Autism spectrum disorder (ASD)

Please, read my thesis paper to do the following
1- Perform proofreading for the result chapter
2- Write the discussion: (The guidelines are written under discussion chapter)
3- Write the conclusion: (The guidelines are written under the discussion) chapter)
4- Write the abstract:
Note: be careful when you follow the guidelines
Please make my thesis logic and easy to follow (same order) and make
the writing style simple

1. Literature Review
1.1: General Background:
Autism spectrum disorder (ASD) is a chronic neurodevelopmental condition characterised by impaired social interactions, decreased communication, repetitive behaviour and/or restricted interests (1). Prior to the last few decades, autism was thought to be fairly rare medical condition with prevalence rate of about 1 per 2000 children. However, ASD is currently a major health issue in the world and it affects about one in 68 children. The condition is at least 4 times more prevalent among boys as compared to girls with data showing that while one in 42 boys has ASD, only one in every 189 girls has the disorder (2, 3).This dramatic increase in the occurrence rate of autism is more likely due to expected improvement of ASD diagnosis, high level public health awareness and expansion of ASD diagnostic classifications to range from mild to very severe condition.

1.1.1 Comorbidity Associated With ASD.
Autistic patients commonly have several comorbid traits such as anxiety, epilepsy, seizures, heightened aggression, motor deficits, sensory processing abnormalities and sleep disturbances. However, up to 90% of autistic children have gastrointestinal dysfunction for which the underlying cause is unknown. This includes chronic diarrhea, constipation and abdominal pain (4, 5).
1.2 The key function of gastrointestinal:
The primary function of gastrointestinal is to break-down food into its nutrients, so that they can be absorbed into the body (6, 7). This is done through several processes. The first one is the initiation of ingestion and propulsion of food from one organ to the other before break down. It allows the squeezing of food along the gastrointestinal tract using the process of peristalsis and segmentation motility. This process involves mechanical digestion so as to prepare the food for additional degradation using enzymes. The second process is to break-down the food into its nutrients inside the stomach so that it can be absorbed. It allows proteins, fats and carbohydrates to be chemically reduced into their rudimentary building blocks such that they can be easily absorbed all through the epithelium of the small-intestines and thus enter the circulation. The third process is absorption whereby the broken down compounds are moved from the lumen to the blood and lymph. Afterwards, the digested food goes into the mucosal-cells found in the small intestines, and which acts as the key absorption location. The last process is defecation which is the removal of indigestible residues through the anus and is usually undertaken by the large intestines (7).
1.3 Function and Structure of Enteric Nervous System (ENS):
The enteric nervous system is one of the key segments of the autonomic nervous system (8). It is involved in the physiological and pathophysiological processes of gastrointestinal tract , and considered to be the second brain of autism (9). The system regulates several processes ranging from motility, to secretion, blood flow to immune responses. ENS integrates all these functions into organized behavioral arrangement through its neural reflexes. The structure of ENS comprises the ganglia, principal inter-ganglionic fiber tract as well as the ancillary and tertiary fibers protruding into the effector systems. The ganglionated plexuses consist of myenteric and submucosal plexuses. The myenteric plexus is located between longitudinal muscle and circular muscle and is responsible for gastrointestinal motility regulation. While, submucosal plexus is situated between circular muscle and submucosa, and plays a major role in absorption control.(10)

Figure1: This diagram shows a model of ENS located in the small intestines illustrating the key components that comprises the double ganglionated plexuses, myenteric plexus along with the submucosal plexus. Each neuron has a role that is determined by the type of chemical coding it is involved in: Source:(8)

1.4 Enteric neurons and neurotransmission:
ENS operates with functional classes of neurons namely the sensory neurons, the inter-neurons, the muscle motor neurons as well as secreto-motor neurons (11). ENS Neurons work together in regulating different functions ranging from gut sensations to intestinal motility to signal-transmissions to secretion of fluids. These neurons use different categories of neurotransmitters in the ENS and they include Acetylcholine (Ach) and tachykinins (TK) which are involved in the excitatory motor neurons (12). Others like NO, vasoactive intestinal-peptide (VIP) and adenosine triphosphate (ATP) are involved in the inhibitory motor-neurons.

1.5 Structure and synthesis of Nitric Oxide:
Nitric oxide is a biological messenger characterized by the double covalently bonded atoms (Nitrogen-oxygen). NO is released by different cells within the mammalian systems to perform different physiological functions (13). For instance, it acts as a neurotransmitter when it is released by central-and-peripheral nervous system neurons, and it is involved in the regulation of blood-pressure and inhibition of blood coagulation when it is released from the endothelium. Synthesis of NO is de novo and is produced when Nitric-oxide synthase enzyme transforms the co-substrate L-arginine along with O2 into NO and L-citrulline (29,30). The Cofactors involved are nicotinamide adenine dinucleotide phosphate (NADN), flavin mononucleotide (FMN) along with flavin adenine dinucleotide (FAD).(13, 14)

Figure2: This diagram shows the structure and synthesis of NO. (A) Nitric oxide has chemical structure that is characterized by the double covalently bonded atoms (Nitrogen-oxygen). (B) Synthesis of NO is de novo and is produced when Nitric-oxide synthase enzyme transforms the co-substrate L-arginine along with O2 into NO and L-citrulline. The Cofactors involved are nicotinamide adenine dinucleotide phosphate, flavin mononucleotide (FMN) along with flavin adenine dinucleotide (FAD). source: (15)
1.6 Regulation of Nitric Oxide synaptic signaling:
The synaptic signaling of NO is mediated by receptors refered to as post-synaptic N-methyl-D-aspartate (NMDA) receptors and the pre-synaptic vesicles regulation. It is also mediated by S-nitrosylation proteins and the release of cyclic guanosine monophosphate (cGMP) (14, 16).When it is released as a neuro-transmitter from the presynaptic terminal, it binds to the postsynaptic N-Methyl-D-Aspartate Receptors (NMDARs) and thus initiate an influx of Ca2+ towards the postsynaptic cell. As a result, massive amounts of Ca2+ and calmodulin complexes are activated which then activates the de-novo synthesis of nitric oxide. Due to its high solubility, nitric oxide diffuses through the pre-synaptic membranes so as to trigger the synthesis of (cGMP) that goes on to activate cGMP reliant protein kinase which results in the initiation of NO-PKG-PIP2 pathway. This pathway is responsible for both exocytosis and endocytosis of the synaptic vesicles, which is very critical in sustaining the continuous conduction of synaptic. (14)

