MEDIATES BRAINSTEM CONTROL OF RATE IN THE

DIVING

A Dissertation

Submitted to

The Temple University Graduate Board

In Partial Fulfillment

of the Requirements for the Degree of

Doctor of Philosophy

By

Fan Yang

May, 2012

Examination committee members: Dr. Nae J Dun (advisor), Dept. of Pharmacology, Temple University Dr. Alan Cowan, Dept. of Pharmacology, Temple University Dr. Lee-Yuan Liu-Chen, Dept. of Pharmacology, Temple University Dr. Gabriela Cristina Brailoiu, Dept. of Pharmacology, Temple University Dr. Parkson Lee-Gau Chong, Dept. of Biochemistry, Temple University Dr. Hreday Sapru (external examiner), Depts. of Neurosciences, Neurosurgery & Pharmacology/, UMDNJ-NJMS.

i

©

2012

By

Fan Yang

All Rights Reserved

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ABSTRACT

AMYLIN’S ROLE AS A IN THE BRAINSTEM

Fan Yang

Doctor of Philosophy

Temple University, 2012

Doctoral Advisory Committee Chair: Nae J Dun, Ph.D.

Amylin, or islet amyloid polypeptide is a 37- member of the

family. Amylin role in the brainstem and its function in regulating heart rates is

unknown. The is a powerful autonomic reflex, however no

have been described to modulate its function. In this thesis study, amylin expression in the brainstem involving pathways between the trigeminal ganglion and the nucleus

ambiguus was visualized and characterized using immunohistochemistry. Its functional

role in slowing and also its involvement in the diving reflex were elucidated

using stereotaxic microinjection, whole-cel patch-clamp, and a rat diving model.

Immunohistochemical and tract tracing studies in rats revealed amylin expression

in trigeminal ganglion cells, which also contained vesicular glutamate transporter 2

positive. With respect to the brainstem, amylin containing fibers were discovered in

spinal trigeminal tracts. These fibers curved dorsally toward choline acetyltransferase

immunoreactive neurons of the nucleus ambiguus, suggesting that amylin may synapse to

parasympathetic preganglionic neurons in the nucleus ambiguus. Microinjection of

fluorogold to the nucleus ambiguus retrogradely labeled a population of trigeminal

ganglion neurons; some of which also contained amylin. In urethane-anesthetized rats,

stereotaxic microinjections of amylin to the nucleus ambiguus caused a dose-dependent iii

that was reversibly attenuated by microinjections of the selective amylin

antagonist, salmon calcitonin (8-32) (sCT (8-32)) or AC187, and abolished by

bilateral vagotomy. In an anesthetized rat diving model, diving bradycardia was attenuated by glutamate receptor antagonists CNQX and AP5, and was further suppressed

by AC187.

Whole-cel patch-clamp recordings from cardiac preganglionic vagal neurons

revealed that amylin depolarizes neurons while decreasing conductance. Amylin also

resulted in a reduction in whole cell currents, consistent with the decrease in conductance.

Amylin is also found to increase excitability of neurons. In the presence of TTX,

spontaneous currents in cardiac preganglionic vagal neurons were observed to decrease in frequency in response to amylin while amplitude remained constant, signifying that amylin reduces presynaptic activity at cardiac preganglionic vagal neurons. Finally, evoked synaptic currents revealed that amylin decreases evoked currents, further demonstrating that amylin depolarization and increase in excitability of cardiac preganglionic vagal neurons is also associated with simultaneous inhibition of presynaptic transmission.

Our study has demonstrated for the first time that the bradycardia elicited by the diving reflex is mediated by amylin from trigeminal ganglion cells projecting to cardiac preganglionic neurons in the nucleus ambiguus. Additionally, amylin results in the depolarization and increased excitability of cardiac preganglionic vagal neurons while

inhibiting presynaptic transmission.

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DEDICATION

I would like to dedicate this thesis to my family, who without their endless love and support, this day would not be possible.

To my loving wife and best friend Irene Hwa Yang, thank you for supporting me and walking by my side through this journey.

To my parents, who I have the highest respect for, thank you for the never ending love and enthusiasm.

To my brother, in whom I see myself, you have taught me more about myself and about life than anyone else. Thank you.

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ACKNOWLEDGEMENTS

I would like to thank everyone who has made helped make my thesis work possible through supporting my professional development and personal growth. I would like to thank my PhD mentor Dr. Nae Dun. Through his thoughtful guidance in my thesis work, he has helped open my mind to science. Dr. Dun has exposed me to a depth of scientific thinking that has made a lasting impact in my life. His unwavering support in my work has given me the ability to develop my intellectual capability immensely. Most of all, as a mentor, Dr. Dun has helped me think out of the box and apply what I have learned not only to furthering scientific knowledge, but also to all areas of life.

The members of the Dun lab have provided a nurturing and supportive environment in which I have spent some of the most memorable years of my life. I want to thank Mrs.

Siok Le Dun for the patience in teaching me immunohistochemistry and helping me navigate through the immense resources available in the Dun Lab. Without her patience, guidance, and support, none of my experiments would be possible. I also want to thank Dr. Cristina

Brailoiu, for serving my PhD committee, and also helping me learn electrophysiology. It is the most technically challenging scientific tool I have ever mastered and it would not have been possible without her help.

I want to thank my fellow PhD students in the Dun Lab whom have taken the quest for science together- Saadet Inan, Xiaofang Huang, and Elena Deliu. Through countless failed experiments we persevered while building lifelong friendships.

I want to thank my thesis committee members for providing me with thoughtful

feedback over the years and helping nurture my love for science.

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TABLE OF CONTENTS

Pages

ABSTRACT ...... iii DEDICATION ...... v ACKNOWLEDGEMENTS ...... vi LIST OF FIGURES: ...... x LIST OF ABBREVIATIONS ...... xi CHAPTER 1: INTRODUCTION ...... 1 1.1 Importance of neural control of heart rate: ...... 1 1.2 Organization of the nervous system: ...... 2 1.3 ...... 3 1.3.1 Somatic ...... 4 1.3.2 Autonomic Reflexes ...... 4 1.3.3 Baroreceptor reflex ...... 5 1.4 Information transmission and processing ...... 7 1.4.1 Excitable membranes ...... 7 1.4.2 Membrane permeability ...... 8 1.5 Synapse ...... 12 1.6 Neurotransmitters ...... 13 1.7 Ionotropic and Metabotropic receptors ...... 15 1.8 Neuropeptides ...... 18 1.9 Neuropeptide synthesis and processing ...... 21 1.10 Neuropeptide Receptors ...... 23 1.11 Amylin: ...... 24 1.11.1 Circulating amylin ...... 24 1.11.2 Amylin the neuropeptide ...... 27 Amylin action on neurons ...... 29 1.11.3 Amylin Genetics ...... 30 1.11.4 Amylin receptor: ...... 31 1.12 Trigeminal‐vagal reflexes ...... 33

vii

1.12.1 Diving reflex and nasopharyngeal reflex ...... 34 1.12.2 Trigemino‐cardiac reflex and ...... 36 1.12.3 Brainstem pathways of Trigeminal‐Vagal Reflexes ...... 38 1.12.4 Neurophysiology of cardiac preganglionic vagal neurons ...... 40 1.13 Knowledge Gap ...... 42 1.14 Aims: ...... 43 CHAPTER 2: MATERIALS AND METHODS ...... 46 2.1 Immunohistochemistry/Immunofluorescence ...... 46 2.2 Stereotaxic microinjection ...... 53 2.3 Rat model of diving reflex ...... 56 2.3.1 Rat diving models ...... 56 2.3.2 Anesthetized rat diving model using MouseOX pulse oximeter ...... 58 2.4 Patch Clamp ...... 59 2.4.1 Introduction to patch clamp electrophysiology‐ ...... 59 2.4.2 Whole‐cel patch‐clamp of cardiac preganglionic vagal neurons ...... 62 2.5 Identification of amylin receptor components using RT‐PCR ...... 67 2.6 Statistical analysis ...... 68 CHAPTER 3: RESULTS...... 68 Amylin distribution in the trigeminal‐vagal pathway ...... 68 Pharmacological effects of amylin in the nucleus ambiguus ...... 79 Amylin neurophysiological effects on cardiac preganglionic vagal neurons ...... 88 CHAPTER 4: DISCUSSION ...... 97 4.1 Amylin in the trigeminal ganglion‐ ...... 97 4.2 Amylin and VGLUT2 in the trigeminal ganglion‐ ...... 98 4.3 Amylin containing fibers in the trigeminal ganglion project to the nucleus ambiguus ...... 99 4.4 Amylin microinjection in the nucleus ambiguus induces dose dependent bradycardia ..... 99 4.5 Amylin receptor blockade attenuates amylin induced bradycardia ...... 100 4.6 Amylin potentiates bradycardia in anesthetized rat diving model ...... 101 4.7 Amylin depolarizes cardiac preganglionic vagal neurons in the nucleus ambiguus ...... 103 4.8 Amylin increases cardiac preganglionic vagal neuron excitability ...... 104 4.9 Amylin decreases whole cell currents in cardiac preganglionic vagal neurons ...... 104 viii

4.10 Amylin decreases spontaneous excitatory post synaptic potential frequency in the nucleus ambiguus ...... 105 4.11 Amylin decreases evoked synaptic currents in the nucleus ambiguus ...... 105 4.12 Future directions ...... 106 4.13 Conclusion ...... 108 REFERENCES: ...... 111

ix

LIST OF FIGURES:

Figure 1: Neuroanatomical components of the baroreceptor reflex...... 7 Figure 2: Signal transmission across the chemical synapse...... 15 Figure 3: Schematic representation of mGluRs at the synapse...... 18 Figure 4: Families of neuropeptides based on structural similarities ...... 21 Figure 5: Schematic of signaling pathways of G-Protein Coupled receptors...... 23 Figure 6: Processing of proamylin into amylin...... 31 Figure 7: Receptor components and G-protein signaling of receptors in the calcitonin peptide family...... 33 Figure 8: Profound bradycardia induced by diving reflex in mammals...... 36 Figure 9: Amylin’s role in brainstem connections mediating the diving reflex is unclear...... 42 Figure 10: Variations of the patch clamp technique...... 61 Figure 11: Patch-clamp recording of rhodamine-labeled CPVN in the nucleus ambiguus in brain stem slices...... 64 Figure 12: Size of amylin immunoreactive neurons in the trigeminal ganglion...... 70 Figure 13: Amylin immunoreactive neurons found in the trigeminal ganglion with their fibers projecting to the nucleus ambiguus in the brainstem ...... 71 Figure 14: Double staining of VGluT2 and amylin immunoreactive neurons in the trigeminal ganglion...... 73 Figure 15: Retrograde tracer fluorogold injection into the nucleus ambiguus in the brainstem...... 74 Figure 16: Fluorogold and amylin immunoreactive neurons in the trigeminal ganglion. 75 Figure 17: Amylin and ChAT immunoreactivity in the nucleus ambiguus...... 76 Figure 18: Colocalization of amylin and rhodamine immunoreactivity in the nucleus ambiguus...... 78 Figure 19: Amylin induces bradycardia in the nucleus ambiguus that is not susceptible to desensitization...... 81 Figure 20: Amylin induced bradycardia is vagal mediated and is reversibly blocked by amylin sCT (8-32)...... 84 Figure 21: Amylin induced bradycardia is mediated by the amylin receptor...... 85 Figure 22: Glutamate and amylin antagonists decrease the amplitude of diving reflex induced bradycardia...... 87 Figure 23: Amylin depolarizes cardiac preganglionic vagal neurons in the nucleus ambiguus under clamp...... 89 Figure 24: Amylin increases excitability of cardiac preganglionic vagal neurons under current clamp with depolarizing current injection...... 91 Figure 25: Amylin induces reduction in steady state whole cell currents in cardiac preganglionic vagal neurons under current clamp...... 92 Figure 26: Amylin decreases spontaneous EPSC frequency but not amplitude...... 94 Figure 27: Amylin decreases amplitude of evoked EPSC in response to stimulating electrode placed at spinal trigeminal fibers...... 95 Figure 28: Summary diagram...... 109

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LIST OF ABBREVIATIONS

AC187 modified salmon calcitonin 8-32(Amylin antagonist) aCSF artificial cerebrospinal fluid ACTH adrenocorticotropic AP5 2-amino-5-phosphonopentanoic acid (selective NMDA antagonist) Ach BSA bovine serum albumin CALCR CALCRL calcitonin receptor like receptor CGRP calcitonin gene-related peptide ChAT choline acetyltransferase CNQX 6-cyano-7-nitroquinoxaline-2,3-dione (AMPA/Kainate receptor antagonist) CN V /cranial nerve 5 CN X /cranial nerve 10 COOH carboxylic acid CPVN cardiac preganglionic vagal neuron DAB 3, 3’-diaminobenzidine EPSC excitatory post synaptic current EPSP excitatory post synaptic potential FDA Food and Drug Administration FITC fluorescein isothiocyanate HSV herpes simplex virus GABA ɣ-aminobutyric acid GPCR G-protein coupled receptor IP intraperitoneal IPSC inhibitory post synaptic current IPSP inhibitory post synaptic potential IV intravenous L-Glu L-Glutamate mGluR metabotropic glutamate receptors mRNA messenger ribonucleic acid NA nucleus ambiguus Na+ Sodium ion NH2 amine NMDA N-methyl-D-asparate NTS nucleus tractus solitaries OCR oculocardiac reflex PACAP pituitary adenylate cyclase-activating polypeptide PBS phosphate buffered saline PC prohormone convertase POMC pro-opiomelanocortin xi

RAMP receptor acitivity modifying protein RT-PCR reverse transcription polymerase chain reaction sCT salmon calcitonin sCT (8-32) salmon calcitonin fragment 8-32(Amylin antagonist) SIDS sudden infant syndrome TCR trigemino-cardiac reflex TG trigeminal ganglion TTX tetrodotoxin (voltage-gated sodium channel antagonist) TX Triton X VGluT vesicular glutamate transporter

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CHAPTER 1: INTRODUCTION

In order to understand how amylin fits in the complex body, we must first

gain an appreciation of the complex nature in which neuropeptides play in regulating

.

1.1 Importance of neural control of heart rate: Healthy neural heart rate regulation is crucial to heart health and overall quality of

life. Understanding the neuropeptide role in mediating heart rate via the autonomic

nervous system is crucial to not only understanding of normal functioning, but will aid in

the understanding and treatment of diseased conditions. Since the vagus nerve dominates

heart rate during resting conditions, studying the vagal control of heart rate is important

to our understanding of heart rate.

Sudden cardiac death due to ventricular tachyarrhythmia is the number one cause of death in developed countries [1, 2]. An imbalance in the otherwise finely tuned control of heart rate due to reductions in cardiac parasympathetic control are associated with an increased risk for sudden death [3-7]. Prevention and termination of has been accomplished by an increase in the cardiac parasympathetic afferents via vagal stimulation in animal models [8, 9] as well as in patients [10]. Additionally, studies have shown that the application of the diving reflex, which causes parasympathetic slowing of heart rate, is an effective treatment of life threatening paroxysmal supraventricular in adults and newborn [11-15]. The diving reflex, which is one of the most

1 powerful autonomic reflexes in mammals, therefore serves as an excellent model to study neural control of heart rate.

1.2 Organization of the nervous system: The function of the nervous system is to process information to ensure survival by responding to environmental stressors and to regulate homeostasis to best achieve the goal of survival. The nervous system accomplishes this task through a hierarchal structure of neurons which serves to both integrate and transmit information. The anatomy of the nervous system is more developed than our understanding of how neurons integrate this information. It is of great importance therefore to understand how neurons integrate information using specific brain pathways and neuromodulators.

The nervous system can be anatomically divided into the central and peripheral nervous system. The central nervous system includes the brain, and the peripheral nervous system includes all the nerves and ganglia outside of the brain and spinal cord.

The peripheral nervous system can be functionally subdivided into sensory division, whose afferent nerve fibers detects stimuli in the environment and motor divisions, whose efferent nerve fibers stimulates effector cells. The sensory and motor divisions can be further divided into somatic division, and visceral divisions. The visceral division functions at an unconscious level to the various organs of the body to regulate homeostasis. The somatic division controls the skeletal muscles for locomotion and manipulation of the external environment.

The visceral motor division can be also referred as the autonomic nervous system, which contains effector neurons that regulates involuntary functions. The autonomic nervous system has three components- sympathetic, parasympathetic, and enteric. The 2

sympathetic nervous system coordinates the various systems for maximum exertion

in a fight or flight reaction. The parasympathetic nervous system organizes the various

organ systems for normal relaxed functions. The enteric nervous system is responsible for

control of digestive functions. The focus of the current study is on the brain pathways

regulating the parasympathetic nervous system in the diving reflex.

1.3 Autonomic Nervous System The autonomic nervous system can be thought of as a set of emergency protocols

that are called upon for predictable stressors in the environment that an organism is likely

to experience throughout its life. When comparing the response to each particular

stressor, the coordinated response by the body is often similar, and shares many

similarities.

The autonomic nervous system is structurally composed of preganglionic neurons that arise in the central nervous system and synapse onto peripheral ganglia that contain the postganglionic neurons which innervates the end organ. The location of the

preganglionic neurons for the parasympathetic nervous system is located in the brainstem

and sacral spinal cord, and synapse onto post ganglionic neurons in ganglia that are

located within the target organ. The preganglionic sympathetic neurons arise from the

thoracic and lumbar spinal cord, and subsequently synapse to postganglionic neurons in

the sympathetic chain ganglion. Many organs receive both parasympathetic and

sympathetic innervation- such as salivary glands, heart, lungs, bladder, and reproductive

organs. Bodily functions that are important during fight or flight conditions contain only

sympathetic innervation, such as the sweat gland, adrenal medulla, piloerector muscles of

the skin, and most blood vessels. 3

In the resting state, the parasympathetic nervous system dominates, and the body’s internal organs have priority to rest and digest. In fight or flight scenarios, the

sympathetic nervous system dominates, and the body is prepared for action. In some

circumstances such as the diving reflex, branches of both the sympathetic and

parasympathetic nervous system are active. The sympathetic nervous system causes

peripheral and hypertension, while the parasympathetic nervous system

causes bradycardia to slow down heart rate.

1.3.1 Somatic Reflexes Predictable stress upon the body may result in activation pre-programmed somatic

or autonomic reflexes. Somatic reflexes are reflexes that involve the voluntary, somatic

nervous system. Examples of these include patellar and achilles reflex, are designed to

unconsciously maintain posture and balance without the input from higher cortical areas.

The sensory neurons in these somatic reflex arcs are activated by afferent neurons in

muscle spindle which senses stretching. The signal is propagated to the spinal cord,

where it activates an efferent motorneuron that results in the contraction of the effector

muscle. This simple reflex arc demonstrates an example of the anatomical simplicity that

can serve the purposes of a specific response to the environmental stress.

