Masters thesis of science intestinal permeability and functional properties of duodenal enteric neurons in a mouse model of autism

49 Figure 13: Small and large intestinal paracellular permeability fasted NL3 R451C and WT mice .... 54 Figure 16: L-Glutamine restored paracellular permeability in NL3 R451C mice to WT

Intestinal Permeability and Functional Properties of Duodenal Enteric Neurons in a Mouse Model of

(RMIT University, Melbourne)

School of Health and Biomedical Science College of Science, Technology, Engineering and Math

RMIT University

Thesis declaration

I certify that except where due acknowledgement has been made, this research is that of the author alone; the content of this research submission is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and ethics procedures and guidelines have been followed

In addition, I certify that this submission contains no material previously submitted for award of any qualification at any other university or institution, unless approved for a joint-award with another institution, and acknowledge that no part of this work will, in the future, be used in a submission in

my name, for any other qualification in any university or other tertiary institution without the prior approval of the University, and where applicable, any partner institution responsible for the joint-award of this degree

I acknowledge that copyright of any published works contained within this thesis resides with the copyright holder(s) of those works

I give permission for the digital version of my research submission to be made available on the web, via the University’s digital research repository, unless permission has been granted by the University

to restrict access for a period of time

I acknowledge the support I have received for my research through the provision of an Australian Government Research Training Program Scholarship

Joshua Kenneth Williams

02 August 2022

Acknowledgements

I would like to express my deepest gratitude to my supervisors A/Prof Elisa Hill and Dr Suzanne Hosie Both Elisa and Suzanne were encouraging, inspiring and supportive throughout my candidature Thank you both for providing me with constructive criticism, along with skills in electrophysiology, permeability, scientific presentations, and scientific writing I would also like to acknowledge my lab members: Jackson, Tanya, Chalystha, Pasindu, Rachele, Samantha and Miti for providing me with invaluable feedback on my presentations and troubleshooting problems with experiments that arose during my candidature I would like to acknowledge my partner Latasha who provided constant emotional support and encouragement throughout my candidature

Table of Contents

Thesis declaration i

Acknowledgements ii

List of Figures vi

List of Tables vii

List of abbreviations and units vii

Abstract 1

Chapter 1: Introduction 3

1.0 Autism Spectrum Disorder (ASD) overview 3

1.1 Gastrointestinal disturbances in Autism patients 4

1.2 The gastrointestinal tract 5

1.3 The enteric nervous system 8

1.4 The submucosal plexus 9

1.5 The myenteric plexus 9

2.0 The gastrointestinal mucosal barrier 10

2.1 Intestinal epithelial cells 12

2.2 Apical junction protein complex 13

2.2.1 Tight junctions 14

2.2.2 Paracellular permeability 16

2.3 Gastrointestinal distress and ASD 17

2.4 Restoration of intestinal permeability 18

2.4.1 The impact of L-glutamine on intestinal permeability 20

2.4.2 The impact of caffeine on intestinal permeability 23

3.0 Classification of enteric neurons 26

3.1 Morphological classification of enteric neurons 26

3.2 Electrophysiological classification of enteric neurons 28

3.2.1 Synaptic-neurons (S-neurons) 28

3.2.2 AH (after-hyperpolarisation) neurons 28

3.3 Functional classification of enteric neurons 29

3.3.1 Intrinsic primary afferent neurons (IPANs) 30

3.3.2 Interneurons 31

3.3.3 Muscle motor neurons 32

3.4 Neurochemical classification of myenteric neurons 33

4.0 ASD genetic mutations 35

4.1 ASD and synaptic cell adhesion molecules 35

4.2 Neuroligins 36

4.3 Neuroligin-3 overview 37

4.4 Neuroligin functionality 37

4.5 NL3 R451C mutation 38

4.6 Expression of Nlgn3 in the gastrointestinal tract 39

5.0 Project rationale 41

5.1 Assessing permeability in an autism mouse model 41

5.2 Action potential characteristics in duodenal myenteric neurons using an autism mouse model 42

5.3 Aims and hypotheses 43

Chapter 2: Measuring intestinal permeability in the Neuroligin-3 R451C mouse model of autism 44

1.0 Introduction 44

2.0 Methods and materials 45

2.1 Animals 45

2.2 Segmentation of the small and large intestine 45

2.3 Preparation of L-glutamine and caffeine stock solutions 46

2.4 Injection of FITC-Dextran 4 47

2.5 Time course permeability experiment 47

2.6 Construction of standard curve using log serial dilutions 49

2.7 Statistical analysis 50

3.0 Results 51

3.1 Small intestinal permeability in non-fasted wild-type and mutant mice 51

3.2 Permeability effects in wild-type and mutant fasted mice 53

3.3 Effects of fasting on NL3 R451C mice 54

3.4 Effects of fasting on wild-type mice 56

3.5 Effects of L-glutamine on fasted NL3 R451C mice 58

3.6 Effects of L-glutamine on fasted wild-type mice 60

3.7 Effects of caffeine on fasted NL3 R451C mice 62

3.8 Effects of caffeine on fasted wild-type mice 64

4.0 Discussion 66

4.1 Understanding how NL3 R451C mutation and feeding conditions effect paracellular permeability 66

4.2 Understanding how L-glutamine and caffeine impact paracellular permeability 70

5.0 Conclusion 77

Chapter 3: Optimisation of the patch-clamp recording technique in the enteric nervous system to examine action potential characteristics in mouse duodenal myenteric neurons 78

