the aboral end illustrating typical waveforms associated with peristalsis (same scale for each). A. Pressure

traces showing peristaltic reflexes (i.e., 4 propulsive contractions) in normal physiological

saline. During control period at pressure of 30 mmH2O. B. Pressure traces showing a sub- peristaltic threshold contraction at a pressure of 40 mmH2O. Flow through of saline is a

common feature at higher pressures (small ripples at right). In presence of hexamethonium, but

no electrical stimulation. C. Pressure traces showing peristaltic contractions in the presence of

hexamethonium and following electrical stimulation. Note that the tissue shows some recovery

at a pressure of 30 mmH2O.

41

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Action Drug Concentrations Nicotinic receptor antagonist Hexamethonium (Hex) 300 µM Alternative nicotinic receptor 3 µM Mecamylamine antagonist

5-HT3 receptor antagonist Granisetron (Gran) 1 µM IKCa++ blocker (excites sensory 100 µM Tram-34 neurons) IKCa++ and nicotinic blockade Tram-34 + Hex Muscarinic M1 receptor antagonist VU 0255035 150 nM M1 and nicotinic blockade VU 0255035 + Hex Cocktail- to block all excitatory RO51 ligand-gated ion channels (RO51: a 10 µM selective antagonist of Hex + Gran + RO51

purinoreceptor subtypes P2X3 and

P2X2/3) Inducing retrograde contractions and Hex + aboral electrical nicotinic blockade stimulation Inhibits conduction of action 100 µM potentials by blocking axonal Lidocaine (Lido) sodium channels Inhibits ionic fluxes, nicotinic Hex + Lido + aboral blockade and retrograde contractions electrical stimulation Non-selective muscarinic receptor 10 µM Hyoscine antagonist

Table 1. Drugs and drug combinations used. The table outlines various actions of the drugs which were used to test the underlying neuronal behaviour during peristalsis. In presence of these drugs, electrical stimulation was applied to overcome the effects of the drugs.

42

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Electrical stimulation

To electrically stimulate the enteric neurons present in the intestinal segments, two electrodes were positioned on either side of the oral or aboral end of the tissue. On most occasions, only one of the two intestinal segments was stimulated and the remaining tissue was used as a control so that any differences between the two differently treated tissues could be determined. The electrical stimulation was initiated by a Master-8 pulse generator (A.M.P.I., Jerusalem, Israel) and given via an SD9 stimulator (Astro Med Inc., Grass, Warwick, USA). Immediately after the peristaltic reflex threshold test, the tissue was electrically stimulated to determine the voltage at which a 20 Hz, 50 pulses train could evoke a peristaltic-like contraction. The voltage was set at 5 V to begin with. The intestinal segments were observed for any peristaltic reflexes or any movements. If there was no reaction, the voltage was increased by 1 V or decreased by 1 V if a full propagating reflex observed. Between each increase or decrease in voltage, approximately 30 seconds of resting time was given. Once determined, the voltage was increased to 150 % of the electrical threshold and was used during the experiment the electrical training protocol (see below). The electrical threshold test was carried out once in control, twice in drug conditions, and once more after the final peristaltic reflex threshold test (i.e. after wash out of drug). This all can be seen in the timeline of the experiment (Figure 4).

As mentioned above, there were two electrical stimulation protocols utilised during the experiment, the test for threshold (above) and the electrical training protocol.

The settings for electrical training were 1 Hz (250 pulses, 1ms duration) at the voltage determined during the electrical test. The electrical training protocol took just over 4 minutes to complete. Electrical training was repeated twice, both during drug conditions.

Electrical training was used in an environment where various neurotransmitter receptors

43

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

and channels were blocked. The purpose of this was to attempt to evoke a change to the types or actions of neurotransmitters involved in the motor pathways (i.e., to evoke neuronal plasticity).

Video recording

Video recordings of the preparation were made throughout the experiment to track the intestinal diameter. A video camera (Logitech Quickcam Pro 9000, Strathfield

South, NSW, Australia) was positioned approximately 5 cm above the dual organ bath.

Any changes in the tissue diameter, pattern of contractions and speed was later identified and analysed by processing the information as a spatial temporal map.

In order to process the raw videos, two programs were used; an edge detection program (Scribble V.2.20) and Matlab R2009a. The edge detection program is an in- house software that detects the edges of the tissue to determine the changes in the diameter. The bottom of the organ bath was covered with a thin piece of black plastic to provide better contrast for the edge detection program. This software finds the edges of the ileum segments and draws a line over each as can be seen in Figure 6. The detection lines (defining the upper and lower edge) are represented in red and green. The detection lines could be adjusted to correspond better to the edges of the tissue segments.

A box was drawn around each piece of intestinal segment to delineate the “region of interest” (Figure 6). Once the diameter information was extracted, it was transformed in to a spatial temporal map. Matlab R2009a (Mathworks, Warwick, USA) was used to produce a spatial temporal map using an in-house script. Each video recorded 30 frames per second at 640x480 pixels which was approximately 20 minutes long. Earlier videos needed to be flipped along the vertical axis as the oral and the aboral end was in a reversed in position due to constraints on the position of the video camera in relation to the organ bath. From the spatial temporal map, information regarding the intestinal 44

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

diameter and pattern of contractions over a defined period of time was determined. On the map, the x-axis represents the length of the ileum and the y-axis represents time in seconds. The colours displayed on the map signify the width of the intestinal segment at a certain time point and at a particular position along the segment. For example, blue represent the dilation of the ileum and red represents the contraction of the ileum. The diameter of the intestinal segment can be determined by looking at the colour legend on the right side of the map. This colour legend contains a range of colour variation between blue and red. An illustration of a spatial temporal map is shown in Figure 7.

The peristaltic reflexes propagate from left (oral) to right (aboral) which allows a diagonal line to be drawn attaining the propagation velocity by the slope.

45

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 6. Example of edge detection software used to find the upper and lower

bounds of the ileal segment. The edge detection program is an in-house software

used to detect the edges of the segments of ileum to determine the changes in

diameter. Oral is on the right and the aboral end is on the left side. Once the “region

of interest” is selected, green and red line appears within the box which defines the

upper and lower regions of the tissue, respectively.

46

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 7. Example of a spatial temporal map generated from diameter. A

spatial temporal map is created from the data obtained by using the edge detection

program. Gut length (in mm) is on the x-axis and the time (in secs) in on the y-

axis. To specify the meaning of the colours on the spatial temporal map, colour

legend is shown which aids in predicting the width of the ileum segments used in

the experiment. Colour blue indicates dilation and red indicates contraction. The

peristaltic reflexes initiates from left to right (oral to aboral end).

47

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Drugs and statistical analysis

Hexamethonium bromide, the nicotinic receptor antagonist was provided by

Sigma-Aldrich Fine Chemicals (Sydney, Australia). All of the drugs that were used in the study were prepared as stock solutions in reverse osmosis (RO) water or dimethyl sulfoxide (DMSO) to concentrations suitable for each drug. Hexamethonium bromide stock solution was made to a concentration of 300 mM. For hexamethonium bromide in particular, on the day of the experiment, the stock solution was diluted 1000-fold in physiological saline. This gave a final concentration of 300 µM. For other drugs, the dilution was mostly done 100-fold in physiological saline. However, the dilution fold was varied according to the drug concentrations. Other drugs used in this experiment were mecamylamine (Sigma-Aldrich Fine Chemicals, Sydney, Australia), granisetron

(SA chemicals International, Penfield, NY, USA), Tram-34 (Sapphire Bioscience,

Redfern, NSW, Australia), VU 0255035 (Sapphire Bioscience), RO51 (Sapphire

Bioscience), lidocaine (Sapphire Bioscience), and hyoscine (Sigma-Aldrich Fine

Chemicals).