1.3.2 Autonomic Reflexes Autonomic reflexes involve a wide range of physiological processes in the body, and are designed to maintain homeostasis. These autonomic reflexes use combination of sympathetic and parasympathetic branches of the autonomic nervous system to coordinate the organs of the body for a specific task. The advantage of studying autonomic reflexes is their reproducible output and relatively simple neuroanatomy, yet 4

has a complexity at the neurotransmitter/neuropeptide and receptor level that still eludes us.

1.3.3 Baroreceptor reflex The baroreceptor reflex is an example of one of the best studied autonomic

reflexes. It operates as a feedback control system that can provide for nearly 90%

compensation for transient changes in blood [16]. In this reflex, blood pressure

increases are sensed by baroreceptors located in the carotid sinus and aortic arch. The

afferent signal is transmitted to the brainstem, where the brainstem integration and

processing takes place. The resulting afferent response activates parasympathetic

slowing of heart rate, and also inhibits of sympathetic pressor response, resulting in

decreased cardiac contractility and peripheral resistance. This reflex highlights the

anatomical complexity of brainstem reflexes, the numerous neurotransmitter and

neuropeptide involvement in brainstem reflexes, and the importance of the nucleus

ambiguus as a regulatory center in heart rate control.

Neuroanatomical connections of the baroreceptor reflex- In the baroreceptor

reflex, blood pressure increases stimulate baroreceptors located in the carotid sinus and

aortic arch, which convey afferent signals to the nucleus tractus solitary (NTS), which

then activates the nucleus ambiguus [17], leading to a slowing heart rate. Fibers from

NTS also activates the caudal ventrolateral medulla (CVLM) [18] which in turn inhibits

the sympathetic center, the rostral ventrolateral medulla (RVLM) [19], resulting in a

depressor response-decreased peripheral resistance and reduced . A

schematic representation of this pathway is illustrated in Figure 1.

5

Neurotransmitter and neuropeptide modulation of the baroreceptor reflex-

This polysynaptic parasympathetic and inhibition of sympathetic nervous system through

the integration of information through discrete brainstem areas is common theme in

autonomic reflexes. Within the baroreceptor reflex, the principal excitatory and inhibitory

neurotransmitters are L-glutamate and gamma-aminobutyric acid (GABA). Other

neuropeptide modulators have been discovered over the years that modulates the activity

of neurons in this pathway, including [20], II [21],

[22], [22], and [22]. These neuropeptides are thought to increase or decrease the sensitivity in which the primary neurotransmitters activate or inhibit the postsynaptic neuron. Although the anatomical pathways for the baroreceptor reflex have been elucidated, the growing list of neuropeptides modulating the baroreceptor reflex introduces a growing complexity into our understanding of one of the most studied autonomic reflexes.

Nucleus ambiguus in the baroreceptor reflex- The cardiac preganglionic vagal neurons in the nucleus ambiguus are responsible for the parasympathetic mediated slowing of heart rate seen in the baroreceptor reflex. These cardiac preganglionic vagal neurons project through the vagus nerve to cardiac ganglia [23]. Lesions in the nucleus ambiguus almost completely attenuates the heart rate responses in the baroreceptor reflex

[24]. This highlights the importance of the nucleus ambiguus as a key brain region of cardiovascular heart rate control.

6

Figure 1: Neuroanatomical components of the baroreceptor reflex. Regulation of the parasympathetic and sympathetic nervous systems by the baroreceptor reflex occurs through the neural pathways illustrated in this schema. Adapted from [25].

1.4 Information transmission and processing

1.4.1 Excitable membranes Excitable membranes allow for signal propagation- The key feature of neurons that allows the propagation of signals is their excitable cell membranes, which are actively maintained at an negative membrane potential of -70 to -80 millivolts (mV), with the interior of the cell having more negative charge compared to the outside. The manipulation of membrane potential is the basis of all neural signals. One of the aims of this thesis project is to study amylin modulation of the excitable membranes of cardiac preganglionic vagal neurons in the nucleus ambiguus.

7

Neurons consumes massive amounts of energy to maintain its excitable membrane- Membrane potential is maintained by differing of the major ions in the cell- K+, Na+, Cl-, and Ca2+. The resting membrane potential is actively maintained using pumps such as the Na+/K+-ATPase, which was discovered by Danish

scientist Jens Christian Skou [26]. The Na+/K+-ATPase pumps 3 sodium ions out of the

cell, and 2 potassium ions inside the cell at the energy consumption of one ATP. It has

been estimated that 40% of the energy expenditure of neurons is due to this pump alone

[27]. This high energy consumption of Na+/K+-ATPase highlights the importance of maintaining excitable membranes as it serves as the underlying infrastructure that neurons use to convey signals.

1.4.2 Membrane permeability Neuronal signals are encoded via changes in membrane permeability- Once

the electrochemical gradient is maintained, the membrane becomes excitable. The

membrane potential can be increased or decreased in a predictable manner to transmit

information from one end of the axon to the other, and also between axons. Release of

neurotransmitters and/or neuropeptides by a presynaptic neuron excites the membrane

potential of a post synaptic neuron. Since the gradient does not change

much over the life of a neuron, it is the permeability differences and resulting membrane

potential changes through which signals are encoded. The effects of amylin on membrane

permeability will be examined as part of this thesis project.

Permeability changes is the sole factor in determining resting membrane

potential changes in healthy neurons- The factors that contribute to the membrane

8

potential can be modeled by the Goldman equation. The Goldman equation listed below

takes into account the relative permeability P of the ions denoted by PK, PNa, and PCl, as

well as the concentrations of the ions K, Na, and Cl inside versus outside of the cell. The

constants in the equation include R-ideal gas constant, T- in Kelvin, and F-

Faraday’s constant. In healthy neurons, the concentrations of the ions maintained at a relatively constant level throughout the life of the cell, and it is the permeability change of individual ions from opening or closing of ion channels that result in the change in the membrane potential and the propagation of signals. When the permeability of an ion increases, the membrane potential moves towards the equilibrium potential of the ion.

The resting membrane potential is primarily determined by K+ equilibrium potential since

K+ permeability is high at the resting state, while its Na+ permeability is low.

Goldman Equation:

Equilibrium potential of ions depends on the concentration and charge- The equilibrium potential of an individual ion depends on its charge, and the concentration difference between the inside and outside of the cell. This equilibrium potential is the membrane potential at which there is a balance between the of and the transmembrane electrical forces, and yields a zero net ion flow through the cell membrane. It can be calculated by the Nernst Equation-, where R-ideal gas constant, T- temperature in Kelvin, z-number of electrons, and F-Faraday’s constant.

Nernst Equation:

9

Each ion’s concentration difference between the inside and outside are the prime

+ determinants of the equilibrium potential. The equilibrium potential of K , Ek is -84 mV

assuming a concentration of 5mM outside the cell and 140mM inside the cell. Na+ equilibrium potential ENa is +40mV assuming 12 mM inside the cell and 140 mM outside

the cell. The equilibrium potential is useful in understanding how opening and closing of

individual ion channels results in membrane potential changes that effect neural signaling.

Since the ionic concentration gradients do not change drastically in a healthy individual or a healthy neuron, likewise, the equilibrium potential does not change during the lifetime of a neuron.

Hyperkalemia causes hemodynamic instability- In diseased states of ionic imbalance such as hyperkalemia where the blood and extracellular concentrations of K+ is higher than normal, the equilibrium potential of potassium is increased, resulting in a depolarization cells. In cardiac , this depolarization results in life threatening arrhythmias and hemodynamic instability, resulting in death if the hyperkalemia is not reversed. This highlights the importance the body places on maintaining its ionic gradient.

Action potentials transmit electrical signals through the length of a neuron-

When these neuron membranes are brought to a more positive resting membrane potential, an action potential can be formed, which can be propagated through the length of an axon. Action potentials are composed of a rapid rise in membrane potential, followed by a decrease to the resting membrane potential. This coordinated all or none response of neuronal membranes allows this signal to travel from the neuron’s cell body down its axon and eventually signal propagation to the next neuron. Once an action

10

potential reaches the end of the axon, it results in a calcium influx which results in

exocytosis and release of neurotransmitters onto the synapse with the post synaptic

neuron’s dendrite. Depending on the neurotransmitter that is released and the ion channel

opened as a result, the postsynaptic neuron will experience a excitatory postsynaptic

potential (EPSP), or a inhibitory postsynaptic potential (IPSP).

Neurotransmitter release by the presynaptic neuron can induce excitatory or

inhibitory postsynaptic potentials- These EPSP and IPSP usually alters the membrane

potential of neurons by a few milivolts, which is not enough to trigger an action potential on the post synaptic neuron. However, since each neuron receives input from up to thousands of other neurons, the summation of all the thousands of superimposed EPSP and IPSP could result in increase in membrane potential past the trigger membrane potential of voltage-gated sodium channels. Once the membrane potential depolarizes past the trigger potential, the voltage-gated sodium channel opens, and the membrane potential is depolarized from -70mV towards the equilibrium potential of sodium of up to

+40. Once an action potential is generated, it is propagated down the axon and the process continues at the thousands of synapses that are formed with the next postsynaptic neuron.

The pattern in which different neurons are excited depends on the neurotransmitter or neuropeptide the presynaptic neuron releases, and also the type of receptors that are present on the post synaptic neuron. For example, the neurotransmitter acetylcholine (ACh) can result in excitation of the postsynaptic neuron, or inhibition of the postsynaptic neuron. The different combinations of neural connections, along with

11

the variety of different neurotransmitters and the wide range of different receptors allows

for the multitude of signals in the nervous system.

1.5 Synapse The synapse is the location in between two neurons that allows for signals to be

communicated between them. The neuron sending the signal is the presynaptic neuron, and the neuron receiving the signal is the postsynaptic neuron. Neurons communicate with one another using two types of synapses- electrical synapse and chemical synapse.

Electrical synapse couples excitable cells via gap junctions- Electrical synapse is composed of connexons, a protein of 6 connexin components which forms a gap junction. These gap junctions are tunnels through which the cytosols of two neurons are directly connected, thereby allowing action potentials to be rapidly and synchronously propagated through the gap junction. These synapses are most commonly found in visceral smooth muscle and cardiac muscle.

Chemical synapse depends on neurotransmitter release and opening of ion channels to transmit signals- Chemical synapses refer to the 20-50nm of space between the presynaptic neuron and the postsynaptic neuron, muscle, or glandular cell where neural signals are transmitted via chemical messengers. The presynaptic neurons release neurotransmitters and neuropeptides into the chemical synapse, which diffuse and bind to receptors on the postsynaptic neuron. This signal propagation takes about 0.5 ms. This may result in changes in membrane potential, permeability, and long term cellular changes. Neurotransmitter released by the presynaptic neuron may also bind to receptors on presynaptic neuron itself, resulting in feedback loops altering the activity of the 12

presynaptic neuron. The many different neurotransmitters can be divided into small

molecule neurotransmitters, and neuropeptides.

1.6 Neurotransmitters All presynaptic neurons in the body release small molecule neurotransmitters that

are central to the chemical transmission of signals in the brain. Small molecule neurotransmitters include acetylcholine; amino acids- glutamate and gamma aminobutyric acid (GABA); biogenic amines- , , and serotonin;

Purines- ATP, ADP, AMP. In the diving reflex, the primary excitatory neurotransmitter is glutamate [28, 29].

Acetylcholine- Acetylcholine is one of the best studied neurotransmitters, and is found throughout the peripheral nervous system and the central nervous system. It is

released at the neuromuscular junction resulting in muscle contraction. It is also found in

neurons in the nucleus ambiguus in the brainstem, which controls the parasympathetic

slowing of heart rate; these fibers project through the vagus nerve to the heart, slowing

heart rate [30].

Glutamate- Glutamate is the most common excitatory neurotransmitter in the

vertebrate brain [31], and is the principal excitatory neurotransmitter in trigeminal

ganglion neurons activating the diving reflex [29].

GABA- GABA is the principal inhibitory neurotransmitter in the brain, and the

effects of alcohol mimic GABA by acting as a potentiator at the GABA receptor

throughout the brain [32].

13

Norepinephrine- Norepinephrine plays a role as a circulating hormone as well as a neurotransmitter in the brain regulating arousal and mood. Dopamine is involved in brain pathways controlling reward, and is therefore of great importance in the study of addiction.

Serotonin- Serotonin is involved in wide range of brain circuits including mood, appetite, and sleep.

Purines- ATP, ADP, and AMP are involved in a wide range of activities, including neuroprotection, neural glial interactions, control of vessel tone and angiogenesis, and pain and mechanosensory transduction.

Research in purine neurotransmitters is evidence of cotransmission, a concept pioneered in the 1970s as it became evident that ATP is always released with other neurotransmitters [33]. In addition to purine neurotransmitters, neuropeptides have also been found to be involved in cotransmission, and act as modulators of small molecule neurotransmitters [34].

14

Figure 2: Signal transmission across the chemical synapse.

Neurotransmitters/neuromodulators may bind to either ionotropic and/or metabotropic receptors. The binding of neurotransmitters (triangles) to ionotropic receptors that act as ligand-gated ion channels causes these channels to open, leading in some cases to a depolarization which in turn may generate an action potential. Neurotransmitters (circles) that bind to metabotropic receptors such as G-protein coupled recptors initiate a complex cascade of chemical events that can produce changes in cell function.

1.7 Ionotropic and Metabotropic receptors Classification- Neurotransmitter receptors can be classified into two broad categories- ionotropic receptors for fast synaptic transmission and metabotropic receptors

15

for slow snaptic transmission through intracellular second messengers. A representative

schematic is illustrated in Figure 2.

Ionotropic Receptors rapidly open ion channels- Ionotropic receptors are also

known as ligand gated ion channels, and result in channel opening upon ligand binding,

allowing ions such as Na+, K+, or Cl- to flow. Ionotropic receptors contain ligand binding

sites on ion channels. Ligand binding to an ionotropic receptor results in opening of the

ion channel, resulting in rapid membrane depolarization or hyperpolarization lasting

milliseconds. The neurotransmitter release results in short millisecond long EPSPs and

IPSPs. Excitatory neurotransmitters tend to open Na+, K+, and Ca2+ freely pass down

their electrochemical gradient. For example, all 3 types of glutamate ionotropic receptors

NMDA receptor, Kainate receptor, and AMPA receptor allow the flow of Na+, K+, and

Ca2+ to depolarize neurons.

Inhibitory neurotransmitters tend to open Cl- channels, resulting in

hyperpolarization of the membrane potential. The primary inhibitory neurotransmitter

GABA binding to the ionotropic GABAA receptor results in hyperpolarization due to

increasing Cl- conductance. This moves the membrane potential closer towards the Cl- equilibrium potential of around -70mV.

Metabotropic receptors results in long term intracellular events mediated by second messengers- Metabotropic receptors, are composed of G protein-coupled receptors, tyrosine kinases, or guanylyl cyclase receptors, which may also result in ion channel opening and closure, but may also results in long term cellular changes.

Metabotropic receptors contain ligand binding sites that are attached to G-protein coupled receptors, tyrosine kinases receptors, or guanyl cyclase receptors with effects that may

16

last much longer than milliseconds. These receptors may be indirectly linked to ion

channels through second messengers, and may also result in signal transduction pathways mediating various cell processes.

GABA may bind to ionotropic receptors, but there also exists an metabotropic receptor for GABA, the GABAB receptor [35]. This G-protein coupled receptor results in

inhibition of both N and P type voltage-gated Ca2+ channels and slow activation of

inwardly rectifying K+ channels, with a time to peak of 50-250 ms, and lasting 100-

500ms [36]. GABAB activation also results in inhibition of adenylyl cyclase, lowering

cAMP levels and resulting in further cell dependent downstream processes.

Glutamate may bind to one of its eight metabotropic receptors, which are all G- protein coupled receptors. These eight metabotropic receptors are classified into three groups. Group I metabotropic glutamate receptors include mGLUR1, mGLUR5, and tend to be located on the post synaptic neuron. Activation will stimulate phospholipase C, resulting in calcium mobilization, activation of protein kinase C, and opening of associated Na+ and K+ channels. Group I metabotropic glutamate receptors has also been found to presynaptically either inhibit [37] or stimulate [38] neurotransmitter release, and also result in long lasting changes in neuronal excitability [39]. Group II and III

metabotropic glutamate receptors tend to be located on presynaptic neurons, inhibiting

presynaptic release of neurotransmitters. As seen in Figure 3, the numerous interactions

by glutamate’s metabotropic receptor highlight the complex chemical signaling that is

possible in the synapse due to myriad of neuromodulators, their receptor, and the receptor

location.

17

Figure 3: Schematic representation of mGluRs at the synapse. Group I mGluRs (green) are typically localized postsynaptically, and promote release of glutamate (yellow circle). Group II (blue) and III (red) receptors are localized in presynaptic locations and typically inhibit release of glutamate or GABA (red circle). Ionotropic glutamate receptors are also shown in the postsynaptic terminal- N-methyl-D- asparate (NMDA), α-amino-3-hydroxyl-5-methyl-4- isoxazole-propionate (AMPA) and kainite. Activating these ionotropic glutamate receptors will lead to increases in intracellular Na+ or Ca2+, promoting cell excitability.

1.8 Neuropeptides Discovery of neuropeptides- Neuropeptides are that have direct effects on neurons in the nervous system through the activation of receptors. The role of neuroactive peptides was first reported by Fitzsimons and Epstein in the 1970s, who studied the central effects of angiotensin and in the brain [40, 41]. Fitzsimons

18

found that thirst could be induced in water satiated rats after the administration of renin

and angiotensin II. Epstein found that the same two peptides, when directly injected into

the cerebroventriclular system of the brain, could also induce thirst. This introduced the

notion that neuroactive proteins may be involved in regulation of brain function.

Neuropeptide function- De Wied and colleagues were the first to use the term

“neuropeptide” to describe these endogenous peptides synthesized in nerve cells and

involved in regulation of neural functions [42]. Since then, neuropeptides have been

discovered to be involved in the regulation and integration of a wide range of vital

functions in the brain, including learning and consolidation of memory, cognitive

processes, emotions, temperature regulation, thirst, sodium appetite, food intake,

, and secretion of hypothalamic , cardiovascular and

respiratory functions. Depending on the neuropeptide receptor location, neuropeptides

in the synapse can act as pre and/or postsynaptic neuromodulators. Therefore the

presence of neuropeptide in the synapse adds to the complexity of possible neuronal signals that can be conveyed between two neurons.

Neuropeptide classifications- Neuropeptides can be grouped based on principal site of action in the body. For example, , , and amylin are all present in

the brain as well as in the , and can therefore be classified as a brain and

pancreatic peptides. Other such as , cortocotropin releasing

hormone, and are present in both the pituitary and .

