Improving the Quality of induced Anaesthesia in Rodents

Prajwal Thakre

Master of Pharmacy ()

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine School of Biomedical Sciences Abstract Laboratory rodents are widely used in a variety of experimental techniques throughout the world, which helps in advancing biomedical research. As this use is likely to continue in the future, it is imperative to prioritize animal welfare so that this research meets ethical standards, facilitates better science and increases the reproducibility of data. Laboratory rodents are regularly anaesthetised as part of biomedical research throughout the world. An anaesthetic agent that is predictable, safe and efficient enough to alleviate pain and distress throughout the duration of anaesthesia is considered ideal. Although there is no ideal anaesthetic available, a constant search for improving current anaesthetic strategies is important. is a neuroactive available as an injectable anaesthetic and used in large animals such as , , horses and swine with safety. However, it often exhibits a significant motor excitation in the form of limb paddling and facial twitching when used in laboratory rodents. We sought to investigate if alfaxalone modulates synaptic to central motor and if there are any underlying second messenger systems that are triggered leading to excitatory behaviour upon alfaxalone administration. We investigated the effects of alfaxalone on inhibitory and excitatory glutamatergic synaptic transmission to rat hypoglossal motor neurons (HMNs). Whole- patch clamp recordings were made from neonatal (P7-14) rat HMNs within the hypoglossal nucleus in a 300µm thick transverse brainstem slice. Spontaneous or evoked excitatory post-synaptic currents (sEPSCs/eEPSCs) or miniature glycinergic inhibitory post-synaptic currents (mIPSCs) were recorded at a holding potential of -60mV using a CsCl-based internal solution. Similarly, (AP) firing was recorded at a holding potential of -65mV with a K+ methyl sulfate-based internal solution. Our results show that alfaxalone significantly reduced mIPSC frequency (-39%) and amplitude (-54%) to HMNs, consistent with a reduction in inhibitory transmission to HMNs, possibly leading to neuromotor excitation exhibited as muscle twitching. There was also a positive shift (+35%) in the baseline holding current (Iholding) upon alfaxalone treatment. It was also noted that alfaxalone, even at higher concentrations (10nM-3µM), failed to significantly alter either spontaneous or evoked (25µM) EPSC frequency, amplitude, half-width, rise-time and

Iholding. Similarly, repetitive action potential firing by HMNs was not altered by alfaxalone. However, the molecular mechanism behind the effect of alfaxalone in inhibiting glycinergic transmission remains unclear. Earlier reports have suggested that and steroid-like molecules can bring about modulation of several TRP channels, which may be physiologically significant. Our

ii data showed a shift in Iholding upon alfaxalone treatment, which could be due to an indirect modulation of transient potential (TRP) channels. Hence, to investigate this further, the effects of capsaicin, a well-known TRPV1 , on synaptic transmission to HMNs was examined. Interestingly, we found that capsaicin increased spontaneous excitatory post-synaptic current (sEPSC) frequency (+115%) and amplitude (+24%) recorded from neonatal rat HMNs. The frequency of miniature excitatory post-synaptic currents (mEPSCs), recorded in the presence of tetrodotoxin (TTX), was also increased by capsaicin, but capsaicin did not alter mEPSC amplitude, consistent with a pre-synaptic mechanism of action. A negative shift in Iholding was elicited by capsaicin under both recording conditions. The effect of capsaicin on excitatory synaptic transmission remained unchanged in the presence of the TRPV1 antagonists, capsazepine or SB366791, suggesting that capsaicin acts via a mechanism which does not require TRPV1 activation. Capsaicin, however, failed to show any effect on eEPSCs, and did not alter the paired- pulse ratio (PPR) of eEPSCs. Repetitive action potential (AP) firing in HMNs was also unaltered by capsaicin, indicating that capsaicin does not change intrinsic HMN excitability. Interestingly, the effect of capsaicin on spontaneous and miniature glycinergic inhibitory post-synaptic currents (sIPSC/mIPSC) was a significant decrease in current amplitude (-62% and 56% respectively) without altering frequency, indicating a post- synaptic mechanism of action. Interestingly, even this effect of capsaicin on inhibitory current amplitude was not blocked by TRPV1 antagonist, capsazepine. These results suggest that modulation of IPSCs by neuroactive steroid, alfaxalone, is unlikely to involve TRPV1 activation. Our results indicate that neuromotor excitation during alfaxalone anaesthesia are most likely to be mediated by decreases in inhibitory synaptic input, without alteration in spontaneous or evoked excitatory synaptic transmission, and that alfaxalone does not excite HMNs sufficiently to cause any changes in action potential firing. We also demonstrate that capsaicin exerts a modulatory effect on glutamatergic excitatory as well as glycinergic inhibitory, synaptic transmission in HMNs via an unknown mechanism independent of TRPV1 channels.

iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis.

iv Publications during candidature Peer-reviewed papers: Thakre PP & Bellingham MC. (2017). Capsaicin enhances glutamatergic synaptic transmission to neonatal rat hypoglossal motor neurons via a TRPV1-independent mechanism. Frontiers in cellular neuroscience 11, 1-18.

Publications included in this thesis Thakre PP & Bellingham MC. (2017). Capsaicin enhances glutamatergic synaptic transmission to neonatal rat hypoglossal motor neurons via a TRPV1-independent mechanism. Frontiers in cellular neuroscience 11, 1-18. – incorporated as Chapter 3 and 4.

Contributor Statement of contribution Prajwal Thakre Conception and design (80%) Analysis and interpretation (70%) Drafting and production (80%) Dr Mark Bellingham Conception and design (20%) Analysis and interpretation (30%) Drafting and production (20%)

v Contributions by others to the thesis No contributions by others.

Statement of parts of the thesis submitted to qualify for the award of another degree None.

Research involving human or animal subjects Title: Neurophysiological investigation of neural and of the neuromotor system AEC Approval Number: SBMS/142/15/NHMRC

vi Acknowledgements “Will changing membrane potential help?”, “Is this current or noise?”, “Is the ground electrode fine?”, “Is this a healthy cell?”, “Is the positive pressure too high?”, “Is this the right internal solution?”, “Why is the baseline dropping?”, “Should I increase concentration?”, … For the past three and half years, I was occupied with these and several similar thoughts & questions. I believe, I have answered most of them and can confidently assert that I understand electrophysiology better. I could not have reached this stage of my career without the guidance, support and motivation from several individuals in my personal and professional life. I take this opportunity to acknowledge them all. First, I would sincerely thank my thesis principal supervisor, Assoc/Prof Mark Bellingham for accepting me as a doctoral student in his lab and for giving me a fantastic opportunity to contribute in the field of Neuroscience. I am truly grateful for his big-hearted decision to take a beat on someone with no prior electrophysiology experience. I deeply appreciate his time, guidance and funding in making my doctoral training a fruitful experience. He has not only taught me how to design good experiments but also how to think like a scientist. I would certainly make a special mention of his calm demeanor while training young researchers and the ability to share his expertise and knowledge with genuine enthusiasm. I have certainly become a better researcher under his guidance. Special thanks to my associate supervisor Assoc/Prof Stuart Mazzone. I deeply appreciate his lightening fast email replies and critical feedback on my thesis. His insights and thoughtful comments have made my thesis better. I would like to thank Assoc/Prof Peter Noakes for granting me access to use his lab equipments, Assoc/Prof Nickolas Lavidis and Dr Shyuan Ngo for agreeing to be part of my thesis committee. I deeply appreciate their time and feedback. I would also like to thank Drs Refik Kanjhan, Arnauld Belmer, John Lee, Matthew Fogarty, Paul Klenowski and Sean Yang for their help, advice and inspiring discussions throughout my Ph.D. I am grateful to Dr Mary-Louise Roy Manchadi for giving me the opportunity to contribute as a tutor at the School of Biomedical Sciences. Tutoring has helped me in developing skills critical to my future ahead. I gratefully acknowledge the funding from The University of Queensland that made my Ph.D. research possible. In particular, Faculty of Medicine Travel Scholarship that supported me to attend domestic and international conferences.

vii My time at The University of Queensland was enjoyable largely due to friendships I made with talented and motivated researchers like Lorenzo Odierna, Haitao Wang, Tesfaye Wolde Tefera, Dengyun Ge, Sophie Wright, Joy Thompson, Qiao Ding, Jonu Pradhan, Andy Lee, Kirat Chand and Erica Mu. I thank you all for your help, advice and support. As I move forward, I will continue to cherish your friendship throughout. I am also grateful to Omkar, Priyank, Lalit, Venkat, Amit and Eshan for the time spent outside lab. Their friendship has been pivotal in my PhD journey. I must acknowledge Mary White, Fiona Parker, James Mather and the UQ Graduate School for their administrative assistance in keeping things organized. Similarly, The University of Queensland's Biological Resources (UQBR) team deserves special thanks for their timely supply of animals and for providing excellent animal housing facilities. Finally, big thanks to my parents, Pradeep and Jyoti, and my little brother, Suprit for believing in me and always supporting my endeavors. I also thank Lucy for keeping everyone busy - you all are fantastic!

viii Financial support This research was supported by The University of Queensland International Scholarship (UQI)

Keywords neuroactive steroid, alfaxalone, anaesthesia, synaptic neurotransmission, slice- electrophysiology, hypoglossal, capsaicin, TRPV1

Australian and Standard Research Classifications (ANZSRC) ANZSRC code: 110902, Cellular , 80% ANZSRC code: 111502, Clinical Pharmacology and Therapeutics, 20%

Fields of Research (FoR) Classification FoR code: 1109, Neurosciences, 80% FoR code: 1115, Pharmacology and Pharmaceutical Sciences, 20%

ix Table of contents

Title Page Abstract ii Declaration by author iv Publications during candidature v Publications included in this thesis v Contributions by others to the thesis vi Statement of parts of the thesis submitted to qualify for the award of another degree vi Research involving human or animal subjects (Ethics approval) vi Acknowledgements vii Financial support ix Keywords ix Australian and New Zealand Standard Research Classifications (ANZSRC) ix Fields of Research (FoR) Classification ix Table of contents x List of figures xiii List of tables xv List of abbreviations used in the thesis xvi Chapter 1 Introduction Laboratory animals in biomedical research 1 Laboratory animal anaesthesia 1 Safety in laboratory rodent anaesthesia 3 Limitations of current anaesthetic regimes 3 Current approaches to anaesthetic drug development 4 as anaesthetics 5 Alfaxalone 7 Neuromotor excitation during anaesthesia 8 Glutamatergic transmission in motor neurons 9 Possible involvement of transient receptor potential (TRP) channels in motor 9 excitation Rationale and aim of study 11 Materials and methods 12

x Chapter 2 Abstract 20 Introduction 20 Results Alfaxalone (25µM) reduces miniature glycinergic IPSC amplitude and 22 frequency to rat hypoglossal motor neurons (HMNs) Alfaxalone does not alter spontaneous excitatory synaptic transmission to rat 25 hypoglossal motor neurons (HMNs) Evoked EPSC parameters and paired-pulse ratio (PPR) remain unchanged 27 after alfaxalone application Action potential (AP) firing of HMNs remains unchanged after alfaxalone 30 application Discussion 32 Chapter 3 Abstract 35 Introduction 36 Results Capsaicin (1µM) increases spontaneous excitatory post-synaptic current 37 frequency to rat hypoglossal motor neurons (HMNs) Capsaicin (10µM) increases spontaneous excitatory post-synaptic current 40 frequency and amplitude to rat hypoglossal motor neurons (HMNs) Effect of capsaicin (10µM) on frequency of excitatory post-synaptic currents 44 is mediated via a TTX-insensitive mechanism TRPV1 antagonists do not block the effect of capsaicin on sEPSC frequency 46 Capsaicin (10µM) does not alter evoked EPSC parameters or paired-pulse 53 ratio (PPR) Capsaicin (10µM) does not affect repetitive action potential (AP) firing of 55 HMNs Discussion 58

xi Chapter 4 Abstract 63 Introduction 63 Results Capsaicin (10µM) reduces spontaneous glycinergic inhibitory post-synaptic 64 current (sIPSC) amplitude to rat hypoglossal motor neurons (HMNs) Capsaicin acts via a TTX-insensitive mechanism to reduce amplitude of 67 glycinergic inhibitory currents in rat hypoglossal motor neurons (HMNs) The TRPV1 antagonist, capsazepine does not block the effect of capsaicin on 70 sIPSC amplitude Discussion 74 Chapter 5 Abstract 77 Introduction 77 Results Alfaxalone (25µM) reduces spontaneous glycinergic IPSC amplitude, half- 79 width and frequency in mouse hypoglossal motor neurons (HMNs) Capsaicin (10µM) increases spontaneous excitatory post-synaptic current 82 (sEPSC) frequency and amplitude to mouse hypoglossal motor neurons (HMNs) Discussion 85 General discussion 88 References 90 Appendices Link for the publication included in thesis 108 Animal ethics approval letter 109

xii List of figures Title Page Figure 1.1 Chemical structure of alfaxalone (3α-hydroxy-5α--11, 20-dione) 7 Figure 1.2 Brainstem slices preparation 14 Figure 1.3 Schematic representation (not to scale) of electrophysiology setup used in 16 this study Figure 1.4 Electrophysiology recordings and data analysis of synaptic currents and 18 action potentials (APs) from hypoglossal motor neurons Figure 2.1 Alfaxalone (Alfa) reduces miniature inhibitory synaptic transmission to rat 24 HMNs Figure 2.2 Spontaneous excitatory post-synaptic currents (sEPSCs) to rat HMNs are 26 unchanged after application of alfaxalone (Alfa) Figure 2.3 Evoked excitatory post-synaptic current (eEPSC) to rat hypoglossal motor 29 neurons (HMNs) remain unchanged after application of alfaxalone (Alfa) Figure 2.4 Repetitive action potential (AP) firing by HMNs is unchanged after 31 alfaxalone (Alfa) application Figure 3.1 1µM Capsaicin (Caps) increases glutamatergic excitatory post-synaptic 39 current frequency to rat HMNs Figure 3.2 10µM Capsaicin (Caps) increases glutamatergic excitatory post-synaptic 42 current frequency and amplitude to rat HMNs Figure 3.3 10µM Capsaicin (Caps) induced increase in sEPSC frequency but not 45 amplitude is mediated via non-TTX sensitive mechanism Figure 3.4 Pre-application of the TRPV1 antagonist, capsazepine (Capz), blocked the 48 effect of capsaicin (Caps) on sEPSC amplitude but not on frequency Figure 3.5 Pre-application of the TRPV1 antagonist, SB366791, blocked the effect of 51 capsaicin (Caps) on sEPSC amplitude but not on frequency Figure 3.6 Evoked excitatory post-synaptic current (eEPSC) to rat hypoglossal motor 54 neurons (HMNs) were unchanged after application of capsaicin Figure 3.7 Repetitive action potential (AP) firing by HMNs is unchanged after 57 capsaicin application Figure 4.1 10µM Capsaicin (Caps) reduces glycinergic inhibitory post-synaptic 66 current amplitude to rat HMNs Figure 4.2 10µM Capsaicin (Caps) reduces miniature inhibitory post-synaptic current 69 amplitude to rat HMNs

xiii Figure 4.3 Pre-application of the TRPV1 antagonist, capsazepine (Capz) failed to 72 block the effect of capsaicin (Caps) on sIPSC amplitude Figure 5.1 Alfaxalone (Alfa) reduces spontaneous inhibitory post-synaptic 81 transmission to WT C57BL/6 mouse HMNs Figure 5.2 Capsaicin (Caps) increases glutamatergic excitatory post-synaptic current 84 frequency and amplitude to mouse HMNs

xiv List of tables Title Page Table 2.1 Miniature IPSC parameters of HMNs upon application of alfaxalone to rat 25 HMNs Table 2.2 Spontaneous EPSC parameters of rat HMNs upon application of alfaxalone 27 Table 2.3 Evoked EPSC parameters of rat HMNs upon application of alfaxalone 30 Table 2.4 Repetitive action potential parameters of HMNs upon application of 32 alfaxalone Table 3.1 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 40 1µM capsaicin Table 3.2 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 43 10µM capsaicin Table 3.3 Miniature EPSC parameters of neonatal rat HMNs upon application of 46 10µM capsaicin Table 3.4 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 49 10µM capsaicin in presence of 10µM capsazepine Table 3.5 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 52 10µM capsaicin in presence of 20µM SB366791 Table 3.6 Evoked EPSC parameters of neonatal rat HMNs upon application of 10µM 55 capsaicin Table 3.7 Repetitive action potential (AP) firing parameters of neonatal rat HMNs 58 upon application of 10µM capsaicin Table 4.1 Spontaneous IPSC parameters of neonatal rat HMNs upon application of 67 10µM capsaicin Table 4.2 Miniature IPSC parameters of neonatal rat HMNs upon application of 10µM 70 capsaicin Table 4.3 Spontaneous IPSC parameters of neonatal rat HMNs upon application of 73 10µM capsaicin in presence of capsazepine Table 5.1 Spontaneous IPSC parameters of neonatal C57BL/6 WT mice HMNs upon 82 application of 25µM alfaxalone Table 5.2 Spontaneous EPSC parameters of neonatal C57BL/6 WT mice HMNs 85 upon application of 10µM capsaicin

xv List of abbreviations used in the thesis 20-HETE 20-hydroxyeicosatetraenoic acid 2-APB 2-aminoethoxydiphenyl borate 5-HT 5-hydroxytryptamine aCSF artificial cerebrospinal fluid Alfa alfaxalone AP action potential Aβ A-beta Ca2+ calcium ion