The data are all represented as mean±SEM. Measurements of diameter, peristaltic threshold, amplitude, frequency and interval between each pressure wave were statistically analysed. The number of animals on which the experiments were conducted was referred to as 'n'. However, in some preparations, the intestinal segments became unresponsive during the experiment as determined by a failure to recover peristaltic activity during the washout period. In such cases, the experimental data were discarded. ezANOVA (ABI, Atlanta, Georgia, United States) was used to assess multiple parameters (such as non-ES versus ES) using an ANOVA with post-hoc T-test to test for statistical significance at a level of P < 0.05 for all experiments.

48

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Results

Measuring the peristaltic reflex threshold

The peristaltic reflex threshold was defined as four consecutive contractions at a certain pressure which started within a 30 seconds window. In our experience, when there are 4 consecutive propulsive contractions, if left at this pressure, contractions will continue until the pressure is lowered (or the tissues fatigue). Thus, four contractions is the minimum number required to measure the peristaltic threshold while preserving as much tissue viability as possible. This procedure was repeated three times which was later used to calculate the average of the pressure wave amplitude (in mmH2O), the time to 80% return to baseline (t80 in s), duration of the pressure wave (s), interval between waves (s) and frequency of waves (Hz or per minute). The average interval was found by measuring the time in seconds from the peak of the initial pressure wave to the peak of the last one and dividing this by the number of waves minus one. As only four consecutive waves were required to establish the threshold, a total of 3 intervals were considered for each trial. Three trials were carried out to confirm the average over the course of time. As for the t80, from the peak to the 80% return to baseline of each pressure wave was measured. This again was averaged later according to the number of trials performed. The pressure wave time course (duration) was measured in seconds, from the beginning of a wave to the peak of a wave (Figure 8). This was evaluated in each of the 4 waves and was averaged.

Time control experiments were performed in order to establish peristaltic reflex threshold over the time course of the experiment so that comparisons could be made to analyse any changing effects on ileum segments due to drug application or electrical stimulation. The second tissue in each bath was designated as a non-stimulated (control)

49

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

tissue and did not receive any electrical stimuli but was otherwise treated identically to the paired tissue that did receive electrical stimulation. The pressure threshold for the peristaltic reflex in control was 31±0.7 mmH2O (n = 24). As for the average amplitude of the pressure wave, in control it was 29.2±3 mmH2O and has a time to peak of 2.2±0.5 seconds. Furthermore, the average interval of 4 consecutive propagating pressure waves was 11.2±1.6 seconds and the average propagation speed was 15.1±3.6 mm per second.

The diameter of the ileum segments on average was 5.1±0.2 mm in normal physiological saline and at 0 mmH2O pressure.

Figure 8. Example of how pressure waves were analysed. Various measurements were made to determine the effects of the drugs. The amplitude (mmH2O), contraction time (time to peak; seconds), t80 (peak to 80% return to base; seconds), interval (time between each waves; seconds) and the frequency (Hz) are the main factors. These measurements were evaluated for each single wave, and were then averaged later.

50

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Effects of blockade of fast excitatory neurotransmitter receptors

Hexamethonium blocks nicotinic receptors

Throughout the control period of the experiment (i.e., before hexamethonium was added), the frequency was 0.085±0.04 Hz (for the tissue which was to be electrically stimulated - ES; n = 20) and 0.1±0.006 Hz (not electrically stimulated - non-

ES; n = 16) which was not significantly different. Similarly, there was no difference between any of the other measurements between the ES and non-ES tissues during the control period. The amplitude of the control average pressure wave peak was 15.8±1.2 mmH2O for ES intestine and 15.2±1.8 mmH2O for non-ES. The time to peak for ES tissues was 1.96±0.1 s and 1.94±0.3 s for non-ES. The average t80 of the ES tissue was

3.6±1.4 s and 2.08±0.21 s for the non-ES. For the interval, ES was 12.07±0.55 s and non-ES was 9.82±0.63 s.

Hexamethonium (HEX; 300µM) was used to examine the effect of nicotinic receptor blockade on peristalsis in the ileum. In the non-ES tissues, the drug caused an inhibition of pressure induced peristalsis (n = 16). In all experiments, there was a loss of peristaltic threshold in the presence of hexamethonium, but in 7 out of 16 experiments there was a partial recovery where a few propulsive contractions were seen (i.e., less than 4 in a row) (Figure 9). Electrical stimulation enhanced the recovery of the peristaltic reflex with 6 out of 20 preparations showing full recovery (i.e., all 6 of 6 attempts to find peristaltic threshold successful). Partial recovery was seen in 4 of 20 while 7 of 20 experiments did not show any recovery (Figure 9). Almost all of the experiments with no recovery demonstrated an effect where the physiological saline passed through the lumen of the ileum without provoking a propulsive contraction - this phenomenon was called flow through (FT) and was seen as a low amplitude ripple in the pressure trace (see Figure 5 above). 51

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

100 Full recovery

Partial recovery 75 n = 6

50 n = 16

Percent recovery (%) 25

0 Time Control Electrical Stimulation

Figure 9. Illustration of recovery status in presence of hexamethonium. The percentage of

partial recovery of both stimulated and non-stimulated tissues are similar. However, the

occurrence of full recovery of ES and non-ES vary significantly. This suggests that neuronal

plasticity may be involved in the recovery process of the intestinal tissues.

Treatment of intestinal segments with hexamethonium changed the overall condition of the guinea pig ileum. It was observed by eye that the colour of the tissue turned from fresh pink with showing blood vessels to a pale grey appearance. The intestinal segments increasingly bloated due to the effect of the drug. This can be seen in Figure 10.

52

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

.

Figure 10. Intestinal segments in the absence or presence of hexamethonium. The above

photo shows the ileum segments in physiological saline (A and B) and in hexamethonium (C

and D). The top tissues (A and C) in both photos had electrical stimuli applied following

hexamethonium. The bottom tissues (B and D) in both photos were not electrical stimulated.

From the photos, there is a width difference in the tissue between C and D. In general, the

tissues dilated in the presence of hexamethonium. A colour change was also evident as the gut

became greyer and paler. Deterioration of the tissues (i.e. debris forming) could be seen once

the ileum segments were present in drug for enough time. In many cases, the tissues

recovered after the organ bath contents were exchanged to normal physiological saline.

53

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Before nicotinic blockade (the control period), for ES tissues the pressure threshold at which 4 consecutive peristaltic pressure waves occurred was 29.2±2.3 mmH2O and for non-ES 32.2±0.1 mmH2O. The amplitude, contraction time, t80 and interval for ES were: 14.6±2.0 mmH2O, 4.16±0.48 s, 2.14±0.15 s and 9.89±0.77 s, respectively. For non-ES tissues in hexamethonium: amplitude, contraction time, t80 and interval were 11.4±0.77 mmH2O, 3.49±0.3 s, 1.77±0.16 s and 9.92±1.4 s, respectively. For both of the intestinal segments, with or without electrical stimulation, the pressure threshold was increased.

During nicotinic receptor blockade, the pressure threshold for ES preparations was increased to 43±4 mmH2O from 33±3 mmH2O in non-ES preparations (n = 6). The t80 was significantly increased (P = 0.03), but other factors were not significantly affected in ES versus non-ES tissues: frequency (P = 0.77), amplitude (P = 0.87), contraction time (P = 0.16) and interval (P = 0.19).

To confirm the effects of hexamethonium on intestinal tissue, the drug was washed out with physiological saline (for 30 minutes) and peristaltic threshold determined again (washout period). The peak pressure for ES preparations after washout of the drug was 14.6±6 mmH2O, contraction time 2.12±0.73 s, t80 1.76±0.16 s, and interval was 11.3±0.33 s. For the non-ES tissue, the amplitude, contraction time, t80 and interval was 11±2.3 mmH2O, 2.34±0.61 s, 1.60±0.21 s, and 8.26±0.33 s, respectively.