1.7 Nitric oxide regulation of smooth muscle relaxation:
Nitric oxide is an inhibitory neurotransmitter that is used by inhibitory motor neurons to hyper-polarize the globular smooth muscle cells. In particular, it is responsible for rhythmic easing of circular smooth-muscle cells found in between the CMMCs (14). Once nitric oxide diffuses from the inhibitory motor neuron to the contractile smooth-muscle cells, it initiates the synthesis of cGMP. The cGMP is then responsible for activating the influx of cGMP-dependent protein kinase that goes on to boost the concentration of intracellular Ca2+ which in return boosts the stimulation of intracellular Ca2+ calcium-sensitive potassium networks that then hyper-polarize the cells (17-19). This process affects the interaction of actin-myosin and thereby helps in soothing the smooth muscle-cell. A decrease in Ca2+ helps to incapacitate calcium sensitive K+ currents and thus results in the depolarization of the smooth muscle cells. NO is also a major ENS non-adrenergic and non-cholinergic neurotransmitter (NANC). Through the neuronal reflexes, it enables the mediation of smooth muscle relaxation in not just the blood-vessels but also in the GI tract, urogenital tract as well as the respiratory tracts. (20)

1.6 Gastrointestinal motor circuit:
An examination of animal models like rats reveals that the gastrointestinal motility reveals patterns which are created by motor circuits instigated by intrinsic enteric sensory neurons (21). For instance in guinea pigs, their small intestines have neurons which are triggered not only by stimuli but also through mucosal deformation and presence of luminal nutrients inside the gut (22). They then transmit the information to the neurons located within the myenteric plexus through ascending-descending reflex pathways resulting in the instigation of motor neurons. However, the motor neurons can be stimulated directly through intrinsic-sensory neurons or indirectly through interneurons, and this results in either the contraction or relaxation of gastrointestinal muscle layers and hence generating the motility patterns. Furthermore, other studies shows that acetylcholine released by cholinergic neurons attaches to the muscarinic receptors within the gastrointestinal muscles and this depolarizes the smooth muscle cells leading to an excitatory-junctional potential, that if it happens to be massive, it often forces the contraction of muscles but this is via an action potential (23). Nonetheless, inhibitory motor-neurons tend to release numerous neurotransmitters like ATP and NO so as to hyperpolarize the smooth muscle cells and thus leading to relaxation.

Figure 3: This diagram shows the motility reflex path way initiated by enteric neurons. The ISN connect synaptically with several types of ascending and descending neurons that interact with excitatory and inhibitory motor neurons in the circular mascle and longitudinal mascle. Source:(15)

1.6.1 Small intestine motility:
Small Intestinal Motility is the coordinated contraction of intestines smooth muscles participate so as to enable both digestion and absorption. It involves mixing of food with digestive enzymes coming from the pancreas and bile-salts originating from the biliary system. After having a meal, the lumen which has chime undergoes two categories of motility that is segmentation contractions and peristalsis. During the interdigestive period or between meals, the lumen undergoes contractions transmitted from the stomach and thus clear out every debris. This motility pattern is the migrating motor complex. Motility is controlled by both excitatory and inhibitory signals coming from ENS. However, the localized nervous signals are controlled by inputs from CNS and some gastrointestinal hormones(24) .
Segmentation motility patterns are also simulated by fatty acids located within the small intestines (25). Several studies have examined the patterns inside the small intestines of guinea-pigs and have revealed that three different contractile events occur. They are stationary contractions, oral propagation and anal propagations and all the three of them are short length contractions but interspersed with different phases of quiescenc (25-27). When there is materialization of sodium-channel blockers reffered to as tetrodotoxin, the motility patterns linked to segmentation are obliterated and this reveals a neural mediation. Nevertheless, the peristalsis that occur in gastric antrum is an outcome of conducted electrical actions refered to as slow waves, and whose foundation is myogenic. (25, 28)

Migrating motor complex is an electromechaminal motility pattern with four different stages of anally proliferating contractions(25, 28). It comprises three major phases that lasts 85 to 115 minutes. They are quiescent phase, intermittent phase and the irregular contractions. Phase I lasts 40 to 60% of the whole cycle length. Phase II takes 20 to 30% and characterized by strong though asymmetrical contractions(28). Phase III takes between 5 to 10 minutes but comprises strong contractions which proliferate caudally (46). Phase III is also characterized by its rhythmic contractions that takes place at maximal frequency. A fourth phase is whereby the activity degenerates into quiescent levels of phase I (25). Most remarkably, impulsive motor complexes have been observed in secluded jejunum, ileum and colon of mouse models (29). It is characterized by a periodic nature that is controlled from CNS and implemented partially by ENS motilin. Commonly refered to as housekeeping because it can be overridden by other more significant processes like ingestion (24).

1.6.2 Colonic Migrating Motor Complex (CMMC) in ENS:
Colonic migrating motor complex is the most important neural facilitated rhythmic ENS propulsive contraction that occurs in the intestines. A neuron known as myenteric 5-hydroxytryptamine (5-HT) is involved in the tonic reservation of the colon, and it is what produces the CMMC including the inflection of the whole ENS like coupling motility during secretion and blood-flow. It has also been observed that mucosal 5-HT is what triggers an effective proliferation of CMMC(30). Recent studies also show that the slow proliferation of CMMC down the colon result from the interaction between the rising excitatory and the downward serotonergic-inhibitory-neural pathways specifically when they interact inside the myenteric plexus and just at the muscle level. This is what makes CMMC propagation to resemble the esophageal peristalsis. It is believed that in ASD patients there is a dysfunction in neuronal generation of nitric oxide which is needed in tonic inhibition between CMMC complexes (31).