CaCl2 calcium chloride Caps capsaicin Capz capsazepine CNS central nervous system

CO2 carbon dioxide CsCl chloride CsOH caesium hydroxide CYP2 DMSO dimethyl sulfoxide eEPSC evoked excitatory post-synaptic current EGTA glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid Fig figure

GABAA gamma (γ) aminobutyric acid type A receptors HEK cells human embryonic cells HEPES N-2-hydroxyethyl-piperazine-N’-2-ethanesulfonic acid HMNs hypoglossal motor neurons HPCD 2-hydroxypropyl-β- Hz hertz

Iholding baseline holding current IM intramuscular IP intraperitoneal IV intravenous KCl potassium chloride KS test Kolmogorov-Smirnov test mEPSC miniature excitatory post-synaptic current Mg2+ magnesium ion

xvi MgCl2 magnesium chloride min minute mIPSC miniature inhibitory post-synaptic current ml millilitre mM millimolar ms millisecond mV millivolt MΩ mega ohm NaCl sodium chloride

NaH2PO4 monosodium phosphate

NaHCO3 sodium bicarbonate NE nNOS neuronal synthases

O2 oxygen pA picoampere PIC persistent inward current POCD post-operative cognitive dysfunction PPR paired-pulse ratio S.D. standard deviation SC subcutaneous SDN sexually dimorphic nucleus sEPSC spontaneous excitatory post-synaptic current sIPSC spontaneous inhibitory post-synaptic current TRP transient receptor potential TRPA transient receptor potential ankyrin TRPC transient receptor potential canonical TRPM transient receptor potential melastatin TRPML transient receptor potential mucolipin TRPP transient receptor potential polycystin TRPV transient receptor potential vanilloid TTX tetrodotoxin WT wild type

xvii Chapter 1 Introduction Laboratory animals in biomedical research Use of animals in laboratories is an integral part of biomedical research. Animal experiments not only help us to understand better, but also help in understanding the changes that occur between normal and diseased states. Animal experiments help in understanding a clinical disease better, and hence, propel the discovery of suitable targets to influence the pathophysiological causes of disease. Use of animals in laboratories has led to improvement of the lives of humans and domestic animals to a great extent, by reducing suffering and promoting health across the world. In the 20th century, with the development of biomedical disciplines such as pharmacology, toxicology and immunology, there has been a sharp increase in the use of animals for research purposes (Baumans, 2004). Among the various animals used in research, laboratory rodents (rats and mice) are the most widely used experimental species. They are routinely used in a variety of research experiments; from fundamental biological research to cardiovascular, neurological, and neurobehavioural studies, laboratory rodents have helped in advancing all key areas of biomedical research. The laboratory rat (Rattus norvegicus), in particular, is a unique model to understand human better, allowing testing of novel therapeutic agents in efficient ways. Traditionally, toxicological studies have also relied more on rats than on mice. Also, when it comes to physiological manipulations, rats are preferred over mice, owing to their larger body size (Krinke, 2000). The prominent importance of rats in research can be easily gauged from the fact that they are the most widely used species in research after humans; no other model system is as widely used as are rats, followed by mice (Jacob, 2010). With the increasing use of laboratory rodents likely to continue in the foreseeable future, it is also equally important to take care of their welfare in order to facilitate better science and ensure the reproducibility of data. With the landmark publication of The Principles of Humane Experimental Technique in 1959 by William Russell and Rex Burch, it was made clear that humane techniques like the 3 R principles of replacement, reduction, and refinement are prerequisites for successful animal experiments (Hubrecht & Kirkwood, 2010). Since then, researchers all over the world follow the 3 R principles.

Laboratory animal anaesthesia One of the key pillars of the 3 R principles is that of refinement, which refers to implementation of modified experimental procedures to minimize suffering, pain and

1 distress to animals (Aronson, 1990). An effective way to minimize pain and suffering in laboratory rodents is by the use of suitable anaesthesia for all procedures likely to cause significant pain. Anaesthesia in laboratory animals can be defined as a state of loss of consciousness and sensation with muscle relaxation (Kohn et al., 1997). Anaesthesia is an important means by which the experimenter makes sure that distress is minimized in animals. In addition to this, research experiments frequently demand either a short duration of immobilization (e.g. retro-orbital blood collection) or prolonged immobilization with sufficient muscle relaxation (e.g. survival surgeries) for which the animal has to be anaesthetised. Critical surgical procedures can only be performed with ease if rodents are successfully subjected to a controlled, deep and relaxed state of anaesthesia for the duration of surgery. Thus, the continued use of animals in biomedical research brings an imperative need to develop a safe anaesthetic protocol. Anaesthetic regimes currently used in common laboratory practices make use of agents that typically fall under two categories, based on their route of administration viz. inhalation and injectable anaesthesia. Inhalation anaesthetics are generally gases or volatile liquids and the popular choices include , , , etc. However, the natural state of these agents makes it necessary to use specialized machines or vaporizers that mixes oxygen with the vaporized anaesthetic and delivers this mixture to the animal. Inhalation anaesthesia involves special instruments making the overall setup expensive, and generally requires specialized training, which makes it less suitable for regular use in standard laboratory settings, making it less preferable. On the other hand, injectable anaesthetics are those that can be easily administered by of the anaesthetic agents. The most prominent advantage of injectable anaesthetics over inhalation anaesthetics is convenience, particularly for personnel who can perform injections with little professional training. They also do not require any special set up; making it the least expensive mode of anaesthesia. Also, most small laboratory rodents can be humanely restrained for anaesthetic injections either via intraperitoneal (IP) or intramuscular (IM) routes, provided they are accustomed to being handled. Intravenous (IV) injections are sometimes difficult in these small species, and hence, administration via intraperitoneal, subcutaneous (SC) or intramuscular routes is usually preferred (Flecknell, 1993; Flecknell, 2016b). Additionally, administration via intraperitoneal and subcutaneous routes is also considered less stressful to the animals as it causes less pain and damage. In general, IP injections are usually preferred because they need very little training and require minimum equipment. Although injectable anaesthesia is relatively simple, there is always a risk when a single bolus preparation is administered, as adjustment of dose is not 2 possible, except for topping up. Considering this, it is empirical to develop injectable anaesthetic agents that are reliable with a high margin of safety. The current popular injectable anaesthetic medications for laboratory rodents include , , , naloxone, or a combination of with or acepromazine or or , etc. The ease and minimal cost associated with injectable anaesthetics make them an agent of choice for anaesthesia in laboratory animals worldwide. However, this current range of anaesthetic agents is fairly limited and not all agents can be safely used in regular laboratory practices without any unwanted side effects.

Safety in laboratory rodent anaesthesia The most critical part of development of any anaesthetic formulation is safety. Safety is usually assessed by a therapeutic window, which compares the amount of drug required for a therapeutic effect to the amount that causes toxicity. Early anaesthetic agents like and ether had a lower therapeutic window when compared to modern anaesthetics like fentanyl and ketamine (Stanley, 2000). The ability to monitor vital signs during anaesthesia and use of combinations of anaesthetics as opposed to single agents has also significantly increased the safety of anaesthesia in laboratory settings. As monitoring of vital signs usually employs specialized equipment to be handled by professionals, it is not the most desired method for laboratory rodent anaesthesia. Hence, in regular laboratory practices, a formulation with an improved therapeutic window, which is predictable and easy to administer, is desired.

Limitations of current anaesthetic regimes Anaesthetic agents currently used in common laboratory practices to induce are not free of adverse events and often show significant side effects, like respiratory and cardiovascular system . However, if these events do not interfere sufficiently with the normal physiology of animals, the use of the anaesthetic agent is considered acceptable. Use of a combination of anaesthetic agents or application of , can sometimes decrease these unwanted events, but usually fails to make anaesthesia completely free of side effects. Reports have suggested potential alteration in the normal physiology of animals following anaesthesia, like post-operative cognitive dysfunction (POCD), altered immune response and other functional impairments, thereby adversely affecting the post-operative outcome. For example, one of the most popular anaesthetic combinations, ketamine-xylazine, which is considered to be the first choice for injectable rodent anaesthesia, has a major drawback when administered by 3 intramuscular route in rats, as it causes muscle necrosis (Smiler et al., 1990; Wellington et al., 2013) and ketamine at its anaesthetic dose also shows prolonged effects (Yilmaz et al., 2002), which is a major limitation especially when collection of behavioural data is planned. Ketamine also causes long-lasting cognitive deficits in young rhesus monkeys (Paule et al., 2011), thus questioning its wide spread use in laboratory research. Also, many of the commonly used anaesthetics like , hydrate, pentobarbital, and urethane affect the basal immune status of rats (Bette et al., 2004). In addition to this, studies also indicate that neuronal apoptosis is particularly increased during the developmental phase of the fetal rodent brain upon exposure to these agents (Loepke & Soriano, 2008; Istaphanous & Loepke, 2009). Similarly, inhalation anaesthetics, which are successfully used in many anaesthetic protocols, are known to cause dose-dependent respiratory depression and increased oligomerization of A-beta (Aβ) peptide that is considered as an early stage of Alzheimer’s dementia (Eckenhoff et al., 2004). These observations make it evident that most anaesthetics cause long-term impairment of brain function and hence can adversely affect the post-surgical outcome, thereby compromising the quality of scientific data. The lack of a suitable anaesthetic protocol was further emphasized when Lau and colleagues conducted a laboratory rodent anaesthetic survey, which identified inadequate depth of anaesthesia to be the most common anaesthetic complication followed by unexpected anaesthetic wake-up, sharp drop in body temperature and apnoea. The survey also found a significant rate of mortality in rodents after the use of popular anaesthetics (Lau, 2013). These findings, along with other reports (Zuurbier et al., 2014; Flecknell, 2016a), clearly show that, although we have come a long way from the earlier anaesthetics like chloroform and ether, modern agents of anaesthesia are not free of unwanted side effects. Clearly, there is an urgent need to improve our understanding of various anaesthetic regimes and to explore the untapped potential of novel, as well as already existing, anaesthetic protocols so that laboratory rodents can be subjected to anaesthesia that is safe, predictable and relatively free of adverse events, ensuring the quality of scientific data collected.

Current approaches to anaesthetic drug development The field of anaesthetic drug development largely relies on modification of chemical structures of already existing anaesthetic agents to enhance their pharmacokinetic and pharmacodynamic profiles. For example, analogues of drugs like midazolam, and are under investigation to obtain a better anaesthetic agent, using similar

4 approachs (Chitilian et al., 2013). However, this traditional approach to drug discovery and development is highly expensive and time consuming. The other approach towards development of better anaesthetic regime is the improvement of current anaesthetic agents in order to get a better anaesthesia outcome. This approach can be undertaken by choosing a suitable combination of agents or by use of specific to enhance or block a particular pharmacological pathway to abolish side effects. In this project, our approach has particularly focused on understanding the mechanism by which alfaxalone leads to neuromotor excitation, knowledge which will then enable us to rationally modify an already existing steroid based anaesthetic regime, in order to provide a safe and reliable anaesthetic formulation.

Neurosteroids as anaesthetics Steroid molecules synthesized within the central nervous system (CNS) that modulate neuronal excitability by rapid non-genomic actions are called neurosteroids (Reddy, 2010). Neurosteroids are known to have organizational as well as activational roles in the CNS (Brinton, 2013). The pharmacological action of neurosteroids and/or their metabolites (including synthetic compounds) occur largely as a result of allosteric potentiation of the ionotropic ligand-gated ion channel gamma (γ) aminobutyric acid type A receptors (GABAA). Neurosteroids have been long-standing candidates known for their involvement in various conditions such as , and depression (Reddy, 2010). Apart from their implication in various psychiatric conditions, certain endogenous steroid molecules like and , as well as synthetic neuroactive steroids, such as alfaxalone, show anaesthetic effects via their action on the GABAA receptor complex (Selye, 1941; Atkinson et al., 1965; Harrison & Simmonds, 1984; Harrison et al., 1987; Reddy & Apanites, 2005). The anaesthetic properties of neurosteroids and its analogs should not come as a surprise, considering their strong modulatory effect on

GABAA receptors to rapidly decrease neuronal excitability (Harrison et al., 1987), which are considered to be a leading candidate for the primary target of general anaesthetics (Arhem et al., 2003; Garcia et al., 2010; Son, 2010). The initial observations that steroid based molecules have very good anaesthetic properties were made by Hans Selye (1941), where he reported the hormone progesterone’s anaesthetic activity, but it was not until 1955, when Laubach and colleagues studied a series of steroids for their anaesthetic property and claimed (21-Hydroxypregnane-3, 20-dione sodium succinate) as the most promising water-soluble anaesthetic steroid (Laubach et al., 1955), that a renewed interest in the

5 anaesthetic properties of steroids was generated. The study showing the anaesthetic property of hydroxydione was particularly important, as it concluded hydroxydione to be superior to anaesthesia, owing to minimal respiratory and cardiac depression; and gives a high ratio between toxicity in animals to that of safety in humans, providing a wide margin of safety (P'An et al., 1955). However, due to its slow onset of action and high tendency to produce phlebitis (venous irritation), use of hydroxydione had to be discontinued (Soliman & Brindle, 1976). The study of structural activity relationships also showed that water-soluble derivatives of steroidal anaesthetics were usually less active and slower-acting (Phillipps, 1975). This led to the development of Althesin in the early 1970s, a water-insoluble combination of two different neurosteroids (viz. alfaxalone and alphadolone) that was devoid of the side effects seen in hydroxydione anaesthesia, while retaining minimal cardiorespiratory depression and wide margin of safety. Alphadolone, although being only half as potent when compared to alfaxalone, was a necessary component of the formulation as it increased the solubility of the formulation when dissolved in Cremophor EL (Swerdlow, 1973). This formulation of alfaxalone in combination with alphadolone was also used with great success as an anaesthetic agent (Saffan) for cats and dogs (Child et al., 1971; Child et al., 1972; Bomzon, 1981; Sear, 1996) and was considered to have bridged the void in anaesthetic formulations available then (Morgan & Whitwam, 1985). Even though this combination had desirable anaesthetic properties, it also caused significant side effects and both Althesin and Saffan were later withdrawn from use in many countries. Limitations such as , coughing, partial laryngeal spasm, cyanosis and postoperative vomiting in cats (Dodman, 1980) and although less fatal, a high incidence of anaphylactoid reactions in dogs were observed (Warne et al., 2015). However, these unwanted side effects of hypersensitivity reactions associated with this combination were a result of the use of Cremophor EL, a non-ionic solubilizing agent which is polyethoxylated , and not the actual neurosteroids (Moneret-Vautrin et al., 1983). Later on, solubilizing alfaxalone in a biologically acceptable formulation was achieved by the use of , which are widely used as solubilizing and stabilizing agents (Stella & He, 2008). This resulted in the current formulation of alfaxalone, Alfaxan CD, containing alfaxalone alone solubilized using a modified cyclodextrin called 2-hydroxypropyl-β-cyclodextrin (HPCD), which is used in veterinary anaesthesia in cats and dogs (Muir et al., 2008; Muir et al., 2009; Zaki et al., 2009).

6

Alfaxalone

Figure 1. Chemical structure of alfaxalone (3α-hydroxy-5α-pregnane-11, 20-dione) (image by Bryan Derksen via Wikimedia Commons).

Among the various neuroactive steroids with anaesthetic properties, one that deserves a special mention is alfaxalone, as it has significant advantage over other agents. A high margin of safety has already been established for alfaxalone in veterinary anaesthesia (Davis & Pearce, 1972). The safety of its earlier formulation i.e. alfaxalone- alphadolone has shown superiority over thiopentone, methohexitone, and ketamine for continuous administration and was considered to be excellent for short as well as long periods of anaesthesia (Green et al., 1978). Alfaxalone, by the virtue of being an analogue of , is deemed to be neuroprotective as it suppresses central nervous system activity by acting as a potent modulator of GABAA receptor system and reducing excitotoxicity (Lambert et al., 1995; Yawno et al., 2009) and hence, use of an alfaxalone based anaesthetic regime seems to be the obvious step forward. At a cellular level, alfaxalone like other anaesthetic molecules depresses the activity of central neurons by enhancing GABA receptor activity in cultured spinal neurons (Barker et al., 1987). In addition to this, at higher concentrations of alfaxalone (comparable to clinical anaesthesia), enhanced membrane Cl- conductance was reported, which is associated with decrease in action potential generation probability, thereby attributing to its action on brain (Barker et al., 1987). Similarly, alfaxalone also potentiates GABA induced currents in cultured cortical neurons (Sasa et al., 1996). However, studies have reported that alfaxalone (as Alfaxan CD) has a major drawback for laboratory rodent anaesthesia, that of prominent neuromotor excitation (Lau, 2013; Siriarchavatana et al., 2016). Rats and mice show neuromotor excitation during the induction and recovery phase of anaesthesia. This excitation is particularly seen as twitching and limb paddling. Muscle twitches are also reported in cats (Mathis et al., 2012), pigs (Keates, 2003) and dogs (Ferre et al., 2006) after alfaxalone anaesthesia. Although 7 regular, these side effects are clinically insignificant in dogs and cats (Jurox, 2011) and hence, the use of the current alfaxalone formulation is still continued. In rats, severe facial and whole body twitches were reported during induction and recovery phases of anaesthesia (Lau, 2013). This phenomenon was also reported in mice earlier when older formulation, Saffan (alphaxalone and alphadalone) was used (File & Simmonds, 1988) and later when improved formulation (alfaxalone alone encased in hydroxypropyl-β-cyclodextrin) was used (Siriarchavatana et al., 2016). These neuro- excitatory effects are the major barrier in developing an effective alfaxalone based anaesthetic regime for laboratory rodents. Despite the potential importance of alfaxalone as an important anaesthetic agent in small laboratory rodents, little is understood about the molecular mechanisms that mediate this neuroexcitation. If the reasons for these neuromotor excitations can be established and then counteracted by pharmacological manipulation, alfaxalone is likely to become a valuable anaesthetic formulation for laboratory rodents.