The pressure threshold for electrically stimulated ileum was 41±2 mmH2O and for tissue with no electrical stimulation was 33±3 mmH2O. Overall, hexamethonium did block peristaltsis where there were at least 4 consecutive contractions. Although electrically stimulated or non-stimulated tissues successfully produced at least some peristaltic pressure waves in presence of nicotinic blockade, the electrically stimulated intestinal tissues were more likely to show either full or partial recovery.

54

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Mecamylamine blocks nicotinic receptors

Mecamylamine (MEC; 3 µM) was used as an alternative nicotinic receptor antagonist to confirm the effects of the nicotinic receptor blockade by hexamethonium.

Mecamylamine inhibited pressure induced peristaltic reflexes (n = 6). Pressure threshold was 35±3.4 mmH2O during the control period while the frequency, amplitude, contraction time, t80 and interval was similar for both ES and non-ES tissues when in the presence of mecamylamine. For ES preparations, the amplitude was 13.2±3 mmH2O, contraction time 4.3±0.6 s, t80 2.49±0.4 s and interval 10.36±0.5 s. Non-ES had amplitude of 29.2±1.5 mmH2O contraction time of 3.54±0.7 s, t80 of 1.88±0.1 s and interval of 9.89±0.5 s. The measurements in the non-stimulated or stimulated ileum segments did not vary in the presence of MEC (contraction time: P = 0.49; t80: P = 0.3; interval: P = 0.16; amplitude: P = 0.88). Overall, 4 of 6 attempts showed full recovery, but at a higher pressure than control, such as 40-50 mmH2O. Furthermore, for the ES tissues 2 of 6 preparations showed full recovery with the peristaltic threshold returning to a near initial pressure of 27±2 mmH2O for ES and 33±2 mmH2O for non-ES. For the ileum which had no electrical stimulation, 3 of 6 preparations showed partial recovery and 3 of 6 had full recovery. This is illustrated in Figure 11. Finally, washout of mecamylamine was done to confirm the drug actions. For ES tissue, amplitude, contraction time, t80, interval was 11.7±8.8 mmH2O, 2.75±0.3 s, 2.29±0.5 s and 10.7±2 s, respectively. For non-ES, amplitude was 10.2±0.9 mmH2O, contraction time was

1.98±0.18 s, and t80 was at 1.87±0.6 s and interval 10.7±1.1 s. In presence of mecamylamine, no preparation failed to show peristaltic reflexes though the pressure at which the 4 consecutive pressure waves occurred was higher than in control.

55

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Figure 11. Graph of partial and full recovery of peristaltic reflexes. A. An illustration of

partial recovery in mecamylamine, Tram-34 with hexamethonium, VU 0255035 with

hexamethonium and hexamethonium, granisetron plus RO 51. Other than mecamylamine,

other drug combinations did not show difference in electrically stimulated or non-electrically

stimulated ileum segments. B. Percentage of fully recovered preparations. The drugs

compared are same as in A. Higher percent of full recovery was seen but the difference in

success rate of electrically stimulated or non-electrically stimulated illustration did not differ.

56

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Blockade of 5-HT3 receptors with granisetron

A selective serotonin 5HT3 receptor antagonist, granisetron (1 µM; n = 6) was used to determine if this receptor was involved in pressure-induced peristalsis. From the data, it was found that granisetron attenuated any spontaneous movement of the tissue however did not completely block the peristaltic reflexes. Incubation with granisetron was extended to one hour instead of 30 minutes to ensure it had time to act as previous experiments have shown that it equilibrates slowly with the tissue (Monro et al., 2002).

The average pressure threshold for control experiments was 36±3.5 mmH2O. The average frequency, amplitude, contraction time, t80 and interval during time control did not show any major variations from other preparations during the control period.

During granisetron application, the frequency in ES tissues was unchanged at

0.088±0.01 Hz compared to 0.087±0.004 Hz in control. The amplitude, contraction time, t80 and interval were 13.5±2.9 mmH2O, 4.96±1 s, 2.07±0.2 s, and 12.01±1.3 s, respectively. For non-ES, frequency was 0.11±0.3 Hz and the interval 12.5±2.8s. The amplitude, contraction time and t80 were 13.2±1.7 mmH2O, 4.1±0.6 s and 1.79±0.2 s, respectively. Furthermore, the tissue which was electrically stimulated had some propulsive contractions triggering at lower pressures such as 30-40 mmH2O, although many were seen at higher pressures (average peristaltic threshold pressure 51.7±4.1 mmH2O). For the non-stimulated intestinal segment, most of the contractions were observed at higher pressures, 50-60 mmH2O (average peristaltic threshold pressure

48.3±9.8 mmH2O). Both of the stimulated and non-stimulated ileal segments in granisetron showed that there was an increase in peristaltic threshold relative to the control period (control: 36±3.5 mmH2O versus ES: 51.7±4.1 mmH2O; and non-ES

8.3±9.8 mmH2O). There were, however, no significant differences between ES and non-

57

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

ES tissue peristaltic parameters during granisetron incubation (amplitude: P = 0.96; frequency: P = 0.88; contraction time: P = 0.33; t80: P = 0.17; and interval: P = 0.36).

To confirm the pharmacological effects of granisetron, the contents of the organ bath were exchanged with normal physiological saline and were equilibrated for 30 minutes. After the washout, the average peristaltic pressure threshold was still increased for ES was 51.7±3 mm while non-ES was 48.3±7 mm suggesting granisetron had not washed out completely. The tissue which was electrically stimulated had a frequency of

0.062±0.003 Hz, amplitude of 9.4±0.3 mmH2O, 6.76±0.72 s for contraction time, t80 of

2.15±0.1 s and interval of 16.4±1s. For the non-ES ileum, the frequency, amplitude, contraction time, t80 and interval were 0.062±0.004 Hz, 11.7±0.9 mmH2O, 4.31±1.4 s,

2.58±0.3 s, and 17.53±0.2 s. Granisetron only the partial blocked peristaltic reflexes and

ES tissues had pressure induced waves occurring at lower pressures than the non-ES tissues.

Effects of blockade of slow excitatory neurotransmitter receptors

Blockade of muscarinic M1 receptors with VU 0255035

VU 0255035 (150 nM), a muscarinic M1 receptor antagonist was used to assess its effects in control and in an attempt to block recovery from nicotinic blockade in ES preparations. When VU 0255035 was used alone (n = 2) to examine its effects in ES and non-ES preparations. During the control period, the pressure threshold was 30 mmH2O, propagation speed was 17.65±8.7 mm/s and the average diameter of ileum was 5.1 mm.

The frequency of a propulsive propagation was 0.075±0.003 Hz, the amplitude

11.7±11.7 mmH2O, contraction time was 2.3±0.1 s, t80 2.4±0.2 s and interval was

13.4±0.6 s. In 2 of 2 preparations, peristaltic reflexes were successful evoked after introduction of VU 0255035 in to the organ bath. The pressure threshold for the tissue

58

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

that was electrically stimulated was 39.2±4.7 mmH2O. The non-ES tissue had a pressure threshold of 40±6.3 mmH2O. The average intestinal segment diameter in ES tissues was measured 4.05 mm and 3.97 mm for non-ES tissues. The average propagation speed for

ES was 5.8±7.2 mm/s and 16.11±3.6 mm/s for non-ES. Furthermore, the frequency, amplitude, contraction time, t80 and interval for ES were 0.12 Hz, 8.8±1.7 mmH2O,

4.4±0.2 s, 1.9±0.2 s and 8.6±0.1 s, respectively. For non-ES, these measurements could not be made as there were not enough values. In presence of VU 0255035, there were no significant variations compared to control: contraction time (P = 0.63) and t80 (P =

0.76), amplitude (P = 0.38), frequency (P = 0.08) and interval (P = 0.24). To confirm the recovery of the intestinal segments from the effects of VU 0255035, washout measurements were taken. The average pressure threshold for the ES was 43.3±5.2 mmH2O and 46.7±4.1 mmH2O in non-ES tissue. The diameter of the ES tissue after washout of the drug was 5.75 mm and the propagation speed was 15.9±9 mm/s. Non-ES tissue propagation speed and diameter could not be analysed.