1.0 Introduction 78

2.0 Methods and materials 80

2.1 Animals 80

2.2 General perfusion/dissecting Krebs solution 80

2.3 Microdissection 81

2.4 Identification of myenteric ganglion 83

2.5 Protease solution 83

2.6 Whole-cell patch recording 83

2.7 External patching solution 84

2.8 Current clamp recording protocol 85

2.9 Statistical analysis 87

3.0 Results 88

3.1 Comparison of success rate of neuronal recordings for older versus younger mice 88

3.2 Recording action potentials in younger mice 90

3.2.1 Comparison of firing properties in 3 myenteric neurons 92

3.2.2 Action potential characteristics in younger mice 92

3.3 Action potential characteristics observed in Neurons 1, 2 and 3 93

3.4 Neuronal profiles in younger mice 97

3.4.1 Comparison of action potentials full trace 98

3.4.2 Comparison of current-voltage (IV) curves 100

3.4.3 Comparison of action potentials and current curves 100

Electrophysiology appendices 119

Appendix Table 1 119

Appendix Table 2 119

Appendix Table 3 120

Appendix Table 4 120

Permeability appendices 121

Appendix Table 5 121

Appendix Table 6 121

Appendix Table 7 121

Appendix Table 8 122

Appendix Table 9 122

Appendix Table 10 122

Appendix Table 11 122

Appendix Table 12 123

Appendix Table 13 123

Appendix Table 14 123

Appendix Table 15 123

Appendix Table 16 124

Appendix Table 17 124

Appendix Table 18 124

Appendix Table 19 125

Appendix Table 20 126

Appendix Table 21 127

Appendix Table 22 128

Appendix Table 23 129

Appendix Table 24 130

Appendix Table 25 131

Appendix Table 26 132

Appendix Table 27 133

Appendix Table 28 134

Appendix Table 29 135

Appendix Table 30 136

Appendix Table 31 136

Appendix Table 32 137

Appendix Table 33 137

Appendix table 34 137

List of Figures

Figure 1: Organization of the mouse gastrointestinal tract 6

Figure 2: The organization of the enteric nervous system 8

Figure 3: The physiology of the mucus layer in the colon is different to the small intestine 11

Figure 4: Cross-sectional image of small intestine depicting the major cell types 12

Figure 5: Tight junctions, desmosomes and adherens junctions, make up apical junctional complexes 13

Figure 6: Intestinal epithelial cells utilise both transcellular and paracellular pathways 16

Figure 7: Potential mechanisms for L- glutamine restoring paracellular permeability in IEC’s 21

Figure 8: Overeating and duration of food deprivation influences intestinal mucus composition 24

Figure 9: Three major functional classes of enteric neurons 28

Figure 10: Setup for measuring the permeability of FITC through the paracellular route 46

Figure 11: Standard curve of known concentrations of FITC with absorbance 47

Figure 12: Small intestinal paracellular permeability in non-fasted NL3 R451C and WT mice 49

Figure 13: Small and large intestinal paracellular permeability fasted NL3 R451C and WT mice 51

Figure 14: Small intestinal paracellular permeability for fasted and non-fasted NL3 R451C mice 52

Figure 15: Effect of fasting and non-fasting on intestinal paracellular permeability in WT mice 54

Figure 16: L-Glutamine restored paracellular permeability in NL3 R451C mice to WT levels in small and large intestinal regions 56

Figure 17: Small and large intestinal paracellular permeability on wild-type mice treated with L-glutamine 58

Figure 18: Caffeine restored paracellular permeability in NL3 R451C mice to WT concentrations in small and large intestinal regions 60

Figure 19: Caffeine decreased paracellular permeability in WT mice 62

Figure 20: Visual representation of obtaining an LMMP preparation 78

Figure 21: Visual representation of the general set up of patch-clamping 81

Figure 22: Custom-made hair cell attached to a glass micropipette 81

Figure 23: A myenteric neuron action potential with measured characteristics 83

Figure 24: Number of times whole cell seal configuration was obtained 84

Figure 25: Number of times whole cell seal obtained and/or AP firing recorded in myenteric neurons using younger and older mice 85

Figure 26: Three duodenal myenteric neurons exhibited action potentials in response to current steps 87

Figure 27: Action potential characteristics of three neurons 92

Figure 28: Characterisation of action potential profiles recorded throughout the full current injection protocol for three duodenal myenteric neurons 93

List of Tables

List of abbreviations and units

AH: After-hyperpolarisation

AHP: After-hyperpolarisation potential

ASD: Autism Spectrum Disorder

ATP: Adenosine triphosphate

BA: Bile acid

cAMP: cyclic adenosine monophosphate

ChAT: Choline acetyltransferase

CM: Circular muscle

Cm: Membrane capacitance

CNS: Central Nervous System

DMEM: Dulbecco’s modified eagle medium

EEC: Enteroendocrine cell

EGF: Epidermal growth factor

ENS: Enteric Nervous System

ER: Endoplasmic reticulum

mTOR: Mechanistic target of rapamycin

FEPSPs: Fast excitatory post-synaptic potential

FITC: Fluorescein Isothiocyanate

GABA: Gamma-Aminobutyric acid

GI: Gastrointestinal

HFD: High-fat diet

HSP: Heat shock proteins

HSF: Heat shock factor

ICC: Interstitial cells of Cajal

IEC: Intestinal epithelial cell

IGF: Insulin-like growth factor

IPAN: Intrinsic primary afferent neurons

ISN: Intrinsic sensory neuron

I-V curve: Current – Volt curve

JAM: Junctional adhesion protein

KO: Knock-out

LM: Longitudinal-muscle

LMMP: Longitudinal-muscle-myenteric-plexus LP: Lamina propria

RMP: Resting membrane potential

SCFA: Short chain fatty acids

S-neurons: Synaptic-neurons

SMP: Submucosal plexus

Tau: Membrane time constant

TGF: Transforming growth factor

TGF- α -: Transforming growth factor α

TJ: Tight junction

TMAO: trimethylamine N-oxide

VAchT: Vesicular acetylcholine transporter

VIP: Vaso-intestinal peptide

WT: Wild-type

ZO: Zonulin

Abstract

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder characterised by impaired social communication and the presence of repetitive behaviours In addition to these behavioural characteristics, as many as ninety percent of individuals with ASD exhibit gastrointestinal (GI) issues A missense mutation at position R451C in the synaptic adhesion protein, neuroligin-3