Neuromotor excitation during anaesthesia A close review of the literature suggests that neuro-excitatory motor responses, including limb rigidity and paddling, after induction of anaesthesia in dogs (Davies, 1991) and horses (Mama et al., 1995) have been observed with various anaesthetics. For example, excitation was observed with etomidate and propofol, which seemed to be due to enhanced spinal and brainstem activity shortly after induction of anaesthesia (Sneyd, 1992). Although these events are rare and occasionally serious, recovery is usually classified as good or acceptable and there is no clinical complication seen post recovery, which is in stark contrast with the neuromotor twitching seen after alfaxalone anaesthesia in rats, where facial and whole body twitches are potentially severe and consistently observed and are seen as alfaxalone’s biggest shortcoming. Previous reports indicate that alfaxalone, particularly at high concentration potentiates the responses to receptor activity, which could possibly contribute to the neuroexcitatory effects seen on alfaxalone anaesthesia. Lau and colleagues showed that inhibitory glycinergic neurotransmission to brainstem motor neurons is progressively suppressed by increasing alfaxalone concentration, potentially causing increased motor neuron excitability (Lau, 2013). However, to date, no studies have examined the effects of alfaxalone on excitatory synaptic transmission to brainstem motor neurons, which will be a key point of investigation in exploring the neuro-excitation seen with alfaxalone anaesthesia.

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Glutamatergic transmission in motor neurons Motor neurons in the brainstem and spinal cord are responsible for transforming central motor commands into a motor activity. The amino acid glutamate is a dominant excitatory in the mammalian CNS (Collingridge & Lester, 1989; Nakanishi, 1992), and is also responsible for motor neuron excitability, thereby controlling involuntary and rhythmic movements. Glutamate receptors are also expressed on motor neurons, which are innervated by glutamatergic inputs from brain systems regulating the state of arousal and motor control (Rekling et al., 2000). Considering the importance of glutamatergic transmission in modulating motor neuron excitability, it becomes imperative to investigate the effect of alfaxalone on excitatory neurotransmission. Interestingly, prior study showed that alfaxalone potentiates synaptic transmission efficacy by enhancing release of excitatory transmitters, an effect associated with depression of excitability in a decerebrated preparation (Morris, 1978). A reduction in synaptic excitability after alfaxalone application was also reported in guinea pig cortical brain slice preparation (el-Beheiry & Puil, 1989). Alfaxalone also showed a depression in synaptic transmission as well as sensitivity of neurones to glutamate in guinea pig olfactory cortex (Richards & Smaje, 1976). These studies clearly indicate a possibility that neurosteroid alfaxalone can indeed change excitatory transmission and intrinsic excitability. However, to our knowledge no study until date has investigated a direct effect of alfaxalone application on excitatory transmission and intrinsic excitability in mammalian motor neurons. Therefore, evaluating the role of alfaxalone on glutamatergic excitatory neurotransmission in hypoglossal motor neurons might lead to key findings that can help in exploring targets for abolishing the neuro-excitatory twitching observed after alfaxalone anaesthesia.

Possible involvement of transient receptor potential (TRP) channels in motor neuron excitation The transient receptor potential (TRP) channel family constitutes of 28 diverse mammalian members divided into 6 subfamilies based on amino acid sequence homology. The subfamilies include vanilloid (TRPV), melastatin (TRPM), canonical (TRPC), ankyrin (TRPA), mucolipin (TRPML), and polycystin (TRPP) (Benemei et al., 2015; Earley & Brayden, 2015) TRP channels. Multiple reports over the years have confirmed that TRP channels can influence cellular function in peripheral tissue as well as central neurons, by causing inward cation currents and directly affecting voltage gated ion channels, thereby bringing rapid changes in intracellular events ranging from contraction of smooth muscle to

9 neuronal depolarization (Nilius et al., 2007). TRP channels are widely expressed throughout the body and are activated by several endogenous and exogenous stimuli, which can be either chemical, physical or physiological in nature. For example, camphor and noxious hot temperatures can activate TRPV1 and TRPV3 (Selescu et al., 2013). TRPV1 can also be partially activated by voltage changes (Matta & Ahern, 2007). 2-aminoethoxydiphenyl borate (2-APB) and laser light of 640 nm, and light at 48 mW can activate TRPV2 (Hu et al., 2004; Zhang et al., 2012). Similarly intracellular calcium is the physiological activator of TRPM5 channels (Prawitt et al., 2003) and ADP-ribose can activate TRPM2 (Rah et al., 2015). Arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), is identified as activator of TRPC6 (Basora et al., 2003) and TRPA1 is activated by noxious cold temperatures (Story et al., 2003). Regulation of Ca2+ is a critical determinant of several physiological functions like muscle contraction, integration of electrical signaling, neuronal excitability, etc. Under normal conditions, activation of TRP channels causes influx of both Na+ and Ca2+ ions, leading to increase in intracellular Ca2+ concentrations and thereby increasing excitability of cells. This further leads to a cascade of events, which can include activation of voltage sensitive Ca2+ channels and several other Na+ dependent processes (Estacion et al., 2006; Abramowitz & Birnbaumer, 2009; Birnbaumer, 2009). TRP channels are broadly considered as non-selective divalent cation channels as they all are more selective to conduct Ca2+ than other monovalent cations, which is largely responsible for their physiological roles (Clapham et al., 2001; Montell, 2001). An exception to this are TRPM4 and TRPM5, which are only permeable to monovalent cations (Nilius et al., 2005a). However, both channels are activated by a rise in internal Ca2+ and hence it plays a regulatory role in their activity. Their activation has reported to induce cell-membrane depolarization and activity of voltage gated ion channels which are strong determinants of cellular physiology (Guinamard et al., 2011). TRPM4 is a Ca2+ activated nonselective cation channel activated by an increase of intracellular Ca2+ and is regulated by several factors like temperature. Previous work has shown characteristic changes in inward current in hypoglossal motor neurons after application of increasing doses of alfaxalone where it induced an inward current, without a significant change in input resistance (Lau, 2013). The persistent inward current (PIC) has an important role to play in motor neuron firing. The characteristic prolonged firing observed in motor neurons even after cessation of input is attributed to the ability of the motoneuronal membrane to generate persistent 10 inward current (Elbasiouny et al., 2010). N- and P-type Ca2+ channels are the major contributors persistent current in juvenile HMNs, with little contribution from L-type Ca2+ channels and the persistent sodium current (Funk et al., 2011). However, another important contributor to PIC is a Ca2+ activated, non-selective cation conductance, that is believed to be mediated by the transient receptor potential melastatin 4 (TRPM4) channel (Nilius et al., 2005b; Funk et al., 2011). It is postulated that the inward shift in current by alfaxalone can be a possible reason for increased excitation in HMNs, which behaviourally manifests as muscle twitching. Also, the fact that these inward currents were blocked by 2-APB, a non-specific blocker of many TRP channels, emphasize the potential importance of TRP channels in causing an inward current and hence the prolonged firing in motor neurons after alfaxalone treatment. In order to identify the role of TRPM channels, in causing an inward shift in current and also to identify the specific channel involved, it is essential to investigate the effects of not only general, but also specific blockers of TRPM channels to see if excitatory effects of alfaxalone are mediated via TRPM channels.

Rationale and aim of study Most or our understanding regarding the modulation of synaptic transmission to central neurons by steroid-based anaesthetic molecules is gathered by electrophysiological studies in brain slice preparations or in cultured neurons. However, all studies focused exclusively on the modulation of GABA receptor activity, as it is the primary target of most general anaesthetics. Alfaxalone, although modulates the activity of

GABAA receptors, it is interesting to note that neuromuscular twitching is most prominently seen in laboratory rodents. Considering this, it is imperative to investigate further the modulation of alfaxalone induced changes in synaptic transmission. Lau and colleagues have provided our first understanding in this direction, by showing that alfaxalone does inhibit inhibitory glycinergic neurotransmission to rat hypoglossal motor neurons, which might be responsible for neuromuscular twitching seen upon alfaxalone anaesthesia in rats (Lau, 2013). Though this study did provide a starting point of investigation, the molecular mechanism behind this effect remains unidentified. The first aim of this study was to further investigate the effect of alfaxalone on miniature glycinergic inhibitory synaptic transmission; this was followed by evaluation of alfaxalone’s effects on excitatory glutamatergic transmission and then intrinsic excitability of HMNs in in vitro brain slices. The second aim was to investigate the cause of shift in membrane current after alfaxalone application and to check the possible involvement of

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TRP channels in modulating motor neuron excitability. The final aim was to see if there is any species related difference between rats and mice in the effect of alfaxalone and TRP channel modulators on HMNs.

Materials and methods Hypoglossal motor neurons (HMNs) to study effects of alfaxalone Motor neurons innervate the skeletal muscle system to evoke a motor response. To investigate if alfaxalone affects synaptic transmission to motor neurons, we used HMNs from acute rat brainstem slices. HMNs are specialized cranial MNs in the brainstem, responsible for various rhythmic motor activities like swallowing, sucking, speech, mastication, chewing and breathing (Berger et al., 1996; Peever & Duffin, 2001). HMNs show intricate dendritic arborizations that receive synaptic inputs from various brain regions, which provide a potential site for integration of various afferent inputs leading to modulation of their activity (Altschuler et al., 1994; Fukunishi et al., 1999; Tarras-Wahlberg & Rekling, 2009). HMNs soma and dendrites receive both glycinergic as well as GABAergic synaptic terminals (Aldes et al., 1988). Apart from this, the HMNs provide some unique features, which are valuable for examining the calcium dynamics. Firstly, HMNs exhibit lower endogenous calcium buffering capacity leading to an increased risk of Ca2+ mediated excitotoxicity (Lips & Keller, 1998). The second important feature of the HMN system is the strong inhibitory input that it receives from glycinergic inputs (Umemiya & Berger, 1995; Singer et al., 1998; Donato & Nistri, 2000; Singer & Berger, 2000). As the effect of alfaxalone has already been studied in detail for GABAergic inputs, we sought to examine its effects on the glycinergic system; as HMNs receive strong glycinergic inputs, it becomes a good model for our investigation. Moreover, a pilot study in our lab has already shown tongue twitching along with other behavioural outcomes like paw paddling and head bobbing upon alfaxalone anaesthesia in rats. This made HMNs an ideal model system to investigate the effects of alfaxalone in in-vitro preparations.

Use of whole-cell patch-clamp technique Excitation during alfaxalone anaesthesia is an unwanted behavioural outcome which indicates significatant modulation of synaptic transmission in the brain. Patch-clamp electrophysiology in slice preparations is a reliable technique to measure inhibitory and excitatory synaptic currents in specific brain regions. This technique can also be employed in measurement of isolated ionic currents which are responsible for synaptic changes and to apply pharmacological agents in studying intracellular signalling pathways. More 12 importantly, this technique also allows for manipulation of voltage (to study voltage dependent ion channels) and current (to study action potential generation and intrinsic excitability) to provide mechanistic insights on the phenomenon of membrane excitability and synaptic transmission. Although slice electrophysiology is a low-throughput technique compared to others, it is still considered a gold standard for studying modulation of ion channels by drug candidates and is a vital tool for drug discovery (Fodor & Nagy, 2007).

Brainstem slice preparation All experimental protocols were performed after approval from The University of Queensland Animal Ethics Committee (SBMS/142/15/NHMRC), and were in accordance with the Queensland Government Animal Research Act 2001, and associated Animal Care and Protection Regulations (2002 and 2008) as well as the Australian Code for the Care and Use of Animals for Scientific Purposes, 8th Edition (National Health and Medical Research Council, 2013). Either neonatal Wistar rats or C57Bl/6 mice (7-14 day old, either sex) were used for all experiments. This particular age of animals was selected because HMNs are electrically mature at this age and their soma is easily accessible for electrophysiological manipulation. Sodium pentobarbitone (Vetcare, Brisbane, ) at dose of 60-80mg/kg, was administered intraperitoneally to anaesthetize the animals, after which the skull was rapidly removed and entire brain was carefully dissected out (Fig. 2A). Further, the brainstem was isolated and placed in ice-cold high Mg2+-low Ca2+ artificial cerebrospinal fluid (aCSF), which contained (in mM) 130 NaCl, 26 NaHCO3, 3 KCl, 5

MgCl2, 1 CaCl2, 1.25 NaH2PO4, 10 D-glucose (Bellingham & Berger, 1996). Care was taken to maintain aCSF at pH 7.4 by constantly bubbling with carbogen (95% O2 + 5%

CO2). The rostral end of the brainstem was fixed on a metal chuck using cyanoacrylate glue. An agar block was glued to the chuck served as a support to the ventral surface of the brainstem (Fig. 2B). The metal chuck with tissue attached was submerged in ice-cold high Mg2+-low Ca2+ aCSF within a cutting chamber, and maintained ice-cold by a slurry of ice and water in a surrounding water bath. Using the central canal as a landmark, transverse brainstem slices (300µm thickness) were cut using a vibratome (Leica VT 1200S, Leica Biosystems). An average of 3-4 slices containing hypoglossal motor nucleus were obtained from each animal. After cutting, slices were transferred to a holding chamber filled with high Mg2+-low Ca2+ aCSF at 37°C, in a temperature-controlled water bath for 45-60min. Finally, slices were transferred to a new holding chamber containing low Mg2+-high Ca2+ recording aCSF (1Mg2+-2Ca2+mM) and equilibrated for at least 30min before recording at room temperature (21-22°C). 13

Figure 2: Brainstem slices preparation. (A) Lateral orientation of brain showing the approximate location of hypoglossal nucleus (dotted circle). After isolation of brainstem, cyanoacrylate glue was used to fix the rostral end of brainstem to metal chuck. Using an agar block support was provided to the ventral surface (B). Horizontal lines denote the slicing plane. 300µm thick transverse slices were cut, giving about 3-4 slices from each brain.

Electrophysiology Individual slices were transferred to a recording chamber and continuously superfused (1-2ml/min) with low Mg2+-high Ca2+ aCSF throughout the experiment (Fig. 3). A custom-made harp was used to stabilize the slice in the chamber. The volume of the recording chamber was approximately 200µL. HMNs were individually identified using a 60X water-immersion objective, an infrared video camera (Hamamatsu, ) and Nomarski optics on a Nikon E600FN microscope (Tokyo, Japan); all HMNs were ventral and lateral to the central canal, within the hypoglossal motor nucleus, and had a large soma. A quick assessment was made of the health of brainstem slices; slices with bright and shiny looking cells were considered healthy. However, slices with darker cells and visible nuclei were avoided. A Sony monitor was used to view neurons and recording electrode. Whole-cell patch-clamp recordings were made using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA, USA). Synaptic current recordings were sampled at a rate of 10kHz, and low-pass filtered at 2kHz, while a higher sampling rate (50kHz) and low-pass filter (10kHz) settings were used for action potential recordings; data was sampled and stored on a Windows XP computer, using PClamp 10.2 software and a Digidata 1332A digitizer (Axon Instruments). The patch electrode was advanced to the cell surface using a micro-manipulator (MPC-200, Sutter Instrument Company). The electrode was directed to the cell body, so that electrode solution ejected by positive pressure produced a small dimple, showing that the electrode was touching the cell surface. Positive pressure was released and mild suction was applied until a stable membrane seal with resistance of >2GΩ was achieved. Stronger suction was then applied until the cell membrane was broken. Cells were voltage-clamped at membrane potentials of -60mV. At 14 least 5min were allowed for the cell to stabilize with the pipette internal solution before starting recordings. The patch pipette internal solution for recording excitatory and inhibitory post- synaptic currents (EPSCs/IPSCs) contained (in mM) 120 CsCl, 4 NaCl, 4 MgCl2, 0.001

CaCl2, 10 Cs N-2-hydroxyethyl-piperazine-N’-2-ethanesulfonic acid (HEPES), 10 caesium ethylene glycol-bis (β-aminoethyl ether)-N,N,N,N-tetra-acetic acid (EGTA), pH adjusted to 7.2 with CsOH. The internal solution for recording action potentials contained (in mM) 135 K methyl sulfate, 8 NaCl, 10 HEPES, and 0.3 EGTA; pH 7.2 with KOH. Pipette solution osmolarity was adjusted to 290-300mOsM with sucrose, using a vapour pressure osmometer (Wescor). 5’-triphosphate (ATP-Mg) and 0.3 guanosine 5- triphosphate-tris (hydroxymethyl) aminomethane (GTP-Tris) were added to the internal solution just before use. For recording glycinergic IPSCs, NBQX (10µM), APV (50µM) and (5µM) were added to the recording buffer. Similarly, for recording glutamatergic EPSCs, HCl (20µM) was added to all external recording solutions to block both glycine and GABAA receptor-mediated inhibitory synaptic currents in HMNs. To isolate miniature synaptic currents, tetrodotoxin (TTX) at a final concentration of 1µM was added to the recording buffer. For evoked EPSC recordings, a bipolar concentric stimulation electrode (Frederick Haer) was placed in the reticular formation ventrolateral to the border of the hypoglossal nucleus, and a pair of stimulus currents of 0.5-1.1mA and 0.1ms duration was applied to reliably evoke an EPSC with consistent first pulse EPSC amplitudes; inter-stimulus interval was 150ms. Action potential (AP) firing was achieved by repetitive depolarizing current steps (2 to 120pA) of 400ms duration from a membrane potential of -65mV in current clamp mode. Input resistance was calculated from the steady state amplitude of a brief (40ms) negative (-10pA) current step prior to the depolarizing step.