VU 0255035 was combined with hexamethonium (blocking both M1 and nicotinic receptors) in an attempt to block recovery in ES preparations (n = 3). The average pressure threshold found in the control period was 26.7±3.3 mmH2O and had an average diameter of 4.56 mm. During the control period, the intestinal segments had an average propagation speed of 23.7±10 mm/s. Other factors such as the amplitude, contraction time, relaxation time and the interval were: 11.7±0.6 mmH2O, 1.9±0.2 s,

2±0.1 s and 8.7±0.4 s, respectively. The frequency with which the propulsive contractions occurred was 0.1±0.004 Hz. The proportion of recovery was the same for both electrically stimulated and non-electrically stimulated ileum segments. In each, 1 of 3 preparations showed full recovery while 2 of 3 attempts to evoke peristaltic reflexes showed partial recovery. The average pressure threshold for ES preparations after the

59

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

drug incubation was 38.8±3 mmH2O. For non-ES tissue, the average pressure threshold was 31.7±5 mmH2O. The diameter and propagation speed for ES was 5.3 mm and

23±12.7 mm/s, respectively. The average propagation speed of non-ES intestinal segment was 21.5±12.3 mm/s and diameter was 5.7 mm. Analysis of the data demonstrated that the interval at which the waves were forming varied between the ES and non-ES ileum segments. The interval for the tissue with electrical stimulus had an average interval of 10.3±0.7 s while 8.05±1.9 s was recorded for the tissue without any electrical stimulation (P = 0.37).

In the presence of the combination of VU 0255035 and hexamethonium, both electrically stimulated and non-stimulated tissues showed changes relative to control in contraction time (P = 0.96), t80 (P = 0.83) and the interval (P = 0.73). The frequency (P

= 0.56) and the amplitude (P = 0.59) did not show great differences between the stimulated and non-stimulated ileum segments throughout the experiment. During washout, the average pressure threshold for ES tissues was 34±4 mmH2O. This value was lower than the average threshold found for non-ES (35±2 mmH2O). The size of the diameter for both intestinal segments decreased to 4.1 mm (ES) and 3.8 mm (non-ES).

Moreover, the propagation speed for ES decreased to 21.8±10.1 mm/s and for non-ES there was an increase to 22.9±10.7 mm/s. The amplitude was higher for ES without the presence of the drug (10.2±1.2 mmH2O to 12.9±2.9 mmH2O). The contraction time was shortened after the wash out for ES (4.64±0.2 s to 2.44±0.4 s) as well as t80 (1.96±0.03 s to 1.5±0.03 s) and the interval (10.3±0.7 s to 7.5±1.6 s). VU 0255035 did not have any effect on preventing recovery in presence of hexamethonium. The only change noted was that a passive distension of the intestinal tissues was lessened in presence of VU

0255035.

60

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Blockade of all muscarinic receptors with hyoscine

Hyoscine (scopolamine, 100 μM; n = 3) was used to block all muscarinic acetylcholine receptors. For this drug, all of the intestinal segments received electrical stimuli following administration of the drug. During the control period, the average propagation speed was 9.5±4.7 mm/s. The average diameter of the tissue was 6.3 mm.

The pressure threshold measured was 36.7±5 mmH2O. The frequency, amplitude, contraction, t80 and interval were 0.1±0.013 Hz, 11.7±2.9 mmH2O, 3.1±0.5 s, 2.3±0.3 s and 10.6±1.3 s, respectively. The effect of hyoscine was very interesting. All 3 out of 3 attempts to evoke peristaltic reflexes were unsuccessful with only passive flow of physiological saline through the lumen (i.e., FT) seen throughout the experiment. The intestinal tissue looked distended and there was no spontaneous movement detected.

Because the waveforms were so small, measurements could not made from the pressure traces, however, by analysing the video via Matlab R2009a the propagation speed and diameter of ileum could be found. The average propagation speed detected in time control was 13.8±11.8 mm/s and diameter was 7.78 mm. Electrical stimulation did not help in the recovery of the intestinal segments which may lead to the speculation that neuronal plasticity was not evoked in presence of hyoscine.

Effects of blocking both fast and slow excitatory neurotransmitter receptors

Hexamethonium, granisetron and RO51 (10 µM; n = 3) were used to block of all ligand-gated ion channels known to participate in fast synaptic transmission in the ENS.

RO51 is a selective antagonist of purinoreceptor subtypes P2X3 and P2X2/3 which has been used to treat pain (Carter et al., 2009). The pressure threshold during the control period was 26.7±3 mmH2O and the ileum diameter was 4.57 mm. The average propagation speed was 24.4±11.8 mm/s. All 3 out of 3 attempts to evoke peristaltic

61

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

reflexes were successful. For ES intestinal segments, all 3 out of 3 preparations showed partial recovery with drug treatment. For non-ES tissue, 1 out of 3 preparations had full recovery and 2 of 3 preparations had partial recovery. ES tissue had propulsive contractions occurring at a frequency of 0.057±0.02 Hz while non-ES showed contractions at 0.12±0.02 Hz. The average interval for the tissue with electrical stimulation with 9.88±0.43 s while for non-ES tissue, the interval was 8.98±1.4 s. For

ES the contraction time and the t80 were 1.9±0.4 s and 2.5±0.3 s, respectively; while for non-ES, the contraction time was 2.5±0.8 s and t80 was 2.2±0.02 s. The pressure threshold for ES and non-ES during the drug treatment was 37.5±2.5 mmH2O and 36±4 mmH2O, respectively. The non-ES ileum segment had a diameter of 6.27mm while the diameter of the electrically stimulated tissue was 5.71 mm. The average propagation speed for ES and non-ES tissue was 20.7±9.7 mm/s and 15.1±7.3 mm/s, respectively.

There was no change in the amplitude (P = 0.87) and contraction (P = 0.87) for both simulated and non-stimulated tissues. Similarly, the frequency (P = 0.16), t80 (P = 0.3) and interval (P = 0.25) did not change due to electrical stimulation. After the washout of the combination of drugs, 3 out of 3 attempts to induce peristaltic reflexes were successful for both ES and non-ES intestinal segments. Pressure threshold for non-ES after washout was 33.3±2.3 mmH2O and 33.3±1.7 mmH2O for ES tissue. The diameter of both tissues decreased back to control values when in normal physiological saline

(ES 4.2 mm; non-ES 4.3 mm). The combination of drugs did not prevent peristaltic reflexes as all of the preparations showed full recovery. There was no critical changes in the measured parameters for either ES or non-ES tissue segments.

62

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Effects of other channel blockers and aboral electrical stimulation

Blockade of intermediate calcium activated potassium channel (IKCa++) with

Tram-34

The IKCa++ blocker Tram-34 (100 µM; n = 3) was used in an attempt to enhance the recovery from nicotinic blockade following electrical stimulation by exciting the sensory neurons. Tram-34 has been shown to increase the excitability of AH neurons in the myenteric plexus (Ferens et al., 2007; Nguyen et al., 2007). During the control period, the average pressure threshold was 30±3.2 mmH2O. The average frequency, amplitude, contraction time, t80 and interval of tissues in time control were 0.1±0.01 Hz,

11.7±1.7 mmH2O, 1.94±0.2 s, 2.04±0.2 s and 9.8±0.5 s, respectively. The average diameter was 5.25 mm and the average propagation speed was 17±9.8 mm/s. In the presence of Tram-34, all 3 of 3 preparations demonstrated full propulsive contractions.