(NLGN3) was previously identified in patients with ASD The Nlgn3 gene is present in both the brain

and the intrinsic neural network of the gut, the enteric nervous system (ENS) Within the ENS, the submucosal plexus (SMP) regulates mucosal barrier functions such as secretion and permeability, while the myenteric plexus predominantly regulates gut motility Clinical studies have shown that individuals with ASD who experience GI disturbances have elevated blood concentrations of simple sugars such as mannitol and lactulose, which is a marker for increased intestinal permeability Previous data from the current laboratory showed faster small intestinal transit time in NL3R451C mice compared to wild-type littermates We hypothesise that the R451C mutation affects intestinal function (i.e., permeability and motility) in mice Duodenal, jejunal, ileal and colonic paracellular

permeability was assessed using an ex-vivo whole tissue assay in non-fasted and fasted mice The

effects of the R451C mutation on functional characteristics of duodenal myenteric neurons were also investigated in wild type mice No significant differences in intestinal permeability were observed between non-fasted NL3R451C mice and wild-type littermates in any intestinal region In a fasted state, however, NL3R451C mice showed increased intestinal permeability in the duodenum, jejunum, ileum and colon when compared to wild-type littermates The addition of L-glutamine and caffeine rescued

recording was greater in younger (2-4-week-old) Swiss white and C57BL/6 mice in comparison to older (6-12-week-old) C57Bl/6 mice Different action potential firing patterns were observed which may correlate with different functional subgroups of myenteric neurons Characterising intestinal permeability and categorizing enteric neurons by their functional profiles may be useful for understanding and reducing GI symptoms, chronic low-grade systemic inflammation, and core neurological symptoms in people with ASD

Chapter 1: Introduction

1.0 Autism Spectrum Disorder (ASD) overview

ASD is a complex neurodevelopmental disorder that results in a range of clinical traits that can differ

in type and severity in each individual (Vahia, 2013) These clinical traits must meet specific criteria outlined in The American Psychiatric Association’s Diagnostic and Statistical Manual, Fifth Edition (Vahia, 2013) For instance, an individual with ASD will often display persistent deficits in social-emotional reciprocity, nonverbal communicative behaviours (hand gestures and eye contact), verbal communication and developing, maintaining and understanding relationships In addition, the individual must have persistent deficits in at least two types of repetitive or restricted behaviour (Vahia, 2013) These include motor movements that are repetitive or stereotyped, difficulties in managing distress at small changes, fixating on interests and hypo- or hyperactivity (Vahia, 2013) The neuropathology underlying the behavioural abnormalities are proposed to include disrupted neurocircuit connectivity, altered synaptic transmission, inhibitory and excitatory imbalance and altered neurochemical signalling Brain regions in which these abnormalities have been observed include the posterior temporal sulcus, amygdala, adjacent anterior cingulate cortex, medial prefrontal cortex and the temporal poles (Etherton et al., 2011, Ha et al., 2015, Tabuchi et al., 2007)

By the age of 8, 1 in 54 United States children are diagnosed with ASD (Baio et al., 2018) An increase

in the prevalence of ASD is thought to be due to implementation of new diagnostic criteria and better monitoring systems (Baio et al., 2018) Although ASD is not specific to ethnic, racial and

1.1 Gastrointestinal disturbances in Autism patients

Historically, ASD research has been overwhelmingly limited to psychological assays and understanding alterations to the central nervous system (CNS) However, more recent advances have identified that gastrointestinal disturbances are commonly experienced by ASD patients The most frequently reported disturbances in Individuals with ASD are diarrhoea, abdominal pain and constipation (Buie et al., 2010, Coury et al., 2012) Other less frequently reported GI disturbances include: bloody stool, flatulence, gastroesophageal reflux, celiac disease, esophagitis, belching and Chron’s disease (Coury et al., 2012) These symptoms are frequently uncomfortable and disturbing

to a person's day-to-day life, and people with ASD have a fourfold increased risk of being hospitalised due to gastrointestinal symptoms compared to the general population (McElhanon et al., 2014) In addition, there is an association between the severity of GI symptoms and core features of autism including social and language impairment (Gorrindo et al., 2012) Alleviating GI disturbances could improve quality of life for patients and carers For example, improved gut health could improve participation in behavioural therapies and sleep patterns leading to potential improvements in mood and behaviour Although GI disturbances are disruptive and more commonly experienced by people with ASD, astonishingly, the awareness of GI dysfunction in ASD is limited and there are no specific treatments available (Rao and Bhagatwala, 2019)

1.2 The gastrointestinal tract

The major aims of this dissertation are to assess for changes in gut function by analysing permeability

in a mouse model of autism and to contribute to enhancing the classification system for enteric neurons in the mouse myenteric plexus based on their action potential firing characteristics The GI tract consists of the oesophagus, stomach, small intestine, and large intestine From the pylorus to the ileocecal valve, the small intestine is divided into three sections: duodenum, jejunum, and ileum (Furness, 2012) The small intestine's major role is nutrient absorption The caecum is the most proximal section of the large intestine, and its exact function in humans (i.e., the appendix) is unknown The large intestine (i.e., the colon), extends from the ileocecal valve to the rectum, and its primary role is in water and electrolyte absorption (Furness, 2012) The small intestine's major role

is nutrient absorption The gut is divided into functional regions, each of which has specific anatomical characteristics Although the general anatomical structure of the mammalian GI tract is highly conserved, the anatomy and physiology of different species differ significantly This could be related to a variety of factors such as diet, metabolic needs, feeding patterns, and body size (Nguyen

et al., 2015) The mouse GI tract, for example, differs from the human GI tract in terms of morphology, physiology, cellular structure, and genetics, despite several commonalities As a result, mouse models are widely used in GI research, as they are a good tool for assessing preliminary differences caused by genetic mutations and as an indication for human research

Figure 1: Organization of the mouse gastrointestinal tract Distinct regions of the gastrointestinal

tract of a mouse The stomach is proximal to the small intestine which is separated into three distinct regions; the duodenum, jejunum and ileum The large intestine is also separated into three distinct regions The caecum is distal to the Ileum with the colon being proximal to the rectum (Furness, 2012)