1.7 Gastrointestinal Dysfunction Associated With ASD:
Although 90% of autistic patients have chronic gastrointestinal dysfunctions, the underline biological mechanisms are not yet clear (32). Some researchers argue that this could be as a result of numerous dysfunctions in the equilibrium between excitatory transmissions and the inhibitory transmissions found within the enteric nervous system The bowel and beyond: the enteric nervous system in neurological disorders (33). This argument is based on recent findings which show that there is increased amount of nitric oxide expressing neurons in two genes known as NL3 R451C and SHANK3 which are found within the enteric nervous system(33, 34). Gastrointestinal dysfunction is not properly understood because it presents variances in factors like different eating habits. Furthermore, it is hard for researchers to generalize the data because they find themselves collecting differing views from parents because parents biased perceptions and cultural beliefs affect their reporting of their children accurate conditions, especially those with ASD kids who cannot express their symptoms due to communication challenges(35). Adams et al study revealed that ASD Children have reduced levels of digestive markers like short-chain fatty acids, acetate, proprionate as well as valerate. They also have severe cases of constipation, abdominal pains and diarrhea due to reduced levels of Bifidobacter and Lysozym but elevated levels of lactobacillus(36).

More recent studies reveal that the gastrointestinal dysfunction could be driven by interferences in GI-related biomarkers and a grouping of genetic exposures which interact with several environmental trigger (37, 38). Campbell et al conducted the first study which showed that gastrointestinal dysfunction in ASD is characterized by dysfunctions in intestinal penetrability and motility such that there is reduction in mucosal immunity, gut microbiome, and disruptions in signaling pathways(39). .

1.8 Genetic Mutations Implicated with Synaptic Function of Autism:
Several factors range from environmental (non-genetic) and genetic have been implicated with ASD. Non-genetic factors include prenatal exposure to thalidomide, maternal diabetes and viral infections like cytomegalovirus. while, genetically factors involve genetic mutation associated with mRNA translation, chromatin transformation, and mutations in the synaptic function belonging to both neuronal and synaptic homeostasis(3, 40, 41).
The genetic mutation associated with synaptic dysfunction negatively affects neuronal communication by altering the functions of proteins located at synapses have in ASD patients(41, 42). Although over 500 gene mutations have been linked to different types of ASD, recent studies show that mutations at synaptic adhesion molecules contributes to ASD incidences(43, 44). One of these studies is that of Sala et al, whereby Shank or ProSAP master-scaffold proteins which are considered critical to synaptic creation, growth, and function are implicated in SHANK 1,2,3 family mutations that causes ASD (45). The study also revealed that Shank3 mutations caused modifications in striatum synapses which resulted in a reduction in cortico-striatal synapses mEPSC frequencies (45). Tong et al study observed that post synaptic mutations at three genes, namely Neurexin, Neuroligin, and CASK results in immobilization of GABAA receptors(46). That immobilization leads to the receptor coming into contact with FRM-3/EPB4.1 and LIN-2A/CASK which leads to alteration of inhibitory transmission that causes ASD cognitive deficits. Another study is that of Giovedí et al which identified Syn a new candidate gene which when there is a deletion of one protein known as Syn there is a manifestation of ASD symptoms(43). Syns are pre-synaptic proteins which control the traffic inside synaptic vesicles, the discharge of neurotransmitter as well as immediate synaptic plasticity. Other ASD mutations involved in synaptic functions are Neuroligin gene mutation which includes NLGN3 and NLGN4X.

1.9.1 Neuroligin protein and NL3 R451C mutation:
Neuroligin proteins are cell-adhesion proteins located at the postsynaptic cell. They have four family members include neuroligin-1 (NL1), neuroligin-2 (NL2), neuroligin-3 (NL3) and neuroligin-4 (NL4). (NL1), (NL2) and (NL3) present at excitatory, inhibitory and excitatory and inhibitory synapses of CNS respectively. While, (NL4) is expressed at glycinergic synapses in the retina (47). Neuroligin proteins are crucial in synapse maturation and preservation(48). Its extracellular domain reveals a 32% to 36% sequence identity which makes it to shape with the globular-domain-of-acetylcholinesterase (AChE), hence it is known to be one of the α/β hydrolase fold-protein superfamily. Neuroligins are involved in the development of synaptic concentrations which are crucial for the transferring of synaptic proteins to freshly developed synapses (48, 49). They work in associations with presynaptic neurexins so as to perfom synaptogenesis. It interacts with several postsynaptic proteins so as to confine neurotransmitter receptors and networks within the post-synaptic density. In particular, it interacts with both PSD-95 and β-neurexins though trans-synaptically with the latter. Expression of neuroligin-1 though bidirectionally is responsible for regulating the generation of steady contacts and this modulates synapse density(49).

NL3 R451C mutation is a point mutation in which arginine is replaced by cystine at 451 positions of neuroligin proteins. This mutation was first discovered in two Swedish brothers with sever gastrointestinal dysfunction. When the mutation occurs in transfected neurons, it results in partial retaining of neuroligin-3 inside the endoplasmic reticulum (48). In addition, it decreases the neuroligin expression by 90 % which lead to lower level of neuroligin protein present at synapses. Furthermore, it impairs the trafficking of neuroligin from endoplasmic reticulum to the cell surface (50).

Figure 1: This diagram shows the interaction of cell adhesion molecules and scaffolding proteins at synapses. Cell-adhesion molecules such as neuroligin are located at postsynaptic cell and function through their interactions with presynaptic neuraxins to intermediate the synaptic signalling. While, scaffolding proteins such as SHANK proteins are located at postsynaptic cell and function thought their interactions with binding partners (e.g. Glutamate receptor) to facilitates the synaptic neurotransmission and maintain the morphology and function of synapse. Source:(51)
1.9.2 SHANK3 proteins and SHANK3 mutation:

Shank 3 protein is the key scaffold located at the density of post-synaptic cells (52). It uses several promoters and differential splicing to create substitute mRNA isoforms together with protein products (53). Along the synapse, it links glutamate receptors to actin-cyto-skeleton but through a sequence of intermediary features (53, 54). It also has promoters and intragenic CpG islets which display tissue-specific patterns of the DNA methylation. Hence, one of its function is to interact with other proteins and complexes so as to coordinate the generation, maturation and upkeep of synapse and dendritic spine. It also interconnects receptors found within the post-synaptic membrane like NMDA-type and metabotropic glutamate but through complexes like GKAP/PSD-95 and the HOMER (54). It is also involved in the physical and functional arrangement of both dendritic-spine and synaptic-junction but this is through its interaction with Arp2/3 and the WAVE1 complexes. Hence, it is involved in the regulation of emerging neurons-growth cone motility as well as NMDA receptor-signaling

The type of mutation in SHANK3 that causes Autism is de novo mutation and deletions. The de novo mutation occurs at the tip of F-actin fibres which disrupts the creation of spine and morphology. Other de novo mutation occurs at 22q13.33 and 20q13.33 which causes a frame shift and loss of language function. It inhibits the localization of binding partners (49). Deletion is heterozygous and occurs at 15 nt inside exon 21 whereby there is removal of 5-aa from a protein and affects greatly the loss of communication and deficiency in social interaction(55). Some ASD patients also experience SHANK3 mutations at the synapses namely, de novo, interstitial and terminal deletion; however, studies on the de novo mutation reveals that altered Shank3 proteins are incapable of being localized at synapses or even control the synapse morphology and functionality (45).