The average propagation speed was 18.8±11.3 mm/s for the intestinal segment that was electrically stimulated. For the non-stimulated tissue, the average propagation speed was

19.9±10.2 mm/s. However, when the diameter of the two ileum segments were analysed, electrically stimulated tissue had a larger average diameter of 5.08mm (the non-ES tissue had a similar average diameter of 4.7 mm; P = 0.79). The intestinal segment with electrical stimulation had an average pressure threshold of 39±3.4 mmH2O and the tissue without any stimuli had an average pressure threshold of 30 mmH2O. The pressure threshold remained constant for ES and non-ES tissue and there was no significant variation in frequency (P = 0.34), contraction time (P = 0.25) and t80 (P =

0.13). For ES tissue, frequency, amplitude, contraction time, t80 and the interval was

0.12±0.7 Hz, 17.5±1.2 mmH2O, 2.1±0.2 s, 1.7±0.4 s, and 8.5±0.6 s. The interval for non-ES was 10.6±0.9 s and the contraction time was 3.3±1.5 s. Frequency, amplitude and t80 were 0.092±0.01 Hz, 12.3±2 mmH2O and 1.8±0.3 s, respectively. During 63

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

washout, the average pressure threshold for ES was 34.4±3.3 mmH2O and non-ES was

37.5±2.5 mmH2O. The average diameter for ES and non-ES was 4.9 mm and 4.5 mm, respectively. Furthermore, the average propagation speed for ES was 15.7±10.5 mm/s.

The non-ES tissue had an average propagation speed of 20.3±9.9 mm/s.

++ Blockade of the IKCa and nicotinic receptors

Hexamethonium was combined with Tram-34 to see if this had any effect on enhancing recovery of pressure induced peristalsis (n = 5). The average pressure threshold in the control period was 31.1±1.1 mmH2O. The average diameter and propagation speed during control was 5.05 mm and 10.3±5 mm/s. The average frequency, amplitude, contraction time, t80 and interval were 0.09±0.003 Hz, 11.7±5.8 mmH2O, 1.6±0.1 s, 1.8±0.2 s and 11.7±0.5 s. For the electrically stimulated intestinal segment, it can be seen that plasticity was not fully evoked as only 1 of 5 preparations showed full recovery and 3 of 5 preparations showed partial recovery. On average the pressure threshold was 40.6±0.6 mmH2O. The average diameter for ES tissues was 4.9 mm and the average propagation speed was 13.7±7.3 mm/s. The non-ES tissue had 1 of

4 preparations which showed full recovery and 3 of 4 attempts showed partial recovery.

The average pressure threshold was 44±2.7 mmH2O. Average diameter was 5.5 mm and the average propagation speed was 12.8±6.6 mm/s. There were no significant changes between electrically stimulated and unstimulated tissues in the presence of the mixture of drugs (frequency: P = 0.22; amplitude: P = 0.36; contraction time: P = 0.08; t80: P =

0.09; and interval: P = 0.17). In washout, the pressure threshold for ES was 38.8±1.3 mmH2O (diameter 4.70 mm). Tissue which was not electrically stimulated had a similar pressure threshold of 38.3±3.3 mmH2O (diameter 4.69 mm). The pressure threshold was increased compared to the initial threshold measured in the control period. Overall,

64

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

these data showed that Tram-34 did not have any effect on improving recovery during nicotinic blockade.

Blockade of neuronal conduction with lidocaine

Lidocaine (100 µM; Sigma) was applied to the serosal side and used to block the sodium channels on axonal membranes thus inhibiting action potential propagation. (n =

2). However, due to technical difficulties and environmental changes, the analysis of data extracted from the pressure traces on BP monitor and spatiotemporal maps in

Matlab was impossible. The technical difficulties and the reasons for them will be discussed further in the Discussion.

Stimulation at the aboral end of the preparation

Retrograde contractions were induced via electrical stimulation on the aboral end of the intestinal segment. The presence of hexamethonium allowed for blockade of nicotinic receptors and examined if anally applied electrical stimulation accelerated the recovery (n = 1) as did oral electrical stimulation. The average pressure threshold during the control period was 30 mmH2O. The average diameter and propagation speed was

5.24 mm and 7.39±3.6 mm/s, respectively. Frequency, amplitude, contraction time, t80 and interval in time control experiment was 0.1±0.02 Hz, 11.7±2.9 mmH2O, 2.5±1.1 s,

1.6±0.05 s, and 10.3±1.5 s, respectively. All attempts to evoke peristaltic reflexes in the presence of hexamethonium were successful for both intestinal tissues. However there was no count of full recovery. FT occurred in place of propagating contractions but the number of FT comparing to the hexamethonium and oral electrical stimulation was less

(1 of 6 attempts for ES had FT and 2 of 6 attempts for non-ES had FT). The average pressure threshold for the tissue with aboral electrical stimulation was 36±2.4 mmH2O.

The control tissue had an average pressure threshold of 40 mmH2O. The diameter of ES

65

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

was 6.8 mm while the diameter for non-ES was 7.1 mm. As for the propagation speed,

ES was 14.7±9.4 mm/s. Non-ES had lower propagation speed of 11.9±5.7 mm/s. There were no differences in other factors such as, amplitude and contraction time. The interval for ES increased to 10.9±0.5 s but for non-ES decreased to 9.29±0.7 s. During washout the average diameter was smaller for ES tissue (5.7 mm) than non-ES (6.5 mm).

For ES intestinal segment in particular showed better propagation speed of 9.8±4.8 mm/s (non-ES 14.1±6.5 mm/s). The frequency, amplitude, contraction, t80 and interval for ES were 0.08±0.006 Hz, 11.7±0.3 mmH2O, 1.9±0.3 s, 1.5±0.2 s and 12.1±0.8 s, respectively. For non-ES the frequency was (0.1±0.02 Hz), the amplitude was

(0.03±0.002 mmH2O), contraction time was (1.6±0.3 s), t80 was (1.5±0.08 s) and interval was (10.7±2.4 s).

66

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Discussion

The major finding of this study was that electrical stimulation can enhance the recovery of peristalsis through neuronal plasticity in the intestine in the presence of nicotinic blockade. Surprisingly, a variety of blockers targeting synaptic transmission in the ENS did not reduce the ability of electrical training to induce full or partial recovery in many of the preparations. Other findings include the observation that the initial susceptibility of the tissue to nicotinic blockade prior to any type of stimulation was one of the chief factors that correlated with recovery after stimulation and that some tissues recovered partially from nicotinic blockade without electrical training.

The drugs used in this study included those which blocked receptors for fast and slow synaptic transmission (ligand-gated ion channel and G-protein coupled receptors, respectively) and that blocked ion channels involved in the excitability of sensory/AH neurons. Overall, no single drug or combination of drugs prevented electrically stimulated recovery from nicotinic blockade. Time control experiments (absent electrical training) did not show any significant variation in factors such as peristaltic pressure threshold, interval, amplitude, contraction time or relaxation time (t80) of the pressure waves.

In this study, some tissues recovered partially, even without electrical stimulation. We can speculate that the fluid passing through the lumen during testing of peristaltic threshold may have acted as a stimulus to the intestine, similar to electrical stimulation. Previous results in the laboratory showed that repeated, rapid pressure changes were an effective stimulus for enhancing recovery during nicotinic blockade (P.

Tay Honours thesis, School of Medical Sciences, UNSW, 2011). In contrast, Spencer et al (1999) found that fluid in the lumen of the intestine was not necessary for inducing a peristaltic motor pattern. However the present findings show that the fluid passing

67

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

through the lumen caused various uncoordinated contractions that may be considered as a contributing factor towards recovery by providing an extra physical stimulus.

Although no extra stimulus was applied, these tissues exhibited some recovery over time (i.e. especially in the second round pressure threshold test of the experimental protocol).