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Figure 3: Schematic representation (not to scale) of electrophysiology setup used in this study. This figure gives an overall view of key components involved in patch-clamp recordings from rodent brainstem slice preparations. In short, brain slices were placed in a tissue chamber (approximately 200µL volume), which had a continuous flow of recording buffer (aCSF) using a gravity fed perfusion system. A low power suction pump was used to drain out excess buffer from the tissue chamber. A micromanipulator was used to manoeuvre the recording glass pipette attached to an electrode holder, which was further connected to Axopatch 1D amplifier, digitizer and later to an oscilloscope and a computer running Windows XP software. Data acquisition was performed using PClamp software. When a stimulation electrode was used, another micromanipulator was installed to firmly hold the stimulation electrode on the opposite side to the recording electrode setup.

Drug application All chemicals were procured from Sigma Aldrich (St Louis, MO, USA) except tetrodotoxin (TTX) which was obtained from Alomone Labs, Israel, and alfaxalone, which was a gift from Jurox Pty Ltd. The final concentrations of all drugs were prepared just before start of experiment. Alfaxalone was dissolved in hydroxypropyl substituted β- cyclodextrin (HPCD, a gift of Jurox Pty Ltd) to a ratio of 1:8, capsaicin in ,

16 capsazepine in and SB366791 in DMSO to make stock solutions, which were then diluted using recording aCSF to final desired bath concentrations just before use. Drug concentrations for all drugs were chosen based on other comparable electrophysiological studies to make sure maximum drug induced responses are obtained. The reservoir containing SB366791 was always wrapped in aluminium foil to protect from light. Final concentration of all solvents was ≤ 0.25% in all experiments.

Data Analysis Spontaneous and miniature synaptic currents were analysed from one or more files of 2min continuous data recordings (Fig. 4). Shape parameter (amplitude, 10-90% rise time, half-width) analysis was performed in Clampfit 10.2 (Axon Instruments), using a template created by averaging visually detected individual synaptic events. Baseline holding current (Iholding) and frequency of EPSCs for spontaneous recordings and input resistance for evoked recordings were also recorded. Paired-pulse ratio (PPR) was calculated as the mean peak amplitude of the averaged second evoked EPSC divided by the mean peak amplitude of the averaged first evoked EPSC. This data was imported into Excel (Microsoft) for further analysis. For action potential analysis, the effects of differences in resting membrane potential of individual HMNs were made relative to changing membrane threshold caused by depolarizing current steps in Clampfit. Results are expressed as mean ± S.D., unless otherwise stated, and a paired two- tailed t-test was used to determine whether differences between data points under different conditions (control, drug, wash, etc.) was statistically significant at P<0.05, except where indicated, using Prism 7 (GraphPad). A two-sample Kolmogorov-Smirnov test was used to compare the cumulative frequency distributions of spontaneous and miniature synaptic currents.

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Figure 4: Electrophysiology recordings and data analysis of synaptic currents and action potentials (APs) from hypoglossal motor neurons. Photograph of a typical brainstem slice containing the hypoglossal nucleus showing several hypoglossal motor neurons (A), identifiable by a large multipolar cell body. A recording electrode is shown approaching one of the motor neurons in the hypoglossal nucleus (B). (C) Shows a representative synaptic current trace recorded from a whole-cell patch clamp recording from a motor neuron. Negative going events were recorded from cells that were voltage-clamped at membrane potentials of -60 mV. Only distinct single currents (*) with a fast rising phase and a slower decay phase were selected for analysis of shape parameters. Example of an averaged current trace (D) showing sections used to calculate amplitude and half-width. Action potentials were evoked by injecting depolarizing

18 current steps from a membrane potential of -65 mV in current clamp mode. (E) Shows a typical AP and sections used to calculate amplitude and half-width.

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Chapter 2 Alfaxalone causes a reduction in inhibitory but not excitatory synaptic transmission or action potential firing in rat hypoglossal motor neurons in vitro Abstract We investigate the effects of alfaxalone on inhibitory glycinergic and excitatory glutamatergic synaptic transmission to rat hypoglossal motor neurons (HMNs). Whole-cell patch clamp recordings were made from neonatal (P7-14) rat HMNs within the hypoglossal nucleus in a 300µm thick transverse brainstem slice. Spontaneous and evoked excitatory post-synaptic currents (sEPSCs/eEPSCs), miniature glycinergic inhibitory post-synaptic currents (mIPSCs) were recorded at a holding potential of -60mV using CsCl-based internal solution. Action potential (AP) firing was recorded at a holding potential of -65mV using K+ methyl sulfate-based internal solution. A significant reduction in mIPSCs frequency (-39%) and amplitude (-54%) to HMNs were recorded indicating a reduction in inhibitory transmission to HMNs even in absence of action potential generation, possibly causing neuromotor excitation observed after alfaxalone administration in rats. Interestingly, this effect was also associated with a positive shift (+35%) in the baseline holding current (Iholding). Further, it was noted that alfaxalone, even at various concentrations (10nM-3µM), fails to alter either spontaneous or evoked (25µM only) EPSC frequency, amplitude, half-width, rise-time and Iholding. Similarly, repetitive AP firing by HMNs was not altered by alfaxalone. These results provide strong evidence for the hypothesis that neuromuscular excitation observed during alfaxalone anaesthesia in rats are most likely mediated by a selective decrease in inhibitory glycinergic synaptic inputs to brainstem motor neurons, without alteration in spontaneous or evoked excitatory synaptic transmission, and that HMNs are not excited sufficiently enough to bring about any changes in AP firing.

Introduction Multiple reports suggest muscular rigidity and motor excitability after alfaxalone anaesthesia in several species like swine (Gonzalez et al., 2013), horses (Wakuno et al., 2017), ponies (Deutsch et al., 2017a), cats (Deutsch et al., 2017b) and dogs (Maney, 2017) which can be seen as limb paddling, psychomotor excitement, hyperaesthesia, minor to violent muscular twitching, stumbling, falling, etc. (Jones, 2012). This type of abnormal neuromotor response was also observed when a pilot study was conducted to evaluate effect of alfaxalone by various route of administrations and with common premedication agents in rats (Lau, 2013) and later in mice (Siriarchavatana et al., 2016). 20

Interestingly, Lau and colleagues report twitching behaviour in rats during induction as well as recovery periods of anaesthesia when administered by intraperitoneal (IP) route (20mg/kg) (Lau et al., 2013). This is a major drawback of alfaxalone anaesthesia in rodents as the twitching was reported to be severe in nature. The same study also suggests that even after use of several common premedications, complete blockage of twitching behaviour was not observed. Despite these shortcomings, when used at the correct dose, alfaxalone is considered a safe and reliable anaesthetic agent for dogs and cats; and has shown good properties when used in combination with other agents in exotic species like wallabies, ferrets and (Jones, 2012). However, attempts to find a dose or suitable premedication for laboratory rodents have remained elusive. Anaesthetic agents in general, act either by potentiating inhibitory or by suppressing excitatory neurotransmission in more than one area of the brain. Traditionally, modulation of inhibitory neurotransmitter gamma (γ) aminobutyric acid type A (GABAA) receptor complex has been the major focus of studies involving alfaxalone, as it is believed to be the major site of action for producing anaesthesia and muscle relaxation (Albertson et al., 1992). However, the severe twitching reported in rodents demands a closer investigation of alfaxalone effects on motor neurons. Interestingly, prior studies show that alfaxalone affects glycinergic currents in a dose-dependent manner. Glycine currents were inactive at low micromolar doses as shown in studies performed in cultured rat spinal neurones (Barker et al., 1987); rat cuneate nucleus slice preparation (Harrison & Simmonds, 1984); transfected HEK 293 cells (Rick et al., 1998) and rat optic nerve preparation (Prince & Simmonds, 1992). However, at higher concentrations alfaxalone when tested on Xenopus laevis oocytes showed to enhance currents mediated by glycine receptors (Weir et al., 2004) via potentiating α-1 function (Mascia et al., 1996). A similar effect was reported when tested in HEK 293 cells transfected with α-1 glycine receptor (Ahrens et al., 2008). Differences in these results point out the possible involvement of different glycine receptors subunits in the biological system. Although these studies did investigate the role of alfaxalone in modulating glycinergic transmission to some extent, none has investigated the direct effect on inhibitory and excitatory synaptic transmission to motor neurons. This led us to investigate the mechanism of possible modulation of synaptic neurotransmission to motor neurons by alfaxalone. Along with GABA, glycine is responsible for fast inhibitory neurotransmission in the central nervous system (CNS). Both cause opening of chloride channels and inhibit the firing of neurons (Jentsch et al., 2002). Interestingly, glycine receptors are predominantly found in caudal 21 areas of the CNS, such as the brainstem and spinal cord, and are involved in the control of motor rhythm generation (Laube et al., 2002). A critical role of glycinergic neurotransmission in regulating muscle tone was also established when pharmacological blockage of glycine receptors by strychnine showed severe neuromotor symptoms, like cramps in skeletal muscles and increased sensitivity to external stimuli like sound (Callister & Graham, 2010; Yevenes & Zeilhofer, 2011). The involvement of glycinergic transmission in producing abnormal behavioural phenotypes is linked to mutations in the glycine receptor gene (Buckwalter et al., 1994; Mülhardt et al., 1994; Ryan et al., 1994). These mutations led to tremors, spasms and excessive startle responses in mutant mice, signs which are similar to those observed in dominant familial startle disease (hyperekplexia) in human subjects (Buckwalter et al., 1994; Koch et al., 1996). However, for rhythmic movements with precise control, the excitatory neurotransmitter, glutamate is also equally important in control of motor neuron excitability (Rekling et al., 2000). These excitatory inputs are responsible for repetitive firing in motor neurons that are in turn are responsible for tonic muscle contractions associated with postural control (Rekling et al., 2000). In light of this knowledge, it is very well possible that there is a alteration of either inhibitory and/or excitatory synaptic transmission to motor neurons, which could manifest as twitching in rats. Hence, to investigate the possible reason behind twitching behaviour in alfaxalone anaesthesia, the following study was undertaken to examine the modulation of inhibitory glycinergic and excitatory glutamatergic synaptic transmission by alfaxalone to brainstem motor neurons.

Results Alfaxalone (25µM) reduces miniature glycinergic IPSC amplitude and frequency to rat hypoglossal motor neurons (HMNs) Earlier reports by Lau and colleagues (Lau, 2013) had suggested that alfaxalone reduces the inhibitory glycinergic synaptic transmission to rat HMNs. However, the study did not differentiate if this is an effect on action potential-independent spontaneous release of glycine or is mediated via action potential-dependent release of neurotransmitter glycine. Therefore, to test the effect of alfaxalone on inhibitory glycinergic synaptic transmission in the absence of spontaneous action potential generation, miniature IPSCs (mIPSCs) where recorded. The recording buffer in addition to NBQX (10µM), APV (50µM) and bicuculline (5µM) contained 1µM tetrodotoxin to block action potentials. Mean mIPSC amplitude was significantly reduced from -95.4 to -43.9pA (-54% change from control, p=0.0014, n=14, Fig 2.1E, Table 2.1). Similarly, there was also a 22 significant decrease in mIPSC frequency from 1.97 to 1.21Hz (-39% from control, p=0.0013, n=14, Fig 2.1C, Table 2.1). Figure 2.1F, G show the distribution of mIPSC amplitudes and inter-event intervals in control and alfaxalone conditions. The amplitude and inter-event interval distribution shows a significant shift toward low amplitude and high frequency events respectively (Kolmogorov-Smirnov test). Figures 2.1H, I show no change in mIPSC half-width and 10–90% rise time after alfaxalone application. It was interesting to note that baseline holding current (Iholding) showed a significant outward (positive) shift from -160.6 to -104.1pA (+35% from control, p=0.0039, n=14, Fig 2.1J, Table 2.1) in presence of alfaxalone which contradicts earlier report where a significant inward (negative) shift was noted when recording spontaneous IPSCs. This result shows that reduction of inhibitory glycinergic synaptic transmission is independent of action potential activity in HMNs. Similarly, a robust decrease in both frequency and amplitude of mIPSCs indicates a possible pre- and post-synaptic mechanism.

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Figure 2.1 Alfaxalone (Alfa) reduces miniature inhibitory synaptic transmission to rat HMNs.

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(A) A time line of the experimental protocol. After baseline control recording, slices were superfused with alfaxalone (25µM) dissolved in aCSF for 2-4min before recording the effect of alfaxalone on miniature inhibitory post-synaptic currents (mIPSCs). (B) Representative mIPSC frequency trace recorded under control and in presence of 25µM alfaxalone from HMNs voltage-clamped at -60mV. (C) Mean mIPSC frequency under control and alfaxalone conditions showing a significant decrease in frequency with alfaxalone. (D) Representative mIPSC amplitude showing a significant decrease after alfaxalone application. (E) Mean mIPSC amplitude significantly reduced after alfaxalone. Averaged cumulative frequency distribution of mIPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test to amplitude and inter-event interval showed a significant difference between control and alfaxalone conditions. Mean mIPSC half-width (H) and rise time (I) remain unchanged after alfaxalone application. Baseline holding current (Iholding) (J), however showed a significant positive shift. All recordings were made with NBQX (10µM), APV (50µM), bicuculline (5µM) and 1µM tetrodotoxin (TTX) in the recording buffer. Data represented as mean ± S.D. for n=14 cells, each from a separate brain slice. Significance is shown as **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control and alfaxalone conditions was statistically significant.

Table 2.1 Miniature IPSC parameters of HMNs upon application of alfaxalone to rat HMNs. Parameter Control 25µM Alfa Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -95.4 60.4 14 -43.9 21.7 14 ** p=0.0014 Half-width (ms) 8.5 4.7 14 7.3 6.6 14 ns, p=0.39 Rise time (ms) 2.4 1.8 14 3.2 2.9 14 ns, p=0.2 Frequency (Hz) 1.97 0.8 14 1.21 0.7 14 ** p=0.0013

Iholding (pA) -160.6 84.5 14 -104.1 50.4 14 ** p=0.0039

Alfaxalone does not alter spontaneous excitatory synaptic transmission to rat hypoglossal motor neurons (HMNs) Our observation that mIPSC amplitude and frequency were reduced by alfaxalone raised the question of whether this effect was specific only to glycinergic neurotransmission or whether glutamatergic neurotransmission was also affected. To

25 investigate this, HMNs were voltage-clamped at a membrane potential of −60mV and were recorded in aCSF containing strychnine HCl (20µM) in order to block both glycine and

GABAA receptor-mediated inhibitory synaptic currents in HMNs. During alfaxalone application period (5min for each concentration, 10nM to 3µM) there was no significant change in any of the shape parameters of sEPSCs like amplitude (Fig 2.2C), half-width

(Fig 2.2D), rise time (Fig 2.2E), frequency (Fig 2.2F) and baseline holding current (Iholding) (Fig 2.2G, Table 2.2). This result shows that effect of alfaxalone is specific on inhibitory synaptic transmission and does not affect excitatory inputs to HMNs.

Figure 2.2 Spontaneous excitatory post-synaptic currents (sEPSCs) to rat HMNs are unchanged after application of alfaxalone (Alfa).

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(A) A time line of the experimental protocol. After baseline control recording, slices were superfused with various concentrations of alfaxalone (10nM-3µM) dissolved in aCSF for at least 5min before recording the effect of alfaxalone on sEPSCs (B) Representative sEPSC amplitude recorded under control and in presence of different concentrations of alfaxalone from HMNs voltage-clamped at -60mV. Mean sEPSC amplitude (C), half-width (D), rise time (E), frequency (F) and baseline holding current (Iholding) (G) remain unchanged after alfaxalone application. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. (n=16 for control, n=13 for 10nM to 300nM, n=15 for 1µM and n=9 for 3µM). To check statistical significance, a linear regression was fitted to the mean and S.D. of different doses, to determine whether the slope was significantly different from zero.