Many neuropeptides can also be grouped into families, based on a common larger precursor pre-prohormones or prohormones that are alternatively spliced or cleaved. For example, the 241 amino acid precursor pro-opiomelanocortin (POMC) can be

19

alternatively cleaved based on the enzymes expressed. In the anterior lobe of the

pituitary, PC1/3 cleaves POMC to form adrenocorticotropic hormone (ACTH) and B-

lipotropin. In the intermediate lobe of the pituitary, PC1/3 also cleaves POMC to form

ACTH and β-lipotropin. The presence of PC2 in addition to PC1/3 in the intermediate lobe further cleaves β-lipotropin to form ɣ-lipotropin and β-endorphin, and cleaves

ACTH to form α-MSH and corticotropin-like intermediate peptide. With respect to the calcitonin peptide family which includes amylin, it is unknown whether amylin or its precursors are cleaved to form any other bioactive peptides.

Neuropeptides are highly conserved across species, with major bioactive motifs preserved across peptide families. When new neuropeptides arise when comparing different species, it is frequently due to gene duplications of preexisting neuropeptides and its associated prohormones and receptors [43]. Gene duplication as a method of generating new neuropeptides may provide clues as to amylin function in relation with other members of the calcitonin gene family. Figure 4 lists a brief list of the numerous neuropeptides that have been discovered, each with a different distribution throughout the brain. Amylin, which will be discussed in depth in a later section, is part of the calcitonin

peptide family and binds to receptors that are a heterodimer of calcitonin receptor and

RAMP. The distribution of amylin in the brainstem and sensory ganglia suggested that it

may play a important role in modulating one of the most powerful autonomic reflexes

known- the diving reflex. For this reason, it is the focus of this thesis project.

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Figure 4: Families of neuropeptides based on structural similarities . 1.9 Neuropeptide synthesis and processing Many neurons synthesize, store, release both classical neurotransmitters and neuropeptides [33]. Neuropeptides tend to be 2-50 amino acids in length, and are considerably shorter than other peptides in the body, with relatively more simple three dimensional shapes. Traditional small molecule transmitters such as acetylcholine or norepinephrine can be synthesized and immediately transported into mature secretory granules [44]. Neuropeptide synthesis typically involves larger prepropeptides that are 21

posttranslational modified using endopeptidase cleavage and removal of the C-terminus using carboxypeptidases into neuropeptides.

Neuropeptides are synthesized as preprohormones- Synthesis begins in the

cytosol by the ribosome with the mRNA as a prepropeptide. The prepropeptide contains a signal peptide, which is a sequence of 15-30 amino acids which guides the insertion of the peptide fragment into the rough endiplasmic reticulum (RER). Once inside the RER, signalase, a highly conserved endopeptidase [45], removes the signal peptide portion of the prepropeptide, resulting in the propeptide. These propeptides are transported to the trans-Golgi network and subsequently the secretory graules, where proteolysis begins.

Amylin is synthesized as a 89 amino acid preprohormone [46].

Prohormone convertases cleaves peptides- Lysis by endopeptidases such as prohormone convertases further cleave the prohormone. PC1/3, PC2, PC4, PACE4, PC5,

PC6, PC7, and furin are among the identified prohormone convertases. Each prohormone convertase has a unique distribution. PC5 and PACE4 are found in the embryo, but in adults [47], PC5 is limited to the gut, endothelial, Sertoli cells, and the adrenal cortex

[48]. PC1/3 and PC2 on the other hand, are mostly expressed in endocrine and neural cells [49]. After endopeptidase cleavage, further cleavage is performed on the newly exposed C-terminal basic residues by carboxypeptidase E within secretory granules.

Amylin is cleaved by prohormone convertases PC2 [50] and PC3 [51].

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Figure 5: Schematic of signaling pathways of G-Protein Coupled receptors. Numerous ligands that can activate G-protein couple receptors. Once the ligand binds, the GDP dissociates and GTP binds to the G protein. G -protein subunits are divided into 4 subfamilies Gs, Gi, Gq and G12. Subsequent dissociation of the each G protein activates a unique set of downstream effectors. Adapted from [52].

1.10 Neuropeptide Receptors Neuropeptide receptors are G-protein coupled receptors- With very few exceptions, neuropeptide receptors tend to be G-protein coupled receptors [53]. The characteristic of these receptors are the second messengers system. Using the second messenger system, the primary signal- the neuropeptide, can result in intracellular signaling cascade to regulate cell functions. Binding of the neuropeptide to the G-protein coupled receptor results in conformation change of the receptor and activation of the G-

23

protein. This active state of the G protein can last a relatively long time- from 3-15 seconds [54]. The G protein can subsequently activate other components of the second messenger system that generate intracellular messengers, such as adenylate cyclase, and phospholipase C. These second messengers each have unique cell specific responses.

The diversity of modes of action by second messengers yields a wide range of effects in the neurons throughout the nervous system. These effects include changes in ionic permeability, as well as long lasting effects resulting from changes in gene expression.

G protein coupled receptor orphans reveal potential unidentified neuropeptides- Using identified orphan G-protein receptors as targets, previously unknown neuropeptides like has been identified [55, 56] that binds to the like G-protein coupled receptor. As the list of neuropeptide and neuropeptide receptors grows, many still remain unknown. There are approximately 550 GPCR in the human genome, and as of 2007, around 25% of which have no known ligand [57]. It is likely that many of these yet unidentified ligands will be revealed as neuropeptides.

Amylin receptor- As detailed in the following section, the amylin receptor is composed of the calcitonin receptor, a G-protein coupled receptor, heterodimerized with a receptor activity modifying protein (RAMP). This heterodimerization modifies the receptor binding affinity of G-protein coupled receptors in the calcitonin peptide family and also may play a role in the receptor trafficking behavior by the cell.

1.11 Amylin:

1.11.1 Circulating amylin Amylin the circulating hormone- Amylin, or islet amyloid polypeptide, is a 37-

amino acid peptide that is co-released with insulin into the bloodstream by pancreatic β

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cells in response to nutrient stimuli. In the blood stream, amylin is a satiety hormone

which reduces appetite [58], slows gastric emptying [59], and decreases postprandial

glucagon secretion [60]. Its association with type II diabetes in has been well

characterized [61-64]. This propensity appears to be species specific, and is seen in a relatively restricted number of mammalian species. Amyloid formation into toxic polymers by amylin in pancreatic beta cells is a characteristic of the progression of clinical diabetes [65]. The toxicity of the amyloid formation has been recently discovered to be due to mitochondrial dysfunction, and shares a common pathway with amyloid formation by Aβ in Parkinson’s disease [66].

Amylin the drug- Due to the hypoglycemic actions of amylin, the development of synthetic amylin- , has been part of clinical trials to treat diabetes [67-72]

and loss [73, 74] and has been approved by the FDA for treatment of type I and

type II diabetes. The treatment of type I diabetes using pramlintide is noteworthy since it is the only non-insulin analog approved by the FDA for treatment of type I diabetes. The extensive research conducted regarding amylin role in diabetes, and pramlintide’s treatment of diabetes has been a driving in positioning amylin in the spotlight; the scientific community has only begun its investigation of amylin role throughout the body.

Circulating amylin and the brain- In the brain, circulating amylin also activates

the circumventricular organs- an area of the brain with an absent blood brain barrier

which is dedicated to transducing the homeostatic signals in the bloodstream [75].

Specifically, post-prandial amylin release leads to activation of the subfornical organ and

the area postrema in the central nervous system [76], resulting in decreased food intake.

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In addition to its effects in the bloodstream as a satiety hormone, amylin has also found to

be synthesized de novo in neurons throughout the nervous system.

Circulating amylin and the blood brain barrier- Amylin has also been found to

cross the blood brain barrier [77, 78]. Studies on the central effects of amylin have found

that amylin may play a role as an adiposity signal informing the brain about the level of

peripheral energy stores. Infusion of amylin into the cerebral ventricles at 2pmol/h for two weeks significantly lowered body weight in rats compared to saline control, and was

found to increase energy expenditure and body temperature [79]. Amylin was also found

to enhance memory in rats [80, 81]. Its effect on locomotor activity, grooming and

rearearings (standing on hind legs) seem to be mixed. Amylin was found to decrease

locomotor activity [82], but increased rearing and grooming behavior [80]. Current

evidence shows that the role of circulating amylin that crosses the blood brain barrier is

involved in long term regulation of homeostasis and not likely to be relevant to the study

of brainstem reflexes.

Circulating amylin and the cardiovascular system- Amylin in the bloodstream

has been found to decrease blood pressure by 35mm Hg over 30 minutes and have rapid

direct vasodilatory effects in rats [83] when given a 100 µg bolus of amylin. These

changes were not associated with any epinephrine or norepinephrine changes in plasma,

nor was it associated with changes in glucose level [83]. CGRP, also in the calcitonin

gene family, causes similar decrease in mean arterial pressure, and has a potency 44 fold

compared to amylin [84]. To address possible binding to the same receptor, salmon

calcitonin (sCT), an amylin that binds to amylin but not CGRP receptors [85], did

26 not result in vasodilation [86] or hypotension [87]. This shows that amylin effects in inducing hypotention is due to binding to CGRP receptors.

Circulating amylin and heart rate- In one study by Gardiner et al [88], Long-

Evans rats infused with 0.25-2.5nmol/kg/min amylin induced dose dependent tachycardia and hypotension. However, in a separate study by Kaygisiz et al [89], tachycardia was observed in isolated rat exposed to an amylin antagonist, amylin (8-37). Further, these positive ionotropic effect were found to be completely blocked by diltiazem, a L- type Ca2+ channel blocker, and ryanodine, a sarcoplasmic reticulum Ca2+ release channel opener [90].

This differing response of amylin inducing tachycardia in whole rats in vivo, and amylin antagonist inducing tachycardia in isolate rat hearts ex vivo is unclear. As discussed previously, amylin induced hypotension is due to actions on the CGRP receptor peripherally. Previous work has also shown that CGRP induced tachycardia is due to reflex activation of the sympathetic nervous system secondary to CGRP induced hypotension [91]. Therefore, it is likely that amylin induce bradycardia, and the amylin induced tachycardia observed was most likely due to CGRP mediated hypotension and subsequent reflex tachycardia.

1.11.2 Amylin the neuropeptide Amylin is found in sensory ganglia and in the brainstem- In addition to circulating amylin, amylin has been found to be synthetized de novo in the brain. In order to determine if a protein is synthesized within a neuron, in situ hybridization can be used to visualize the distribution of mRNAs of the neuropeptide or its receptor. This

27

process uses complementary portions of RNA or DNA that is tagged with a labeled probe.

If the target protein is made by a cell, the tagged RNA or DNA will bind to the complementary precursor DNA or RNA segments of the protein of interest. In situ hybridization studies with amylin found distribution in small to medium sized nerve cell bodies in dorsal root ganglia from all levels and in the jugular-nodose and trigeminal ganglion [92]. Immunohistochemistry studies have also found amylin in the trigeminal system within the brainstem [93].

Amylin is found in sensory ganglia and may play a role as a sensory neuropeptide- Using immunohistochemical techniques, Mulder et al. discovered amylin- immunoreactivity in the brain as well as sensory ganglia in the peripheral nervous system

[92]. Using immunocytochemistry, and in situ hybridization, it was found that amylin is expressed in small to medium sized nerve cell bodies in dorsal root ganglia from all levels and in the jugular-nodose and trigeminal ganglion. The trigeminal ganglion is the sensory ganglion for the trigeminal nerve. The trigeminal nerve is responsible for sensation in the face, and therefore amylin likely mediates sensory processes sensed by the trigeminal nerve. These neurons were also found to contain the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide (PACAP). Substance

P and PACAP are both known to coexist with CGRP in sensory neurons [94, 95]. Since amylin was expressed in sensory neurons of small size that are known to give rise to C- type unmyelinated fibers, it was thought that amylin may mediate transmission of nociceptive transmission [92]. This was one of the first studies to show that amylin was not only present, but was synthesized by sensory neurons in peripheral sensory ganglia.

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However, it is not known what sensory processes amylin may mediate in these sensory

ganglia.

Amylin in the brainstem may mediate reflexes- In the brainstem, D’Este et al

[93] explored the distribution of amylin in the brainstem. Polyclonal antiserum against the peptide fragment 25-37 of the COOH-terminal of human amylin was used in the rat brainstem. It was found that amylin immunoreactive cell bodies were observed in vestibular, cochlear, trapezoid, and inner cerebellar nuclei and mesencephalic nucleus of the trigeminal nerve. Nerve fibers containing amylin were also found in the spinal trigeminal tract, NTS, and the area postrema. Amylin effects in the area postrema are

related to its anoretic effects. It was hypothesized that amylin presence in these various

nuclei of the may indicate involvement as regulator of some local

reflex [93]. For instance, various types of alarm or uncomfortable inputs may provoke

common responses, including nausea, vomiting, and food refusal. Amylin role in the

brainstem is still unclear, and amylin has not been associated with any reflexes.

Amylin action on neurons Several studies have been published on the effects of amylin on beta cells in the

pancreas, however only one study has examined the effects of amylin on neurons [96].

Due to the amyloid forming nature of amylin and Aβ, Jhamandas et al [96] examined the

effects of amylin and Aβ on rat cholinergic basal forebrain neurons under patch-clamp.

Neuronal activity of dissociated neurons from the diagonal band of Broca, a cholinergic

basal forebrain nucleus was examined. Application of 1 nM to 5 µM of human amylin

resulted in a dose-dependent reduction of whole-cell currents in a voltage range from -30

mV to +30 mV (voltage-clamp), reduced Ca2+ activated K+ conductance, and also

29

depressed transient outward and delayed rectifier K+ currents. These effects were

blocked by the amylin receptor antagonist AC187. Diagonal band of Broca contain

cholinergic and GABAergic neurons. However it was found using single cell reverse

transcription polymerase chain reaction (RT-PCR) that amylin effects were only seen in

cholinergic neurons and not GABAergic neurons.

These results suggest that amylin depolarizes cholinergic neurons in the forebrain

of rats, and that this depolarization is associated with closure of several different types of

potassium channels. Since this study was conducted in the basal forebrain neurons, it is

not known whether amylin acts via the same mechanisms in cardiac preganglionic vagal

neurons in the nucleus ambiguus.

1.11.3 Amylin Genetics Amylin is part of the calcitonin gene family, which includes calcitonin, calcitonin gene- related peptide (CGRP), , and intermedin/adrenomedullin 2. Calcitonin receptor stimulating peptide has recently been added to this family, but is not found in humans. Amylin gene [97] is on the short arm of human chromosome 12. Amylin is synthesized as a 89 amino acid preprohormone [46], which is further cleaved to form

amylin by prohormone convertases PC2 [50] and PC3 [51]. Further posttranslational

processing includes amidation at the C terminal, and formation of a disulphide loop

between cysteine-2 and cysteine-7 [63].

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Figure 6: Processing of proamylin into amylin.

The amino acid sequence of human pro-amylin with the cleavage site for PC2 at the NH2 terminus and the cleavage site for PC1/3 at the COOH terminus, indicated by arrows. The KR residues (blue) that remain at the COOH terminus after PC1/3 processing are removed by carboxypeptidase E. This event exposes the glycine residue that is used for COOH-terminal amidation. Above is a cartoon of amylin in blue with the intramolecular S-S bond between residues 2–7 and the amidated COOH terminus. Adapted from [98].

In humans, amylin is 43% similar to CGRP, and 20% homologous with adrenomedullin [99]. Calcitonin and CGRP are alternatively spliced products of the same calcitonin gene [100]. Although human amylin is not homologous to calcitonin, sequence similarities exist between human amylin and salmon calcitonin in the N and C terminal regions [87]. This similarity with salmon calcitonin has been exploited in the development of amylin receptor antagonists, which are based on salmon calcitonin fragment 8-32.

1.11.4 Amylin receptor: RAMP modifies the calcitonin receptor to bind preferentially amylin- The amylin receptor is composed of a calcitonin receptor heterodimerized with RAMP1 and

RAMP3 [101]. Receptor activity modifying protein (RAMP) are single transmembrane proteins that modifies the receptor affinity of the calcitonin receptor to bind amylin. 31

RAMP proteins modifies the calcitonin receptor (CALCR) and calcitonin receptor likereceptor (CALCRL) binding specificity and may modulate other functions such as receptor internalization and recycling and strength of activation of downstream signaling pathways [102]. As shown in Figure 7, receptors for the calcitonin peptide family are composed of combinations of CALCR or CALCRL with RAMP1, RAMP2, and RAMP3.

RAMP involved in receptor trafficking- In addition to modifying receptor binding affinity in the calcitonin peptide family, RAMP have also been found to be involved in receptor trafficking. In a study of adrenomedullin receptors 2 (CALCRL+

RAMP3), the PDZ type I motif at the C terminus of RAMP3 has been found to interact with N-ethylmaleimide-sensitive factor and cause the CALCRL/RAMP3 complex to be targeted for recycling after internalization [103]. RAMP has been found to be required for the forward trafficking of the calcium sensing receptor to the plasma membrane [104].

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Receptor Components G protein

Calcitonin CALCR Gs/Gq

Amylin1 CALCR + RAMP1 Gs

Amylin2 CALCR + RAMP2 Gs

Amylin3 CALCR + RAMP3 Gs

Calcitonin gene CALCRL + RAMP1 Gs/Gq related peptide

Adrenomedulin1 CALCRL + RAMP2 Gs

Adrenomedullin2 CALCRL + RAMP3 Gs

Figure 7: Receptor components and G-protein signaling of receptors in the calcitonin peptide family. The members of calcitonin gene family bind to receptors that all share the calcitonin gene receptor (CALCR), calcitonin receptor like receptor (CALCRL), and one of the receptor activity modifying proteins- RAMP1, RAMP2, of RAMP3. These G-protein coupled receptors also all signal through Gs and/or Gq

1.12 Trigeminal‐vagal reflexes Trigeminal-vagal reflexes include four physiological reflexes which are among

the most powerful autonomic reflexes observed in the human body. Diving reflex, nasopharyngeal reflex, trigemino-cardiac, and oculo-cardiac reflexes all result in profound bradycardia. Diving reflex and nasopharyngeal reflex serve to maintain oxygenation of the brain and heart during environmental conditions during which is not possible or is not safe for the organism, such as diving underwater or under exposure to toxic fumes. The trigemino-cardiac and oculo-cardiac reflexes have been observed during cranial-facial surgeries where the trigeminal nerve is inadvertently stimulated or irritated, resulting in life threatening arrhythmias or asystole. 33

1.12.1 Diving reflex and nasopharyngeal reflex Cardiovascular system under stress enacts emergency protocols- The

cardiovascular system maintains homeostasis by perfusing the body tissue with a constant supply of oxygenated blood. When external factors alter the supply or the internal metabolic demands of the body, the cardiovascular system must provide adequate oxygenation to crucial organs in the midst of changing supply of oxygen. The nervous system is designed to ensure that vital organs such as the heart and the brain maintain an adequate oxygenation. The neurons in the nervous system are therefore organized and

programmed in a manner which guarantees maximum survival of the individual. The

sympathetic nervous system and parasympathetic nervous system are designed to regulate

this system to meet changing metabolic demands.