Electrical stimulation enhanced the peristaltic motor pattern in the presence of nicotinic receptor blockade

Most attempts to evoke a single peristaltic pressure wave in the presence of hexamethonium were successful for both stimulated and non-stimulated intestinal tissues. However, it was difficult to produce the four consecutive pressure waves needed to calculate a peristaltic threshold in the presence of nicotinic blockade. For the electrically trained intestinal segments, most preparations either showed full recovery or partial recovery. Bartho et al (1987) found that 110 µM hexamethonium blocked the peristaltic reflex in all 20 of their preparations. In contrast, Nicholas and Spencer (2010) found recovery of the intestinal tissues occurred in the presence of hexamethonium over time. They found that the concentration of hexamethonium was not a significant factor as the same result was obtained using 500 µM and 1 mM hexamethonium (however, there was no initial block at 100 µM). In the present study, even though the electrical stimulation enhanced the recovery of the peristaltic reflexes, the elevation of the pressure threshold was common in the presence of hexamethonium, an effect also seen by Bartho et al (1987).

Mecamylamine was used as an alternate nicotinic receptor antagonist to confirm the pharmacological characteristics of nicotinic receptor blockade. This drug was used by Bartho et al (1987) as an alternative to hexamethonium and was successful in

68

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

preventing peristaltic activities. However in the present experiments, it only reduced spontaneous pressure threshold in both stimulated and non-stimulated tissues but as with hexamethonium there were no failed attempts to evoke a single peristaltic presure wave. From an analysis of the data in mecamylamine, electrical training tended to show improved recovery from nicotinic receptor blockade but there was not a great difference compared to unstimulated tissue segments.

Distension location and degree of distension may be another reason that contributes to the inhibition of the propagating reflexes. From the experiments carried out by Holzer et al (1993), it was found that in the case of nicotinic receptor blockade, the site of distension determined whether peristaltic activity was inhibited or not. This is in line with the results of the present study where higher pressures and electrical stimulation at higher voltages was required to induce peristaltic reflexes in the presence of hexamethonium. It was also observed that with stronger electrical stimulation, the tissue recovered faster.

Finally, it should be noted that not all of the blockers used in previous and recent studies on peristalsis are specific; many have actions at other receptors in the ENS. A study done by Wijngaarden et al (1993) showed that there may be a high or weak affinity displayed by ondansetron, cilansetron and granisetron for the 5-HT3 receptor.

Juarez and colleagues (2013) showed that ondansetron, picrotoxin and bicuculline were non-specific and concentration dependent while only hexamethonium showed high selectivity towards nACh receptors.

Blockade of 5-HT3 receptors did not have an effect on recovery

One idea of how recovery might occur is that stimulation of the tissue might cause the ENS to produce more 5-HT or ATP. Monro et al (2004) stated that 5-HT3

69

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

receptors were required to mediate the descending excitation during a propulsive contraction and, as 5-HT3 receptors contribute to fast EPSPs in the myenteric plexus

(Zhou & Galligan, 1999), it was thought that this might underlie the block of peristalsis- like activity seen by Monro et al. On the other hand, Nicholas et al (2010) showed that in guinea pig distal colon, neither serotonin nor nicotinic receptors were essential for peristalsis suggesting that fast EPSPs mediated by these receptors are not critical for a propulsive motor pattern.

Granisetron, the 5-HT3 receptor antagonist, was found in the present experiments, to only partially block peristalsis on its own. Though the underlying mechanisms may differ it is worth noting that these data are in contrast to a previous study where the frequency of the MMC in mice was reduced by the 5-HT3 receptor antagonist alosetron (Bush et al., 2001). In the present study, the electrically stimulated tissue in granisetron recovered more rapidly with some propulsive contractions triggered at lower pressures compared to 50-60 mmH2O. Furthermore, fewer failures to evoke peristalsis were seen for the tissue with electrical stimuli. It can be postulated that the IPANs may be in action while the normally recognised route for IPAN production was hindered by block of 5-HT3 receptor (Bertrand et al., 2000) which explain the existence of peristaltic reflexes after granisetron administration. According to Monro et al (2004), granisetron had no effect on 17 of 20 submucosal neurons tested and only 3 of 20 preparations showed decreased fast EPSP amplitude. Furness et al (2002) also confirmed that granisetron did not reduce reflex responses.

Hexamethonium, granisetron and RO 51 were used to block all of the ligand- gated ion channels but all of the preparations had either full or partial recovery in presence of the drugs. This showed that the blockades of neural pathways using fast synaptic transmission are not effective in preventing peristaltic activity.

70

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Excitation of AH/sensory neurons may enhance the peristaltic reflex

The main finding of the experiment with Tram-34 was that the excitation of

AH/sensory neurons may have enhanced recovery from nicotinic blockade. All 3 of 3 experiments demonstrated full peristaltic activities whether intestinal tissue was electrically stimulated or not. No partial recovery or failures were observed throughout the study with Tram-34. The drug did increase factors overall such as propagation speed, diameter and pressure threshold. Moreover the electrically stimulated tissue had lower data values. The electrical stimulation at low frequencies to AH/sensory neurons may cause prolonged excitation which may be postsynaptic (Clerc et al., 1999). Continuous electrical training to the intestinal tissue reduced the diameter compared to non- stimulated tissue and more spontaneous movements were noted. This may be explained by the excitation of the AH/sensory neurons. The study done by Clerc et al (1999) which involved low frequency stimulation stated that the AH/sensory neurons are “non- adapting” meaning that continuous stimuli will maintain AH/sensory neuron excitability.

The excitation of AH/sensory neurons in the presence of nicotinic blockade seemed to enhanced recover with only one attempt to evoke peristalsis unsuccessful.

An M1 muscarinic receptor antagonist did not have an effect on recovery

In an attempt to prevent recovery from nicotinic blockade in electrically stimulated experiments, the muscarinic M1 receptor antagonist, VU 0255035 was tested.

First, it was used alone to assess its effects on peristalsis. All 2 of 2 attempts to generate propulsive contractions were successful following superfusion with VU 0255035.

Compared to the non-stimulated tissue, the tissue with electrical stimuli showed relatively lessened values of propagation speed and interval between each 4 consecutive contractions. When VU was used in the presence of nicotinic blockade, there was no

71

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

reduction in recovery suggesting M1 muscarinic receptors are not crucial to the recovery of the peristaltic reflex. VU 0255035 seemed to reduce the bloating of ileal segments seen in the presence of hexamethonium. This was examined by eye and the peristaltic reflex activities observed by the tissues throughout the experiment. The average pressure threshold for both electrically stimulated (38.8±3 mmH2O) and non- electrically stimulated (31.7±5 mmH2O) tissues were lower compared to other ileum segments present in other combination of drugs (including hexamethonium).

Interestingly, the non-specific muscarinic receptor antagonist hyoscine did not show any full peristaltic reflexes. All 3 attempts to evoke peristaltic reflexes were unsuccessful. This result was incongruous to the findings by Bartho et al (1982) and

Tonini et al (1981) as it was found that hyoscine delayed but did not abolish peristalsis.

The electrical stimulation did not have any effect on the intestinal tissue after hyoscine administration. Most likely, the muscarinic receptors on the smooth muscle were the target of hyoscine.

Overcoming experimental problems and issues

There were several unforeseen problems which had to be overcome throughout the course of this study. The main problem was the laboratory had to move in late April.

The third floor of Wallace Wurth building had to be vacated and we were relocated to the second floor. The room that was provided was very limited and small compared to the original laboratory. Only critical equipment, drugs and chemicals were allowed into the new room. The whole process took about one month. Luckily, there was no major disruption or damage to the organ bath or any other equipment but there still needed to be maintenance done.

72

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

As well as the move of the laboratory from the third floor, the whole Wallace

Wurth building was going under reconstruction. There was constant drilling and banging on the third floor and throughout the whole building and this affected the organ bath set up. The organ bath and other equipment is connected by very sensitive electrical wires. Especially the pressure transducers were very vulnerable to extremely small noise and trembling. Due to this, several experiments could not be recorded properly and some experiments had to be stopped due to technical troubles. To block out the noise from the shaking ground and trembles above, the sensitive wires were wrapped with foil. Furthermore, there was several power failures which interrupted the experiment.