1.10 NL3 R451C mouse model:
Researchers use mouse models to study autistic behaviors because mice express almost similar homologs to human beings and are effective when trying to objectively quantify all of the behavioral assays. Most of the research has emphasized on monogenic aberrations especially the loss of functions due to gene mutations.
In a study done by Tabuchi et al in NL3 R451C-mutant mice, the mutation causes diminished social interactions but improved spatial learning capabilities but with a surge in inhibitory synaptic broadcast. However, removal of neuroligin-3 did not result in any change; hence R451C-substitution is a gain-of-role mutation(50). Burrows et al showed that when the mice having that mutation are treated using risperidone their aggression level decreases to wild-type levels.
A preliminary data from current laboratory have shown that mice with NL3 R451C mutation have an increase in the numbers of NO synthase neuron, increased small intestinal length and have more broken villi in compared to WT mice. In the same study, NL3R451C and WT mice were assessed for changes in jejunal activity using video imaging of organ bath motility however small intestinal motility was notoriously difficult to analyse due to its irregular characteristic of jejunal motility. Therefore, setting up a new luminal pressure protocol to increase the consistency of Propagating Contracting Complexes (PCCs) is significantly needed as it will enable the analysis of jejunal motility.

Figure 4: Increased number of nNOS-IR cell bodies and overall proportion of nNOS neurons in NL3R$51C mice (A) & (B) Confocal images showing nNOS (green) in adult mouse proximal jejunum. In NL3R451C mice, at the level of the myenteric plexus appeared to contain a higher number of nNOS-IR cell bodies (B) compared to the WT (A).

Figure 5: NL3R451C mice had more damaged villi. Villi marked † were counted as broken and * identifies full villi. NL3R451C mice coronal sections displays a greater number of broken villi (†) versus full villi (*), compared to the WT. Scale bars = 100 μm
1.11 Shank3 mouse model of autism
Using SHANK3 KO mouse model, Wang et al manipulated the mice mGluR5 activity which resulted in extreme grooming and learning but lessened striatal synaptic plasticity. The results reveal that lack of Shank3 impair Homer scaffolding leading to aberrations in cortico-striatal circuit(56). Speed et al created a new model by inserting only one guanine nucleotide into the mice exon 21 Shank3. This caused a frame shift that led to a premature HALT codon and removal of key molecular weight. The results revealed that the mice with Shank3G lacked hippocampus dependent spatial learning abilities because of diminished motor coordination as well as deficits in sensory processing. (57)
1.12: Thesis hypothesis:
In this research, I hypothesize that the Neuroligin-3 R451C and Shank3 KO mutations alter gastrointestinal structure and function in mice.

1.13: Thesis objectives:
This study aims to examine for differences in jejunal histological structure of NL3R451C and SHANK3 KO autism mouse models with different genetic background to the previous studies models, in comparison to wild type mice. It also aims to establish an increased luminal pressure protocol using the organ bath and video imaging setup to reduce the variability of motility patterns in the jejunum of small intestine. The last goal of this study is to apply N-nitro-L-arginine (NOLA; an inhibitor of NO synthesis) onto ex vivo NL3R451C mouse jejunal segments to investigate effects of NO on jejunal motility in comparison to wild type mice.
Studying histological changes in autism mouse models, developing a new method to study jejunal motility of small intestine and investigating the effect of NO on jejunal neuronal activity in NL3R451C mice will provide insight into biological mechanisms underlying gastrointestinal dysfunction in autism

Chapter2: Method and Materials:
2.1: Increased luminal pressure video imaging experiment:
In this experiment, WT and NL3R451C mice were used to obtain the optimal luminal pressure that induce the presence of consistent PCCs cluster in the spatiotemporal maps. This was performed to enable analysis of jejunal motility.

2.1.1: Experimental animal:
These mice were initially bred on a mixed background (Sv129/ImJ/ C57BL/6) when obtained from the Jackson Laboratory (Bar Harbour, Main USA) to University of Melbourne at the Biological Sciences Animal Facility (BSAF). Subsequently mice were bred with C57BL/6 mice for more than 10 generations and maintained on a C57BL/6 genetic background. Then, pups were bred by mating WT female and hemizygous NL3R451C to produce male offspring hemizygous for NL3R451C mutation or WT mice. In this experiment, only male NL3 R451C and WT mice (aged 11-14 weeks) were used. This is because preliminary data shown that there was no significant difference in the motility of homozygous or heterozygous of female NL3R451C and WT mice.
Table1: This table shows general data of the experimental mice (5 WT& 9 NL3R451C) used in increased luminal pressure video imaging experiment.

2.1.2: KREB’s stock solution preparation:
2.1.2.1: Materials and equipment:

Table2: Chemicals and relevant quantities (g) used for KREB’s x10 stock solution preparation (2L).

2.1.2.2: Preparation procedure:
Fill about 2/3 of 2L conical flask with distal water. Then, use weighing boat and weighing device to separately weigh needed quantity of relevant chemical. after addition of each chemical, make well mixing using magnetic tablet and magnetic mixing device to ensure chemicals completely dissolved. After that, add distal water up to 2L mark and make last mixing for the solution(2L). Finally, store the prepared KREB’s solution at cold room for optimum stability.