The GI tract is comprised of distinctly different cellular layers The innermost layers (i.e., in close proximity to the host organs) are the longitudinal muscle (LM), adjacent to the myenteric plexus (MP), with the next layer being the circular muscle (CM) alongside the SMP muscularis mucosae (MM) and the mucosa layer (Etherton et al.) (Figure 2) The intestinal mucosa is a single layer of epithelial cells acting as the interface between the external gut environment and the internal environment of the body Epithelial cells absorb nutrients, water, and electrolytes, produce a variety of digestive secretions and act as a physical barrier to potentially hazardous luminal substances Enteroendocrine cells (EECs) and other modified epithelial cell subtypes produce regulatory proteins that have local paracrine or neurocrine effects Paneth cells, for example, release antimicrobial mediators which prevent invasions of potential pathogenic microbes into the epithelium (Allaire et al., 2018, Elphick and Mahida, 2005) Similarly, mucus is secreted by goblet cells to lubricate the epithelial lining and form a protective barrier against some microorganisms in the gut The distribution of secretory cell types varies by gut area and is intimately linked to the function of each gut region Within the SMP is

a network of cells that coordinate the absorption, secretion, and immune activity of the mucosa The SMP consists of blood vessels, smooth muscle cells, neurons, and glial cells (Allaire et al., 2018)

The GI tract, in broad terms, protects the host from harmful substances that pass through the intestinal epithelium Neurons from the SMP mostly innervate the mucosa (Neunlist et al., 2013) Immune cells and neurons work together to orchestrate inflammatory responses and evoke neuronal signalling pathways that help the body eliminate infections Neural activity regulates gut contractile function as well as impacting permeability and secretion (e.g., of ions and water) Running the entire length of the GI tract are three clearly defined smooth muscle layers, the CM, LM and the MM As their names suggests, the cells of the CM layer are oriented in a circular direction around the gut and change gut diameter, while the cells in the outer LM layer are oriented along the gut's longitudinal direction and change the gut length Outside the lamina propria (Elphick and Mahida, 2005), the MM

is a thin layer of muscle that serves to separate it from the submucosa Coordinated contractions and relaxation of the CM and LM layers shape the complex motility contractions of the GI tract These patterns range from non-propulsive mixing movements to highly propulsive peristaltic contractions (Costa et al., 2000, Furness, 2012, Furness et al., 2014) Embedded within the muscle layers of the GI tract are two ganglionated plexus known as the SMP and the MP Together these make up the ENS,

an intrinsic neural network that can regulate intestine activities independently of the CNS (Furness

et al., 2014) The ENS governs practically all gastrointestinal activities, including mucosal barrier function, secretion, motility, and blood flow, for efficient food digestion and absorption Although the ENS can function independently of the CNS, it receives extrinsic innervation from the brain and spinal cord to coordinate vital GI activities (Furness et al., 2014)

Figure 2: The organization of the enteric nervous system In humans and large animals, the mucosa

is the intestine's exterior covering, which separates the lumen from the internal structures The submucosal and myenteric plexuses are the two ganglionic plexuses of the ENS The sub-mucosal plexus (SMP) runs between the mucosa layer and the circular muscle (CM) layer, and its ganglia are divided into one or three layers The SMP innervates the mucosa and regulates mucosal barrier functions as secretion and permeability (Furness et al., 2014)

1.3 The enteric nervous system

The ENS is the largest division of the autonomic nervous system, with more than 100 million neurons and 400 million neuron-supporting glial cells This intricate neural system is crucial for maintaining proper digestive function (Furness, 2012) Enteric nerve cells and glia are grouped together to form ganglia in the ENS A neural plexus is formed when ganglia are connected by nerve fiber bundles The SMP and the MP are two ganglionated plexuses in the ENS The MP connects the LM and CM layers

of the external musculature, while the SMP connects the CM layer to the mucosa The MP regulates gut motility, while the SMP regulates mucosal barrier function, however, both plexuses work together to ensure optimal GI functionality Even though the ENS can perform functions independent

of the CNS, it is not autonomous To govern local enteric reflexes, the integrated network of the ENS and CNS mediates neuronal regulation of the GI system Vagal nerve routes, the spinal thoracolumbar

spinal cord, and the pelvic pathway all play a role in neural communication between the CNS and the ENS (Furness et al., 2013)

1.4 The submucosal plexus

The SMP is predominantly responsible for regulating water and electrolyte secretion along with regulating localised blood flow (Furness, 2012) These processes are governed by a variety of enteric neurons and elucidating the function of specific neuronal subtypes can offer insight into therapeutic treatments for GI disorders In the SMP of mice and guinea pigs, there are two pharmacologically and neurochemically separate populations of neurons Cholinergic neurons express choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine Non-cholinergic neurons possess vasoactive intestinal peptide (VIP) but lack the ability to express ChAT Many VIP neurons in humans and rats, on the other hand, express ChAT SMP neurons express an array of neurochemicals that provide them with a unique neurochemical code in addition to VIP and ChAT as two primary neurotransmitters (Bornstein and Foong, 2018)

1.5 The myenteric plexus

The MP is located between the LM and CM layers and predominantly regulates GI motility including peristaltic contractions of the smooth muscles to facilitate the transit of luminal contents (Furness, 2012) The MP comprises approximately 16 enteric neuronal subtypes which release a range of excitatory (e.g Acetylcholine and tachykinins) or inhibitory (i.e nitric oxide, ATP (adenosine tri-phosphate)-like transmitters and VIP) neurotransmitters to contract or relax the intestinal smooth