Future directions

The analyses of these data suggest that electrical stimulation enhanced recovery of the peristaltic reflex in the presence of nicotinic blockade. However, attempts to find out the mechanism for this by trying to prevent recovery using blockers for ligand-gated or G-protein coupled receptors known to be involved in neurotransmission were not successful. Some of these outcomes were dissimilar to the known pharmacological properties of the blockers. Further investigations are required to determine the basis for the enhancement of recovery by electrical stimulation as the traditionally known neurotransmitters and receptors did not appear to be critical for recovery.

The electrical stimuli applied to the intestinal segments require further examination. Considering the anatomical features of the intestinal segments, it is unclear whether the propagating contractions were occurring due to stimulated neuron activity or to muscle activity.

73

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Conclusions

In conclusion, these data suggest that electrical stimulation improved the recovery of peristalsis in the presence of nicotinic receptor blockade. The first hypothesis was that recovery might be caused by other ligand-gated receptors involved in fast synaptic transmission such as 5-HT3 and P2X, but recovery was unaffected in the presence of these blockers. The second hypothesis was that G-protein coupled slow synaptic transmission via muscarinic receptors was responsible, but similarly there was no reduction in recovery. The final hypothesis was that AH/sensory neuron networks might become more excitable after electrical stimulation and thus enhance recovery.

Although no direct evidence for this was found, Tram-34 did enhance recovery of peristalsis in a way that was similar to electrical stimulation. Further investigations are necessary in order to determine the basis of recovery and provide direct evidence for or against a role of the AH/sensory neuron network.

74

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

References

Barbara G, Stanghellini V, De Giorgio R & Corinaldesi R. (2006). Functional

gastrointestinal disorders and mast cells: implications for therapy.

Neurogastroenterol Motil 18, 6-17.

Barbezat GO & Grossman MI. (1971). Intestinal secretion: stimulation by peptides.

Science 174, 422-424.

Bartho L, Holzer P, Donnerer J & Lembeck F. (1982). Effects of substance P,

cholecystokinin octapeptide, bombesin, and neurotensin on the peristaltic reflex

of the guinea-pig ileum in the absence and in the presence of atropine. Naunyn-

Schmiedeberg's archives of pharmacology 321, 321-328.

Bartho L, Holzer P & Lembeck F. (1987). Is Ganglionic Transmission through

Nicotinic Receptors Essential for the Peristaltic Reflex in the Guinea-Pig Ileum.

Neuropharmacology 26, 1663-1666.

Bayliss WM & Starling EH. (1899). The movements and innervation of the small

intestine. The Journal of physiology 24, 99-143.

Bell RRaDR. (2013). Small Intestinal Motility. In Medical Physiology: Principles of

Clinical Medicine 4th edition. Wolters Kluwer Philadelphia

Bennett A & Whitney B. (1966). A pharmacological study of the motility of the human

gastrointestinal tract. Gut 7, 307-316.

Bertrand PP, Kunze WA, Furness JB & Bornstein JC. (2000). The terminals of

myenteric intrinsic primary afferent neurons of the guinea-pig ileum are excited

by 5-hydroxytryptamine acting at 5-hydroxytryptamine-3 receptors.

Neuroscience 101, 459-469. 75

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Bornstein JC. (2008). Purinergic mechanisms in the control of gastrointestinal motility.

Purinergic signalling 4, 197-212.

Bornstein JC, Costa M & Grider JR. (2004). Enteric motor and interneuronal circuits

controlling motility. Neurogastroenterology and motility : the official journal of

the European Gastrointestinal Motility Society 16 Suppl 1, 34-38.

Brandtzaeg P. (1998). Development and basic mechanisms of human gut immunity.

Nutr Rev 56, S5-18.

Brehmer A & Stach W. (1998). Regional structural differences in the neuronal

composition of myenteric ganglia along the pig small intestine. The Anatomical

record 250, 109-116.

Brookes SJ, Song ZM, Steele PA & Costa M. (1992). Identification of motor neurons to

the longitudinal muscle of the guinea pig ileum. Gastroenterology 103, 961-973.

Bush TG, Spencer NJ, Watters N, Sanders KM & Smith TK. (2001). Effects of

alosetron on spontaneous migrating motor complexes in murine small and large

bowel in vitro. American journal of physiology Gastrointestinal and liver

physiology 281, G974-983.

Carter DS, Alam M, Cai H, Dillon MP, Ford AP, Gever JR, Jahangir A, Lin C, Moore

AG, Wagner PJ & Zhai Y. (2009). Identification and SAR of novel

diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X(3)/P2X(2/3)

antagonist for the treatment of pain. Bioorganic & medicinal chemistry letters 19,

1628-1631.

76

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Clerc N, Furness JB, Bornstein JC & Kunze WA. (1998). Correlation of

electrophysiological and morphological characteristics of myenteric neurons of

the duodenum in the guinea-pig. Neuroscience 82, 899-914.

Clerc N, Furness JB, Kunze WA, Thomas EA & Bertrand PP. (1999). Long-term effects

of synaptic activation at low frequency on excitability of myenteric AH neurons.

Neuroscience 90, 279-289.

Costa M, Brookes SJ & Hennig GW. (2000). Anatomy and physiology of the enteric

nervous system. Gut 47 Suppl 4, iv15-19; discussion iv26.

Costa M, Brookes SJ, Steele PA, Gibbins I, Burcher E & Kandiah CJ. (1996).

Neurochemical classification of myenteric neurons in the guinea-pig ileum.

Neuroscience 75, 949-967.

Dogiel A. (1899). Über den Bau der Ganglien in den Geflechten des Darmes und der

Gallenblase des Menschen und der Säugetiere. Arch Anat Physiol (Leipzig) 130.

Dooley CP, Dilorenzo C & Valenzuela JE. (1992). Variability of Migrating Motor

Complex in Humans. Digest Dis Sci 37, 723-728.

Drossman D, Morris CB, Hu Y, Toner BB, Diamant N, Whitehead WE, Dalton CB,

Leserman J, Patrick DL & Bangdiwala SI. (2007). Characterization of health

related quality of life (HRQOL) for patients with functional bowel disorder

(FBD) and its response to treatment. The American journal of gastroenterology

102, 1442-1453.

77

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Ferens D, Baell J, Lessene G, Smith JE & Furness JB. (2007). Effects of modulators of

Ca(2+)-activated, intermediate-conductance potassium channels on motility of

the rat small intestine, in vivo. Neurogastroenterology and motility : the official

journal of the European Gastrointestinal Motility Society 19, 383-389.

Fiocchi C. (1998). Inflammatory bowel disease: etiology and pathogenesis.

Gastroenterology 115, 182-205.

Furness JB. (2000). Types of neurons in the enteric nervous system. Journal of the

autonomic nervous system 81, 87-96.

Furness JB. (2006). The organisation of the autonomic nervous system: peripheral

connections. Auton Neurosci 130, 1-5.

Furness JB. (2012). The enteric nervous system and neurogastroenterology. Nature

reviews Gastroenterology & hepatology 9, 286-294.

Furness JB, Bornstein JC & Trussell DC. (1988). Shapes of nerve cells in the myenteric

plexus of the guinea-pig small intestine revealed by the intracellular injection of

dye. Cell and tissue research 254, 561-571.

Furness JB, Costa, M. (1987). The Enteric Nervous System. Churchill Livingstone, New

York.

Furness JB, Kumano K, Larsson H, Murr E, Kunze WA & Vogalis F. (2002).

Sensitization of enteric reflexes in the rat colon in vitro. Autonomic

neuroscience : basic & clinical 97, 19-25.

Furness JB, Kunze WA, Bertrand PP, Clerc N & Bornstein JC. (1998). Intrinsic primary

afferent neurons of the intestine. Progress in neurobiology 54, 1-18.

78

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Galligan JJ. (2002). Ligand-gated ion channels in the enteric nervous system.