Ischemia tolerance via the diving reflex- The brain and heart are two of the

most metabolically active parts of the body that are extremely sensitive to . It is

therefore crucial for the body to employ strategies to maintain oxygenated blood flow to

these areas to preserve life. The most powerful autonomic response in the body that acts

as an ischemic tolerance mechanism is known as diving reflex. The diving reflex involves

concurrent activation of the parasympathetic and sympathetic branches of the autonomic

nervous system. The diving reflex elicits involuntary to prevent inhalation of water,

parasympathetic mediated bradycardia [105], and non-reciprocal sympathetic mediated

vasoconstriction to reduce peripheral blood flow and to increase cardiac contractility. The

result of this synergistic activation of both branches of the autonomic nervous system is

reduced blood flow to non-vital organs through selective vasoconstriction and decreased

cardiac metabolism mediated by bradycardia. This leads to a systemic decrease in oxygen

34

usage and redistribution of oxygen rich blood to the heart and brain [106-110]. As an

example of the magnitude of the dive reflex in mammals, the heart rate of the gray seal

can decrease from 120bpm to average of 6.5bpm for 14 minutes (Figure 8).

Water and smoke triggers the diving reflex- The diving reflex is stimulated with cessation of respiration and maximally stimulated with concurrent face immersion in cold water [105]. The nasopharyngeal reflex is a variant of the diving reflex that is elicited by nasopharyngeal stimulation such as by smoke or irritant gases, and results in apnea, hypertension, and bradycardia [111, 112]. Although the environmental conditions triggering the nasopharyngeal reflex (smoke) differ from the diving reflex (cold water), the autonomic changes that are elicited are very similar. In both reflexes, trigeminal fibers are stimulated, resulting in apnea and bradycardia. Despite the natural protective functions of the diving reflex, there has been mounting evidence that exaggerated activation of the diving reflex in infants is one of the primary causes of sudden infant death syndrome (SIDS) [113, 114]. This area of research highlights the importance in understanding the diving reflex so that we may better treat disease processes such as

SIDS.

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Figure 8: Profound bradycardia induced by diving reflex in mammals. The figure above shows an example of the profound heart rate changes in gray seal during diving at sea obtained by radiotelemetry. Arrows mark the start and end of the dive. During this particular dive, heart rate averaged 6.5 beats per min and was below 4 beats per min for 90% of the time.

1.12.2 Trigemino‐cardiac reflex and oculocardiac reflex Reflexes seen in craniofacial surgeries mimic the diving reflex- The trigemino- cardiac reflex (TCR) and the oculocardiac reflex (OCR) are physiologically similar to the diving reflex and are seen in various types of craniofacial surgeries and ocular surgeries.

In both reflexes, the afferent branch of the reflex is mediated by the trigeminal nerve, and the efferent branch of the reflex both result in bradycardia.

TCR and OCR may result in life threatening arrhythmias and asystole- The

TCR was first seen in rabbits and is manifested by a predominant parasympathetically mediated bradycardia up to asystole, arterial hypotention, apnea, and gastric hypermotility [115]. The oculocardiac reflex (OCR) is a variant of the TCR. The OCR is observed mainly in strabismus surgery, and may cause bradycardia, arrhythmias [116,

117], and cardiac arrest [118-121]. The cardiac arrest has been observed at a rate as high 36

as 1 in 2200 cases during strabismus surgery [122, 123]. The OCR may be induced by

mechanical stimulation of ocular and periocular structures innervated by the ophthalmic

division of the trigeminal nerve [122]. While the absence of arterial hypertension

suggests the lack of a sympathetic component to the TCR and OCR, ectopic beats

suggestive of sympathetic activation is seen in patients undergoing squint surgery whose

vagal tone is pharmacologically suppressed [124].

Diving, nasopharyngeal, TCR, OCR all result in trigeminal-evoked

bradycardia- At first glance, the TCR and OCR seen in surgical procedures in humans

may not appear physiologically related to the diving reflex, however, both reflexes excite

similar autonomic pathways resulting in pronounced bradycardia. This similarity

highlights the importance of the brainstem pathways which underlie both set of reflexes.

A better understanding of these reflexes may specifically help in preventing the

dangerous bradycardia and cardiac arrest that may result from TCR and OCR during

cranial-facial surgeries.

Trigeminal-vagal reflexes as a model for studying the neural control of

cardiovascular system- These trigeminal-vagal reflexes are part of a set of organized

ischemic survival strategies seen in humans and other mammals to help tolerate hypoxia.

While the trigeminal stimulation and vagal outflow is known, the brainstem pathways

mediating these reflexes are not known. Not only will deciphering these pathways help in

understanding of normal ischemic tolerating mechanisms, but will also equip us with the

knowledge to develop treatments and cures for hypoxic disease states such as

cerebrovascular accidents, myocardial ischemia, sleep apnea, chronic bronchitis, emphysema, recurrent in premature babies, and sudden infant death syndrome.

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1.12.3 Brainstem pathways of Trigeminal‐Vagal Reflexes Trigeminal nerve stimulation activates the diving reflex- Although the afferent

input and the efferent output for the trigeminal-vagal reflexes are currently known, the

intermediate brainstem pathways mediating these reflexes are unknown. Studies of the

diving reflex and trigemino-cardiac reflex have shown that the sensory afferents travel

exclusively by the trigeminal nerve [111, 122, 125, 126]. It has also been shown that the

circuitry for the diving reflex is intrinsic to the brainstem because cardiorespiratory

responses were maintained in decerebrate preparations [127]. Although it is known that

the trigeminal nerve mediates neurotransmission that activates the diving reflex, the

brainstem pathways mediating this reflex are not clear.

Anterograde transneuronal tracing in the diving reflex- To elucidate the

brainstem pathways mediating the diving reflex, Panneton et al [128] used Herpes

simplex virus-1(HSV-1) strain 129 as an anterograde transneuronal tracer. The HSV-1 was introduced to the anterior ethmoid branch of trigeminal nerve in muskrats. After 2-

3 days, trigeminal projections were found in numerous brain regions, including the

nucleus ambiguus [128]. This study suggests that the nucleus ambiguus may play a role

in mediating brainstem lowering of heart rate. Since the nucleus ambiguus is the site of

cardiac preganglionic vagal neurons that project through the vagus to the cardiac ganglia

[23], this result supports nucleus ambiguus playing a role in the bradycardia in the diving

reflex.

Vagal mediated diving bradycardia key to hypoxia tolerance- While both

sympathetic and parasympathetic systems are excited by the diving and trigeminal-

38

cardiac reflexes, the heart rate component is dominated by the parasympathetic slowing

of the heart through the vagus nerve. Pharmacological blockade of the vagus nerve using

in diving muskrats has been observed to completely block diving bradycardia,

and also drastically reduce the endurance of dives [129]. This result demonstrates that not only is diving bradycardia vagal mediated, but it is crucial to the hypoxia tolerance that the diving reflex serves the body.

Cardiac preganglionic vagal neurons in the nucleus ambiguus mediates bradycardia in the diving reflex- Vagal control of heart rate originates from cardiac preganglionic vagal neurons that are localized within both the dorsal vagal nucleus and the nucleus ambiguus [130]. Cardiac preganglionic vagal neurons within the dorsal vagal nucleus have non-myelinated axons [131, 132], while the cardiac preganglionic vagal neurons within the nucleus ambiguus have myelinated axons [133]. Recent studies have found the nucleus ambiguus to be responsible for the bradycardia induced by the diving reflex [114, 134]. Evidence supporting nucleus ambiguus-induced bradycardia was found due to incremental electrical stimulation of the vagus, from which can be concluded that bradycardia was solely due to myelinated efferent fibers [135] located predominantly in the nucleus ambiguus. The nucleus ambiguus is therefore the major brainstem nucleus which is responsible for the parasympathetic vagal control of heart rate. Unfortunately, it is not known how the numerous inputs into the nucleus ambiguus are preferentially activated or inhibited during diving.

Immunohistochemistry in the diving reflex- A study using HSV-1 strain 129 as an anterograde transneuronal tracer found trigeminal projections to the nucleus ambiguus

[128]. Initial studies that spawned this thesis work using rabbit polyclonal antiserum

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directed against the human amylin have found amylin immunoreactive fibers from the

trigeminal ganglia that project to the nucleus ambiguus. Amylin immunoreactive boutons

were found juxtaposed to neurons in the nucleus ambiguus. This finding verified the

HSV-1 anterograde transneuronal tracer study that there exists a trigeminal-vagal connection that is mediated by amylin. However, many questions remain as to the role amylin and other neuropeptides may play in this trigeminal-vagal pathway.

1.12.4 Neurophysiology of cardiac preganglionic vagal neurons Retrograde tracers crucial to study of cardiac preganglionic vagal neurons-

The study of the neurophysiology of cardiac preganglionic vagal neurons in the nucleus ambiguus is made possible through the use of fluorescent retrograde tracers such as rhodamine injected into the pericardial sac of the heart 2-3 days prior to electrophysiology study [136]. Using anterograde tracing studies, these cardiac preganglionic vagal neurons project directly to cardiac ganglia [23]. Cardiac preganglionic vagal neurons have been found to control cardiac function to decrease heart rate (negative chronotrope), decrease AV nodal conduction speed (negative dromotrope), and decrease cardiac contractility (negative ionotrope) [137].

Firing properties of cardiac preganglionic vagal neurons- Mendelowitz et al.

[138] characterized the firing properties of cardiac preganglionic vagal neurons. Patch clamp was performed on brain slices with retrogradely labeled cardiac preganglionic vagal neurons with or without depolarizing currents induced. The neurons were found to be intrinsically silent. When depolarizing currents were induced, these neurons did not exhibit spike frequency adaptation. Lack of spike frequency adaptation demonstrates that action potentials can be induced without decay over prolonged periods of time. This data

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shows that cardiac preganglionic vagal neurons are silent and could be activated

synaptically for prolonged periods of time without desensitization or decay of signal intensity. This electrophysiological finding is consistent with nucleus ambiguus control of diving reflex since diving bradycardia may last for seconds up to minutes.

Nucleus ambiguus function impaired in diabetes and chronic intermittent hypoxia- Baroreceptor reflex also activates cardiac preganglionic vagal neurons in the nucleus ambiguus. Domoic acid lesions of the nucleus ambiguus were found to abolish control of heart rate [24]. Other disease processes that modify baroreflex mediated vagal control of heart rate were examined. It was found that diabetes [139], and chronic intermittent hypoxia (model for sleep apnea and aging) [140-142] impairs baroreflex function by altering nucleus ambiguus function.

Cardiac preganglionic vagal neurons in diabetic rat model- It was found that in mouse with diabetic (OVE26 mice) mothers, cardiac preganglionic vagal neurons had augmented potassium currents that contributes to after hyperpolarization and attenuated excitability [143]. However since amylin concentrations in the bloodstream are higher in diabetes [144] as well as gestational diabetes [145], the OVE26 mouse diabetes model might not be applicable since it is unknown whether amylin levels are similarily elevated in the mouse model. Current studies of cardiac preganglionic vagal neuron focusing on the effects of diabetes may not be applicable to our understanding of amylin role in the nucleus ambiguus.

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1.13 Knowledge Gap Amylin role in circulation as a hormone is well described. In the brain, the

neuropeptide amylin has been found in sensory ganglia and also in the brainstem,

however amylin role as a neuropeptide is unknown. Our initial studies found amylin in the trigeminal ganglion projecting to nucleus ambiguus neurons in the brainstem. This pathway is involved in control of bradycardia in the trigeminal-vagal reflexes including the powerful diving reflex. Understanding the diving reflex may aid in our understanding of life threatening arrhythmias. As shown in Figure 9, this reflex’s afferent and efferent limb has been established, but the brainstem connections are mostly unknown. No neuropeptide has been shown to modulate the diving reflex, and therefore this project was undertaken to further understand amylin role in the diving reflex.

Figure 9: Amylin’s role in brainstem connections mediating the diving reflex is unclear.

Figure 9: Amylin role in brainstem connections mediating the diving reflex is unclear. The afferent limb of the trigeminal-vagal reflexes involve the trigeminal nerve (CN V) and trigeminal ganglion. The bradycardia seen in the trigeminal-vagal reflexes is mediated through the vagus nerve (CN X).

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1.14 Aims: The overall aim of this thesis project is to uncover the unique role of amylin in the nucleus ambiguus and its role in modulating the diving reflex. The proposed research will use immunohistochemistry, stereotaxic microinjection, electrophysiology, and a rat diving model to study the physiologic and pharmacologic aspects of amylin in the nucleus ambiguus. The findings will further the understanding of brainstem pathways involved in the trigeminal-vagal reflexes and will also aid in the understanding of amylin in modulating these pathways. Understanding these brainstem reflexes will help reveal the body’s unique strategies used to tolerate natural states of ischemia, as well as neural control of heart rate.

Specific Aim 1: To establish the presence of amylin in the nucleus ambiguus

a. Identify amylin-immunoreactive fibers in the nucleus ambiguus.

b. Determine if cell bodies of the amylin immunoreactive fibers originate from the

trigeminal ganglion.

c. Establish the cholinergic phenotype of nucleus ambiguus neurons that receives

amylin projections.

d. Establish that amylin fibers projecting to the nucleus ambiguus synapse onto

cardiac preganglionic vagal neurons.

e. Verify if amylin immunoreactive neurons use amylin as a neurotransmitter in the

nucleus ambiguus to modulate cardiac preganglionic vagal neuronal function.

Specific Aim 2: To establish the pharmacological role of amylin in the nucleus ambiguus.

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a. Determine if amylin activates the cardiac preganglionic vagal neurons in the

nucleus ambiguus and the duration of such effects.

b. Determine if the neurons activated by amylin in the nucleus ambiguus project

directly to the heart through the vagus nerve.

c. Determine if desensitization of amylin receptors occurs with repeated treatment of

amylin in the nucleus ambiguus.

d. To determine if the pharmacological affects of amylin can be blocked by the

putative amylin receptor antagonist AC 187.

Specific Aim 3: To determine the effect of amylin on diving reflex in rats

a. Determine if diving bradycardia can be repeatedly activated in anesthetized rats

using facial immersion in water.

b. Determine if diving bradycardia is vagal mediated.

c. Determine if amylin antagonists and/or glutamate antagonists injected into the

cisterna magna will modulate diving bradycardia.

Specific Aim 4: To determine the effect of amylin on cardiac preganglionic vagal neurons in the nucleus ambiguus.

a. Determine if amylin changes the resting membrane potential of cardiac

preganglionic vagal neurons.

b. Determine if amylin modulates cardiac preganglionic vagal neuron excitability

c. Determine the current-voltage relationship of amylin-induced response on

cardiac preganglionic vagal neurons

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d. Determine if amylin modulates the signaling of synaptic inputs to cardiac

preganglionic vagal neuron in the nucleus ambiguus.

e. Determine if amylin alters the properties of pre or post synaptic membranes.

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CHAPTER 2: MATERIALS AND METHODS

2.1 Immunohistochemistry/Immunofluorescence

Immunohistochemical distribution of neuropeptides- Modern in vitro imaging of neuropeptides and its receptors are made possible using immunohistochemistry [146] and immunofluorescence [147] of fixed tissue slices. Antibodies play a central role in both immunohistochemistry and immunofluorescence, and are used for specifically binding the antigen of interest. There are direct and indirect methods, where direct uses one antibody, and indirect methods using two antibodies. In the direct method, one antibody recognizing the antigen of interest is tagged with a colored compound or fluorescent compound, which allows it to be visualized. This method is unpopular due to the lack of signal amplification compared with the indirect method, and the resulting low signal. In the indirect method, a primary antibody is used against the target antigen. This primary antibody is made by a one host animal such as a rabbit, against a target compound such as amylin. A second antibody made in a second animal such as goat, is tagged with either a chemical compound such as biotin, or a fluorescent compound. The purpose of the second antibody is signal amplification, which greatly increases the signal and ability to detect the antigen of interest. A second benefit of the indirect method is due the economical nature of being able to use different secondary antibodies tagged with different compounds to visualize the antigen. This makes highly detailed images using light microscopy possible.

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Laser confocal microscopy aids in visualizing distribution of neuropeptides-

Laser scanning confocal microscopy has a spatial resolution on the order of 100

nanometers [148], and is therefore extremely useful for visualizing the distribution of

neuropeptides within individual neurons and nerve fibers. An advantage of confocal

microscopy compared to conventional microscopy is the ability to optically excite only

one narrow X-Y plane in the Z-axis, reducing scatter and aiding in the noise free

visualization of neuropeptide distribution in the brainstem. An added benefit of using

lasers with different emission wavelengths in conjunction with confocal microscopy is

the ability to use fluorescent secondary antibodies each with its own fluorescent

excitation and emission spectra. Multiple lasers of different wavelengths are capable of

exciting the fluorescence of each antibody sequentially in isolation without disruption

from excitation of fluorescence from other secondary antibodies.

Retrograde and anterograde axonal transport- In healthy neurons, a large

fraction of total energy expenditure by the cell is devoted to managing intracellular

movement of molecules and organelles from the neuron cell body to and from the axon

terminals. Anterograde transport towards the axon terminal is mediated by kinesins and

is used in translocation of membranous organelles such as mitochondria, as well as

vesicles of macromolecules such as actin, myosin, and enzymes of neurotransmitter

synthesis. Retrograde transport towards the cell body from the axon is mediated by

dyneins and is used in translocation of neurofilaments, microtubules, and solule enzymes/materials taken up by endocytosis.

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Axonal transport used by neuronal tracers to visualize neural pathways-

Since the 1970s, neuronal tracing has revolutionized neurobiology by helping scientists

visualize neuronal pathways between brain structures. Endocytosis by axons is the cellular process which is used by neuronal tracers to be incorporated and anterograde or retrogradely transported. In addition to single neuron tracing, neurotropic alpha- herpesviruses have been used to tracing multisynaptic pathways or circuits of several neurons.

Neuronal tracing reveals cardiac preganglionic vagal neurons in the nucleus ambiguus- Retrograde neuronal tracing was also used by Izzo et al [149] to verify that cardiac preganglionic vagal neurons originating in the nucleus ambiguus controls parasympathetic slowing of heart rate [149]. In a separate study by Binder et al.[150], retrograde tracers were injected into cardiac ganglia in cats, and retrograde labeled cells were exclusively found in the nucleus ambiguus.

Trigeminal-vagal pathway and the nucleus ambiguus- While the trigeminal- vagal connection and the location of cardiac preganglionic vagal neurons has been established, no one has yet verified that these trigeminal fibers to the nucleus ambiguus synapse onto cardiac preganglionic vagal neurons. Since this trigeminal-vagal pathway is of great impotance in understanding the bradycardia seen in the diving reflex, we will use retrograde labeling to identify cardiac preganglionic vagal neurons, and examine its relationship with amylin in the diving reflex.

Animals

This investigation conforms to the “Guide for the Care and Use of Laboratory

Animals published by the United States National Institutes of Health (NIH Publication No.