2.0 The gastrointestinal mucosal barrier

The intestinal epithelial wall of the human Gl tract covers an estimated 400 m2 of mucosal surface area in an adult individual Microorganisms, digestive enzymes and acids, digested food particles, microbial by-products, and food-associated toxins all penetrate the mucus layer, which serves as the GI tract's first line of defence This mucus layer coats the surface of the GI tract, lubricates the luminal contents, and acts as a physical barrier to bacteria and other antigenic chemicals present in the lumen The moist, nutrient-rich mucus layer next to the epithelial barrier of the GI tract is also critical for intestinal homoeostasis because it contains a robust biofilm including both beneficial and pathogenic bacteria species, reviewed by Herath and co-authors (Herath et al., 2020) The GI tract is continually under attack by pathogens, medications, nutrients, and bacterial toxins Not only must the host distinguish between commensal bacteria and potential pathogens, but the host must also prevent these species and secreted molecules from crossing the epithelial barrier while

allowing nutrients to be absorbed Thus, the intestinal epithelium functions as a selective barrier to luminal substances This is accomplished in part by the innate epithelial defence system of the mucosa, which operates via a responsive biological system composed of constitutive and inducible mechanisms Therefore, if the intestinal epithelial barrier's function is impaired, the host may be more vulnerable to a variety of GI disorders.Consequently, this thesis will discuss how increased paracellular permeability may be a possible explanation for why the NL3R451C mouse model exhibits

a dysfunctional epithelial barrier and how natural therapeutic treatments/agents such as, fasting from solid foods, L-glutamine and caffeine supplementation may restore a compromised epithelial barrier in (ASD)

Figure3: The physiology of the mucus layer in the colon is different to the small intestine (A) the

small intestine contains a single layer of mucus that is loosely linked to the epithelium and easily penetrable Anti-microbial modulators effectively repel microorganisms from the small intestinal epithelium (B) The distal colon has two mucus layers including a stratified adhering inner mucus layer (which is not penetrable by molecules greater than 1 μm diameter) The inner mucus layer of the colon is virtually sterile, whereas the outer mucus layer contains intestinal bacteria (Herath et al., 2020)

2.1 Intestinal epithelial cells

The intestinal mucosal barrier comprises a variety of extrinsic and intrinsic components that regulate mucosal homeostasis The intestinal epithelial cell layer and the tight junctions that connect these cells to form a continuous single layer throughout the GI tract are essential components of the mucosal barrier (Peterson and Artis, 2014) The intestinal epithelial cells (IECs) sense and respond to microbial stimuli to strengthen barrier function and coordinate appropriate immune responses, ranging from anti-pathogen immunity to tolerance As a result, IECs play a critical immunoregulatory role in the formation and homeostasis of mucosal immune cells The intestinal epithelial cell layer and the tight junctions that connect these cells to form a continuous single layer are essential components of the mucosal barrier throughout the GI tract

Figure 4: Cross-sectional image of small intestine depicting the major cell types The intestinal

epithelium is made up of secretory and absorptive cell lineages; enterocytes perform the absorption function, while three secretory cell types, including hormone-releasing EECs, antimicrobial peptide-producing Paneth cells and mucus-producing goblet cells, are responsible for secretion (Kong et al.,

2018)

2.2 Apical junction protein complex

Apical junction protein complexes in the lateral membranes of neighbouring cells maintain epithelial cells in a tightly packed confirmation The apical junctional complex is formed by three types of intercellular junctions: desmosomes, tight junctions and adherens junctions (Figure 5)

Figure 5: Tight junctions, desmosomes and adherens junctions, make up apical junctional complexes At the tight junction, claudin, junctional adhesion molecule (JAM), occludin, Zonular

occluden and F-actin interact to merge the lateral and apical plasma membranes of two adjacent cells Adherens junctions are formed when E-cadherin, β-catenin α-Catenin and F-actin interact Desmoplakin, desmoglein, desmocollin and keratin combine to produce desmosomes (Neunlist et al., 2013)

2.2.1 Tight junctions

The primary driver of mucosal permeability is the apical membrane tight junction complex Tight junctions act as a rate-limiting factor for paracellular permeability by selectively limiting solute flux through the intestinal epithelium There are two major paths for transporting substances through the epithelium in the intestine: the transcellular pathway, which allows material to flow through cellular membranes, and the paracellular pathway, which allows substances to pass via tight junction complexes (Shen et al., 2011) This project is focused on understanding the paracellular pathway in the GI tract in a mouse model of autism

The paracellular pathway is selectively permeable due to the presence of different types of proteins

at tight junction complexes, which separate molecules traveling through intercellular spaces based

on their size and charge The three main types of tight junctional proteins are transmembrane proteins, scaffolding proteins such as Zonular occudens and regulatory proteins (Odenwald and Turner, 2017) Transmembrane proteins comprise the pore-forming elements of tight junctions The well-studied adhesion molecules occludin, claudins and tricelluin are integral transmembrane proteins (Furuse et al., 1998, Furuse et al., 1993, Ikenouchi et al., 2005) Claudins are considered the most critical transmembrane proteins contributing to mucosal permeability, as they form a pore between neighbouring cells to govern tight junctional ion selectivity (Van Itallie and Anderson, 2006) Scaffolding proteins (including occludins) play an important role in cell signalling pathways by bringing together many binding partners to enhance their coordinated interactions and functions The zonula occludens (ZO) proteins, which have three different forms in mammals, are some of the most well-studied scaffold proteins located on the cytoplasmic side of the epithelial tight junction (ZO-1, ZO-2, ZO-3) (Stevenson et al., 1986) Through binding to claudins and other occludins, as well

as phosphoinositides located within the plasma membrane, ZO proteins are targeted to the plasma membrane and assist in regulating pore size (Stevenson et al., 1986) Although the precise function

of occludin proteins in barrier modulation is unknown, both cell culture and animal model studies show that occludin is critical for tight junction construction and barrier function (Findley and Koval, 2009) This is evident from recent research using siRNA to reduce translation of all tight junction occludin mRNA in male C57BL/6 mice This enabled intestinal permeability to be investigated which demonstrated a selective increase in intestinal epithelial cell paracellular permeability in the absence

of occludins These findings suggest that occludins play a role in maintaining tight junctions (Al-Sadi

et al., 2011)

Figure 6: Intestinal epithelial cells utilise both transcellular and paracellular pathways Highlights

the two main pathways that metabolites can translocate in gut epithelial cells Finger-like projections represent microvilli on the luminal side of the cellular membrane The flat basolateral membrane is illustrated at the base of this diagram Note that the transcellular pathway enables small molecules such as mannitol (182 Da) to pass across the epithelium whereas the paracellular pathway is permeable to larger molecules (e.g., lactulose which is approximately 340 Da in size) (Vojdani, 2013)