2.1.3: KREB’s solution preparation (2L):
2.1.3.1: Material:
Table3: Chemicals and relevant quantities used for KREB’s solution preparation(2L):

2.1.3.2: preparation procedure:
Fill about 2/3 of 2L conical flask with distal water and then add 200 mL of KREB’s x10 solution. After that, weight 4 g of dehydrates glucose (D-glucose) and 4.2g of sodium carbonate (NaHCO3) using weighing device and weighing boat. After addition of each consumable, make a proper mixing until chemicals completely dissolved. Next, add distal water up to 2 L mark and mix it again. Finally, keep the prepared solution at room temperature to settle down before being use it for video imaging experiment.
2.1.4: Charcoal black stain Preparation:
2.1.4.1: Material:
Table 4: Consumables and relevant quantities used for preparation of Charcoal black stain:
No Consumables Quantity Company name/City/Country
1 SYLGARD 184 SILICONE ELASTOMER CURING AGENT 27g DOW CORNING CORPORATION / USA
2 RTV Sealant 3g DOW CORNING CORPORATION / USA
3 TEETH WHITING POWDER
(White GIO) 3g Barros Laboratory PTY Ltd /Sydney/Australia

2.1.4.2: Preparation procedure:
Mix 27 g of RTV Sealant with 3 g of Silicon Elastomer Curing Agent in plastic plate using nasal swap. Then, while mixing, gradually add Teething Whiting Powder until black colour background is obtained to make a contrast for organ bath chambers (black) against jejunum tissue colour (white).

2.1.5: Organ bath assembly:
2.1.5.1: Experimental protocol:
1- prior to the organ bath assembly, use standard laboratory retort stand to position inflow reservoir (400 mL bottle) 30 cm over organ bath and then connect it to organ bath via 3-ways stopcock connected to inflow tubing (2.00 x 3.00 mm PE tube).
2- Use sewing needle and cotton thread to insert the one end of inflow tube through basement part to chambers part of organ bath.
3- To avoid future liquid leakage issues, apply sealant on the top of the basement part using nasal swab to spread it over and attach it together with the chamber part. Then, use screw to firmly attach the basement part to the chamber part with 6 screws positioned at bottom side of basement part.
4- Finally, apply charcoal black stain to organ bath cumbers until it just touches the lower side of inflow tube holes and leave it to dry at room temperature.

Figure 6: This diagram displays Schematic of organ bath. (A) Top view, (B) Underside, (C) Front view, (D) Side view, of a two chambered organ bath set up. Dimensions in mm.

2.1.6: Organ bath and video imaging experimental setup:
2.1.6.1: Experimental protocol:
1- Position front pressure reservoir (5 mL) on adjustable stand retort on the right side of organ bath and back pressure (5mL out tube) on the left side. Then, connect them to in/outlet tube (2.00 x 2.00 mm PE tube) via three ways stopcock.
2- Use a sealant gel to make about 1 cm wall of sealant surrounding the top of organ bath chambers. This will increase the height of chambers and hence makes isolated tissue well submerged. After that, use a blade to make paths exactly fits for in/outlet tube to be held at the top of organ bath chambers.
3- To connect chambers to carbogen source, attach one end of carbogen tube (. 2.00 x 2.00 PE) to chambers base using insect tips and connect the other end to carbogen tank tube via a turbo needle.
4- connect vacuum flask (3L) to one end of vacuum tube (2.00 x 2.00 mm PE tube) via 3-way stopcock and attach the other end to the top of chambers using insect tips.
5- To warm up organ bath to (32-36 c°), attach thermoregulatory device to the top of water container (5L) and then connect it to water heater metal of organ bath via PE tube (heat resistant) Master Flex tube L/S 15).
6- Finally, position Logitech HD Pro webcam camera C910 (Logitech Co., California, USA) at about 10 to 15 cm over organ bath using adjustable retort stand and then, connect it to computer system via inhouse Virtual Dub software (Version. 1,10,4 AMD64).

2.1.7: Organ bath Preparation:
1.Fill water bath container (5 mL) with 4 mL tap water and turn on thermoregulatory device at 82 c°. This will warm up running KREB’s solution (25c °) in the chambers to the optimal temperature (33 c°- 37c °) to maintain tissue health.
2. Fill 10 mL syringe with KREB’s and flush any debris that could block the tubing of inflow and front/back pressure reservoir. Then, fill the inflow and front pressure reservoir with KREB’s solution and securely close their opening using rubber stoppers.
3. Turn on vacuum and carbogen and immediately allow KREB’s solution in the inflow and front pressure reservoir to flow down into chambers by turning on 3 ways stopper
2. Once chambers are filled with KREB’s solution, wait for five minutes to check organ bath temperature (33 – 37 c °) using a temperature probe.

2.1.8: Experimental mouse dissection:
1.8.1 Dissection protocol:
1. Perform cervical dislocation to Kill adult mice and then, record total mouse weight.
2.Use hypodermic needles (Size: 20 G) to pin the four paws of mouse body to a dissection board making ventral side exposed to experimenter
3. Use dissecting scissors and forceps to open the abdominal cavity along the midline of the abdomen to the sternum.
4.Pour (Krebs) solution previously bubbled at RT with carbogen gas (95% O2 and 5% CO2) onto abdominal contents during the dissection process every one minute. This will prevent tissue to be dehydrated.
5. Cut the adjoining mesentery by fine dissecting scissor to separate small intestine from abdominal bowl (be careful not to stretch, handle or cut the jejunal tissue while trimming the adjoining mesentery).
6. Hold the cecum (located adjacent to the ilium) with dissecting forceps to separate the small intestine from colon using dissecting scissor
7. Hold up the stomach with forceps to separate small intestine and stomach from oesophagus using dissecting scissor.
8. Place the full-length small intestine with stomach into a beaker filled with KREB’s solution (previously carboxygenated). Then, put it on a plate to collect the jejunum tissue
9. Leave about 5-6cm of (ilium) to collect about 5 to 7cm jejunum length. Then, mark its oral end using insect tip and immediately submerged it into organ bath chamber filled with KREB’s solution.

2.1.9: Jejunal Tissue Preparation and Experimental Set Up for Video Imaging:
1.Cannulate the oral end of isolated jejunum into submerged inlet tubes and then use standard cotton sewing thread and forceps to make two tight knots capable to prevent leakages.
2.Turn on 3-way stopcock of front pressure reservoir to allow KREB’s solution to flow through inlet tube down to jejunum lumen. This will allow the faecal content to be flushed out into the organ bath chamber.
3.To facilitate anal end cannulation, turn of saline flow from front pressure reservoir. Then, Cannulate the anal end to the submerged outlet tube, (connected via a 3-way stopcock to a vertical outflow tube; 6 cm in height).
4. Remove mesentery located at basal side of the cannulated jejunum using spring scissors This is done to
prevent mesentery tissue to disturb the edge detection process.
5. Turn on the front and back pressure reservoir to apply luminal pressure to the cannulated jejunum.
5. Finally, record the length of cannulated jejunum using 30 cm ruler as it will be utilized for image width calculation during scribble process.