Table 2.2 Spontaneous EPSC parameters of rat HMNs upon application of alfaxalone. Parameter Control 10nM Alfa 30nM Alfa Mean ±SD n Mean ±SD n Mean ±SD n Amplitude (pA) -26.8 9.01 16 -24.1 6.64 13 -24.07 5.2 13 Half-width (ms) 2.2 0.56 16 2.4 0.85 13 2.4 1.2 13 Rise time (ms) 1.09 0.46 16 1.3 0.74 13 1.2 0.61 13 Frequency (Hz) 2.33 1.24 16 3.029 1.29 13 2.5 1.06 13

Iholding (pA) -127.3 101.5 16 -149.3 108 13 -155.1 113.5 13

100nM Alfa 300nM Alfa 1µM Alfa 3µM Alfa Mean ±SD n Mean ±SD n Mean ±SD n Mean ±SD n -22.5 6.3 13 -25.2 8.1 13 -26.1 8.9 15 -28.4 11.7 9 2.5 0.1 13 2.4 0.9 13 2.7 0.7 15 2.7 0.9 9 1.2 0.6 13 1.2 0.6 13 1.4 0.6 15 1.3 0.5 9 2.57 0.8 13 2.12 1.2 13 2.061 0.93 15 2.1 0.9 9 -134.7 70.06 13 -161.5 140.7 13 -156.2 144.7 15 -188.4 140.5 9

Evoked EPSC parameters and paired-pulse ratio (PPR) remain unchanged after alfaxalone application An earlier report (Lau, 2013) had suggested that alfaxalone, in addition to reducing spontaneous IPSCs, also reduced evoked IPSC amplitude in a dose dependent manner,

27 suggesting that there is a modulation of activity dependent inhibitory neurotransmitter release in the post-synaptic neuron. However, it remained unclear if activity dependent excitatory neurotransmitter release can also be modulated by alfaxalone application. To further investigate this, a pair of electrical stimuli in the reticular formation lateral to the hypoglossal nucleus were applied to evoke two excitatory currents separated by 150ms (Fig 2.3B). Fig 2.3C shows no change in the mean amplitude of the first evoked EPSC before and after application of alfaxalone (n=8, Table 2.3). Similarly, no change was observed in other shape parameters of evoked currents like half-width (Fig 2.3D) and rise time (Fig 3.3E) indicating that the post-synaptic excitatory neurotransmitter response is not altered by alfaxalone. The paired pulse ratio (PPR) of the the amplitude of the second to first eEPSCs was unchanged by alfaxalone (Fig 2.3G), providing further evidence that alfaxalone does not modulate action potential-dependent pre-synaptic release of glutamate. Similar to spontaneous recordings, the baseline holding current (Iholding) (Fig 2.3F, Table 2.3) in evoked recordings remained unchanged. Our observations that alfaxalone alters only inhibitory synaptic transmission but fails to modulate spontaneous and activity-dependent excitatory currents emphasizes the fact that twitching seen during alfaxalone anaesthesia is solely because of its effect on inhibitory glycinergic synaptic transmission in the doses tested.

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Figure 2.3 Evoked excitatory post-synaptic current (eEPSC) to rat hypoglossal motor neurons (HMNs) remain unchanged after application of alfaxalone (Alfa). A pair of stimulus currents of 0.5-1.1mA and 0.1ms duration was applied to evoke a pair of eEPSC. (A) Time line showing experimental protocol. After baseline control recording, slices were superfused with alfaxalone (25µM) dissolved in aCSF for 2-4min before recording the effect of alfaxalone on evoked excitatory post-synaptic currents (eEPSCs). (B) Examples of averaged eEPSCs traces in control or in presence of 25µM alfaxalone from HMN voltage-clamped at -60mV. Black triangles represent the time of stimulus current. (C) Mean eEPSC amplitude showed no change during alfaxalone treatment. Similarly, no significant change was observed in (D) half-width, (E) rise-time, (F) baseline holding current (Iholding) and (G) paired pulse ratio (EPSC2/EPSC1). 20µM strychnine HCl was present in the bath solution during all recordings. Data represented as mean ± S.D. for n=8 cells for all parameters. A paired two-tailed t-test was used to determine whether the difference between data points under control and alfaxalone conditions was statistically significant. 29

Table 2.3 Evoked EPSC parameters of rat HMNs upon application of alfaxalone. Parameter Control 25µM Alfa Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -231.9 99.8 8 -215.4 122.4 8 ns, p=0.43 Half-width (ms) 10.1 5.4 8 8.5 7.1 8 ns, p=0.35 Rise time (ms) 5.5 1.9 8 4.9 1.4 8 ns, p=0.098

Iholding (pA) -47.3 61.6 8 -40.06 52.5 8 ns, p=0.38 Paired Pulse Ratio 2.3 1.6 8 1.7 0.2 8 ns, p=0.31

Action potential (AP) firing of HMNs remains unchanged after alfaxalone application Effect of alfaxalone on sIPSCs (Lau, 2013) and mIPSCs (present study) strongly suggest that it acts by reducing the pre-synaptic release of glycinergic neurotransmission. Furthermore, using evoked IPSC recordings, Lau and colleagues also suggest that there might be a post-synaptic modulation of glycine receptor activity by alfaxalone (Lau, 2013). Similarly, a negative and positive shift in baseline holding current during sIPSCs and mIPSCs respectively meant that there might be a modulation of post-synaptic HMN excitability after alfaxalone application. Endogenous steroids like can reduce membrane excitability (Zakon, 1998) and this led us to investigate if alfaxalone, which shares structural similarity to the endogenous neurosteroid allopregnanolone, might also cause changes to membrane excitability and therefore to action potential firing. To make direct measurements of the effect of alfaxalone on post-synaptic HMN excitability, we evoked repetitive action potential (AP) firing from HMNs current clamped at −65mV, using a series of progressively increasing depolarizing current steps (Fig 2.4B). Frequency of repetitive AP firing remained unchanged from control to alfaxalone conditions (Fig 2.4C, H) (n=8, Table 2.4), nor did alfaxalone change the number of APs evoked by maximum current injected in individual cells (Fig 2.4E) (n=8, Table 2.4). Similarly, analysis of shape parameters of APs under control and alfaxalone conditions showed no significant change in AP amplitude (Fig 2.4D), half-width (Fig 2.4F) and rise slope (Fig 2.4G) (n=8, Table 2.4).

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Figure 2.4 Repetitive action potential (AP) firing by HMNs is unchanged after alfaxalone (Alfa) application. (A) Time line showing experimental protocol. After baseline control recording, slices were superfused with alfaxalone (25µM) dissolved in aCSF for 2-4min before recording the effect of alfaxalone on repetitive action potential firing. (B) Depolarizing current steps caused steady-state membrane depolarization in the sub-threshold range (grey trace) and repetitive action potential firing in the supra-threshold range (black trace). (C,H) Alfaxalone (25µM) had no effect on the number of action potentials generated at the maximum amount of current injected neither the firing frequency was altered. (D) Quantification of mean AP amplitude showed no change after alfaxalone treatment. (E) Maximum number of APs generated at the highest injected current also remained unchanged. Other AP parameters like half-width (F) and max rise slope (G) were also unaffected by alfaxalone. Data represented as mean ± S.D. for n=8 cells, each from separate brain slice. A paired two-tailed t-test was used to determine whether the difference between data points under control and alfaxalone conditions was statistically significant.

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Table 2.4 Repetitive action potential parameters of HMNs upon application of alfaxalone. Parameter Control 25µM Alfa Statistical Mean ±SD n Mean ±SD n Significance AP Amplitude (mV) 110.5 17.21 8 108.3 22.8 8 ns, p=0.68 AP Half-width (ms) 1.463 0.165 8 1.491 0.2204 8 ns, p=0.59 AP Max Rise slope (mV/ms) 98.5 12.08 8 97.51 26.15 8 ns, p=0.89 Max AP count 27.13 6.792 8 25 4.84 8 ns, p=0.12

Discussion Alfaxalone-induced neuromotor excitation in rats is seen as a major limitation, as spontaneous muscle contractions and muscle rigidity are unwanted side effects in any anaesthetic regime. These behavioural effects can also be compared to those observed in conditions like hyperekplexia, tetanus poisoning, and poisoing with inducing compounds like strychnine. Abnormal suppression of glycine receptors or glycine release mechanisms are implicated in all the above mentioned conditions. In the case of alfaxalone, it is possible that a disruption of the inhibitory-excitatory balance to motor neurons is brought about by reduction of glycinergic transmission to motor neurons. The aim of our present study was to investigate if alfaxalone causes selective reduction of glycinergic inhibitory neurotransmission without altering excitatory glutamatergic neurotransmission. As a previous report showed a reduction in spontaneous IPSCs upon alfaxalone application in a dose dependent manner, we investigated if this holds true even after blockage of action potential generation. Hence, a study was designed where, in addition to a NBQX (10µM), APV (50µM) and bicuculline (5µM) cocktail, 1µM tetrodotoxin was also added to recording buffer to block action potentials. A high dose of alfaxalone (25µM) was selected for this study to observe full activation of all available receptor systems. As per traditional quantal analysis, a change in amplitude of miniature synaptic currents is taken as a change of post-synaptic receptor activity. Similarly, a change in the frequency is taken as a measure of pre-synaptic release probability (Redman, 1990; Stevens, 1993; Isaacson & Walmsley, 1995). As alfaxalone reduced mIPSC amplitude along with frequency, we conclude that alfaxalone causes both a decrease in post- synaptic glycine responses and also a reduction in action-potential independent pre- synaptic glycine release probability. Finally, as spontaneous currents include action

32 potential-independent miniature currents, and action potential dependent currents, we conclude that the decrease in sIPSC frequency (Lau, 2013) is due to a decrease in mIPSC frequency (present study). HMNs receive inhibitory (Lowe, 1978; Takagi et al., 2017) as well as excitatory (Koizumi et al., 2008; Revill et al., 2015) inputs from other regions in the brain. It was hence interesting to investigate whether the effect of alfaxalone is selective for glycinergic transmission, as in intact animals (as for the condition of anaesthesia) there is a possibility of feedback mechanism from other neurotransmitter systems. Hence, to investigate this, a dose response study was designed to check for any possible modulation of glutamatergic synaptic transmission to HMNs at increasing doses of alfaxalone (10nM to 3µM). No significant change was observed in any of the shape parameters recorded at all doses tested. This shows that there is a selective modulation of inhibitory glycinergic synaptic transmission to HMNs. However, it remained unclear if alfaxalone along with selective modulation of spontaneous glycinergic release also changes activity-dependent post-synaptic glutamate release machinery. Hence, in order to investigate this, evoked excitatory currents were recorded under control and alfaxalone conditions. However, no significant effect of alfaxalone on evoked EPSCs were observed, strongly suggesting that alfaxalone does not affect action potential-dependent post-synaptic glutamate release.

Finally, we investigated the modulation of baseline holding current (Iholding) upon alfaxalone application. It was interesting to note that in the absence of tetrodotoxin (sIPSCs), a negative shift in holding current was observed which was not consistent with the one observed in presence of tetrodotoxin (mIPSCs), where a positive shift was observed which we believe, might be an effect of blockage of voltage gated sodium (Na+) channel current by TTX as they block the large transient currents during action potentials. Although the reasons behind this varying effect were not investigated further, it led to a new line of investigation, as a direct effect of changes in holding current could be a change in intrinsic excitability and therefore action potential firing rate of motor neurons. Hence, we tested if alfaxalone altered repetitive action potential (AP) firing by HMNs. Alfaxalone failed to alter repetitive firing in HMNs nor were there any changes in action potential shape parameters. These results indicate that alfaxalone does not affect motor neuron intrinsic excitability and the differences in the modulation of Iholding is perhaps due to activation of yet unidentified either cationic or anionic currents. Our study shows a decrease in action potential-independent release mechanism involved in the activity of alfaxalone, which do not involve modulation of voltage-gated sodium channels. In addition, no change in 33 spontaneous and evoked EPSC parameters was observed suggesting that alfaxalone does not directly modulate either pre-synaptic glutamate release or postsynaptic glutamate receptor activity. In conclusion, the synthetic neuroactive compound, alfaxalone, significantly reduces glycinergic inhibitory synaptic transmission to HMNs in brainstem slice preparation without affecting glutamatergic transmission. We also show that alfaxalone does not change intrinsic excitability of HMNs. This study expands our understanding regarding the modulation of synaptic transmission to motor neurons by alfaxalone, which can be a possible cause of excitatory responses observed in alfaxalone anaesthesia.

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Chapter 3 Capsaicin, a TRPV1 agonist, enhances excitatory synaptic transmission to rat hypoglossal motor neurons along with modulation of membrane holding current

(Iholding) Abstract Our data from synaptic current recordings during alfaxalone application consistently showed a shift in Iholding, indicating an indirect modulatory effect. Steroids and steroid-like molecules can bring about modulation of several physiologically significant TRP channels.

However, whether TRP channels are involved in the actions of alfaxalone that cause Iholding changes remains unknown. Hence, to further investigate this, the effects of capsaicin, a well-known TRPV1 agonist, on modulation of Iholding and synaptic transmission to HMNs were examined. Interestingly, we found that capsaicin at 1µM dose selectively increased only spontaneous excitatory post-synaptic current (sEPSC) frequency (+38%) without significantly affecting sEPSC amplitude. However, a higher concentration (10µM) caused a significant increase in both sEPSC frequency (+115%) as well as amplitude (+24%) recorded from neonatal rat HMNs. We further analysed miniature excitatory post-synaptic currents (mEPSCs), recorded in the presence of tetrodotoxin (TTX); capsaicin also increased mEPSC frequency, but mEPSC amplitude remained unchanged, indicating a pre-synaptic mechanism of action. Interestingly a negative shift in membrane current

(Iholding) was elicited by capsaicin under all three recording conditions. The effect of capsaicin on sEPSC frequency remained unchanged in the presence of two different TRPV1 antagonists, capsazepine and SB366791, suggesting that capsaicin acts to increase EPSCs via a mechanism which does not require TRPV1 activation. Capsaicin, however, failed to alter evoked excitatory post-synaptic currents (eEPSCs) or the paired- pulse ratio (PPR) of eEPSCs. Repetitive action potential (AP) firing in HMNs was also unaltered by capsaicin, indicating that capsaicin does not change HMN intrinsic excitability. Overall, these results show that capsaicin-like TRPV1 can cause modulation of Iholding. However, in the present study; this effect is more likely due to an increase in glutamate release, rather than a direct effect of capsaicin on TRPV1 receptors, thereby indicating that alfaxalone induced changes in Iholding are mediated by mechanism that do not involve TRPV1.

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Introduction A consistent observation upon alfaxalone application while recording synaptic currents in rat HMNs was a change in membrane holding current (Iholding), which we believe might drive the membrane potential to a more positive value, thereby contributing to motor neuron depolarization and thus may contribute to neuromotor excitation. In addition to this, there is also a possibility of generation and release of endogenous , as a rise in intracellular Ca2+ upon depolarization is one of the triggering mechanisms for - endocannabinoid release. A shift in Iholding can be brought about by either Cl efflux or cation influx. Although the source of this current remains unidentified, activation of the hyperpolarisation-activated cation (IH) current or modulation of several transient receptor potential (TRP) channels may be implicated as its source. Several neurotransmitters like 5-HT (5-hydroxytryptamine), NE (norepinephrine) and glutamate can directly modulate the membrane current in motor neurons (Rekling et al., 2000). Similarly, IH current, which is reported in juvenile rat HMNs (Bayliss et al., 1994), is a rectifying cationic current that produces an increased inward membrane conductance during hyperpolarisation from resting membrane potential. However, another more interesting source of modulation of

Iholding by steroids involve the activation of TRP channels, which constitute a large family of non-selective cation permeable channels and can bring about changes in intracellular calcium concentrations (Clapham et al., 2001; Moran et al., 2004; Desai & Clapham, 2005; Pedersen et al., 2005). Wagner and colleagues show that there is a direct and rapid activation of a specific TRP channel, TRPM3 by neuroactive steroid to cause the release of insulin from pancreatic cells (Wagner et al., 2008). This leads to the possibility that similar modulation of TRP channels by alfaxalone might be possible, considering the wide distribution of several families of TRP channels in the brain.