2.3 Gastrointestinal distress and ASD

Increased intestinal permeability has been observed in both Individuals with ASD and animal models To date, the majority of clinical evidence comes from sugar permeability tests, which examine the urine collection of two sugars (lactulose and mannitol) with distinct molecular sizes and absorption pathways (D'Eufemia et al., 1996, de Magistris et al., 2010, Souza et al., 2012) Mannitol

is due to abnormal levels of intestinal tight junction proteins and a dysfunctional paracellular pathway (de Magistris et al., 2010) Even in the absence of GI problems, children with autism reportedly have relatively high intestinal permeability (i.e., compared to age-matched controls) (D'Eufemia et al., 1996) Sugar permeability experiments also reveal changes in intestinal permeability in first-degree relatives of people with ASD, implying the presence of genetic factors in these families that could affect tight junction protein levels (de Magistris et al., 2010) ASD patients and animal models of ASD have altered expression levels of genes that encode tight junction proteins, suggesting that this contributes to a disruption in mucosal barrier functionality at the cellular level For example, ASD patients have been found to have altered expression of tight junction proteins in the blood-brain barrier and in the intestinal epithelial apical junction complexes (Fiorentino et al., 2016) Claudin-12 and claudin-5 levels were higher in cerebellar and post-mortem cortex tissue samples from those patients, while MMP9, Claudin-3 and tricellulin were higher in cerebral cortex tissue Tricellulin, claudin-1 and occludin expression levels were decreased, while claudin-15, claudin-2 and claudin-10 expression levels were elevated in the gastrointestinal tract (Fiorentino et al., 2016) A decrease in expression of genes encoding occludin, zonulin-2, claudin-8, and zonulin-581, as well as increased claudin-15 expression, was observed in offspring of a maternal inflammatory activation mouse model of autism, similar to findings in patient tissue samples (Hsiao

et al., 2013) Despite reports of hyperpermeability, no research has been done to determine which region of the small or large intestine may demonstrate increased permeability in transgenic mouse models of ASD Understanding which region of the GI tract contributes to alterations in permeability can shed light on biological mechanisms contributing to GI dysfunction (for example in the context

of ASD), given that each gut region has different anatomical and physiological characteristics

2.4 Restoration of intestinal permeability

The mucosa of the digestive tract is lined with multifunctional, rapidly growing epithelial cells They serve as the principal gateway between luminal contents and interstitial tissue These cells receive nourishment from both luminal and systemic sources and are influenced by intra- and extra-luminal nutrient intake as reviewed in (Herath et al., 2020) During a typical lifetime, 60 tonnes of food travel through the gastrointestinal tract, providing a constant threat to the integrity of the gastrointestinal tract and the host organism overall (Kårlund et al., 2021) Proteases, dietary components, medications, bacteria, intestinal ischemia, bacterial toxin exposure, microbial degradation, cytotoxic agents and the presence of pro-inflammatory cytokines such as IFN and TNF typically cause mild damage to the tight junction proteins (Anderson and Van Itallie, 1995, Kårlund et al., 2021, Mitic and Anderson, 1998) An inability to increase tight junction protein expression can be harmful as it can result in multiple organ dysfunction, chronic low-grade inflammation and sepsis (Blikslager et al.,

2007, Gonzalez et al., 2015) Thus, to identify potential therapeutic targets, it is critical to understand the mechanisms that regulate tight junction complexes and gene expression during intestinal epithelium repair Numerous innovative medications have recently been investigated relevant to restoration of tight junction protein expression For example, monoclonal antibodies with high affinity and specificity for claudin receptors promote claudin protein production (Singh et al., 2017) Commonly used, safe, and affordable supplements have also been assessed to ascertain if these can reduce intestinal permeability For instance, decaffeinated coffee has been shown to stimulate the expression of tight junction proteins and aid in weight loss in a rat model of obesity (Caporaso et al., 2016) Additionally, the herb known as marshmallow root (Althaea officinalis) that is indigenous to

protective GI tract barrier through increased mucus production (Ruszczyński et al., 2014, Zaghlool et al., 2019) Specifically, it is unknown if mice expressing the R451C mutation encoding Neuroligin-3 have enhanced intestinal permeability or if this is restricted to specific gut regions

2.4.1 The impact of L-glutamine on intestinal permeability

L-glutamine is the most prevalent amino acid in human blood, and skeletal muscle (Achamrah et al.,

2017, Decker, 2002, Deters and Saleem, 2021, Rao and Samak, 2012b) It is involved in numerous physiologically significant metabolic processes; as an intermediary in energy metabolism and as a substrate for the production of peptides and non-peptides such as nucleotide bases, glutathione, and neurotransmitters (Albrecht et al., 2010, Amores-Sánchez and Medina, 1999) Under normal physiological conditions, L-glutamine is a non-essential amino acid and the body produces sufficient amounts In the case of severe infections, physical trauma, specific disease states, radiation-induced damage, and serious burns, however, physiological L-glutamine levels are insufficient and should be supplemented with dietary L-glutamine Once glutamine reserves are exhausted, the gastrointestinal lining becomes more susceptible to injury (Rao and Samak, 2012a) Additionally, L-glutamine aids in the elimination of ammonia and in maintaining a healthy acid-base balance in the body (Patience, 1990) The gut consumes around 30% of total L-glutamine (Wu, 1998), demonstrating that it is a critical nutrition source for the intestine Three-quarters of enterally administered L-glutamine is absorbed into splanchnic tissues, and the majority of absorbed L-glutamine is processed in the small intestine (Newsholme and Carrié, 1994) When plasma L-glutamine passes through the small intestine, one-fourth of it is absorbed (Hankard et al., 1995) The intestine competes with other tissues for L-glutamine obtained from the body's amino acid pool and dietary sources (Evans and Shronts, 1992) L-glutamine protects tight junction proteins in three key ways including: i) preserving intestinal tissue integrity, ii) producing anti-inflammatory mediators, and iii) preventing apoptosis

and cellular stress (Figure 7) (Kim and Kim, 2017) As previously described, tight junctions connect adjacent epithelial cells to form a physical barrier between epithelial and endothelial cells (Bjerknes and Cheng, 2005) Evidence suggests that a sub-population of individuals with ASD have increased intestinal permeability, however drugs or supplements that may restore increased intestinal permeability in this population are understudied and correlations of increased GI permeability in different intestinal regions in animal models of ASD are not well established