2.1.10: Luminal pressure setup:
1. Prior to the beginning of the experiment, ensure a stable luminal pressure at (6 cm H2O) by measuring the difference in the vertical distance between tissue cannulated in organ bath chambers and the meniscus height of KREB’s solution inside the glass tube of front pressure reservoir and outflow tube (back pressure reservoir) (figure 4).

Figure 7: This diagram illustrates measurement of luminal pressure performed for video imaging experiments. This was done by changing the difference in the vertical distance between the meniscus height of KREB’s solution the inside front/back pressure reservoir and the height of cannulated tissue inside the organ bath.(10)

2.1.11: Image Capture and Experimental Protocol:
1.Before recording jejunal motility, leave the cannulated jejunum to equilibrate in KREB’s solution for 30 min.
2.Use Webcam with (30 frames/sec, 640 x 840 pixels) feature connected to Virtual Dub software to record real time jejunal motility as Avi video form. Make sure to compressed imaging video utilising ‘Div X6.9.2. Codec’ compression and disable audio capture to reduce using of storage space. Then, select stop condition option to set recording duration at (10 minutes).

3.Make sure that cannulated tissue is completely visible and centred within video frame by manual adjustment of camera location or via the video capture software window. Then, use cardboard shield to cover the camera and organ bath as it will reduce light reflections. After that, use the camera software interface to adjust the exposure settings, contrast and brightness for video quality optimization.
5.Before capturing, name and localize recording video into the computer system
6.During video image recording, check saline level in the inflow reservoir, organ bath temperature and luminal pressure stability (6cm H2O).
7.Record jejunal motility for (2hrs and 20 min) (includes (20 min) under equilibrating condition and (40 mints) under each control, drug and washing condition. 10 (mins) per each recorded video.

2.1.12: Data Processing and Spatiotemporal Maps Generation:
1.Process captured Avi video files offline into Scribble software (scribble, version 2.1) utilising edge detection algorithm function to converts Avi video of jejunum motility to a summary file of analytical data.
2- Then, highlight the region of interest (ROI) and calculate the Image width which represent jejunum length (mm) in spatiotemporal map.

3-Manipulate edge detection upper and lower threshold value until they become adjacent to the upper and lower boundaries of cannulated jejunum.

4- Use Scribble software to convert the proportion of black background pixel (organ bath chamber base) against pale pixel (cannulated jejunum) in every frame captured during motility video recording to colour-coded rows of pixel (A). During the analytical process, scribble software specifies (yellow-red) colour when the proportion of pale pixel against black background pixel is high while contracting and specifies (blue-green) colour when the proportion of pale pixel is low when compared to black background colour during jejunum resting (B) (figure 6)

Figure 9: This chart exhibits colour coded horizontal rows assigned by Scribble software during frames capturing based on differences in proportion of pale pixel (cannulated tissue) against black background pixel (organ bath) (A). It shows (yellow-red) coded colour assigned when gut contracting and (blue-green) coded colour when dilating. (B)
5- Process summary file generated by Scribble to MATLAB software (version, 7.14.0.739, R2016b, The MathWorks Australia Pty Ltd, Massachusetts, USA.) with (analyse2 software). This is done to generate three-dimensional spatiotemporal map presenting jejunum PCCs in (red-yellow) colour vertical bands and dilation in (blue-green) colour along isolated jejunum length within 10 minutes recording time frame.

2.2: Pharmacology (NOLA):
In this experiment, NOLA drug (NO inhibitor) was administered to ex vivo jejunum to compare NO effect on jejunal motility of NL3 R451C and WT mice. This was performed to examine for possible alteration in jejunal motility of NL3 R451C mice. (the second goal of my study).
.
2.2.1: Experimental animal:
For NOLA study only 3 WT and 2 NL3R451C mice were used due to time constrain
Table 5: This table shows data of experimental mice used to in NOLA study.

2.2.2: NOLA Stock solution (100mM/mL) preparation:
1.Add 160.01 g of N-Nitro-L-Arginine (NOLA) drug (SIGMA ALDRICH / VIC / Australia) to 5 mL of distal water and mix.
2.Add I00 µL of HCL as a dissolvent and make strong mix for one minute until completely dissolved.
3. add 2.2 mL of distal water followed with gentle mix and then store at cold room (6 c°).

2.2.3: NOLA solution (100µM/ mL) preparation
2.2.3.1 preparation procedure of NOLA drug solution (100µM/mL):
NOLA drug (100µM/ mL) solution was prepared by mixing (800 µl) of NOLA Stock solution (100 mm/mL) to the desired volume of KREB’s solution (800 mL).

2.2.4: Experimental protocol:
Once the luminal pressure was optimised at (6 cm H2O), video imaging experiment was run under each experimental condition (control. NOLA and washout). In this experiment, NOLA drug was administered to ex vivo jejunum of NL3 R451C and WT mice via the inflow reservoir. Then, it was replaced with KREB’s solution to record jejunal motility under washout condition. This was performed to compare NO effect on jejunal motility of NL3R451C and WT by examining the significant changes in the PCCs frequency and gut resting diameters under each experimental condition.

2.2.5: Parameter measurement:
Before parameter measurement, make sure that scale colour, jejunum length (mm) and recording time frame are the same for the video imaging captured in the same experimental condition (figure 9).

Figure 12: representative spatiotemporal maps show the consistency of scale colour, jejunum length (mm) and recording time frame as there were generated in the same experimental condition.

2. PCCs frequency was measured in four Spatiotemporal maps generated under control condition. This was performed by manually counting of each PCCs that propagates 50 % or more along jejunum segment for 10 minutes video recording of jejunal motility (figure10).

Figure 13: Representative spatiotemporal map shows measurement of PCCs frequency. This was done by manual counting of PCCs clusters induced duration 10 minutes recording of jejunal motility (PCCs/10 minutes)
4.To measure gut resting diameter, click on “Take cross section” button on the Heat map to make a cross horizontal line intersects with all PCCs on spatiotemporal map. Then, gut width will be shown in a new window. Now, click the cursor on the straight line to obtain the gut resting diameter (figure11).