We therefore hypothesis that the modulation of Iholding elicited by alfaxalone was due to TRP channel activation. Hence, we investigated the role of a well-known TRP vanilloid subtype-1 (TRPV1) agonist, capsaicin in modulating synaptic transmission to HMNs and particularly to check if Iholding is affected after its application. TRPV1 is a calcium permeable non-selective cation channel that is activated by the vanilloid capsaicin (Caterina et al., 1997; Messeguer et al., 2006), acidic pH (<6) (Tominaga et al., 1998), heat (Caterina et al., 1997) or membrane depolarization (Voets et al., 2004). In the periphery, capsaicin stimulates TRPV1 channels in a subset of sensory afferent neurons, causing excitation and local release of inflammatory mediators (Caterina et al., 1997; Caterina et al., 2000). Activation of TRPV1 by capsaicin is known to initiate depolarization by the influx of sodium and calcium ions in peripheral sensory nerves, causing action potential generation and 36 propagation to the brainstem and spinal cord, ultimately giving rise to burning or itching sensations (Anand & Bley, 2011). The presence of TRPV1 on the central terminals of sensory afferents in the spinal cord and brainstem, as well as in several brain regions, including substantia nigra, locus coeruleus, hippocampus, nucleus tractus solitarius, periaqueductal grey and amygdala (Mezey et al., 2000; Roberts et al., 2004; Tóth et al., 2005; Sun et al., 2009; Aguiar et al., 2015) is consistent with the results of electrophysiological studies demonstrating TRPV1- mediated effects of capsaicin in these areas (Marinelli et al., 2002; Marinelli et al., 2003; Zschenderlein et al., 2011; Lu et al., 2017). These studies indicate that activation of TRPV1 by capsaicin can either modulate or directly release neurotransmitters in central neurons. However, whether TRP channels can modulate these excitatory inputs to HMNs remains a relatively unexplored question. In anesthetized rats, the administration of capsaicin directly into the right atrium to excite vagal pulmonary C fibres, has shown to lower hypoglossal nerve activity at low doses, while higher doses reduce phasic hypoglossal activity but increase tonic hypoglossal discharge (Lee et al., 2003), while capsaicin administration in the jugular vein suppresses HMN activity (Lee et al., 2007). Moreover, capsaicin has reported to rapidly increase inhibitory synaptic transmission to an adjacent brainstem structure, the dorsal motor nucleus of the vagus (DMVs) (Xu & Smith, 2015) via a pre-synaptic mechanism. Similarly, capsaicin lowered respiratory motor output in medullary slice preparations in neonatal rats (MorgadoValle & Feldman, 2004). Most recently, Ren and collegues (Ren et al., 2017) showed that acting via TRPV1 receptors, capsaicin brings about an inhibition of the respiratory central pattern generator when tested in rhythmically active brainstem-spinal cord preparation. However, the direct effects of capsaicin on synaptic inputs to HMNs and changes associated with it are yet to be reported.

Results Capsaicin (1µM) increases spontaneous excitatory post-synaptic current frequency to rat hypoglossal motor neurons (HMNs) Earlier capsaicin studies showed an increase in glutamatergic synaptic transmission in central neurons (Marinelli et al., 2002; Marinelli et al., 2003; Sikand & Premkumar, 2007). We therefore examined whether capsaicin altered spontaneous glutamatergic excitatory synaptic transmission (sEPSCs) to HMNs. During capsaicin application (8-14 min, 1µM),

37 there was a significant increase in sEPSC frequency without any change in amplitude (Fig 3.1B-F). An increase in sEPSC frequency is shown by the representative traces in Fig 3.1B. Fig 3.1C show mean sEPSC frequency increased from 2.5 to 3.5Hz (+38% from control, p=0.019, n=10). Mean sEPSC amplitude remained unchanged (Fig 3.1F). Distribution of sEPSC amplitude showed no shift to higher amplitude events in the presence of capsaicin (Fig 3.1D). However, the distribution of sEPSC inter-event intervals shows a significant shift towards smaller inter-event intervals representing a higher frequency (Fig 3.1E). Other sEPSC shape parameters, such as half-width (Fig 3.1G) and 10-90% rise time (Fig 3.1H) remained unchanged after application of capsaicin. Baseline holding current (Iholding), however, showed a significant inward shift from -193.9 to -245.5pA (+27% from control, p=0.018, Fig 3.1I, Table 3.1).

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Figure 3.1 1µM Capsaicin (Caps) increases glutamatergic excitatory post-synaptic current frequency to rat HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (1µM) dissolved in aCSF for 8-14min before recording the effect of capsaicin (Caps) on spontaneous excitatory post-synaptic currents (sEPSCs). (B) Representative sEPSCs recorded in the absence (control), and presence of 1µM capsaicin from a HMN voltage-clamped at -60mV. (C) Mean sEPSC frequency during control and capsaicin conditions showing a significant increase in sEPSC frequency with capsaicin. Averaged cumulative frequency distribution plots of sEPSC amplitude (D) and inter-event interval (E). Kolmogorov-Smirnov test of inter-event interval shows a significant difference between control and capsaicin conditions. Mean sEPSC amplitude (F) half-width (G) and rise time (H) remain unchanged after capsaicin application. Baseline holding current

(Iholding) (I) did show a significant change in inward direction after capsaicin. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. for n=10 cells, each from a separate brain slice. Significance is shown as *p<0.05. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 3.1 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 1µM capsaicin. Parameter Control 1µM Caps Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -26.4 4.7 10 -33.5 12.8 10 ns, p=0.08 Half-width (ms) 2.5 1.3 10 2.7 1.2 10 ns, p=0.34 Rise time (ms) 1.15 0.64 10 1.2 0.7 10 ns, p=0.56 Frequency (Hz) 2.58 1.09 10 3.57 1.26 10 *p=0.019

Iholding (pA) -193.9 139.6 10 -245.5 129.4 10 *p=0.018

Capsaicin (10µM) increases spontaneous excitatory post-synaptic current frequency and amplitude to rat hypoglossal motor neurons (HMNs) An increase in sEPSC frequency upon application of 1µM capsaicin was also associated with an increase in amplitude of currents. This effect, although not significant, showed a strong trend towards an increase in amplitude of currents (+27% from control). This result may mean that there was an insufficient activation of all available receptors in

40 the brainstem slice preparation. Earlier studies used capsaicin in a safe and reliable manner even at much higher concentration during patch-clamp recordings (Morgado-Valle & Feldman, 2004; Gavva et al., 2005; Benninger et al., 2008). Hence, in order to maximally activate all available receptors and to obtain a robust effect after application, a higher concentration of capsaicin was selected (10µM). sEPSCs were again recorded, this time with a bath application of 10µM capsaicin. During capsaicin application (9-11min, 10µM), there was a significant increase in both sEPSC frequency and amplitude (Fig 3.2B-G). Increased sEPSC frequency is illustrated by the representative traces in Fig 3.2B. Mean sEPSC frequency was also increased from 1.34 to 2.89Hz (+115% from control, p=0.013; Fig 3.2C). A washout of 15min partially restored sEPSC amplitude and frequency towards control values. Fig 3.2D represents averaged spontaneous EPSCs from the three time periods shown, illustrating increased sEPSC amplitude with capsaicin and a partial washout of this effect. Mean sEPSC amplitude increased from -20.3 to -25.3pA (+24% from control, p=0.018, n=7; Fig 3.2E). The distribution of sEPSC amplitude showed a shift to higher amplitude events in the presence of capsaicin (Fig 3.2F). Similarly, the distribution of sEPSC inter-event intervals shows a significant shift towards smaller inter-event intervals (equating to higher frequency), which returned toward control values upon washout (Fig 3.2G). Other sEPSC shape parameters, such as half-width (Fig 3.2H) and 10-90% rise time (Fig 3.2I) remained unchanged during capsaicin application. Baseline holding current (Iholding), however, did show a significant inward shift from -95.75 to -116.6pA (+21% from control, p=0.0003, Fig 3.2J), and this effect also partially recovered after washout.

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Figure 3.2 10µM Capsaicin (Caps) increases glutamatergic excitatory post-synaptic current frequency and amplitude to rat HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 9-11min before recording the effect of capsaicin (Caps) on spontaneous excitatory post-synaptic currents (sEPSCs). Followed by a 15min wash with aCSF devoid of Caps. (B) Representative sEPSCs recorded in the absence (control), and presence of 10µM capsaicin, and after 15min wash from a HMN voltage-clamped at -60 mV. (C) Mean sEPSC frequency during control, capsaicin and wash conditions, showing a significant increase in sEPSC frequency with capsaicin. (D) Representative averaged amplitude traces of sEPSCs before (control), during capsaicin and after wash. (E) Mean sEPSC amplitude during control, capsaicin and wash conditions, showing a significant increase in sEPSC amplitude with capsaicin. Averaged, cumulative frequency distribution plots of sEPSC amplitude (F) and inter-event interval (G) for HMNs in C and E. Kolmogorov-Smirnov test of inter-event interval shows a significant difference between control and capsaicin conditions. Mean sEPSC half-width (H) and rise time (I) remain unchanged after capsaicin application. (J) baseline holding current (Iholding) was significantly altered in inward direction by capsaicin. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. for n=7 cells, each from a separate brain slice. Significance is shown as *p<0.05, ***p<0.001. A paired two-tailed t-test was used to determine whether the difference between data points under control, capsaicin and wash conditions was statistically significant.

Table 3.2 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps 15 min Wash Mean ±SD n Mean ±SD n Mean ±SD n Amplitude (pA) -20.3 4.6 7 -25.3 6.1 7 -22.5 7.1 7 Half-width (ms) 2.8 1.0 7 3.1 1.5 7 3.6 1.0 7 Rise time (ms) 1.6 0.8 7 1.4 0.9 7 1.8 0.5 7 Frequency (Hz) 1.34 0.5 7 2.89 1.3 7 1.90 0.6 7

Iholding (pA) -95.75 47.1 7 -116.6 51.1 7 -102.6 39.2 7

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Parameter Statistical Significance Statistical Significance Control vs 10µM Caps Control vs 15 min Wash Amplitude (pA) * p=0.018 ns, p=0.24 Half-width (ms) ns, p=0.42 ns, p=0.12 Rise time (ms) ns, p=0.54 ns, p=0.48 Frequency (Hz) * p=0.013 ns, p=0.11

Iholding (pA) *** p=0.0003 ns, p=0.47

Effect of capsaicin (10µM) on frequency of excitatory post-synaptic currents is mediated via a TTX-insensitive mechanism Capsaicin-induced changes in sEPSCs can either be an effect on action potential- independent spontaneous release of glutamate, or may, in part, be mediated via action potential-mediated release of neurotransmitters. Hence, to further test the effect of capsaicin on excitatory synaptic transmission in the absence of spontaneous action potential generation, we analysed miniature EPSCs (mEPSCs) where, in addition to strychnine, 1µM tetrodotoxin (TTX) was added to completely block action potentials. Mean mEPSC frequency was still significantly increased by capsaicin, from 2.65 to 7.5Hz (+183% from control, p=0.0026, n=10, Fig 3.3C). However, there was no change in mEPSC amplitude after capsaicin application (Fig 3.3F). Fig 3.3D and E show the distribution of mEPSC amplitudes and inter-event intervals in control and capsaicin conditions. The amplitude distribution shows no significant change (Fig 3.3D), however, inter-event interval distribution shows a significant shift towards higher frequency (Fig 3.3E, Kolmogorov-Smirnov test). Similar to sEPSCs, there were no significant changes in mEPSC half-width (3.3G) and 10-90% rise time (Fig 3.3H). Baseline holding current

(Iholding) again showed a significant inward shift from -55.99 to -81.72 pA (+45% from control, p=0.014, n=10, Fig 3.3I). Together, these effects of capsaicin on sEPSCs and mEPSCs show that capsaicin acts by increasing the release probability of pre-synaptic terminals or by modulating the excitability of pre-synaptic neurons making excitatory inputs to HMNs.

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45

Figure 3.3 10µM Capsaicin (Caps) induced increase in sEPSC frequency but not amplitude is mediated via non-TTX sensitive mechanism. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 9-11min before recording the effect of capsaicin (Caps) on spontaneous excitatory post-synaptic currents (sEPSCs). All recordings were made in the presence of 1µM tetrodotoxin (TTX) and 20µM strychnine HCl. (B) Representative mEPSCs recorded in the absence (control) and presence of 10µM capsaicin from a HMN voltage-clamped at -60mV. (C) Mean mEPSC frequency shows a significant increase during capsaicin application. Averaged cumulative frequency distribution plots of mEPSCs amplitude (D) and inter-event interval (E). Kolmogorov- Smirnov test applied to inter-event interval shows a significant difference between control and capsaicin. (F-H) shows amplitude, half-width and rise-time of mEPSCs which were unchanged during capsaicin application. (I) baseline holding current (Iholding) showed an inward shift with capsaicin. Data represented as mean ± S.D. for n=10 cells, each from a separate brain slice. Significance is shown as *p<0.05, **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 3.3 Miniature EPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -17.6 4.9 10 -17.8 7.7 10 ns, p=0.94 Half-width (ms) 2.0 0.4 10 2.0 0.3 10 ns, p=0.89 Rise time (ms) 1.4 1.0 10 0.7 0.2 10 ns, p=0.07 Frequency (Hz) 2.65 1.4 10 7.50 3.6 10 ** p=0.0026

Iholding (pA) -55.99 60.1 10 -81.72 71.0 10 * p=0.014

TRPV1 antagonists do not block the effect of capsaicin on sEPSC frequency Next, we investigated if the actions of capsaicin are mediated via TRPV1 activation, by testing whether pre-application of the TRPV1 antagonist capsazepine (Urban & Dray, 1991) could block the effect of capsaicin on glutamatergic excitatory transmission to rat HMNs. Earlier reports show that application of capsaicin in brain slice preparations (Marinelli et al., 2002) and cultured neurons (Sikand & Premkumar, 2007) increased

46 excitatory transmission through activation of TRPV1 receptors and that this effect was abolished by capsazepine. Application of capsaicin in the presence of capsazepine still increased sEPSC frequency (+123% from control, p=0.0038, n=9; Fig 3.4C) and Iholding (+25% from control, p=0.0047, n=9; Fig 3.4J), but had no effect on amplitude (Fig 3.4H). Fig 3.4B represents the increase in frequency after addition of capsaicin in presence of capsazepine. Fig 3.4D shows averaged amplitude under 3 different conditions (control, capsazepine and capsazepine + capsaicin) respectively. It was interesting to note that capsaicin in presence of capsazepine increases the half-width of spontaneous currents (+30% from control, p=0.032, n=9; Fig 3.4E). The amplitude distribution of sEPSC showed a significant shift towards lower amplitude upon capsazepine application, but showed no further change after capsaicin application (Fig 3.4F). Also, the distribution of inter-event intervals showed no change between control and capsazepine, but showed a significant shift towards shorter inter-event intervals upon addition of capsaicin (Fig 3.4G). Capsazepine alone did not cause any significant effect on sEPSC frequency (Fig 3.4C), half-width (3.4E), rise time

(3.4I) and Iholding (3.4J); however, there was a significant decrease in sEPSC amplitude (- 19% from control, p=0.028, n=9, Fig 3.4H), indicating a partial tonic effect of TRPV1 on sEPSC amplitude. We therefore used a structurally different, selective, and potent TRPV1 antagonist, SB366791 (Gunthorpe et al., 2004; Varga et al., 2005), in order to further test the involvement of TRPV1 receptors in the actions of capsaicin. Earlier reports have shown that SB366791 antagonized capsaicin-induced changes in brain slice preparations (Gunthorpe et al., 2004; Varga et al., 2005). As for capsazepine, SB366791 did not alter the effect of capsaicin on sEPSC frequency (+94% from control, n=6, p=0.063; Fig 3.5C), but did block the effect of capsaicin on sEPSC amplitude (Fig 3.5B) and Iholding (Fig 3.5H). The averaged sEPSC amplitude distribution showed no significant change between either control-SB366791 or control-capsaicin conditions (Fig 3.5D). Similarly, the averaged inter- event intervals distribution showed no change with SB366791, but showed a significant shift in events towards higher frequency when capsaicin was added (Fig 3.5E). No significant changes were observed in sEPSC half-width (Fig 3.5F) and 10-90% rise time (Fig 3.5G) under all conditions tested. By contrast to capsazepine, which decreased sEPSC amplitude, SB366791 by itself had no effect on sEPSC amplitude, indicating absence of any tonic SB366791-sensitive activity.

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Figure 3.4 Pre-application of the TRPV1 antagonist, capsazepine (Capz), blocked the effect of capsaicin (Caps) on sEPSC amplitude but not on frequency. (A) Experimental protocol timing. After recording baseline control activity, slices were superfused with capsazepine (Capz, 10µM) in aCSF for 5min before recording capsazepine-induced changes in sEPSCs. Finally, capsaicin (10µM) in aCSF was added for 9-11min after which capsaicin induced sEPSC responses were recorded. Representative traces for sEPSC frequency (B) under control, capsazepine and capsazepine + capsaicin conditions. (C) Averaged frequency plot showing significant increase after capsaicin application even in presence of capsazepine. (D) Representative amplitude for sEPSC frequency under all 3 conditions. (E) Mean half-width plot showing significant increase after capsaicin. Averaged cumulative frequency distribution plots of sEPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test shows significant difference between control vs capsazepine condition in amplitude plot and a significant difference between control and capsaicin condition in inter-event interval plot. (H) Mean sEPSC amplitude plot shows a significant decrease in amplitude after capsazepine alone, but no change after capsaicin application. (I) Mean sEPSC rise time was not altered after capsazepine or capsaicin application. (J) Mean baseline holding current (Iholding) was significantly altered after capsaicin, but not after capsazepine application alone. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. for n=9 cells, each from a separate brain slice. Significance is shown as *p<0.05, **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control, capsazepine and capsaicin + capsazepine conditions was statistically significant.