Figure 7: Potential mechanisms for L- glutamine restoring paracellular permeability in IEC’s

Glutamine preserves intestinal tissue integrity by facilitating enterocyte proliferation, activating mitogen-activated protein kinases (MAPKs) (JNK and ERK1/2), optimisation of growth factors insulin-like growth factor (IGF), (TGF)-α), transforming growth factor (TGF), (epidermal growth factor (EGF) and stimulating the expression of tight-junction (TJ) proteins (zonula occludins (ZO)-1, ZO-2, and ZO-

3, claudin-1, occludin and claudin-4 Glutamine inhibits pro-inflammatory signal transduction factors such nuclear factor-B (NF-B) and signal transducers and activators of transcription Glutamine inhibits widespread apoptosis by contributing to the production of glutathione (GSH) and by modulating the heat shock factor-1-(HSF-1) induced expression of heat shock proteins (HSPs) Glutamine reduces endoplasmic reticulum (ER) stress and induces autophagy by suppressing the mechanistic target of rapamycin (mTOR) pathway, thereby protecting enterocytes against stressful cellular conditions The

T bars denote inhibition, whereas the arrows reflect activation Kim and Kim, (2007)

2.4.2 The impact of caffeine on intestinal permeability

Caffeine is a common component in coffee, energy beverages, and food supplements Caffeine stimulates the CNS, increasing alertness and producing anxiety and restlessness in susceptible individuals It relaxes smooth muscle, promotes cardiac muscle contraction, and improves athletic performance (Nehlig et al., 1992) Caffeine stimulates gastrointestinal motility and stomach acid

secretion (Liszt et al., 2017) In vitro research (e.g., in animal models or cell systems) has mostly

examined the complex pharmacology of caffeine's effects and identified that caffeine is an adenosinergic antagonist via a non-selective pathway (Institute of Medicine Committee on Military Nutrition, 2001) In addition, caffeine raises the amount of cyclic adenosine monophosphate (cAMP)

in tissue via decreasing the action of phosphodiesterases in numerous cell types (Institute of Medicine Committee on Military Nutrition, 2001) Caffeine has been used to investigate the contractile and/or electrical properties of the various gut wall components involved in motor function along the gastrointestinal tract including the myenteric plexus (both neurons and glial cells), smooth muscle cells and ICCs, as well as their dependence on intracellular calcium dynamics (Ito et al., 1974) Various approaches, including organ baths and electrical recordings of single cultured

smooth muscle cells, have been used to examine the in vitro effects of caffeine on the gastrointestinal

tract smooth muscle in mice (Tokutomi et al., 2001) It has been demonstrated that caffeine promotes the secretion of anions by enterocytes This occurs via the RyR/Orai1/Ca2+ signalling pathway These findings show components of the RyR/Orai1/Ca2+ pathway regulated by caffeine may provide novel potential therapeutic targets for the regulation of intestinal anion secretion (Wei

2.4.3 The impact of fasting on intestinal permeability

Short-term fasting has comparatively few negative side effects and can be advantageous for persons who are generally healthy and wish to control their weight (Alscher et al., 2001) During short-term fasting by utilising the nutrients and electrolytes received by the intestinal epithelium, the requirements of the body's metabolism are met (Ferraris and Carey, 2000) However, fasting deprives the body of essential nutrients and can cause electrolyte imbalances, both of which affect physiological homoeostasis The digestive system is the first organ system to be impacted by changes

in nutrient intake, and undergoes the most rapid and severe adaptations in response to dietary shortage (Ferraris and Carey, 2000) These modifications may increase or decrease intestinal permeability Before evaluating the effect of fasting on gastrointestinal permeability in humans, it is necessary to ascertain the duration of abstinence from eating Recent reports indicate that the amount of protective mucus coating the lumen of the digestive tract is correlated to the duration of time spent fasting (Alscher et al., 2001, Ferraris and Carey, 2000, Mohr et al., 2021) In contrast, it is commonly accepted that permeability is increased when the mucus barrier is weakened (Figure 8), such as following overfeeding or after consuming a high-fat diet which results in elevated inflammatory effects and alterations in the mucus barrier (Mohr et al., 2021) If a person fasts for less than two days, mucus production has been shown to increase Consequently, under these conditions, IECs will be better shielded from physical damage and potential pathogenic invasion and

GI permeability will be reduced (Mohr et al., 2021) When an individual fasts for more than two days, endogenous bacteria in the stomach begin to break down mucus in order to meet the energy needs

of microorganisms, resulting in increased intestinal permeability (Mohr et al., 2021)

Figure 8: Overeating and duration of food deprivation influences intestinal mucus composition

The potential impact of fasting/feeding cycles on gut function include increased permeability as a result of high fat diets and overeating whereas short term fasting and potentially longer term fasting can lead to reduced GI permeability SCFA: short chain fatty acids; HFD: high-fat diet; LPS: lipopolysaccharide; TJ: tight junction; LPS: lipopolysaccharide; BA: bile acid; TMAO: trimethylamine N-oxide; GI: gastrointestinal Mohr et al., (2021)

3.0 Classification of enteric neurons

The enteric nervous system is the largest division of the autonomic nervous system, with over 400 million neuron-supporting glial cells and over 100 million neurons that lie within the walls of the gallbladder, biliary tree, small and large intestines, oesophagus, stomach, pancreas as well as nerve fibres that link these nerve fibres ganglia and that supply the mucosal epithelium, gut wall muscle and arterioles (Furness, 2006) The normal functioning of the digestive system is dependent on this complex neuronal system Enteric neurons are classified based on their morphology, electrophysiology, function and neurochemistry (Furness, 2006)