Figure14: The plot A shows Spatiotemporal map cross sectioned at a certain point of jejunum length. The blot B is taken from the cross sectioned heatmap (A) where inflection indicates for gut diameter during contraction. While, straight line indicates for gut diameter during dilation. This was performed to measure the gut diameter in the resting stage (dilation) by the annotation function of MATLAB software.

2.2.6: Statistical analysis:
The average values of this parameters were utilized by GraphPad Prism 6 software (GraphPad Software, California USA) to perform unpair- nonparametric- statistically analysis in two-tailed Student t-test format. This was performed to examine for significant differences in the jejunal motility of both (NL3R451C &WT) mice as it considered to be significant as P value < 0.05.

2.3: Histological study:
In this study, the histological structure of SHANK 3 KO mouse was compared to WT mouse to examine for possible alteration in the jejunum histological structure of KO mice. this was performed by investigating significant differences in the height and width of villi and height of crypts. (The third goal of the study).

2.3.1: Experimental animal:
These mice were initially bred on a mixed background (Sv129/ImJ/ C57BL/6) when obtained from the Jackson Laboratory (Bar Harbour, Main USA) to Pasteur institution in Paris. Subsequently mice were bred with C57BL/6 mice for more than 10 generations and maintained on a C57BL/6 genetic background. Then, pups were bred by mating WT female and hemizygous SHANK3 KO to produce male offspring hemizygous for SHANK3 KO mutation or WT mice.

Table 6: This table shows data of SHANK3 KO and WT jejunum tissue samples used for histological study

2.3.2: Freezing preparation of SHANK3 KO jejunum tissue:
This method utilized Liquid Nitrogen to allow very rapid tissue freezing Preventing ice crystal formation (tissue artefact) due to water expansion with slow freezing.
2.3.2.1: Material:
Table 7: This table shows chemicals used for tissue freezing preparation of SHANK3 jejunum samples.
2.3.2.2: Experimental protocol:
1-Fixation and sucrose incubation:
Prior to tissue preparation start, SHANK3 jejunum tissue specimens (KO & WT) were obtain from Paster institution in Paris where they have been fixated with 4% formaldehyde and cryoprotected in 30% sucrose solution at 4c°.
To start, fill a Dewar flask with liquid nitrogen (take serious care using liquid nitrogen and wear proper PPE!!) and then Transport immediately LN filled Dewar flask to standard fume hood
2-Labelling:
Label standard micromolds and storage bag with identification information (specimen ID, preparation date and initial) before OCT embedding process.
3- Tissue preparation:
Horizontally hold jejunum tube on dissecting board using standard forceps and cut it into about 1 cm x 1 cm x 4 mm) using dissecting forceps.

4- OCT Embedding:
Fill Cryomolds with OCT (avoid air bubbles) and then immerse each specimen into it. Once submersed in OCT, orient jejunum tube than vertically exactly in the centre. Submerged jejunum tube usually stays stable as a result of medium viscosity.
5- Tissue freezing:
1- Add about (100 mL) of 2-methyl butane to Plastic baker (500mL). This amount of liquid will cover about 1 cm of the plastic baker allowing the cryomold to be floated and not fully submerged during freezing process.
2-Carefully insert the plastic bake into the liquid nitrogen. Methyl butane will be quickly cold causing vapour formation. Wait for 3 minutes, so liquid will reach freezing point generating solid white colour at the base of the plastic container.
3- Quickly Place cryomold into LN frozen 2-Methyl butane and then immediately transfer it into LN using a forceps and PPE. Wait for 1 minute, so a complete freezing is achieved.
4- Use forceps to remove blocks from 2-methyl butane and examine the top centre of the block for characteristic bump.
6-Storage:
Finally, leave frozen blocks at room temperature to allow LN to evaporate before transferred to dry ice filled Styrofoam to be stored in relevant labelled storage bag at -80 c° freezer).

2.3.3: Cryosectioning of SHANK 3 KO jejunum tissue
2.3.3.1 Experimental protocol:
Five jejunum tissue samples for each mouse type (KO & WT) were cryosectioned at 6-μm thickness under –18 C temperature using Cryostat instrument (Leica CM1950, Fronine Laboratory Supplies, VIC, Australia). Then, all cryosectioned slices were mounted into electrostatic charged slides (SuperFrostPlus, Menzel-Glacer, Braunschweig, Germani) and left at room temperature for one hour to dry before being stained with H&E stain.

2.3.4: H&E stain:
3.4.1 Experimental protocol:

2.3.5: Microscopy:
The H&E stained jejunum tissue sections of SHANK3 KO and WT mice were processed under Olympus Slide Scanner Microscope (Olympus Australia LTD.; Melbourne, Australia) for jejunum imaging. The jejunal images were captured at 40 magnification power and then saved as VSI images. Three sections (free of architecture) per each jejunum tissue sample were scanned and areas of interest like villus and crypts were focused utilizing edit focus function.
2.3.6: Parameters measurement:
The VSI jejunum images were processed offline to ImageJ software (ImageJ 1.53a, NIH, USA) with the BIOP plugin to measure the height and width of villi and the height of crypts. The measurement scale of image J was utilized to measure the height of villi from its tip point to the midpoint of the villi base, and the villi width was measured from the area which has the largest width. While, crypts heights were measured from the tip point to the midpoint of crypt base (figure11). In each jejunum sample, the average height and width of villi as well as crypts height were taken from three villi and crypts of each section.

Figure 15: representative jejunum image of SHANK3 KO and WT mice show parameters measurement (height and width of villi as well as crypts height) utilizing the measurement tool of image J software. The arrows and black line are arbitrary lines used for parameters measurement and scale bar represents 200 μm.

2.3.6: Statistical analysis:
The average values of this parameters were utilized by Prism software to perform unpair- nonparametric- statistically analysis in form of two-tailed Student t-test. This was performed to investigate for significant differences in the jejunal histological structure of both (KO&WT) mice as it considered to be significant as P value < 0.05

Chapter 3: Result
3.1: Increased luminal pressure video imaging setup:
In this experiment, I managed to find out the optimal luminal pressure that can induce the presence of consistent and well-organized clusters of PCCs in spatiotemporal maps. This was performed by changing the vertical distance of the meniscus height inside both front/ back pressure reservoir and the height of cannulated jejunum. The optimal luminal was determined to between at 6 cm H2O (Figure 12).
Figure 16: Representative spatiotemporal maps generated by MATLAB software at different luminal pressure (3- 8 cm H2O) to find out the optimal luminal pressure that induce consistent PCCs clusters. Spatiotemporal maps generated at lower luminal pressure (3,4 &5cm H2O) display overlapping and irregular PCCs clusters (A, B&C). Spatiotemporal maps generated at higher pressure (7&8 cm H2O) exhibit diminished clusters of PCCs (E&F cm H2O). While, the spatiotemporal map generated at 6 cm H2O shows clear and well-organized and consistent clusters of PCCs.