Table 3.4 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin in presence of 10µM capsazepine. Parameter Control 10µM Capz 10µM Caps Mean ±SD n Mean ±SD n Mean ±SD n Amplitude (pA) -23.3 7.4 9 -18.8 4.3 9 -27.9 9.9 9 Half-width (ms) 2.8 0.9 9 2.4 0.8 9 3.6 1.0 9 Rise time (ms) 1.5 0.5 9 1.4 0.6 9 1.6 0.2 9 Frequency (Hz) 1.40 0.7 9 1.24 0.8 9 3.14 1.1 9

Iholding (pA) -80.26 92.5 9 -85.4 92.6 9 -100.7 78.9 9

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Parameter Statistical Significance Statistical Significance Control vs 10µM Capz Control vs 10µM Caps Amplitude (pA) * p=0.029 ns, p=0.17 Half-width (ms) ns, p=0.18 * p=0.033 Rise time (ms) ns, p=0.53 ns, p=0.75 Frequency (Hz) ns, p=0.31 ** p=0.0038

Iholding (pA) ns, p=0.32 ** p=0.0047

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Figure 3.5 Pre-application of the TRPV1 antagonist, SB366791, blocked the effect of capsaicin (Caps) on sEPSC amplitude but not on frequency.

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(A) Experimental protocol timing. After recording baseline control activity, slices were superfused with SB366791 (20µM) prepared in aCSF for 5min before recording SB366791 induced sEPSC responses. Finally, capsaicin (10µM) prepared in aCSF was added for 9- 11min, and then capsaicin induced sEPSC activity was recorded. (B) Mean amplitude scatter plot shows no difference between control, SB366791 or capsaicin conditions. (C) Mean frequency scatter plot shows a significant change after capsaicin, but no change with SB366791 application alone. Averaged cumulative frequency distribution plots of sEPSC amplitude (D) and inter-event interval (E). Kolmogorov-Smirnov test of inter-event intervals shows a significant difference between control vs SB366791 and control vs capsaicin conditions. sEPSC half-width (F), rise time (G) and baseline holding current

(Iholding) (H) remained unchanged under SB366791 or capsaicin conditions. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. for n=6 cells, each from separate brain slice. Significance is shown as **p<0.01. A paired two- tailed t-test was used to determine whether the difference between data points under control, SB366791 and capsaicin + SB366791 conditions was statistically significant.

Table 3.5 Spontaneous EPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin in presence of 20µM SB366791. Parameter Control 20µM SB366791 10µM Caps Mean ±SD n Mean ±SD n Mean ±SD n Amplitude (pA) -23.1 4.1 6 -21.8 2.6 6 -21 6.2 6 Half-width (ms) 1.9 0.7 6 2.0 0.6 6 2.3 1.2 6 Rise time (ms) 1.0 0.6 6 1.1 0.2 6 1.2 0.4 6 Frequency (Hz) 2.55 0.8 6 1.98 0.7 6 4.96 1.5 6

Iholding (pA) -58.89 65.6 6 -58.96 56.4 6 -78.54 62.0 6

Parameter Statistical Significance Statistical Significance Control vs 20µM SB366791 Control vs 10µM Capsaicin Amplitude (pA) ns, p=0.18 ns, p=0.49 Half-width (ms) ns, p=0.53 ns, p=0.44 Rise time (ms) ns, p=0.55 ns, p=0.55 Frequency (Hz) ns, p=0.063 ** p=0.0016

Iholding (pA) ns, p=0.99 ns, p=0.34

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Capsaicin (10µM) does not alter evoked EPSC parameters or paired-pulse ratio (PPR) As capsaicin increased both sEPSC and mEPSC frequency, capsaicin may act via a pre-synaptic mechanism that is not mediated via classic TRPV1 activation. To further differentiate between modulation of activity-dependent and -independent transmitter release, we analysed the effects of capsaicin on evoked excitatory post-synaptic currents (eEPSCs). Two stimuli in the reticular formation lateral to the hypoglossal motor nucleus evoked a pair of short-latency multi-synapse excitatory currents (average first eEPSC amplitude=237.9 ± 68.8pA, n=11) separated by 150ms. As shown in Fig 3.6, the first eEPSC before and after application of capsaicin showed no change in amplitude (Fig 3.6C), rise time (Fig 3.6D) and half-width (Fig 3.6E), suggesting that activity-dependent release is not altered by capsaicin. This interpretation was strengthened by a lack of effect of capsaicin on the PPR of the first and second eEPSCs (Fig 3.6G). As for our observations of sEPSCs and mEPSCs, there was a negative shift in Iholding while recording eEPSCs, but we found no associated change in HMN input resistance (Fig 3.6H).

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Figure 3.6 Evoked excitatory post-synaptic current (eEPSC) to rat hypoglossal motor neurons (HMNs) were unchanged after application of capsaicin. (A) Experimental protocol timing. A pair of stimulus currents of 0.5-1.1mA and 0.1ms duration was applied to evoke a pair of eEPSC. After recording baseline control responses, slices were superfused with capsaicin (10µM) in aCSF for 9-11min before 54 recording the capsaicin induced eEPSC responses. (B) Examples of averaged eEPSCs traces in control or in presence of 10µM capsaicin from HMN voltage-clamped at -60mV. Black triangles represent the time of stimulus current. (C) eEPSC amplitude showed no change during capsaicin treatment. Similarly, no significant change is observed in (D) rise- time, (E) half-width, (G) paired pulse ratio (EPSC2/EPSC1) and (H) input resistance. However, there was a significant shift in the baseline holding current (F). 20µM strychnine HCl was present during recordings. Data represented as mean ± S.D. for n=8 cells for input resistance and n=11 cells for all other parameters. Significance is shown as **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 3.6 Evoked EPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -237.9 68.8 11 -213.9 68.3 11 ns, p=0.25 Half-width (ms) 9.9 6.5 11 8.3 3.8 11 ns, p=0.42 Rise time (ms) 6.7 2.6 11 7.4 4.0 11 ns, p=0.61 Paired Pulse Ratio 1.46 0.1 11 1.576 0.2 11 ns, p=0.17

Iholding (pA) -56.63 76.9 11 -103 103.4 11 ** p=0.0033 Input Resistance (MΩ) 124.7 47.9 08 130.9 55.0 08 ns, p=0.13

Capsaicin (10µM) does not affect repetitive action potential (AP) firing of HMNs The effects of capsaicin on sEPSCs and mEPSCs strongly suggests it acts by increasing the release probability of pre-synaptic neuron terminals, with little post-synaptic effect on HMN excitability, although capsaicin consistently evoked an inward current in all conditions tested. To directly measure the effect of capsaicin on post-synaptic HMN excitability, we evoked repetitive action potential (AP) firing from HMNs current clamped at -65mV, using a series of progressively increasing depolarizing current steps (Fig 3.7B). Capsaicin did not change AP firing frequency in HMNs (n=9, Fig 3.7C and D), nor did capsaicin change the number of APs evoked by maximum current injected in individual cells (Fig 3.7F). Analysis of shape parameters of APs under control and capsaicin conditions showed no significant change in AP amplitude (Fig 3.7E), rise slope (Fig 3.7G), half-width (Fig 3.7H) and after-hyperpolarisation amplitude (Fig 3.7I). Capsaicin also had

55 no effect on the minimum amount of current required to generate the first AP under both conditions tested (Fig 3.7K). However, it was interesting to note that under the recording conditions used for APs (holding potential, -65mV and potassium methyl sulfate-based internal solution), there was no significant shift in Iholding (Fig 3.7J).

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Figure 3.7 Repetitive action potential (AP) firing by HMNs is unchanged after capsaicin application. 57

(A) Experimental protocol timing. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 9-11min before recording the effect of capsaicin (Caps) on repetitive action potential firing. (B) Depolarizing current steps caused steady-state membrane depolarization in the sub-threshold range (grey trace) and repetitive action potential firing in the supra-threshold range (black trace). (C) Capsaicin (10µM) had no effect on the number of action potentials generated at the maximum amount of current injected. (D) Quantification of mean firing rate at the maximum current injected showed no change during capsaicin. All other action potential parameters, including amplitude (E), max AP count (F), rise slope (G), half-width (H), after- hyperpolarization amplitude (I) remained unchanged. Baseline holding current (Iholding) (J) and minimum current required to generate first AP (K) also remained unchanged during capsaicin. Data represented as mean ± S.D. for n=9 cells, each from separate brain slice. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 3.7 Repetitive action potential (AP) firing parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps Statistical Mean ±SD n Mean ±SD n Significance AP Amplitude (mV) 115.8 16.8 9 105.3 23.0 9 ns, p= 0.088 AP Half-width (ms) 1.4 0.4 9 1.5 0.3 9 ns, p= 0.27 AP Max Rise slope 102.0 30.5 9 90.2 23.3 9 ns, p= 0.11 (ms) After Hypolarization -11.01 3.7 9 -11.27 4.3 9 ns, p= 0.68 Amplitude (mV)

Iholding (pA) -41.25 11.6 9 -36.08 17.7 9 ns, p= 0.19 Max AP count 8.963 3.8 9 9.185 4.1 9 ns, p= 0.59 Min. Current required 29.33 20.48 9 27.22 19.22 9 ns, p= 0.32 to generate 1st AP

Discussion Our present study shows that capsaicin, a TRPV1 receptor agonist, increases glutamatergic synaptic transmission to rat HMNs at two different concentrations (1 and 10µM). This effect was associated with a negative shift in the baseline holding current

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(Iholding). When tested in presence of TTX, an increase in frequency of excitatory currents remained unchanged, as did a change in Iholding. Evoked excitatory synaptic currents showed no change in the presence of capsaicin, consistent with pre-synaptic enhancement of action potential-independent release probability. Further testing of the effect of capsaicin on excitatory synaptic current frequency in presence of two TRPV1 antagonists showed no blockage of effects on sEPSC frequency, suggesting that this effect is via a mechanism that does not require TRPV1 receptor activation. We also show that capsaicin application did not change repetitive AP firing in HMNs. However, it was interesting to note that capsaicin consistently caused a shift in membrane current during mEPSC, sEPSC and evoked EPSC recordings. The most conservative mechanism for this inward current is an increase in tonic activation of glutamate receptors, presumably secondary to increased mEPSC frequency. This raises the question of where this input comes from. HMNs receive strong glutamatergic inputs from several regions, including respiratory central drive from Dbx1 positive interneurons in the reticular formation (Koizumi et al., 2008; Revill et al., 2015). It is likely that these last order premotor interneurons also integrate inputs from vagal afferent-driven NTS neurons (Bailey et al., 2002; Bailey & Fregosi, 2006; Beaumont et al., 2017), as well as TRPV1 positive trigeminal somatosensory neurons (Cavanaugh et al., 2011), as in vivo studies show that respiratory rhythm drives and modulates both whisking and sniffing behaviours (Moore et al., 2013; Moore et al., 2014). Recently, capsaicin, acting via TRPV1 receptors, has shown to cause potent inhibition of the respiratory central pattern generator in the rhythmically active brainstem-spinal cord in vitro preparation and in plethysmographic in vivo recordings, but had no effect in a rhythmically active brainstem slice (Ren et al., 2017). Interestingly, respiratory apnoea was often accompanied by increased tonic motor activity (Ren et al., 2017), which is consistent with our interpretation, and with a study by Lee and colleagues, where increased tonic hypoglossal discharge was reported (Lee et al., 2003) at higher concentrations of capsaicin in anaesthetised rats. However, other structures may provide capsaicin-sensitive afferent inputs to HMNs. TRPV1-positive vagal nerve afferents arborize extensively in structures adjacent to the hypoglossal motor nucleus, including the dorsal motor nucleus of the vagus, and the nucleus of the solitary tract (Kalia & Sullivan, 1982; Hermes et al., 2016). Synaptic transmission to neurons in these structures is modulated by capsaicin, increasing excitatory spontaneous release (Shoudai et al., 2010) and reducing miniature inhibitory post-synaptic currents (Xu & Smith, 2015). It is possible that these modulated vagal afferents may contribute to the enhancement of glutamatergic transmission by capsaicin in 59

HMNs. As capsaicin took around 9-11mins to produce effects in our study, it is also possible that the effect seen in HMNs may be mediated by spread of a secondary messenger, such as endocannabinoids or endovanilloids. The vanilloid capsaicin is one of the five major capsaicinoids present in chili peppers (Barbero et al., 2014). The effects of capsaicin are usually attributed to activation of TRPV1 channels (Caterina et al., 1997; Messeguer et al., 2006), and are blocked by specific antagonists of TRPV1. In our study, we used capsazepine, an antagonist of TRPV1 channels (Marinelli et al., 2002; Marinelli et al., 2003), to see if its pre-application can block the effect of capsaicin on sEPSCs. Although capsazepine is a competitive antagonist of TRPV1 receptors (Docherty et al., 1997), capsazepine can act on several other ion channels and receptors, including voltage-activated calcium channels (Docherty et al., 1997), nicotinic receptors (Liu & Simon, 1997), and hyperpolarisation- activated cyclic nucleotide-gated channels (Gunthorpe et al., 2004). Capsazepine also shows species-related variable effects on calcium influx (Savidge et al., 2002) and hence may be a less potent TRPV1 antagonist in mouse and rat (Correll et al., 2004). Although capsazepine did block the effect of capsaicin on sEPSC amplitude, it did not block the effects on sEPSC frequency and Iholding. These results suggest capsaicin increases action potential-independent glutamatergic transmission by a mechanism other than TRPV1 activation. Our interpretation is further supported by reports that capsaicin elicited increases in sEPSC frequency in both wild type and TRPV1 knock-out mice (Benninger et al., 2008), indicating that modulation of excitatory synaptic transmission by capsaicin is not always mediated by TRPV1 channels. Although capsaicin is a highly potent agonist for the TRPV1 receptor, some of the actions of capsaicin may not be mediated by TRPV1 receptors. Capsaicin is reported to directly inhibit voltage-gated sodium (Balla et al., 2001; Liu et al., 2001; Lundbaek et al., 2005; Cao et al., 2007; Wang et al., 2007) as well as calcium (Köfalvi et al., 2006) channels. As we saw pre-synaptic effects of capsaicin in the presence of TTX, we can rule out direct modulation of voltage-gated sodium channels, while the absence of any change in evoked EPSC amplitude or paired-pulse ratio strongly suggests that capsaicin does not directly affect pre-synaptic Ca2+ channels. Other potential off target effects of capsaicin also include inhibition of transient (type A) and sustained (delayed rectifier type) voltage gated potassium channels in mouse trigeminal ganglion neurons, and these effects of capsaicin persisted in TRPV1 knockout mice (Yang et al., 2014). Both type A and delayed rectifier potassium currents contribute to regulation of rat HMN firing (Viana et al., 1993),

60 which was unaltered by capsaicin. However, it remains possible that capsaicin reduces the pre-synaptic potassium channel activity through a TRPV1-independent action, and this increases glutamatergic release probability. Furthermore, in human embryonic kidney (HEK) cells expressing TRPV1, capsaicin inhibited protein synthesis and depolymerized microtubules (Han et al., 2007). Additional effects of capsaicin at high doses are direct inhibition of the mitochondrial respiratory chain (Shimomura et al., 1989), significant decrease in mitochondrial membrane potential (Dedov et al., 2001), and actions as mitochondrial inhibitors to activate apoptosis and/or necrosis (Dedov et al., 2001). It was interesting to note that capsazepine alone had significant effects; it decreased sEPSC amplitude, indicating that there might be tonic activation of TRPV1 channels, or that there are non-specific actions of capsazepine (Yamamura et al., 2004). Another interesting finding was that sEPSC half-width was significantly increased by capsaicin in the presence of capsazepine, an effect that was not observed following capsaicin application alone. To address this issue, we tested the effect of another TRPV1 antagonist, SB366791. As for capsazepine, SB366791 did not block the effect of capsaicin on sEPSC frequency; however, SB366791 did block the change in sEPSC amplitude and in Iholding. No changes were observed on any of the sEPSC shape parameters after SB366791. It seems likely that any tonic effect on sEPSC amplitude is largely by post-synaptic TRPV1 modulation. We confirmed this by testing the effect of capsaicin on eEPSCs. Capsaicin did not change any eEPSC shape parameters or the eEPSC paired-pulse ratio. Only Iholding was altered significantly, consistent with prior results on spontaneous and miniature EPSCs. We note that there is a scattered population of TRPV1 positive interneurons in the reticular formation (Mezey et al., 2000), raising the possibility that our electrical simulation protocol failed to activate these TRPV1-sensitive neurons.