Neurogastroenterology and motility : the official journal of the European

Gastrointestinal Motility Society 14, 611-623.

Groneberg D, Konig P, Koesling D & Friebe A. (2011). Nitric oxide-sensitive guanylyl

cyclase is dispensable for nitrergic signaling and gut motility in mouse intestinal

smooth muscle. Gastroenterology 140, 1608-1617.

Gutzwiller JP, Degen L, Heuss L & Beglinger C. (2004). Glucagon-like peptide 1

(GLP-1) and eating. Physiol Behav 82, 17-19.

Hansen MB. (2003). Neurohumoral control of gastrointestinal motility. Physiological

research / Academia Scientiarum Bohemoslovaca 52, 1-30.

Hirst GD, Holman ME & Spence I. (1974). Two types of neurones in the myenteric

plexus of duodenum in the guinea-pig. The Journal of physiology 236, 303-326.

Holzer P, Lippe IT, Bartho L & Saria A. (1987). Neuropeptide-Y Inhibits Excitatory

Enteric Neurons Supplying the Circular Muscle of the Guinea-Pig Small-

Intestine. Gastroenterology 92, 1944-1950.

79

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Holzer P, Schluet W & Maggi CA. (1993). Ascending enteric reflex contraction: roles

of acetylcholine and tachykinins in relation to distension and propagation of

excitation. The Journal of pharmacology and experimental therapeutics 264,

391-396.

Horina JH, Holzer H, Reisinger EC, Krejs GJ & Neugebauer JS. (1992). Elderly

patients and chronic haemodialysis. Lancet 339, 183.

J.C. Bornstein JBF, W.A.A. Kunze and P.P. Bertrand. (2002). Enteric Reflexes that

Influence Motility.

Jang HJ, Kokrashvili Z, Theodorakis MJ, Carlson OD, Kim BJ, Zhou J, Kim HH, Xu X,

Chan SL, Juhaszova M, Bernier M, Mosinger B, Margolskee RF & Egan JM.

(2007). Gut-expressed gustducin and taste receptors regulate secretion of

glucagon-like peptide-1. Proc Natl Acad Sci U S A 104, 15069-15074.

Juarez EH, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano LM &

Barajas-Lopez C. (2013). Selectivity of antagonists for the Cys-loop native

receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons.

Autonomic & autacoid pharmacology.

Kunze WA, Furness JB, Bertrand PP & Bornstein JC. (1998). Intracellular recording

from myenteric neurons of the guinea-pig ileum that respond to stretch. The

Journal of physiology 506 ( Pt 3), 827-842.

Langley JN. (1921). The Autonomic Nervous System.

80

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Lennard-Jones JE. (1989). Classification of inflammatory bowel disease. Scand J

Gastroenterol Suppl 170, 2-6; discussion 16-19.

Messenger JP, Bornstein JC & Furness JB. (1994). Electrophysiological and

morphological classification of myenteric neurons in the proximal colon of the

guinea-pig. Neuroscience 60, 227-244.

Monro RL, Bertrand PP & Bornstein JC. (2002). ATP and 5-HT are the principal

neurotransmitters in the descending excitatory reflex pathway of the guinea-pig

ileum. Neurogastroent Motil 14, 255-264.

Monro RL, Bertrand PP & Bornstein JC. (2004). ATP participates in three excitatory

postsynaptic potentials in the submucous plexus of the guinea pig ileum. J

Physiol-London 556, 571-584.

Nguyen TV, Matsuyama H, Baell J, Hunne B, Fowler CJ, Smith JE, Nurgali K &

Furness JB. (2007). Effects of compounds that influence IK (KCNN4) channels

on afterhyperpolarizing potentials, and determination of IK channel sequence, in

guinea pig enteric neurons. Journal of neurophysiology 97, 2024-2031.

Nicholas S & Spencer NJ. (2010). Peristalsis and fecal pellet propulsion do not require

nicotinic, purinergic, 5-HT3, or NK3 receptors in isolated guinea pig distal colon.

American journal of physiology Gastrointestinal and liver physiology 298,

G952-961.

Olsson C & Holmgren S. (2001). The control of gut motility. Comparative biochemistry

and physiology Part A, Molecular & integrative physiology 128, 481-503.

81

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Puddu A, Storace D, Durante A, Odetti P & Viviani GL. (2010). Glucagon-like peptide-

1 counteracts the detrimental effects of Advanced Glycation End-Products in the

pancreatic beta cell line HIT-T 15. Biochem Biophys Res Commun 398, 462-466.

Ren J & Bertrand PP. (2008). Purinergic receptors and synaptic transmission in enteric

neurons. Purinergic Signal 4, 255-266.

Sanger GJ. (1996). 5-Hydroxytryptamine and functional bowel disorders.

Neurogastroenterology and motility : the official journal of the European

Gastrointestinal Motility Society 8, 319-331.

Schwartz GJ & Moran TH. (1998). Duodenal nutrient exposure elicits nutrient-specific

gut motility and vagal afferent signals in rat. The American journal of

physiology 274, R1236-1242.

Spencer N, Walsh M & Smith TK. (1999). Does the guinea-pig ileum obey the 'law of

the intestine'? J Physiol-London 517, 889-898.

Steinert RE, Meyer-Gerspach AC & Beglinger C. (2012). The role of the stomach in the

control of appetite and the secretion of satiation peptides. American journal of

physiology Endocrinology and metabolism 302, E666-673.

Stevenson C. (1999). Australia: YLD summary. Australian Institute of Health and

Welfare.

Tache Y & Perdue MH. (2004). Role of peripheral CRF signalling pathways in stress-

related alterations of gut motility and mucosal function. Neurogastroenterology

and motility : the official journal of the European Gastrointestinal Motility

Society 16 Suppl 1, 137-142.

82

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Thomas PA, Akwari OE & Kelly KA. (1979). Hormonal control of gastrointestinal

motility. World journal of surgery 3, 545-552.

Thompson WG, Longstreth GF, Drossman DA, Heaton KW, Irvine EJ & Muller-

Lissner SA. (1999). Functional bowel disorders and functional abdominal pain.

Gut 45 Suppl 2, II43-47.

Tonini M, Frigo G, Lecchini S, D'Angelo L & Crema A. (1981). Hyoscine-resistant

peristalsis in guinea-pig ileum. Eur J Pharmacol 71, 375-381.

Uematsu S & Akira S. (2008). Toll-Like receptors (TLRs) and their ligands. Handb Exp

Pharmacol, 1-20. van Wijngaarden I, Hamminga D, van Hes R, Standaar PJ, Tipker J, Tulp MT, Mol F,

Olivier B & de Jonge A. (1993). Development of high-affinity 5-HT3 receptor

antagonists. Structure-affinity relationships of novel 1,7-annelated indole

derivatives. Journal of medicinal chemistry 36, 3693-3699.

Vantrappen G, Janssens J, Peeters TL, Bloom SR, Christofides ND & Hellemans J.

(1979). Motilin and the Interdigestive Migrating Motor Complex in Man. Digest

Dis Sci 24, 497-500.

Wattchow DA, Brookes SJ & Costa M. (1995). The morphology and projections of

retrogradely labeled myenteric neurons in the human intestine. Gastroenterology

109, 866-875.

Wisero. (2010). NHMRC funded research into bowel disease and disorders 2000-2010.

Australian Government Department of Health and Ageing

83

Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways

Yamamoto M, Rennert P, McGhee JR, Kweon MN, Yamamoto S, Dohi T, Otake S,

Bluethmann H, Fujihashi K & Kiyono H. (2000). Alternate mucosal immune

system: organized Peyer's patches are not required for IgA responses in the

gastrointestinal tract. J Immunol 164, 5184-5191.

Zhou X & Galligan JJ. (1999). Synaptic activation and properties of 5-

hydroxytryptamine(3) receptors in myenteric neurons of guinea pig intestine.

The Journal of pharmacology and experimental therapeutics 290, 803-810.

84