3.1.2: Experiment repeatability:
To test if the experiment is repeatable, video imaging experiment was repeated three times per each mouse type (WT&NL3R451C) at 6 cm H2O luminal pressure. They show repeatable results as all PCCs clusters induced in each experiment were consistent and easy to measure.

Figure 17: Representative spatiotemporal maps of 3 NL3R451C and WT mice generated at optimal luminal pressure (6cm H2O) to test repeatability of luminal pressure experiment. These heatmaps show repeatable results of consistent PCCs clusters

3.2: Pharmacology (NOLA):
In this experiment, NOLA drug was administered to ex vivo jejunum tissue to compare the effect of NO on jejunal motility of WT and NL3 R451C mice by measuring the changes in PCCs frequency and gut resting width under each experimental condition. This was performed to examine for alteration in the jejunal motility of NL3R451C
According to the statistical analysis, there was no significant difference in PCCs frequency of NL3R451C and WT jejunum preparation under control condition with P value of 0.7 and average value of 7.25 and 6.83 respectively (figure 14 and table 8). While there was a dramatic increase in PCCs frequency under NOLA and washout condition (figure 15 and 16). As a result, PCCs clusters were uncountable and hence were incomparable under drug and washing conditions.
In addition, there was no significant difference in the gut resting diameter of NL3 R451C and WT jejunum preparation under control, drug and washout conditions with P value of (0.8, 0.8 and 0.9) respectively and average value of (4.1, 4 and 4, 2 NL3 R451C and 4.3, 4.3 and 4.3, 3 WT respectively) (figure 16, 17, 18 and table 9).

Table 8: This table shows the average values of PCCs frequency obtained from NL3 R451C and WT jejunum preparation and measured under control condition.

Figure 18: This graph shows a comparison between NL3R451C and WT mice with regard to PCCs frequency under control condition. It shows no significant differences as P value (0.7) was less than 0.05

Figure 19: Representative spatiotemporal map shows the changes in PCCs frequency after NOLA drug administration into the jejunum preparation of NL3-49A mouse. (A) heatmap shows consistent and countable clusters of PCCs under control condition. While (B & C) heatmaps display overlapped and uncountable clusters of PCCs under drug and washout due to deactivation of inhibitory motor neurotransmission of NO neurotransmitter by NOLA drug.

Figure 20: Representative spatiotemporal maps show the changes in PCCs frequency after NOLA drug administration into the jejunum preparation of WT-62 A mouse. (A) heatmap shows consistent and countable clusters of PCCs under control condition. While (B & C) heatmaps display overlapped and uncountable clusters of PCCs under drug and washout due to deactivation of inhibitory motor neurotransmission of NO neurotransmitter by NOLA drug.

Table 9: This table shows the average resting gut width of WT and NL3R451C mice in each experimental condition.

Figure 21: This graph shows a comparison between NL3R451C and WT mice with regard to gut resting diameter under control condition. It shows no significant differences as P value (0.8) was less than 0.05

Figure 22: This graph shows a comparison between NL3R451C and WT mice with regard to gut resting diameter under drug condition. It shows no significant differences as P value (0.8) was less than 0.05

Figure 23: This graph shows a comparison between NL3R451C and WT mice with regard to gut resting diameter under washout condition. It shows no significant differences as P value (0.9) was less than 0.05

3.3 Histology:
in this experiment, the jejunal histological differences of NL3R451C and WT mice was examined by investigating significant differences in the height and width of villi and height of crypts in five jejunum tissue samples of SHANK3 KO and WT mice.
In this study, there was no significant difference in the villi height of SHANK3 KO and WT mice with P value of (0.41) (figure 19) and average value of (355, 486, 453, 432 and 482, KO and 411, 433, 378 ,420 and 451, WT respectively) (table 10 and 11).
For jejunal width. there was no significant difference in the villi height of SHANK3 KO and WT mice with P value of (0.46) (figure 20) and average value of (93, 66, 78, 72 and 83, KO and 83, 79, 86, 75 and 90, WT respectively) (table 10 and 11).
In addition, there was no significant difference in the crypts height, of SHANK3 KO and WT mice with P value of (0.59) (figure 21) and average value of (39, 45, 66, 51 and 60) and (53, 51, 50, 54 and 67) respectively (table 10 and 11).
Table 10: This table shows the average values of villi height and width and crypts height in the jejunum preparation of five WT mice.

Table 11: This table shows the average values of villi height and width and crypts height in the jejunum preparation of five SHANK3 KO mice.

Figure 24: This graph shows a comparison of villi height between SHANK3 KO and WT mice. It shows no significant differences as P value (0.41) was less than 0.05

Figure 25: This graph shows a comparison of villi width between SHANK3 KO and WT mice. It shows no significant differences as P value (0.46) was less than 0.05

Figure 26: This graph shows a comparison of crypts height between SHANK3 KO and WT mice. It shows no significant differences as P value (0.59) was less than 0.05

Before writing the discussion, please perform proofreading for the result section
Chapter 4: Discussion: (1500) words
The Thesis Structure: An Overview
• Discussion
Summarise background – issue, rationale and research question
What have you done to answer each aim?
Summarise the findings in order of how you presented your Results
Table and/or diagram?
•  Have your findings addressed the aims and hypothesis? Explain.
•  Discuss your results in the context of other similar studies
•  Derive a theory based on the discussion of your results A further hypothesis?
•  Limitations
•  Further studies

Conclusion: (200) words
The Thesis Structure: An Overview
• Conclusion
Provides a summary:
•  One statement summarising research problem and research questions
•  Re-state the hypothesis
•  Briefly list the techniques used for data generation/collection to address the hypothesis
•  Main findings and significance – answer the aims
•  A general conclusion based on the interpretation of your findings
•  Did your findings support or challenged your hypothesis?

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