3.1 Morphological classification of enteric neurons

Enteric neurons were initially characterised by morphological traits as three types of neurons: Dogiel type I, II and III neurons (Brehmer et al., 1999, Dogiel, 1895) Dogiel type I neurons have small cell bodies with short dendrites and a single axon and include ascending and descending interneurons,

as well as inhibitory and excitatory motor neurons (Furness, 2006) In mice, Dogiel type II neurons make up 10-20% of myenteric neurons and are multiaxonal Dogiel type II neurons could be absent from the sub-mucosal plexus (Mongardi Fantaguzzi et al., 2009) which could impact intestinal permeability due to the several innervations However, other authors suggest Dogiel type II neurons are present in the submucosal plexus (Furness et al., 2003) Dogiel type III neurons feature large smooth cell bodies that are oval or spherical, with numerous axons that run circumferentially (Bornstein et al., 1991, Foong et al., 2012, Furness, 2006, Nurgali et al., 2004) Use of other methods such as dye injections and immunohistochemistry enabled the identification of additional neuronal morphologies, thus expanding on Dogiel's classification to include type IV, V, VI, and VII neurons, as well as mini neurons (Brehmer et al., 1999) Type IV neurons are uni-axonal and have short, branching, tapering dendrites that extend vertically Type V neurons are uni-axonal and have

long branched dendrites, forming clusters Dogiel neurons of type VI have a single axon and fine dendrites (Furness, 2006)

3.2 Electrophysiological classification of enteric neurons

Enteric neurons are divided into two broad groups based on their electrical features: synaptic neurons (S-neurons) and after-hyperpolarisation (AH) neurons S-neurons display short action potentials lacking slow after-hyperpolarizing potentials and rapid excitatory postsynaptic potentials

AH neurons are characterised by large action potentials with an inflection on the falling phase, followed by extended after-hyperpolarizing potentials (Hirst et al (1974)

3.2.1 Synaptic-neurons (S-neurons)

S-neurons exhibit fast excitatory post-synaptic potentials (fEPSPs) (Hirst et al., 1974) Other characteristics that distinguish S-neurons include: i) a larger proportion of membrane potential depolarisation events than AH-neurons ii) hyperexcitability (due to lack of afterhyperpolarisation) iii) when depolarising pulses are applied, lengthy spike trains of action potentials are elicited that are amplitude dependent (Nurgali 2009)

3.2.2 AH (after-hyperpolarisation) neurons

Microelectrodes were initially used to study the electrical properties of sensory neurons in the ENS which were named intrinsic primary afferent neurons (IPANs, or intrinsic sensory neurons; ISNs) IPANs were dubbed AH-type neurons due to their action potentials exhibiting a significant AHP component Functional and expression features typical of AH neurons include: i) a greater hyperpolarized membrane potential than S-neurons ii) expression of IK channels, and a iii) hump in

the action potential falling phase (Li, 2022))In AH neurons the rising phase of action potentials are

carried by Na+ and Ca2+ ions and a shoulder are evident in the falling phase (North 1973) Similar to many neuron subtypes in the CNS, the falling phase is modulated by voltage- and time-dependent K+channels The presence of the AHP component, which stops continued excitation of the neuron for

up to 30 seconds, is critical to the action potential properties of AH neurons

3.3 Functional classification of enteric neurons

There are approximately 20 distinct types of enteric neurons, with the numbers varying slightly between intestinal regions and animal species Each type is defined by a combination of characteristics (projections to targets, functional roles, morphology, neurochemical properties, cell physiology and electrophysiological properties) Three functional classifications can be distinguished among the twenty types; i) IPANs (or ISNs), ii) motor neurons and iii) interneurons IPANs can detect both the physical state of organs (for instance, tension in the intestinal walls) and the chemical composition of luminal contents IPANs respond to these signals by initiating reflex regulation of blood flow, motility and secretion IPANs communicate with one another and directly with motor neurons and interneurons Interneurons interact with motor neurons and other interneurons Muscle motor neurons, motor neurons to enteroendocrine cells (EECs) and neurons innervating lymphoid follicles, secretomotor neurons, secretomotor/vasodilator neurons are all examples of motor neurons (Furness et al., 2014) This research project aimed to assess the functional characteristics of myenteric neurons of the mouse duodenum using whole cell patch clamp recording

as an effort to contribute to future more detailed classification of neuronal subtypes using multiple methodological approaches

Figure9: Three major functional classes of enteric neurons (Intrinsic primary afferent neurons; also

known as Intrinsic Sensory Neurons (ISNs), interneurons, and motor neurons To influence GI

function, IPANs synapse with other interneurons, motor neurons, and interneurons Interneurons and motor neurons also communicate with one another via synapses (Furness, 2012)

3.3.1 Intrinsic primary afferent neurons (IPANs)

Intrinsic primary afferent neurons (IPANs) or intrinsic sensory neurons (ISNs) sense the state of the

GI tract By encoding information about the chemical environment of the gut lumen and the state of the tissue they innervate, these neurons communicate with the enteric neuronal circuitry to modulate gut function IPANs detect a number of stimuli, including luminal chemicals, intestinal wall force/strain, mucosa deformation, and activate intrinsic reflex pathways to regulate physiological activities such as gut motility, secretion, and blood flow (Furness et al., 2014) IPANs have a large smooth cell body and numerous axons and correspond to a Dogiel type II morphology Electrophysiological features of these neurons include extended after hyperpolarizing potentials (AHPs) (Kunze et al., 1995, Bertrand et al., 1997) IPANs form synaptic connections with various types

of myenteric neurons, including interneurons and motor neurons, as well as with other IPANs (Furness et al., 2004) Within the submucosal plexus, Dogiel type II neurons communicate with neurons in the myenteric plexus and other submucosal plexus neurons According to a study using combined electrophysiological and neurochemical techniques, IPANs are not detected in the mouse