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80-23, revised 1996). Experimental protocols have been reviewed and approved by

Temple University Institutional Animal Care and Use Committee. Adult male Sprague-

Dawley rats (Ace Animals Inc., Boyertown, PA), weighing 300-350 g, were used in

stereotaxic microinjection experiments, and male rats weighing 200-250 g were used for

immunohistochemical studies and anesthetized rat diving model.

Perfusion and preparation of brain tissue for immunohistochemistry

In all immunohistochemistry and immunofluorescence experiments, animals were anesthetized with urethane (1.2 g/kg, intraperitoneal injection, I.P.). The thorax was surgically opened, and a 20 gauge needle inserted into the heart. The rat was then intracardially perfused with 0.1M phosphate buffered saline (PBS) followed by freshly prepared 4% paraformaldehyde/0.2% picric acid in PBS. The inferior vena cava was cut to allow for drainage of the perfused . Rat brains and brainstem were removed, postfixed for 2 hrs, and stored in 30% sucrose/PBS solution overnight.

Amylin immunohistchemistry

In single staining, tissues were processed for amylin-immunoreactivity (irAMY) by the avidin-biotin complex procedure. Sections were incubated in a rabbit polyclonal directed against human amylin (1:1,000 dilution; Phoenix Pharmaceuticals, Burlingame,

CA) for 48 hrs, as previously described [151]. The sequence homologies between human amylin and rat amylin are significantly high at 84%, which justifies the appropriate use of human amylin. Tissues were incubated in amylin-antiserum pre-absorbed with rat amylin

(1 μg/ml) overnight as negative staining controls.

Detailed steps for indirect antibody staining of amylin using the Avidin Biotin Complex

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1. Wash for 10 min with phosphate buffered saline (PBS), then second wash with PBS+ 0.4% Triton X-100 (TX) for 20 minutes. 2. Tissues were treated with 3% H2O2 for 10 minutes to lyse and remove red blood cells 3. Wash with PBS for 10 minutes (perform this step twice). Wash with PBS + 0.25% bovine serum albumin (BSA) + 0.4% TX for 20 minutes. 4. Block nonspecific staining with 10% normal serum (dependent on animal the secondary antibody) diluted with PBS+0.5% BSA+0.4% TX for 2 hours. 5. Incubate with primary anti-amylin antibody (diluted with PBS+0.5% BSA+0.4% TX) for 2 days 6. Repeat step 3 wash. 7. Incubate with secondary antibody (diluted with PBS+0.5%BSA+0.4% TX) 8. Repeat step 3 wash. 9. Incubate 1.5 hours with avidin biotin complex diluted to 1:100 with PBS+0.4% TX 10. Develop the section with 3, 3’-diaminobenzidine (DAB) solution for 5-10 minutes to visualize staining 11. Stop DAB reaction with tris buffer, repeat wash with tris buffer overnight 12. Mount tissue with 0.25% gel alcohol subbed slide after passing through the following for 10 minutes each to dehydrate the tissue- 50%, 70%, 95%, 100% ETOH, xylene (3 times). 13. Apply cover slide with citiflour mounting medium.

Immunohistochemistry double staining

Sequential labeling with primary antibodies from different hosts was used for double-staining [151]. Immunofluorescence using different excitation and emission wavelengths allows the visualization of two different antibodies with fluorescent molecules.

To avoid non-specific cross-reactivities, none of the primary or secondary antibodies chosen were generated from rats. Medullary sections were incubated with choline acetyltransferase (ChAT) antiserum (1:200; a goat polyclonal, Chemicon) and amylin antiserum (1:500). Trigeminal ganglion sections were double stained with fluorogold antiserum (1:2000, Chemicon) and amylin antiserum (1:500), or guinea pig polyclonal vesiclar glutamate transporter 2 (VGLUT2;1:100, Chemicon) and amylin antiserum

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(1:500). Tissues were then incubated in appropriate Texas Red-conjugated IgG or fluorescein isothiocyanate (FITC)-conjugated IgG antibodies, washed with PBS, and mounted in Citifluor.

Detailed steps for double staining with FITC and Texas Red:

1. Wash for 10 min with PBS, then second wash with PBS+ 0.4% TX for 20 minutes. 2. Tissues were treated with 3% H2O2 for 10 minutes to lyse and remove red blood cells 3. Block nonspecific staining of the first primary antibody with normal horse serum diluted 1:30 with PBS+0.5% BSA+0.4% TX for 2 hours. 4. Repeat step 1 wash. 5. Incubate with first primary biotinylated antibody (antigoat, diluted with PBS+ 0.5% BSA+0.4% TX) for 2 days 6. Repeat step 1 wash. 7. Incubate with aviding FITC (diluted 1:50 with 0.1M sodium bicarbonate + 0.15M NaCl, pH=8.5) for 3 hours 8. Repeat step 1 wash. 9. Block nonspecific staining of the second primary antibody with normal donkey serum diluted 1:30 with PBS+0.5% BSA+0.4% TX for 2 hours. 10. Incubate with second primary antibody (antigoat, diluted with PBS+0.5% BSA+0.4% TX) for 2 days 11. Repeat step 1 wash. 12. Incubate with donkey antirabbit TexasRed (1:50, diluted with 0.1M sodium bicarbonate+0.15M NaCl, pH=8.5) for 4 hours. 13. Wash with PBS for 10 minutes 3 times. 14. Apply cover slide with Citiflour mounting medium.

Visualization of immunofluorescent antibodies:

Sections were examined under a confocal laser scanning microscope (Leica TCS SP2), with excitation/emission wavelengths set to 488/520 nm for FITC and 543/620 nm for

Texas Red in the sequential mode.

Retrograde tracer fluorogold microinjection to the nucleus ambiguus

Stereotaxic microinjection of the retrograde tracer fluorogold into the nucleus ambiguus was performed to identify the trigeminal neurons with fibers that project to the

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nucleus ambiguus. The detailed procedure for stereotaxic microinjection is listed in

chapter 3. Briefly, a two barreled glass micropipette containing 5mM L-Glu and 4%

fluorogold (Biotium) was stereotaxically positioned in the nucleus ambiguus. Injection of

10 nL of 5 mM L-Glu was used to verify the location of the pipette in the nucleus ambiguus – bradycardia with absence of blood pressure changes verified the location of the pipette in the nucleus ambiguus. A volume of 5 nL of 4% fluorogold solution was deposited to the nucleus ambiguus area. The surgical opening was packed with small pieces of gel foam, and the rats were surgically sutured and given 50mg/kg/day pentobarbital (Sigma-Aldrich) for five days for sedation. On the 5th day post-operation,

animals were re-anesthetized and perfused intracardially, as described above. Brainstem

and trigeminal ganglia were removed and sections were processed for

immunohistochemistry. Brainstem slices were viewed under UV light to verify injection

of fluorogold. The trigeminal ganglion sections were double-labeled with anti-amylin and

anti-fluorogold antiserum.

Quantification of size of amylin immunoreactive neurons in the trigeminal ganglion

Quantification of irAMY neurons in the trigeminal ganglion were conducted

using image analysis software (ImageJ 1.41). Images were captured at 40x magnification.

Eight hundred and seventy four neuron profiles were captured from five trigeminal

ganglion sections and were subjected to analysis. The long axis of each neuron was

measured, and mean and standard error were calculated.

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Rhodamine retrograde labeling of cardiac preganglionic vagal neurons in the

brainstem:

Retrograde labeling by injection of a fluorescent tracer into the pericardial sac of the heart has been an established method for visualization of the subset of neurons in the nucleus ambiguus that are cardiac preganglionic vagal neurons [136, 152]. Adult male rats 200-250g were anesthetized using urethane (1.2g/kg) via intraperitoneal injection. A right sided thoracotomy was performed to expose the heart. A needle was inserted into the pericardial sac and 20µL of rhodamine (XRITC, 2mg, 1% solution in saline,

Molecular Probes, Eugene, OR) was topically applied to the epicardial surface of the heart that contains parasympathetic ganglia. The incision sites were subsequently closed, and the animal was allowed 3 days to recover while the retrograde tracer was allowed to be retrograde transport up the vagus nerve.

After three days, the brainstem underwent preparation and processing for immunofluorescence analysis as described above. Amylin immunofluorescence and rhodamine immunofluorescence were visualized under confocal microscopy described above at excitation/emission spectra of 488/520 nm for amylin FITC and 572/596 nm for

Rhodamine.

2.2 Stereotaxic microinjection

Stereotaxic microinjection

Stereotaxic microinjection is the primary method to test the effect of nanoliter volumes of pharmacological compounds in localized brain areas with minimal disruption

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to brain tissue. A three dimensional stereotaxic coordinate system is used to ensure proper anatomical location of pipette location during experiments, which is adjusted in the coordinate system using a micromanipulator. Animals are fixed using the stereotaxic holder, and landmarks such as the calamus scriptorius in the brainstem are used for calibration of pipette location. Nanoliter amounts of pharmacologically active compounds are microinjected into brain areas of interest, simulating the natural physiological release of these compounds.

Microinjection of neuropeptides and neurotransmitters into the nucleus ambiguus

Microinjection of pharmacological compounds such as neuropeptides and

neurotransmitters has been used extensively in the study of cardiovascular control of

brainstem regions, including the nucleus ambiguus. Microinjection of nociceptin [153]

and glycine [154] into the nucleus ambiguus has been found to induce tachycardia in rats.

Microinjections of -1 [155], alpha- stimulating hormone [156],

dopamine [157] have been found to induce bradycardia. Heart rate in response to these pharmacological compounds provides evidence of pharmacological effects of these compounds in the nucleus ambiguus, many of which are neuropeptides.

Stereotaxic microinjection of amylin into brainstem

In order to study the effect of amylin microinjected into the nucleus ambiguus, animals were first anesthetized with isofluorane (2% in 100% oxygen). One femoral vein was cannulated for intravenous administration of urethane (1.2g/kg) and one femoral artery was cannulated for monitoring pulsatile blood pressure via a pressure transducer

(Statham P23 Db). Urethane was injected intravenously in 6-7 aliquots at 2 min intervals.

Inhalation of isofluorane was discontinued after the administration of the third aliquot of

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urethane. The depth of anesthesia was checked by pinching the hind paw of the rat. The

absence of heart rate or blood pressure change verified that the rat was properly

anesthetized. Heart rates (HR) were monitored by a tachograph (Grass 7P4), which was

triggered by blood pressure waves. The pulsatile arterial pressure, mean arterial pressure,

and heart rate were recorded on a polygraph (Grass Instruments, model 7D). The trachea

was cannulated PE 240 polyethylene tubing and the animal was artificially ventilated

using a rodent respirator. Rectal temperature was monitored and maintained at 37±0.5°C

using an infrared lamp connected to a digital thermometer (Physitemp model BAT-8).

In experiments of suboccipital craniotomy, rats were placed in a supine position

in a stereotaxic apparatus (David Kopf Instruments model 1430, Tujunga, CA, USA,)

with the head flexed 18mm below the level of the interaural line. Suboccipital craniotomy

was performed to expose the medulla. The dorsal neck muscles were removed, an

incision of the atlantooccipital membrane was made, and part of the occipital bone and dura were removed to expose the calamus scriptorius. Three to four barreled glass micropipettes (tip size 20-40 µm) were used for microinjections. The first barrel contained artificial cerebrospinal fluid (aCSF), the second contained L-Glutamic acid (L-

Glu) (5mM), the third contained amylin (Phoenix Pharmaceuticals) at various concentrations, and a fourth containing the amylin antagonist sCT (8-32) (Phoenix

Pharmaceuticals). L-Glu (10nL, 5mM) was used as a positive control in all experiments to identify the correct pipette location in the nucleus ambiguus. A drop in heart rate with little or no changes of blood pressure after glutamate microinjection provided evidence of proper location of the pipette in the nucleus ambiguus. All of the for microinjections were freshly prepared with aCSF (pH 7.4), which itself was used as the

55 negative control in all experiments. Each barrel of micropipettes were connected to polyethylene tubing connected to different channels of a picospritzer (General Valve

Corp), and solutions were ejected (30nL) by air pressure. The coordinates used to locate the nucleus ambiguus were 0.4 mm caudal to the calamus scriptorius, 1.8-1.9 mm lateral to the midline, and 1.9-2.0 mm deep from the dorsal surface of the medulla. A volume of

30 nL microinjected to the nucleus ambiguus was selected because it elicited maximum cardiovascular responses in the nucleus ambiguus with minimal activation of the cardiovascular responses of the neighboring ventral lateral medulla [153].

Vagotomy

Bilateral vagotomy was performed in several animals during stereotaxic microinjection. Since cardiac preganglionic vagal neurons synapse to cardiac ganglia via the vagus nerve, severing the vagus will verify if heart rate changes from pharmacological compounds injected to the nucleus ambiguus is due to activation of cardiac preganglionic vagal neurons. Prior to placement of animals in the stereotaxic instrument, silk sutures were loosely placed around the vagi bilaterally. Vagotomy was performed by gently pulling on the silk sutures to expose the vagi, which were cut with a pair of fine scissors.

2.3 Rat model of diving reflex

2.3.1 Rat diving models Experimental diving models have been studied in rats and muskrats. McCulloch et al [29] used an anesthetized rat diving model. Rats were anesthetized with

56 and droperidol with blood pressure and heart rate monitored via femoral artery cannulation. Muscles were paralyzed, and the rat was placed on a ventilator. Diving reflex was simulated via cessation of ventilator with nasal infusion of water.

Interventions to study the diving pathway involved anesthetizing the trigeminal nerves, ligating the trigeminal nerves, electrolytic lesioning of the spinal trigeminal nucleus, or infusing glutamate receptor antagonist into the spinal trigeminal nucleus.

McCulloch et al [158] further developed a rat diving model by training rats to swim underwater for brief periods of time. Heart rate and blood pressure were assessed via implantable biotelemetric transmitters, and trained and untrained rats were subjected to voluntary and forced diving. Both voluntary and forced diving in both trained and untrained rats revealed similar bradycardia of 78%, however the mean arterial pressure was higher in forced dives. While this diving model simulates real dives in rats, no pharmacological interventions were used.

Signore et al [129] studied the pharmacological responses of heart rate in diving muskrats. An implantable electrocardiogram transmitter was surgically placed in the periotoneal cavity. Muskrats received pharmacological intervention of muscarinic blocker atropine to pharmacologically block the vagus nerve, α-adrenergic blocker phentolamine to assess for peripheral vasocontstriction, or β-adrenergic blocker nadolol and propranolol to block sympathetic control of heart rate via subcutaneous injection prior to forced or voluntary dives. It was found that atropine abolished diving bradycardia, nadolol and propranolol reduced predive and postdive tachycardia.

Although phentolamine did not result in heart rate changes, both atropine and

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phentolamine reduced the endurance of muskrats underwater, showing that bradycardia

and peripheral vasoconstriction are key to the hypoxia tolerance of the diving reflex.

2.3.2 Anesthetized rat diving model using MouseOX pulse oximeter In our lab, we developed an anesthetized rat diving model using novel non-

invasive pulse oximeter measurements of blood oxygen concentration and heart rate.

Pharmacological intervention was achieved via injection of glutamate and amylin

antagonists into the cisterna magna. Intracisternal injection of pharmacological

compounds is an established method of modulating brainstem pathways [159].

Male Sprague-Dawley rats weighing 200-250g were anesthetized with 1.5 g/kg urethane (Sigma Chemicals) via I.P. injection, and MouseOx pulse oximeter sensor

(STARR life sciences) were secured to the rats’ hind paw. Heart rate, blood oxygen saturation, and respirations were monitored in real time and recorded using MouseOx software. The apnea component of the diving reflex prevented rats from choking in water.

Anesthesia depth was determined by apnea without water inhalation, and lack of movement during procedure. The diving bradycardia was assessed via a 20 second immersion of the rat’s nostril in cold ice water. This duration was comparable to other diving rat models [160], and was selected for its length in allowing for stable bradycardia to develop while allowing for a rapid recovery of overall oxygenation status once resumed. The diving bradycardia value recorded was the lowest heart rate observed during the dive. In between diving reflex recordings, the animal’s head was dried with a towel and allowed to rest on a warm heated blanket (STARR life sciences) for 5 minutes. To access the cisterna magna the dorsal neck muscles were removed, and the atlanto-occipital membrane was exposed. A 30 G needle and a 10µL Hamilton

58 syringe (Hamilton Company USA) were used for intracisternal injections. The needle was gently inserted 2 mm deep and horizontally into the cisterna magna to inject the following compounds: glutamate receptor antagonists (2R)-amino-5-phosphonovaleric acid, AP5 [100μM in 0.9% saline, 1% dimethyl sulfoxide (DMSO), 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX; 100μM in 0.9% saline, 1% DMSO), and amylin receptor antagonist AC187 (500μM in 0.9% saline, Phoenix Pharmaceuticals).

Intraperitoneal injection of atropine (1mg/kg in 0.9% Saline) was used to block the vagal- mediated bradycardia. In all experiments, controls were established prior to surgical exposure of the atlantooccipital membrane and also after intracisternal injection of 0.9% saline/1% DMSO.

2.4 Patch Clamp

2.4.1 Introduction to patch clamp electrophysiology‐ Patch clamp is the single most powerful tool to study the activity of single neurons- Developed in the 1970s, it has since revolutionized our understanding of action potentials, ion channels, and even how pharmaceutical drugs cause arrthymias.

Developed by two German electrophysiologists- Erwin Neher and Bert Sakmann [161], the patch clamp allowed the study of the activation of ion channels in real time. The patch clamp involves using a pulled glass pipette with a 5 µm diameter or less opening and coupling it with either a single patch of membrane, or the entire cell. The pipette and the membrane are electrically coupled with a giga-ohm seal, which functionally results in isolation of the inside of the pipette electrically from the outside of the pipette. This

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electrical isolation allows the study of current flows and voltage changes in the cell that

occur in real time with very little electrical noise. Prior to the development of the patch

clamp, activity of individual neurons could only be measured using giant axons in

invertebrates such as snails and squids, and large cells such as oocytes of sea urchins.

Types of patch clamp- There are 4 basic patch clamp modes, where the type of

attachment to the cell or membrane varies. As see in the Figure 10 below, the four

variations of the patch clamp technique are achieved once the giga-ohm seal is formed

and the on-cell patch mode is achieved. The inside-out patch (Figure 10B) and the

outside-out patch mode (Figure 10 B, D) both involve small portions of the cell

membrane, allowing the study of currents and voltages of possibly single channels under

the pharmacological manipulation of the intracellular and extracellular solutions. The

whole-cel patch-clamp mode is the most common mode, and allows recording of the

electrical activity of the entire neuron.

Patch clamp can also be visual or blind. Visual patch clamp involves usin light

microscopy under high magnification to identify cells of interest in order to patch.

Under blind patch clamp light microscopy under low magnification is used to identify the

general area to path, but the cells patched are not individually visually identified. An

advantage of the blind patch is the more heterogenous and less biased sampling of

neurons in the nucleus ambiguus. For the purposes of the current project however since cardiac preganglionic vagal neurons require retrograde labeling and identification using rhodamine, blind patch clamp would not be ideal.

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Figure 10: Variations of the patch clamp technique.

Figure 10: Variations of the patch clamp technique. A- on cell patch is achieved when there is a giga-ohm seal between the inside of the pipette and the extracellular space. B- inside out patch is achieved by pulling the pippete away from on-cell patch, with extracellular side of the cell membrane facing the inside lumen of the pipette. C-whole cell clamp mode is achieved by applying light negative pressure until the membrane inside the pipette ruptures. D- Outside-out patch clamp mode is achieved by pulling away from the whole-cell clamp mode, achieving intracellular side of the cell membrane facing the inside lumen of the pipette. Adapted from [162].

Voltage and current of a cell cannot be both measured simultaneously using patch clamp- Due to the nature of the recording equipment of patch clamp, either the current or the voltage can be modified and the other recorded from the cell, not both simultaneously. Recordings can be made by clamping the current constant and observing the voltage change, or clamping the voltage constant and observing the current changes.

Both are powerful techniques that reveal different types of information about the neuron.

Observation of current changes under voltage-clamp- Voltage clamp electrically clamps the voltage of the cell, and measures the cell currents that pass

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through ion channels. This allows the study of how the current flows in response to

changing voltages. Historically, the use of voltage-clamp to study giant squid axons by

Alan Hodgkin and Andrew Huxley in 1952 [163] revolutionized the understanding of

action potentials. The study examined sodium and potassium currents at different

clamped voltages, and found that these changing ionic currents at different voltages occur

in sequence to form an action potential.

During voltage-clamp, the current measured is linearly proportional to membrane

conductance, which is directly proportional to ion-channel activity. Many ion channels are also voltage-gated, and therefore under voltage-clamp, these ion channels can be studied under controlled conditions. Voltage clamp will be used to determine amylin effect on current flows in cardiac preganglionic vagal neurons in the nucleus ambiguus.

Observation of voltage changes under current clamp- Under current clamp, the current that flows through the cell’s ion channels are fixed, and the resulting voltage changes can be observed. This method allows the study of depolarizing or

hyperpolarizing current effects on neurons. Neuronal excitability can be measured as

fixed depolarizing currents, resulting in varying amount of action potentials in response

to a pharmacological compound. Currently, amylin effect on neuron excitability is

unknown.

2.4.2 Whole‐cel patch‐clamp of cardiac preganglionic vagal neurons

Rhodamine labeling of cardiac preganglionic vagal neurons- In an initial

surgery 1-2 days prior to the day of the experiment, a right thoracotomy was performed

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on Sprague-Dawley rat pups were placed on ice to anesthetize them and slow down heart

rate. A needle was inserted into the pericardial sac and 20µL of rhodamine (XRITC, 2mg,

1% solution in saline, Molecular Probes, Eugene, OR) was topically applied to the

epicardial surface of the heart that contains parasympathetic ganglia. The incision sites

were subsequently closed, and the animal was allowed 3 days to recover while the

retrograde tracer was allowed to be retrograde transport up the vagus nerve.

Brainstem slice preparation and maintenance under oxygenated modified

high sucrose artificial CSF solution- Visualized whole-cel patch-clamp was performed

on brain slices from male Sprague-Dawley rats 3-8 days old. On the day of the

experiments, rats were anesthetized with urethane (1.2g/ kg IP), and the brainstem was

rapidly removed and placed in chilled, oxygenated high sucrose artificial CSF solution of

the following composition (in mM) : 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7.0

MgCl2, 25 NaHCO3, 75 sucrose, and 25 glucose. The high sucrose modified artificial

CSF is to decrease neuronal death due to dissection and vibratome slicing and to increase

overall viability of neurons. The solution was saturated with 95% O2-5% CO2. The brainstem was dissected and the pia mater was carefully removed with a pair of fine forceps. The brainstem was then fixed onto an agar block with cyanoacrylic glue and sectioned in the slicing chamber of a Vibratome (series 1000).

Brainstem slice incubation

Brainstem slices of 250 µm containing nucleus ambiguus will be incubated in an

oxygenated Krebs solution at room temperature (21 ± 0.5°C) for at least 1h before the

start of experiments. This slice thickness is a good balance between slick thickness

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needed to maintain viability of neurons, and thin enough to allow visual identification of neurons under the microscope. One slice will be transferred to the recording chamber

(RC-22, Harvard Apparatus), held in place by the grid of a fine nylon mesh, and perfused with oxygenated artificial CSF solution at a rate of 3-6 ml/min (Artificial CSF solution

[164]: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 CaCl2, 1.0 MgCl2, 25 NaHCO3, and 25 glucose. All experiments will take place at room temperature.

Figure 11: Patch-clamp recording of rhodamine-labeled CPVN in the nucleus ambiguus in brain stem slices. A: DIC imaging of CPVN. B: fluorescent imaging shows XRITC-labeled CPVNs in the nucleus ambiguus that were labeled by injection of the fluorescent tracer XRITC (2% solution, 4 ml, Molecular Probes) into pericardial sac 2 days before experiment. Scale bar: 40 μm. Adapted from [165].

Patch clamp pipette preparation

Patch pipettes will be pulled from thin-walled fiber-filled borosilicate glasses

(WPI, OD 2.0 mm) and filled with intracellular recording solution of the following

+ composition (in mM): 130 K gluconate, 1 MgCl2, 2 CaCl2, 4 ATP, 10 EGTA and 10

HEPES, with a resistance of 5-7 MΩ.

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Cardiac Preganglionic vagal neuron identification and giga-seal formation

Cells will be visualized using a Nikon (Eclipse E600FN) upright microscope.

Rhodamine labeled CPVN will be identified by excitation between 350-395 nm and an

emission band of 530-600 nm. The CPVN will be approached by a positive pressure

patch electrode. Once the patch electrode is in contact with the cell, the positive pressure

will be released and a giga-ohm seal will be made by applying light suction to the patch

electrode. Recordings will be made using a CV-4 headstage (Axon Instruments) attached

to an Axopatch 1D amplifier (Axon Instruments).

Whole-cel patch-clamp configuration

The patch pipette will be voltage-clamped at -65 mV and the cell membrane will be ruptured under negative pressure, resulting in a whole cell configuration of the CPVN.

The patched CPVN can be studied under voltage-clamp or current clamp mode.

Pharmacological compounds will be administered to the CPVN either by bath

at a rate of 1mL/min, or by pneumatic Picospritzer (General Valve Corp.) pressure

injection at 20 psi from a patch pipette positioned within 30 μm from the patched CPVN

for 500 ms to 1 sec. The data will be digitized and stored for later analysis using a

Digidata 1322a analog-digital converter (Axon Instruments).

Voltage Clamp experiments

To measure amylin effects on whole cell currents, cardiac preganglionic vagal

neurons will be voltage-clamped at a resting membrane potential of -60 mV.

To measure the steady state currents of cardiac preganglionic vagal neurons, the

neuron can be subjected to voltage ramps of -120mV to +40mV in 10mV increments,

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each lasting 300 ms and returning to -60 mV prior to the next voltage ramp. Currents will be measured during this protocol before and after the administration of amylin.

Previous studies have firmly established that electrical stimulation of the trigeminal nerve induces vagal mediated bradycardia that is seen in the diving and trigeminal-vagal reflexes [166-170]. Since our current study has found amylin to be present in this pathway, it is unknown the effects of amylin on evoked synaptic transmission in the trigeminal-vagal pathway. In these experiments requiring the stimulation of the trigeminal nerve, a bipolar stimulating electrode (outer diameter 0.25 mm, Frederick Haer and Co.) will be placed near the trigeminal fibers of medullary brain slices. Synaptic currents will be evoked at a low frequency of 0.03-0.1Hz with duration of 50-200 ms and intensity of 3 to 15V. Amylin (1mM) will be applied via picospritzer.

The cell will be observed under voltage-clamp in order to measure the current response to synaptic activation of cardiac preganglionic vagal neurons.

In experiments requiring measurement of spontaneous EPSC frequency and amplitude, 0.1µM of TTX was administered by bath 10 min prior to administration of amylin in order to abolish synaptic transmission. This method allows for the study of amylin effects on presynaptic and postsynaptic membranes, corresponding with spontaneous EPSC frequency and amplitude changes. Digitized data with EPSC were automatically detected and characterized using MiniAnalysis software (Synaptosoft, Fort

Lee, NJ). The EPSC frequency and amplitude were further analyzed using Graphpad

Prism (GraphPad Software, La Jolla, CA) statistical analysis software.

Current clamp experiments

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To measure amylin effects on cardiac preganglionic vagal neuron excitability,

cells were current clamped and the voltage could be observed.

In experiments measuring cardiac preganglionic vagal neuron excitability,

neurons were first current clamped with zero current. Increasing depolarizing current

injections of 100-500 pA were performed lasting 500 ms each in order to observe the

excitability of these neurons. The number of action potentials in response to these

depolarizing current injections will be observed before and after the administration of

amylin.

2.5 Identification of amylin receptor components using RT‐PCR Amylin receptor mRNA analysis by reverse transcription polymerase chain reaction

To verify amylin receptor presence in the brainstem for patch clamp and

microinjection experiments, amylin receptor components were detected in the brainstem

of adult and neonatal rats using reverse transcriptase polymerase chain reaction (RT-

PCR) with primers designed for amylin receptor components. Adult male rats (200g) and neonatal rat pups (3 day old) were anesthetized with urethane (1.2g/kg, i.p.), and decapitated with a guillotine. The brainstem was removed and the total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). First strand cDNA was synthesized using SuperScript II (Invitrogen, Carlsbad, CA). PCR was performed using the following primers: CT-R 5’ primer (TGGTTGAGGTTGTGCCCAATGGA), CT-R

3’ primer (TTCATGGGTTTGCCTCATCTTGGTC)[171], RAMP1 5’ primer

(ACTGCACCAAACTCGTGGCA), RAMP1 3’ primer

(AGCAGGATGCAATGTGGGGTCT), RAMP2 5’ primer 67

(AGCAACCTGCGGTATTGCTTGG), RAMP2 3’ primer

(ATCCTGGTTCCAGCAGGGTACA), RAMP3 5’ primer

(CCCTCTGTTGCTGCTGCTTTGT), RAMP3 3’ primer

(TCAGTGCGCTTGCTACGCCATA). The amplified products were loaded into a 1% agarose gel for electrophoresis, and subsequently stained with ethidium bromide. The image was acquired with FujiFilm Las-1000 imaging system (Fujifilm Medical Systems,

Stamford, CT).

2.6 Statistical analysis All statistical analysis were performed with GraphPad Prism (GraphPad

Software). The means and standard error of the means were calculated for all data sets including trigeminal ganglion cell size, maximum changes in heart rate during stereotaxic microinjection, and pulse oximeter heart rate measurements. Comparison of the bradycardia induced by various concentrations of amylin was made by using a one-way analysis of variance followed by a Tukey-Kramer multiple comparison test. Comparison of bradycardia induced in the rat diving model was made by matched student’s t-test analysis.

CHAPTER 3: RESULTS

Amylin distribution in the trigeminal‐vagal pathway Amylin immunoreactive neurons identified in the trigeminal ganglion

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We first examined the distribution of amylin in the brainstem. Using high

specificity anti-amylin antibodies to perform immunohistochemical analysis in brain

tissue sections, the location of amylin immunoreactivity was characterized. Examination

of medullary sections labeled with amylin antiserum from five rats revealed networks of

nerve fibers in the spinal trigeminal tract, some of which traversed the ventral medulla

and projected dorsally to terminate in the nucleus ambiguus (Figure 13A and B). Amylin

immunoreactive fibers of varying densities were detected throughout the entire neuraxis

of nucleus ambiguus. Particularly dense cell processes and/or nerve terminals were

observed and appeared to come in contact with the compact formation of the nucleus

ambiguus (Figure 13B and C). In addition to the spinal trigemenial tract, amylin

immunoreactive fibers were detected in the nucleus of the solitary tract (not shown).

Amylin immunoreactive cells in the trigeminal ganglia were mostly small to medium in

diameters (Figure 13D, E), with an average diameter along its long axis of 34.9±0.4 μm

(n=874 from sections from 6 trigeminal ganglion from 3 rats, Figure 12). In contrast, no amylin immunoreactivities were found in medullary sections labeled with amylin-

antiserum preabsorbed with the peptide (1 µg/ml), and a representative section is

illustrated in Figure 13F.

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Diameter of Amylin Immunoreactive Trigeminal Ganglion Neurons (n=874) 200

150

100 Count

50

0 15 25 35 45 55 65 75 Diameter(m)

Figure 12: Size of amylin immunoreactive neurons in the trigeminal ganglion. Amylin immunoreactive neurons in the trigeminal ganglion were small to medium sized neurons with an average diameter along its long axis of 34.9± 0.4μm (n=874 from sections from 6 trigeminal ganglion from 3 rats.)

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Figure 13: Amylin immunoreactive neurons 71 found in the trigeminal ganglion with their fibers projecting to the nucleus ambiguus in the brainstem Top panel shows the schematic diagram of the experiment. A-C, amylin immunoreactive fibers are present in spinal trigeminal tract (Sp5). Fibers are also seen projecting to the nucleus ambiguus. D-E ,small to medium sized neurons (single arrow) in the trigeminal ganglion show amylin immunoreactivity. Large cells can be seen without amylin (double arrows). F, Negative control showing preabsorption with Amylin in the brainstem. Calibration bar: A, 250 µm; B,D, F 100 µm; C,E 50 µm

Amylin containing neurons colocalize with VGluT2

A large majority of rat trigeminal ganglion cells are vesicular glutamate transporter 2-positive (VGLUT2)[172], which suggests that these neurons are glutamatergic. Since an intact glutamatergic pathway is needed for the diving reflex, we suspect that amylin immunoreactivity must be at least partially overlapped with VGLUT2 positive trigeminal ganglion cells. Thus, double-immunostaining was conducted to determine whether or not amylin immunoreactive cells were VGLUT2-positive. Nearly all amylin immunoreactive cells found in trigeminal ganglia were VGLUT2-positive, however, not all VGLUT2-positive cells were amylin immunoreactive cells (Figure 14).

Hence, these results suggest that amylin immunoreactive ganglion cells constitute a subset of neurons immunoreactive for VGluT2 (Figure 14), and supports our hypothesis that amylin is a modulator for the diving reflex.

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Figure 14: Double staining of VGluT2 and amylin immunoreactive neurons in the trigeminal ganglion. Top panel shows the schematic diagram of the experiment- amylin and VGluT2 immunoreactivity is examined. Panel A shows VGluT2 immunofluorescence in the trigeminal ganglion. Panel B shows amylin immunofluorescence in the trigeminal ganglion. Panel C shows colocalization, signifying that amylin containing neurons also contain VGLUT2, a marker of glutamatergic neurons. Calibration bar 75 µm.

Amylin fibers project directly to the nucleus ambiguus

To determine whether amylin immunoreactive cell bodies in the trigeminal ganglion project directly to the nucleus ambiguus, retrograde fluorescent dye fluorogold

(4%) was microinjected stereotaxically into the nucleus ambiguus five days prior to tissue staining. The injection site in the brainstem shows Fluorogold is localized to a small area surrounding the nucleus ambiguus (Figure 15). Microscopic examination of trigeminal ganglion sections double-labeled with fluorogold antiserum (Figure 16B) revealed that nearly all amylin immunoreactive cells contained fluorogold (Figure 16C) and that not all

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fluorogold-positive cells were amylin immunoreactive. These results suggest that amylin

immunoreactive trigeminal neurons directly project fibers to the area of the nucleus

ambiguus.

Figure 15: Retrograde tracer fluorogold injection into the nucleus ambiguus in the brainstem. Medullary slices are shown with fluorogold injected into the region of the nucleus ambiguus. Five days passed prior to immunohistochemical analysis of trigeminal ganglion to allow for fluorogold retrograde transport from the nucleus ambiguus to the trigeminal ganglion.

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Anti‐amylin and Anti‐fluorogold FG

ophthalmic maxillary CN V CN X mandibular TG NA

Figure 16: Fluorogold and amylin immunoreactive neurons in the trigeminal ganglion. Top panel represents the schematic diagram of the experiment, showing amylin and fluorogold immunoreactivity in the TG after fluorogold injection into the NA. Panel A shows trigeminal neurons retrogradely labeled with fluorogold originally injected in the nucleus ambiguus. Panel B shows amylin immunoreactivity in the nucleus ambiguus. Panel C shows colocalization of amylin with fluorogold, demonstrating that amylin containing fibers project directly to the nucleus ambiguus. Calibration bar: 75 µm

Amylin fibers project to cholinergic neurons in the nucleus ambiguus

The bradycardia component of the diving reflex is parasympathetically mediated by the nucleus ambiguus. We therefore hypothesized that amylin immunoreactive fibers projecting to the nucleus ambiguus have direct contact with parasympathetic preganglionic neurons, which are cholinergic neurons. To support this hypothesis, medullary sections were double-labeled with amylin antiserum and choline acetyltransferase immunoreactivity, a marker for cholinergic neurons. Figure 17A, D illustrates a cluster of choline acetyltransferase immunoreactive neurons in the compact

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formation of nucleus ambiguus, some of which were opposed by amylin immunoreactive

cell processes (Figure 17 B, E) ramifying among amylin immunoreactive cells. Figure

17C, F is a merged amylin immunoreactivity and cholineacetyltransferase

immunoreactivity image, where amylin containing bouton-like elements abut onto

cholineacetyltransferase immnoreactive neurons. Taken together, our results demonstrate that amylin containing fibers identified in the medulla synapse onto cholinergic neuons in the nucleus ambiguus.

Figure 17: Amylin and ChAT immunoreactivity in the nucleus ambiguus. 76

Top panel represents the schematic diagram of the experiment, showing amylin and ChAT immunoreactivity in the NA. Panels A and D show ChAT immunoreactivity in the NA. Panel B and E show amylin immunoreactivity in the NA. Panel C and F show colocalization between amylin containing fibers or endings from the trigeminal ganglion, and ChAT containing neurons in the nucleus ambiguus. Calibration bar 75 µm.

Amylin fibers project to cardiac preganglionic vagal neurons in the nucleus ambiguus

Cardiac preganglionic vagal neurons are the neurons primarily responsible for the parasympathetic mediated slowing of heart rate. The retrograde tracer rhodamine was injected into the pericardial sac of the heart five days prior to immunohistochemical analysis of brainstem tissue. The retrograde tracer rhodamine retrogradely labeled the cardiac preganglionic vagal neurons in the nucleus ambiguus (Figure 18B, E). Amylin immunoreactivity is shown in Figure 18A, D. Amylin immunoreactivity was found to be a subset of the rhodamine immunoreactive neurons in the nucleus ambiguus.

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Figure 18: Colocalization of amylin and rhodamine immunoreactivity in the nucleus ambiguus.

Top panel represents a schematic diagram of the experiment showing amylin immunoreactivity and rhodamine fluorescence in the nucleus ambiguus after rhodamine injection to the pericardial sac of the heart. Panels A and D represent amylin immunoreactivity in the nucleus ambiguus. Panels B and E represent rhodamine immunofluorescence from rhodamine that was retrogradely transported from the heart. Panels C and F represent colocalization of amylin and rhodamine. Rhodamine containing neurons in the nucleus ambiguus represent parasympathetic cardiac vagal neurons. Cardiac vagal neurons colocalize with a subset of amylin containing fibers and boutons in the nucleus ambiguus. Solid arrow shows amylin + rhodamine immunoreactive cell; open arrow indicate amylin immunofuloresence cell only, and open arrowhead indicates rhodamine containing cell only. 78

Pharmacological effects of amylin in the nucleus ambiguus

Stereotaxic microinjection of amylin to the nucleus ambiguus induces bradycardia

We hypothesized that amylin immunoreactive neurons with projections to the nucleus ambiguus modulates heart rate. To test this, we injected amylin in vivo into the nucleus ambiguus using stereotaxic microinjection. In urethane-anesthetized rats, the average resting values for MAP and heart rate were 98.1± 2.6 mm Hg and 386±6.8 bpm, respectively. As shown in Figures 19 and 20 below, unilateral microinjections of L-Glu

(5 mM) to the nucleus ambiguus caused a brisk bradycardia with a mean of fall of

105±8.1 bpm (n=38), which also served to verify the pipette location in the nucleus ambiguus. Figures 19 and 20 below also demonstrate microinjections of artificial CSF as a negative control did not elicit any significant responses.

We evaluated the dose-response of amylin microinjection to the nucleus ambiguus.

In one group of rats (n=24), microinjections of various concentrations of amylin (50, 100

500, 1000, and 1,500 µM) into the nucleus ambiguus elicited decreases in heart rate

(Figure 21B). Microinjection of 1mM amylin elicited the maximal heart rate response, and was thus selected for the subsequent tachyphylaxis, vagotomy, and receptor blockade studies. The onset and duration of bradycardia induced by 1mM amylin were 9.8±1.9 sec and 38±3.8 min with return to the baseline value recorded before injection(n=5). Amylin microinjections into neighboring areas around the nucleus ambiguus did not elicit any bradycardic responses, reinforcing our hypothesis that amylin containing fibers project to cholinergic neurons that modulate bradycardia in the nucleus ambiguus.

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To test whether the bradycardic response desensitizes to amylin, the nucleus

ambiguus was repeatedly microinjected in vivo with amylin at an interval of 40 min. The decreases in heart rate in response to three consecutive microinjections of 1mM amylin at

40 min intervals were 68.8±12.4, 65±14.3, and 68.4±14.8 bpm, respectively (n=5, p>0.05) (Figure 19B). These results indicate that a detectable tachyphylaxis was not observed with repeated microinjections of amylin, suggesting that amylin does not induce desensitization of the amylin receptor that induces bradycardia.

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Figure 19: Amylin induces bradycardia in the nucleus ambiguus that is not susceptible to desensitization. In all studies, L-Glu was used as a positive control to identify the pipette location in the nucleus ambiguus and the artificial CSF vehicle served as the negative control. A. Microinjection of 1mM amylin into the nucleus ambiguus resulted in a bradycardia with an onset of 9.8 ± 1.9 sec and duration of 38 ± 3.8 min(n=5). B. Repeated injections of 1mM amylin into the nucleus ambiguus did not show tachyphylaxis (p<0.05, n=5). The bradycardia observed in response to three consecutive microinjections of 1mM amylin at

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45 min intervals were 68.8 ± 12.4, 65 ± 14.3, and 68.4 ± 14.8 bpm, respectively (n=5, p>.05).

An intact vagus nerve is required for amylin induced bradycardia

Since nucleus ambiguus neurons that mediate parasympathetic slowing of heart rate projects to the heart via the vagus nerve, we hypothesized that an intact vagus nerve is essential for amylin induced bradycardia. In rats (n=4), the effect of bilateral vagotomy

on heart rate responses to amylin was studied (Figure 20A). Microinjections of 5 mM L-

Glu and 1mM amylin resulted in bradycardia of 105±10.6 bpm and 75.5±15.819 bpm

respectively(n=4), suggesting that amylin and L-Glu are putative signaling molecules for

inducing bradycardia. Bilateral vagotomy increased heart rate from a resting value of

408.8±5.2 bpm to 477.5±6.3 bpm(p<0.05, n=4). Post-vagotomy microinjection of 5mM

L-Glu did not elicit bradycardia, and subsequent microinjection of amylin (1 mM) failed

to elicit a detectable fall in heart rate (Figure 20A).

Amylin receptor blockade also blocks the amylin induced bradycardia in nucleus

ambiguus

We hypothesized that amylin induces bradycardia via its interaction with the

amylin receptor. sCT (8-32) was reported to be a selective antagonist to the amylin

receptor [173]. Attenuation of a bradycardic response was not observed at doses lower

than 4mM sCT (8-32), which was used in subsequent experiments. Microinjection of 4

mM sCT (8-32), which by itself did not evoke a detectable bradycardia, blocked the

bradycardic responses to subsequent (within 5 min) microinjections of 1mM amylin into

the nucleus ambiguus (n=5; p<.05) (Figures 20B and 21C). When 4mM sCT (8-32) was

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given, no heart rate or blood pressure changes were observed. After five minutes, amylin

was again microinjected and an attenuated bradycardia response of 14±7.5bpm was

observed. After a 2 hour recovery, amylin (1mM) microinjection resulted in a

bradycardia of 50±8.0bpm, which was not statistically significantly different compared to

that elicited by amylin administered prior to sCT (8-32) injection (p>0.05) (Figure 20B), suggesting that the blocking effect of sCT is reversible.

Amylin receptor components in the brainstem are detected using RT-PCR

RT-PCR of amylin receptor components consisting of both isoforms of the calcitonin receptor CT-Ra and CT-Rb, and RAMP 1, RAMP2, and RAMP3 were analyzed in rat brainstem homogenates with β-actin serving as control (Figure 21A). In both adult and neotnate brainstems, all components were present, although there were higher levels of CT-Ra compared to CT-Rb. RAMP1, RAMP2, and RAMP3 were also

present, although RAMP1 and RAMP2 levels were higher than RAMP3.

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Figure 20: Amylin induced bradycardia is vagal mediated and is reversibly blocked by amylin receptor antagonist sCT (8-32). A. Microinjections of 5mM L-Glu and 1mM amylin resulted in a bradycardia of 105 ± 10.6 bpm and 75.5 ±15.82 bpm, respectively (n=4). Bilateral vagotomy increased heart rate from a resting value of 408.8 ± 5.2 to 477.5 ± 6.3 bpm. B. Microinjection of 4mM of the amylin receptor antagonist, sCT (8-32) blocked the bradycardic responses to subsequent microinjection of 1mM amylin into the nucleus ambiguus. Amylin was first microinjected to the nucleus ambiguus to establish a basal response. After a 40 minute recovery period, the amylin receptor antagonist sCT (8-32) was microinjected, which itself did not result in a change in heart rate or blood pressure. Following a brief 5 minute

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waiting period, amylin was microinjected once more, and the bradycardia was attenuated by sCT (8-32) (p<0.05). After a 2 hour recovery period, amylin induced bradycardia returned to baseline.

Figure 21: Amylin induced bradycardia is mediated by the amylin receptor. A. RT-PCR of the rat brainstem in adults and neonates show the presence of both isoforms of the calcitonin receptor as well as all 3 RAMP subtypes. The presence of CT- R along with RAMP1 and RAMP3 show the brainstem contains the amylin receptor. B. Microinjection of varying doses of amylin (50uM, 100uM, 500uM, 1mM, 1.5mM) into the nucleus ambiguus elicited varying degrees of bradycardia (n=5 for each dose). 1mM amylin resulted in the largest bradycardia (p<0.05), and was used in all subsequent bradycardia studies. C. Microinjection of 4mM sCT (8-32) partially blocked the bradycardic responses to 1mM Amylin.(n=5, P<.05).

Amylin-induced bradycardia plays an important role in mediating the diving reflex

We hypothesized that amylin modulates bradycardia seen in the diving reflex. To

examine this hypothesis, we used an anesthetized rat diving model with intracisternal

injections to assess the effects of amylin and glutamate receptor blockade on brainstem

pathways in the diving reflex. During the dive, heart rate and oxygen saturation rapidly

decreased, stabilized, and then subsequently recovered shortly after dive completion

(Figure 22A). In all experiments, baseline heart rate and diving bradycardia were

assessed before surgical exposure of the atlanto-occipital membrane and served as 85

controls. The baseline heart rate was established at 381.8±2.78 bpm. Diving bradycardia

was established at a baseline value of -212±7.17 bpm (56% decrease) (n=21), which is

comparable to control saline intracisternal injections of -208±8.78bpm (54% decrease)

(Figure 22 B, n=21, p>0.05). To ascertain that diving bradycardia acts through the

nucleus ambiguus, atropine (1 mg/kg) was administered intraperitoneally to block vagal

innervation to the heart. This resulted in significant attenuation of the diving bradycardia

from -235±23.1 bpm in saline controls to -7.82±1.94 bpm (n=5, p<0.05)(Figure 22 B,C).

An intact glutamatergic trigeminal pathway is essential for the bradycardic component of

the diving reflex [29]. We therefore administered the glutamate non-NMDA antagonist

CNQX (100μM) and NMDA antagonist AP5 (100μM) via intracisternal injection. In these rats, the diving bradycardia was attenuated from -199±12.9 bpm (51% decrease) to

-130±22.2 bpm (34% decrease)(n=6, p<0.05)(Figure 22 D) 10 minutes post-intracisternal injection.

To determine amylin role in diving bradycardia, we injected intracisternally the amylin antagonist AC187 (500μM). Diving bradycardia was slightly attenuated from -

236±12.9 bpm (61% decrease) to -197±12.0bpm (51% decrease)(n=5, p<0.05)(Figure

22E) 15 min post AC187 injection. Glutamate and amylin antagonists significantly

attenuated diving bradycardia more than either antagonist alone, from -174 ± 25.6 bpm

(46% decrease) to –101±28.1bpm (27% decrease; n=5, p<0.05) (Figure 22F),

demonstrating that both glutamate and amylin mediate bradycardia in the diving reflex.

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Figure 22: Glutamate and amylin antagonists decrease the amplitude of diving reflex induced bradycardia. Urethane anesthetized rat diving model using pulse oximeter heart rate measurements. A. An example of a 30s dive, the duration of which is shown by the black horizontal bar. B. Maximum bradycardia (percentage heart rate decrease) observed during the diving reflex after intracisternal injection of various pharmacological antagonists showing the statistically significant effects (p<0.05) of the glutamate and/or amylin receptor antagonists in attenuating diving induced bradycardia. C. Diving bradycardia after intraperitoneal injection of atropine shows chemical vagotomy abolishes the diving induced bradycardia(p<0.05, n=4). D. Diving bradycardia after intracisternal injection of 87 glutamate receptor antagonists CNQX and AP5(p<0.05, n=5). E. Diving bradycardia after Intracisternal injection of amylin receptor antagonist(p<0.05, n=6). F. Intracisternal injections of both the glutamate receptor and amylin receptor antagonists(p<0.05, n=5). All data displayed as mean ± SEM.

Amylin neurophysiological effects on cardiac preganglionic vagal neurons

Amylin depolarizes cardiac preganglionic vagal neurons

Amylin was found to depolarize neurons in the nucleus ambiguus under current clamp mode as shown in Figure 23. 25% of CPVN tested responded to 1mM picospritzer administration of amylin. The average depolarization was 6.4 ± 1.1mV and the average duration was 662 ± 160 sec (n=36). The top two panels in Figure 23 show 1mM amylin picosprizter administration for 500 ms and 1 s. The lower panel in Figure 23 shows 150 nM amylin superfusion over the course of 10 min. During the experiment, hyperpolarizing current injections were given every 10 seconds to assess the resistance

(or conductance) of the membrane. Membrane resistance was found to increase after administration of amylin.

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Figure 23: Amylin depolarizes cardiac preganglionic vagal neurons in the nucleus ambiguus under current clamp.

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25% of CPVN tested responded to picospritzer administration of 1mM amylin. The average depolarization from 1mM picospritzer of amylin was 6.4 ± 1.1mV and the average duration was 662 ± 160 sec (n=36). The top two panels show amylin responses to picosprizter administration for 500ms and 1s. The lower panel shows 150 nM amylin superfusion over the course of 10 min.

Amylin increases excitability of cardiac preganglionic vagal neurons

In response to depolarizing current injection under current clamp, cardiac preganglionic vagal neurons depolarized, resulting in action potentials as seen in Figure

24 left panel. This depolarizing currents lasted 500 ms, and varied from 100-500pA. The membrane depolarized, and triggered action potentials. The number of action potentials formed is a measure of the excitability of the neurons. As seen in right panel in Figure 24, amylin picospritzer resulted in an increased excitability in response to 100-500pA of

depolarizing current (n=6, P<0.05). At 100pA, excitability increased from 1.43± 0.869 to

3.57±1.29 action potentials. At 200pA, excitability increased from 3.57±1.73 to 7.14±

1.52. At 300pA, excitability increased from 6.143±1.738 to 9.86±1.57. At 400pA, excitability increased from 8.00±2.08 to 12.4±1.51. At 500pA, excitability increased

from 9.14± 2.143 to 15.286±1.48.

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Figure 24: Amylin increases excitability of cardiac preganglionic vagal neurons under current clamp with depolarizing current injection. In the left panel, the voltage tracing can be seen in mV displaying a membrane potential show several action potentials in response to 0.2 nA depolarizing current pulse. Five minutes after 1mM amylin picospritzer, the same amount of depolarizing current resulted in greater number of action potentials displayed as mean ± SEM (n=6, p<0.05).

Amylin decreases whole cell steady state currents in cardiac preganglionic vagal neurons

To study the effects of amylin on whole cell currents, cardiac preganglionic vagal neurons were observed under voltage-clamp from -120mV to +40mV at 10mV intervals.

Prior to each voltage ramp, the neuron was returned to the resting -60mV for 40ms prior to being exposed to voltage-clamp at the set potential for 300ms. Figure 25 top panel shows the superimposed current responses of control state and after 1mM amylin picospritzer administration. Figure 25 bottom panel shows the current-voltage plot, with the membrane potential on the x-axis, and the current displayed as the y axis. Amylin currents can be seen to decrease at higher membrane potentials compared to control. The amylin induced current is calculated by subtracting the control current from the amylin

91 current. The amylin induced current is seen as an inward current at positive voltages, which decreases as the membrane potential decreases.

Figure 25: Amylin induces reduction in steady state whole cell currents in cardiac preganglionic vagal neurons under current clamp.

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Current-voltage plot of cardiac preganglionic vagal neurons before and after administration of amylin displayed as mean ± SEM. Amylin can be seen to induce an inward current associated with a decrease in conductance that increases at positive voltages(p<0.05, n=8).

Amylin decreases spontaneous excitatory post synaptic potential frequency in the nucleus ambiguus

To study the effect of amylin on presynaptic membrane release of neurotransmitters and neuropeptides and post synaptic neuron, TTX (0.1µM) was administerd in the artificial CSF bath in order to abolish synaptic transmission between neurons. The resulting membrane currents of cardiac preganglionic vagal neurons were observed under voltage-clamp. As shown in Figure 26 left panel, the spontaneous excitatory post synaptic current (EPSC) frequency was decreased after amylin administration. The frequency of spontaneous EPSC at baseline was

4.38±1.30counts/min during the 5 minutes prior to amylin. The frequency of spontaneous EPSC increased to 2.45±0.83counts/min 10 minutes after 1mM amylin picospritzer and recovered to 10.45±2.52counts/min after 30 minutes recovery (n=13, p<0.05). The amplitude of EPSC was 53.79±2.983pA at baseline, and increased to

55.95±4.239pA 5 minutes after amylin administration, and recovered to 55.62±3.507 after 30 minute recovery (n=13, p>0.05).

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Figure 26: Amylin decreases spontaneous EPSC frequency but not amplitude. Figure 26: Amylin decreases spontaneous EPSC frequency but not amplitude.

Under administration of 0.1µM TTX to abolish all synaptic release of neurotransmitters, Amylin administration via picospritzer decreases spontaneous EPSC frequency from mean±SEM of 4.38±1.30counts/min during the 5 minutes prior to amylin to 2.45±0.83counts/min during 10 minutes after amylin(p<0.05, n=13). Spontaneous EPSC frequency recovered after 30 minutes to 10.45±2.52counts/min. Amylin administration via picospritzer does not significantly change the spontaneous EPSC amplitude(p>0.05, n=13).

Amylin decreases evoked synaptic currents in the nucleus ambiguus

In order to examine the effects of amylin on evoked synaptic currents in the

nucleus ambiguus, a bipolar stimulating electrode was placed in the spinal trigeminal

tract with stimulating pulses set to induce evoked synaptic currents in cardiac

preganglionic vagal neurons (Figure 27). Prior to administration of amylin, evoked inward current were observed mean±SEM of -81.48±23.99pA. After 5 min of administration of amylin, evoked synaptic currents decreased to -24.70±3.37pA (n=5,

p<0.05). Finally after a 30 min recovery, evoked synaptic currents recovered to -

50.20±6.87pA.

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Figure 27: Amylin decreases amplitude of evoked EPSC in response to stimulating electrode placed at spinal trigeminal fibers.

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The EPSC amplitude decreased from a mean±SEM of -81.48±23.99pA to -24.70±3.37pA after administration of amylin. Evoked EPSC recovered to -50.20±6.87pA after 30 min. n=5, P<0.05

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CHAPTER 4: DISCUSSION

4.1 Amylin in the trigeminal ganglion‐

Amylin is well characterized as a circulating hormone that is co-secreted with insulin from pancreatic islet β cells into the bloodstream. Since it has been detected in the nervous system including the trigeminal ganglia [92] and spinal trigeminal tract [93], questions remain concerning its function in the brain. In addition, the effects of amylin expressed in these neurons resulting in cardiovascular regulation are unknown. Our immunohistochemical and tract-tracing studies unveil a direct pathway of amylin immunoreactivity in trigeminal ganglion fibers which traverse through the spinal trigeminal tract to the parasympathetic choline acetyltransferase immunoreactive neurons as well as cardiac preganglionic vagal neurons located in the nucleus ambiguus.

The amylin immunoreactive neurons in the trigeminal ganglion were found to be small to medium sized neurons with an average diameter along its long axis of

34.9±0.4μm. This size is comparable to previous findings using in situ hybridization with amylin mRNA probes in the trigeminal ganglion showing a mean diameter of 30±0.7μm

[92]. Small neurons in the trigeminal ganglion give rise to C-type unmyelinated fibers, which are known to convey nociceptive transmission to the spinal cord and brainstem.

Thus, our finding that amylin is expressed in small to medium sized neurons supports the notion that amylin is a neuropeptide synthesized in neurons that modulates sensory transmission in the trigeminal system.

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4.2 Amylin and VGLUT2 in the trigeminal ganglion‐ VGluT2 confers glutamatergic phenotype in neurons and is responsible for

vesicular transport of L-glutamate [174]. In previous studies, VGluT1 and VGluT2 is

coexpressed in more than 80% of trigeminal ganglion neurons [172]. In our study,

colocalization of VGluT2 and amylin suggests that a subset of VGluT2 containing

neurons in the trigeminal ganglion also expresses amylin (Fig. 2C). The diving reflex is mediated by an intact glutamatergic trigeminal pathway [29] and may be mediated by

NMDA receptors [28]. Our colocalization study with VGluT2 and amylin in the

trigeminal ganglion suggests that amylin exists in a subset of trigeminal glutamatergic neurons, some of which may be responsible for mediating the cardiac responses seen in

the diving reflex.

This colocalization between amylin and VGluT2 suggests that amylin and

glutamate may be involved in cotransmission. Amylin and glutamate as cotransmitters is

consistent with cotransmission by glutamate with other neuropeptides such as

releasing hormone [175], substance P [176], [176]. Glutamate

cotransmission with nonpeptide neurotransmitters have also been found including

dopamine [177], ATP [178], nitric oxide [179], Throughout the central nervous system,

neurons which release more than one neurotransmitter has been increasingly been found

and has been accepted as the rule rather than exception [180]. Our anesthetized rat diving

model supports the notion of cotransmission of amylin with glutamate by showing that

intracisternal injection of the glutamate receptor blockade diminishes the bradycardia

seen in the diving reflex, which is further attenuated via amylin antagonist.

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4.3 Amylin containing fibers in the trigeminal ganglion project to the nucleus ambiguus The nucleus ambiguus, dorsal motor nucleus, and an intermediary zone in the

medulla, contains cardiac preganglionic vagal neurons (CPVN), which regulates

parasympathetic slowing of heart rate [181]. However, only the CPVN in the nucleus

ambiguus are responsible for the bradycardia seen in the diving reflex [182]. The diving

reflex is elicited by a glutamatergic trigeminal-vagal pathway previously described as

polysynaptic [182]. However, our studies support a direct projection of glutamate/amylin- containing fibers from the trigeminal ganglion to the nucleus ambiguus. We observed a nearly 100% colocalization rate between irAMY neurons and fluorogold immunoreactive neurons in trigeminal ganglia. Our finding of a monosynaptic trigeminal-vagal pathway defined by the presence of glutamate and amylin does not exclude previously described polysynaptic pathways that may utilize glutamate and/or other putative signaling molecules from nerve fibers originating from trigeminal ganglion cells or other brain areas [182].

4.4 Amylin microinjection in the nucleus ambiguus induces dose dependent bradycardia The novel amylin containing pathway extending from the trigeminal ganglion to

cholinergic neurons in the nucleus ambiguus leads us to the hypothesis that amylin may

physiologically modulate bradycardia. We therefore evaluated the effects of stereotaxic

injection of amylin into the nucleus ambiguus to simulate the synaptic release of amylin

from these trigeminal fibers. Amylin caused a dose-dependent drop in heart rate in

urethane-anesthetized rats. This dose response has been reported with other chemical

agents [153]. Maximal heart rate decrease was seen with microinjection of 1mM amylin.

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While this concentration is about 106 times higher than the plasma amylin concentration

which is in the picomolar range [183], it is comparable to the concentrations used in other

neuropeptide microinjection studies in the nucleus ambiguus [153].

4.5 Amylin receptor blockade attenuates amylin induced bradycardia Tachyphylaxis to amylin was assessed by repeated injections of the peptide at 40

min intervals to allow for recovery to baseline. Repeated injections did not result in a

statistically significant difference in the degree of bradycardia observed (n=5, p>.05).

The absence of a significant tachyphylaxis justified the validity of subsequent vagotomy and receptor blockade studies. Vagotomy abolished the amylin induced bradycardia, leading to the conclusion that the bradycardia is mediated by cardiac preganglionic vagal neurons in the nucleus ambiguus with fibers projecting to the vagus.

Receptor blockade using the putative amylin receptor blocker sCT (8-32) was used to evaluate whether amylin-induced bradycardia is pharmacologically mediated through its binding to the amylin receptor. Microinjection of 4mM sCT (8-32) prior to microinjection of 1mM amylin resulted in a statistical significantly attenuated amylin response. This attenuation was reversible and disappeared after a 2 hr recovery period, therefore demonstrating that amylin induced bradycardia reversibly acts on amylin receptors.

In order to verify the presence of the amylin receptor in the brainstem, RT-PCR was performed to detect calcitonin receptor, and RAMP 1, RAMP2, and RAMP3. The mRNA for all the components for the amylin receptor was found in the brainstem.

Microinjection studies shows amylin induced bradycardia is blocked by sCT (8-32), we

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RT-PCR studies in the brainstem found amylin receptor to be present, and it is likely that diving bradycardia is due to activation of the amylin receptor.

4.6 Amylin potentiates bradycardia in anesthetized rat diving model To assess the role of amylin in the diving reflex, we established an anesthetized rat diving model similar to the forced awake rat diving model [160]. The MouseOX pulse oximeter system used for heart rate measurement is a novel noninvasive technique developed to allow measurements of heart rate changes during simulated diving prior to any surgical or pharmacological interventions. In the current study, rats had an average heart rate of 381bpm and an average bradycardia of 211bpm (56%) following immersions of the rat nostril in cold water. These values are not significantly different than the average bradycardia of 208bpm (54%) observed after saline intracisternal injection.

These control diving bradycardia is less profound than the 76% decrease previously described in forced diving using unanesthetized rats [160] and may be due to the cardioinhibitory effects of urethane [184]. In a study of urethane-anesthetized rat responses to ammonia vapor, which activates the nasopharyngeal reflex, bradycardia of

36% [185] were observed while using 1.3g/kg urethane. This amount of urethane is comparable to our current study where 1.5g/kg urethane was used.

Pharmacological vagotomy using atropine administered I.P. abolishes the bradycardic component of the diving reflex in rats [186] and muskrats [129]. We verified this finding in our urethane-anesthetized rat diving model. Subsequently, glutamate antagonists were injected to the cisterna magna space near the nucleus ambiguus to evaluate the glutamatergic activation of the trigeminal-vagal bradycardia. The reflex was significantly attenuated from a 51% decrease in heart rate to a 34% decrease after 101 application of glutamate antagonists in one group of rats. In another group of rats, amylin antagonist sCT (8-32) resulted in a statistically significant attenuation of diving bradycardia from a 61% heart rate decrease in saline controls to 51% after sCT (8-32) intracisternal injection, indicating that release of amylin from trigeminal nerves to vagal preganglionic neurons in the nucleus ambiguus modulates heart rate. Lastly, the injection of amylin antagonists and glutamate antagonists conjointly resulted in a 46% to 21% heart rate decrease, a larger attenuation than injecting glutamate or amylin antagonists alone. Viewed in this context, our result raises the hypothesis that amylin enhances the synaptic activity of glutamate, resulting in a more sustained bradycardic response seen in the diving reflex.

Amylin role in the nucleus ambiguus is not only relevant to the diving reflex, but also has therapeutic implications for treating arrhythmias. Reductions in cardiac parasympathetic control are associated with an increased risk for sudden death [3].

Studies to increase cardiac parasympathetic control via vagal stimulation have resulted in the prevention and termination of arrhythmias in animal models [8] as well as in patients

[10]. Additionally, studies have shown that application of the diving reflex is an effective treatment of paroxysmal supraventricular tachycardia [13]. Trigeminal-vagal reflexes also include two reflexes elicited during brain surgery that may result in cardiac arrest: trigemino-cardiac [166] and oculo-cardiac [117, 122] reflexes which also result in apnea and bradycardia. However, these reflexes are triggered by accidental irritation of trigeminal nerves [187] during deep skull base surgeries and in rare cases, may result in cardiac arrest [166]. The current study not only for the first time links a neuropeptide

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with the diving reflex, but it paves the way for future research in diseased states of heart

rate control and ischemia tolerance.

4.7 Amylin depolarizes cardiac preganglionic vagal neurons in the nucleus ambiguus In whole-cel patch-clamp of cardiac preganglionic vagal neurons, amylin was

found to depolarize only 25% of neurons tested (n=36). This finding is supported by our

immunofluorescent study showing amylin immunoreactive fibers synapsing on a subset

of cardiac preganglionic vagal neurons identified using the retrograde tracer rhodamine.

The slight depolarization of 6.4 ± 1.1mV over the course of almost 10 minutes shows that

amylin does not likely act on ionotropic receptors which directly acts on ion channels.

The time course is consistent with amylin activating the amylin receptor, which is the

calcitonin receptor, a G-Protein coupled receptor, coupled with RAMP 1 or 3.

The hyperpolarizing current injection is used to measure conductance. When

excitable membranes experience a conductance decrease, the membrane potential will

move away from the equilibrium potential of the ionic conductance decrease. It is likely

that in the case of amylin depolarization is due to closure of potassium channels, resulting

in a shift away from potassium’s equilibrium potential of around -84mV. A decrease in

potassium conductance is thought to be the primary mechanism underlying

depolarizations elicited by slow excitatory neurotransmitters including 5-HT [188] and

neuropeptides such as neurotensin [189]. It is also possible that superimposed increase in

cation conductance is also involved. Cation conductance increase has been suggested to be involved in excitatory effects of neurotransmitters such as acetylcholine, noradrenaline, and 5-HT [188].

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4.8 Amylin increases cardiac preganglionic vagal neuron excitability Excitability of cardiac preganglionic vagal neurons were elicited under current

clamp by injecting depolarizing current lasting 500ms and of variable amplitude (100-

500pA). Amylin was found to increase excitability across the amplitudes tested. This result is consistent with our stereotaxic microinjection studies, which shows that amylin results in an extended period of increased activation of cardiac preganglionic vagal neurons. While amylin itself may not be responsible for the prolonged activation, but it

increases the likelihood that other synaptic inputs into the nucleus ambiguus would activate cardiac preganglionic vagal neurons.

Small conductance Ca2+ activated K+ channels were found to be responsible for regulating firing properties and excitability in cardiac preganglionic vagal neurons [165].

As discussed in the following section, amylin was found to decrease these Ca2+ activated

K+ channels in dorsal forebrain neurons. It is likely that the same mechanism is used by

amylin in the nucleus ambiguus to modulate excitability.

4.9 Amylin decreases whole cell currents in cardiac preganglionic vagal neurons Amylin effect on whole cell currents was examined under voltage-clamp. The

cell was exposed to 300ms long voltage ramps to measure the current response. Whole

cell currents were found to decrease, especially at positive voltages. This decrease in

conductance by amylin is consistent with its effects in increasing excitability and

depolarization. Reduction in whole cell currents in this voltage range is associated with

activation of calcium-dependent conductances [96]. Previous work in rat cholinergic

basal forebrain neurons in the diagonal band of Broca found characteristically similar

current-voltage relationship by amylin [96]. In that study, it was also found that this 104

whole cell current reduction is due to reduction of small conductance Ca2+ activated K+ channels in cholinergic dorsal forebrain neurons. As mentioned in the previous section, excitability of cardiac preganglionic vagal neurons were found to be modulated by small conductance Ca2+ activated K+ channels [165]. Amylin reduction in whole cell currents in

our study is therefore likely also mediates via small conductance Ca2+ activated K+ channels, which results in an increase in excitability.

4.10 Amylin decreases spontaneous excitatory post synaptic potential frequency in the nucleus ambiguus In whole-cel patch-clamp of cardiac preganglionic vagal neurons under the

suppression of synaptic transmission by TTX, amylin was found to decrease spontaneous

excitatory post synaptic current frequency, but not amplitude. A reduction in frequency

but not amplitude is associated with a presynaptic modulation of membrane function,

resulting in decreased likelihood that neurons will release neurotransmitter packets to

activate the post synaptic neuron, which in this case are cardiac preganglionic vagal

neurons. This novel finding by amylin reveals that in addition to depolarization and

inducing increased excitability, amylin may have a silencing effect on other synaptic

inputs to cardiac preganglionic vagal neurons, reducing the likelihood that other synaptic

inputs are transmitted to cardiac ganglia.

4.11 Amylin decreases evoked synaptic currents in the nucleus ambiguus In whole-cel patch-clamp of cardiac preganglionic vagal neurons under voltage-

clamp, a bipolar stimulating electrode was placed nearby in the spinal trigeminal tract.

Stimulating pulses were given, evoking synaptic release of neurotransmitters from the

spinal trigeminal tract. Amylin treatment resulted in a decrease in amplitude of evoked 105

synaptic responses. This shows further evidence that amylin inhibits not only presynaptic

release of neurotransmitters, but also inhibits synaptic transmission to cardiac

preganglionic vagal neurons.

4.12 Future directions Current techniques are somewhat limited in being able to determine under which

circumstances these neuropeptide modulators are released into the synapse, and how the

numerous neuropeptides interact with one another. Studies involving anesthetized rat

diving model with microdialysis may be useful in definitively confirming if amylin is

indeed released during diving bradycardia.

Since amylin receptor is calcitonin receptor heterodimerized with RAMP, it is

unknown whether the presence of another member of the calcitonin peptide family in the

synapse may alter amylin effects in neurons. Future work may clarify this interaction

between the different members of the calcitnonin peptide family, especially if these peptides are involved in cotransmission at a common synapse.

Some mammals such as the gray seal and muskrats are expert divers compared to

rats and humans. It would be valuable to examine the interspecies differences in

neuropeptide amylin mRNA and protein expression in the brainstem. By correlating

expression level with dive length, we may be able to determine amylin role in mediating

heart rate in the brainstem.

Human, monkey and cat amylin can form insoluble polymeric form in pancreatic

beta cells, which are associated with diabetes[190]. It is not known how this toxic form of amylin may play in the brain. Future work may need to be conducted to compare feline, human, and rat amylin possibly differential effects on neurons. 106

Previous intracerebroventricular administration of amylin resulted in thermogenesis, , and tachycardia that are reversed by intravenous β-

adrenergic blockade [191]. Although the current study focuses on characterizing amylin

novel role in mediating bradycardia in the brainstem, it remains to be shown whether

amylin thermogenic and tachycardia actions in the cerebral hemispheres are related to

brainstem reflexes.

Diabetes impairs nucleus ambiguus function [139]. Patients with diabetes

developed less bradycardia in response to cold facial stimulus [192]. Amylin

concentrations in the bloodstream are higher in diabetes [144]. These studes may link

amylin concentration with altered cardiac function in diabetes and the diving reflex.

Future studies may be conducted to determine if this impairment in nucleus ambiguus

function and diving bradycardia seen in diabetes is mediated by amylin.

Impaired activation of cardiac preganglionic vagal neurons in the nucleus

ambiguus has been implicated in sudden infant death syndrome [193, 194]. It has also

been found in infants with home cardiorespiratory monitors that apnea, bradycardia were

spontaneously recorded within the first month of life [195]. Amylin potential role in the

pathogenesis of sudden infant death syndrome is unknown.

The magnitude of diving bradycardia in infants seems to decrease after birth [196].

In a separate study, it was found to decrease from -38.8% bradycardia in 4-5 month old

infants, to -18.6% in 10-12 month old infants [197]. This decrease in diving bradycardia

after birth may signify a role of diving bradycardia during birth or during life in the

womb. Further, amylin is found in the placenta of both human and rat placenta relative to

nutritional status [198]. This finding shows that amylin may regulate metabolic functions

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in the placenta. The combination of amylin role in diving bradycardia and amylin

presence in the placental regulation of metabolic functions suggests that there may be a

central theme in amylin role in the body related to situations involving metabolic stress such as birth. Future work in this area may hold the key to our understanding of amylin.

4.13 Conclusion As shown in the new working model seen Figure 28, our study shows the

functional role of amylin in nerve fibers projecting from the trigeminal ganglia to neurons

in the nucleus ambiguus. These amylin containing trigeminal fibers were found to

project to ChAT positive neurons in the nucleus ambiguus. Amylin containing trigeminal

fibers were also found to project to a subset of cardiac preganglionic vagal neurons identified in the nucleus ambiguus. In vivo, amylin microinjected directly to the nucleus ambiguus caused a reversible, vagal mediated bradycardia in anesthetized rats, which was partially blocked by the amylin receptor antagonist sCT (8-32). Furthermore, our study uses the anesthetized diving rat model to show that amylin modulates the glutamatergic transmission of diving-triggered bradycardia. Amylin was found to depolarize neurons while decreasing whole cell currents and increasing excitability. Amylin also decreased spontaneous excitatory post synaptic current frequency, a sign that amylin decreases presynaptic neurotransmitter release. Amylin was also found to decrease synaptic potentials and decrease evoked synaptic transmission. Taken together, these data suggest that amylin plays an essential role in the brainstem integration of sensory input from the trigeminal ganglia resulting in parasympathetic control of heart rate. Not only is our current study the first time amylin has been associated with a brainstem reflex, the 108

current study greatly improves the understanding of neural control of heart rate in one of

the most powerful autonomic reflex. Due to its association with diabetes and its

involvement in metabolic functions, amylin important role in the diving reflex may only play a small part in a more unified understanding of global patterns in amylin control.

Figure 28: Summary diagram. Trigeminal-vagal reflexes including the diving reflex are activated by stimulation of trigeminal nerve (Star 1). The bradycardia component is mediated by the vagus nerve (Star 4). The aim of this thesis project was to examine amylin in the trigeminal-vagal pathway (Star 2) and the mechanism through which amylin activates cardiac preganlionic vagal neurons in the nucleus ambiguus (Star 3). Amylin mediates diving bradycardia via the trigeminal-vagal pathway (Star 2). Amylin is found in small to medium sized trigeminal ganglion neurons (Star 1) that are also VGluT2 positive (Star 6). These amylin containing neurons in the trigeminal ganglion project fibers directly to the area of the nucleus ambiguus in the brainstem (Star 3), where the fiber boutons are observed to directly oppose ChAT (Star 9) containing neurons. These amylin containing (Star 5) trigeminal fibers (Star 2) were also found to project to a subset of cardiac preganglionic vagal neurons (Star 3). Microinjection of amylin into the nucleus ambiguus to simulate the pharmacological effects of amylin release shows a dose dependent bradycardia that is vagal mediated and is reversibly blocked by the amylin receptor blocker (Star 7). In an 109

anesthetized rat diving model, glutamate receptor antagonists are found to diminish the bradycardia seen in the diving reflex (Star 8), with further attenuation by the amylin receptor antagonist (Star 7). Whole-cel patch-clamp found that amylin depolarizes cardiac preganglionic vagal neurons (Star 3), and also increases excitability. Whole cell currents were reduced after amylin administration in cardiac preganglionic vagal neurons (Star 3). Amylin reduced presynaptic release of neurotransmitter after administration of amylin (Star 10). Amylin also reduced evoked synaptic currents.

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