The negative shift in baseline holding current (Iholding) elicited by capsaicin might alter HMN intrinsic excitability and action potential firing rate. We thus tested whether capsaicin altered repetitive action potential (AP) firing by HMNs. Capsaicin did not affect repetitive firing in HMNs nor were there any changes in action potential shape parameters. These results indicate that capsaicin does not strongly modulate motor neuron intrinsic excitability. Both capsazepine and SB366791 blocked the increase in sEPSC amplitude elicited by capsaicin, indicating that this effect of capsaicin is mediated by TRPV1 activation. Noxious peripheral stimulation causes rapid translocation of GluR1-containing AMPA receptors to the cell membrane (Galan et al., 2004; Larsson & Broman, 2008) and 61 phosphorylation of GluR1 subunits (Fang et al., 2003a; Fang et al., 2003b), suggesting possible mechanisms driving increases in sEPSC amplitude. However, increased sEPSC and mEPSC frequency after capsaicin were not blocked by prior application of TRPV1 antagonists, ruling out a role for TRPV1 in this effect. In conclusion, our hypothesis that a neuroactive steroid like alfaxalone might directly modulate the activity of specific or several TRP channels situated in the plasma membrane of motor neurons to bring about a change in Iholding was not supported by the present study. Although capsaicin, a classical TRPV1 agonist, significantly increased the

Iholding this effect is more likely due to an increase in glutamate release and not a direct effect on TRPV1. This conclusion is also highlighted by the fact that it took around 8-14 min for capsaicin to show its effect on excitatory synaptic transmission. Finally, we show that capsaicin significantly increased glutamatergic synaptic transmission to HMNs and caused a change in Iholding. However, this effect of capsaicin was not sufficient to change the intrinsic excitability of HMNs. We also show that these effects of capsaicin are largely not mediated by classic TRPV1 activation. This study adds to our present understanding of mechanism of action of capsaicin in central neurons and the possibility of non-TRPV1- mediated effects of capsaicin.

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Chapter 4 Capsaicin, a TRPV1 agonist reduces glycinergic inhibitory synaptic transmission to rat hypoglossal motor neurons (HMNs) Abstract Our observations that sEPSC and mEPSC frequency was consistently increased by capsaicin application raised the question of whether this effect was specific to excitatory glutamatergic presynaptic terminals. As capsaicin has already shown to strongly increase spontaneous inhibitory post-synaptic current (sIPSC) frequency and amplitude in spinal neurons, we decided to investigate whether capsaicin can also regulate inhibitory synaptic transmission to HMNs. Glycinergic IPSCs to HMNs were recorded after pharmacological isolation by perfusing brainstem slices with a cocktail of NBQX (10µM), APV (50µM) and bicuculline (5µM). Interestingly, capsaicin significantly decreased sIPSC amplitude without changing the frequency, indicating a post-synaptic mechanism of action. The amplitude of miniature inhibitory post-synaptic currents (mIPSCs), recorded in the presence of tetrodotoxin (TTX), was also reduced by capsaicin, without changing mIPSC frequency, consistent with a post-synaptic mechanism of action. No significant change in membrane current (Iholding) was recorded by capsaicin under both recording conditions, however there was a trend towards an outward shift in holding current during sIPSC recordings. The effect of capsaicin on inhibitory synaptic transmission also remained unchanged in the presence of the TRPV1 antagonist, capsazepine, suggesting that capsaicin acts to reduce IPSCs via a mechanism that does not require TRPV1 activation. Here we also show that capsaicin reduces glycinergic inhibitory synaptic transmission in HMNs by a post-synaptic mechanism. These results expand our understanding regarding the extent to which capsaicin can modulate inhibitory synaptic transmission to central neurons.

Introduction Our studies on hypoglossal motor neurons after capsaicin application showed a very robust increase in excitatory glutamatergic synaptic neurotransmission (Chapter 3). However, HMNs not only receive excitatory (serotonergic/noradrenergic) but also inhibitory inputs from different brain regions. HMN somata and dendrites receive both glycinergic as well as GABAergic synaptic terminals (Aldes et al., 1988). Furthermore, HMNs also receive strong inhibitory inputs, predominantly mediated by glycinergic neurotransmission (Umemiya & Berger, 1995; Singer et al., 1998; Donato & Nistri, 2000; Singer & Berger, 2000), making this a good model system to study glycinergic neurotransmission. Considering this, we became interested in investigating if capsaicin also modulates 63 inhibitory glycinergic transmission to HMNs, along with increasing glutamatergic transmission. Interestingly, prior reports have suggested varying degrees of modulation of inhibitory transmission to central neurons by capsaicin. For example, Xu and Smith have reported an increase in inhibitory currents to neurons of the dorsal motor nucleus of the vagus (Xu & Smith, 2015). However, in substantia gelatinosa, capsaicin facilitated excitatory without altering inhibitory synaptic transmission (Yang et al., 1998). HMNs receive several direct and indirect inputs from different brain structures like locus coeruleus (McBride & Sutin, 1976), reticular formation (Rekling et al., 2000), raphe nuclei (Manaker & Tischler, 1993), nucleus tractus solitarius (NTS) and area postrema. Interestingly, NTS and area posterma receive pulmonary and airway vagal afferents (Beaumont et al., 2017), which show expression of the TRPV1 receptors (Doyle et al., 2002; Hermes et al., 2016). Although, TRPV1 expression is reported in several brain structures, including cortex, dentate gyrus, , thalamus, trigeminal somatosensory neurons and spinal cord (Mezey et al., 2000; Roberts et al., 2004; Cavanaugh et al., 2011), no TRPV1 expression has been reported in the hypoglossal motor nucleus. Hence, it becomes an intriguing question to investigate if capsaicin, which is classically known to activate TRPV1 receptors, affects the inhibitory synaptic transmission to HMNs. The aim of this study was to investigate the actions of capsaicin on spontaneous inhibitory glycinergic synaptic transmission to HMNs in the neonatal rat brainstem slices.

Results Capsaicin (10µM) reduces spontaneous glycinergic inhibitory post-synaptic current (sIPSC) amplitude to rat hypoglossal motor neurons (HMNs) The effects of capsaicin on several central and peripheral neuronal types have been already studied to varying degrees. However, the effects of capsaicin on inhibitory synaptic transmission to HMNs remain uninvestigated. This was particularly important, as we have already reported an increase in excitatory synaptic transmission by capsaicin (Chapter 3). The possibility of dual modulation of inhibitory as well as excitatory synaptic transmission to HMNs by capsaicin led us to investigate this effect. During the capsaicin application period (10µM, 9-11min) mean sIPSC amplitude showed a significant reduction from -74.7 to -27.9pA (-63% change from control. p=0.0009, n=11, Figure 4.1D and E, Table 4.1). Similarly, there was also a significant decrease in sIPSC half-width from 10.8 to 5.9ms (-45% change from control, p=0.0009, 64 n=11, Figure 4.1H, Table 4.1). However, this effect was not associated with any significant change in the frequency of sIPSCs (Figure 4.1B and C). Figure 4.1F and G show the distribution of sIPSC amplitudes and inter-event intervals in control and capsaicin conditions respectively. The amplitude and inter-event interval distribution shows a significant shift toward low amplitude and low frequency events respectively (Kolmogorov- Smirnov test). No change was observed in sIPSC 10–90% rise time (Figure 4.1I) and baseline holding current (Iholding) (Figure 4.1J). It was interesting to note that although the change in Iholding was not significant, it shifted toward more positive levels, opposite to the change observed during recordings of sEPSCs.

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Figure 4.1 10µM Capsaicin (Caps) reduces glycinergic inhibitory post-synaptic current amplitude to rat HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 9-11min before recording the effect of capsaicin on spontaneous inhibitory post-synaptic currents (sIPSCs). (B) Representative sIPSC traces recorded in (control) and in 10µM capsaicin from HMNs voltage-clamped at -60 mV. (C) Mean spontaneous IPSC frequency during control and capsaicin conditions, showing no significant change in frequency with capsaicin. (D) Representative averaged amplitude traces of spontaneous IPSCs before (control) and during capsaicin. (E) Mean spontaneous IPSC amplitude during control and capsaicin, showing a significant decrease in amplitude with capsaicin. Averaged cumulative frequency distribution of sIPSC amplitude (F) and inter-event interval (G). Kolmogorov- Smirnov test to amplitude and inter-event interval showed a significant difference between control and capsaicin conditions. Mean sIPSC half-width (H) also showed a significant reduction after capsaicin application. Rise time (I) and baseline holding current (Iholding) (J) remain unchanged after capsaicin application. All recordings were made with NBQX (10µM), APV (50µM) and bicuculline (5µM). Data represented as mean ± S.D. for n=11 cells, each from separate brain slice. Significance is shown as ***p<0.001. A paired two- tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 4.1 Spontaneous IPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -74.7 39.4 11 -27.9 11.1 11 *** p=0.0009 Half-width (ms) 10.8 4.2 11 5.9 1.7 11 *** p=0.0009 Rise time (ms) 3.0 1.5 11 3.1 1.4 11 ns, p=0.8 Frequency (Hz) 1.37 0.2 11 1.26 0.6 11 ns, p=0.57

Iholding (pA) -122.3 66.6 11 -93.18 36.5 11 ns, p=0.067

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Capsaicin acts via a TTX-insensitive mechanism to reduce amplitude of glycinergic inhibitory currents in rat hypoglossal motor neurons (HMNs) Reduction of sIPSC amplitude is consistent with a postsynaptic reduction in glycinergic currents; however, it is also possible that it could be due to a reduction in presynaptic glycine release. Hence, to further differentiate between these possible mechanisms underlying the effect of capsaicin on inhibitory synaptic transmission, miniature IPSCs (mIPSCs) were analysed where, in addition to NBQX, APV and bicuculline, 1µM tetrodotoxin (TTX) was added for complete blockage of action potentials (Figure 4.2B). During the capsaicin application period (10µM, 4-6min), mean mIPSC amplitude still showed a significant decrease from -57.8 to -25.6pA (-56% from control, p=0.05, n=7, Figure 4.2D and E, Table 4.2). The effect of capsaicin on half-width in presence of TTX also persisted and showed a significant reduction from 9.3 to 5.2ms (- 44% from control, p=0.025, n=7, Figure 4.2H, Table 4.2). Figure 4.2F and G show the distribution of mIPSC amplitudes and inter-event intervals in control and capsaicin conditions. The amplitude and inter-event interval distribution shows a significant shift toward low amplitude and low frequency events respectively (Kolmogorov-Smirnov test). Similar to sIPSCs, no significant changes were observed in mIPSC 10–90% rise time

(Figure 4.2I) and baseline holding current (Iholding) (Figure 4.2J).

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Figure 4.2 10µM Capsaicin (Caps) reduces miniature inhibitory post-synaptic current amplitude to rat HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 4-6min before recording the effect of capsaicin on miniature inhibitory post-synaptic currents (mIPSCs). (B) Representative mIPSC frequency trace recorded under control and in presence of 10µM capsaicin from HMNs voltage-clamped at -60mV. (C) Mean mIPSC frequency under control and capsaicin conditions showing no significant change in frequency. (D) Representative mIPSC amplitude trace showing a significant decrease after capsaicin application. (E) Mean mIPSC amplitude significantly reduced after capsaicin. Averaged cumulative frequency distribution of mIPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test to amplitude and inter-event interval showed a significant difference between control and capsaicin conditions. Mean mIPSC half-width (H) also showed a significant reduction under capsaicin condition. Rise time (I) and baseline holding current (Iholding) (J) remain unchanged after capsaicin application. All recordings were made with NBQX (10µM), APV (50µM), bicuculline (5µM) and 1µM tetrodotoxin (TTX). Data represented as mean ± S.D. for n=7 cells, each from separate brain slice. Significance is shown as *p<0.05. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 4.2 Miniature IPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin. Parameter Control 10µM Caps Statistical Mean ±SD n Mean ±SD n Significance Amplitude (pA) -57.8 38.9 7 -25.6 5.4 7 *p=0.05 Half-width (ms) 9.3 3.6 7 5.2 1.1 7 *p=0.025 Rise time (ms) 3.02 1.4 7 2.8 0.63 7 ns, p=0.7 Frequency (Hz) 1.56 0.38 7 1.05 0.39 7 ns, p=0.11

Iholding (pA) -142.2 105.5 7 -133.4 133 7 ns, p=0.5

The TRPV1 antagonist, capsazepine does not block the effect of capsaicin on sIPSC amplitude Several reports suggest that the effects of capsaicin application on central neurons is via TRPV1 receptors, as capsaicin effects were successfully blocked by capsazepine

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(Savidge et al., 2001; Zschenderlein et al., 2011). However, our studies on glutamatergic excitatory synaptic transmission show that TRPV1 activation is not involved in the actions of capsaicin (Chapter 3). This led us to investigate if the effect of capsaicin on glycinergic inhibitory transmission is also independent of TRPV1 activation. Application of capsaicin (10µM, 4-10min) in the presence of capsazepine still showed a reduction in sIPSC amplitude (-46% from control, p=0.0016, n=8, Figure 4.3D and E, Table 4.3). It was interesting to note that only in the presence of capsazepine, capsaicin also showed a significant reduction in sIPSC frequency (-40% from control, p=0.044, n=8, Figure 4.3B and C, Table 4.3), which was not observed when capsaicin was applied alone (Figure 4.1 and 4.2). Figure 4.3F and G show the distribution of mIPSC amplitudes and inter-event intervals in control and capsaicin conditions. The amplitude and inter-event interval distribution shows a significant shift toward low amplitude and low frequency events respectively when tested for control and capsaicin condition (Kolmogorov-Smirnov test). Capsazepine interestingly blocked the effect of capsaicin on sIPSC half-width (Figure 4.3H). No changes were observed in 10-90% rise time (Figure

4.3I) and baseline holding current (Iholding) (Figure 4.3J). Capsazepine alone did not have a significant change in either of the parameters tested; however, there was a trend towards lower frequency and amplitude events.

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Figure 4.3 Pre-application of the TRPV1 antagonist, capsazepine (Capz) failed to block the effect of capsaicin (Caps) on sIPSC amplitude. (A) Experimental protocol timing. After recording baseline control activity, slices were superfused with capsazepine (Capz, 10µM) in aCSF for 5min before recording capsazepine-induced changes in sIPSCs. Capsaicin (10µM) in aCSF was added for 4- 10min after which capsaicin induced sIPSC responses were recorded. Representative traces for sIPSC frequency (B) under control, capsazepine and capsaicin conditions. (C) Averaged frequency plot showing significant decrease after capsaicin application in presence of capsazepine. (D) Representative amplitude for sIPSC frequency under all 3 conditions. (E) Mean amplitude plot showing significant reduction after capsaicin. Averaged cumulative frequency distribution plots of sIPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test shows significant difference between control and capsaicin condition in both plots. Mean sIPSC half-width (H), rise time (I) and baseline holding current (Iholding) (J) were unaffected under both conditions. All recordings were made in the presence of NBQX (10µM), APV (50µM) and bicuculline (5µM). Data represented as mean ± S.D. for n=8 cells, each from a separate brain slice. Significance is shown as *p<0.05, **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control, capsazepine and capsaicin + capsazepine conditions was statistically significant.

Table 4.3 Spontaneous IPSC parameters of neonatal rat HMNs upon application of 10µM capsaicin in presence of capsazepine. Parameter Control 10µM Capz 10µM Caps Mean ±SD n Mean ±SD n Mean ±SD n Amplitude (pA) -50.5 15.7 8 -45.8 15.4 8 -27.4 8.3 8 Half-width (ms) 6.2 2.9 8 6.4 3.2 8 5.3 2.6 8 Rise time (ms) 2.1 1.1 8 2.4 1.2 8 2.8 1.2 8 Frequency (Hz) 2.21 0.94 8 1.65 0.78 8 1.33 0.81 8

Iholding (pA) -168 113 8 -226.2 286.5 8 -214 275.3 8

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Parameter Statistical Significance Statistical Significance Control vs 10µM Capz Control vs 10µM Caps Amplitude (pA) ns, p=0.39 **p=0.0016 Half-width (ms) ns, p=0.66 ns, p=0.42 Rise time (ms) ns, p=0.093 ns, p=0.11 Frequency (Hz) ns, p=0.62 *p=0.044

Iholding (pA) ns, p=0.41 ns, p=0.49

Discussion This study was aimed at determining whether capsaicin also modulates inhibitory glycinergic synaptic transmission to rat HMNs, as along with glutamatergic (O'Brien et al., 1997; Del Caño et al., 1999), they also receive strong glycinergic inputs (Singer et al., 1998; Donato & Nistri, 2000). In contrast to its effects on sEPSCs, capsaicin selectively and significantly decreased sIPSCs amplitude and half-width, without altering sIPSC frequency. It was intriguing to note that Iholding was not significant altered, and indeed tended to shift toward more positive value, opposite of the significant negative shift observed when sEPSCs were recorded. This result suggests that capsaicin significantly reduces glycinergic inhibitory synaptic transmission to rat HMNs by a post-synaptic mechanism of action. We further tested if this activity is dependent on action potential mediated release of inhibitory neurotransmitter. We observed that the effect of capsaicin on glycinergic transmission is not dependent on spontaneous action potential generation, as there was a significant decrease in mIPSC amplitude even in presence of TTX. As action potential- independent miniature currents and action potential-dependent currents constitute the sum of spontaneous synaptic currents, we conclude that the decrease in sIPSC amplitude is due to a decrease in mIPSC amplitude. Prior slice electrophysiology studies investigating capsaicin and its effect on inhibitory transmission to central neurons have reported varying effects, with different mechanisms of action. Study on dorsal motor nucleus of the vagus by Xu and Smith show an increase in inhibitory current frequency (Xu & Smith, 2015). On the other hand, capsaicin facilitated excitatory but not inhibitory transmission in substantia gelatinosa neurons (Yang et al., 1998). Similarly, a study performed on mouse spinal neurons, a model system closer to the one used in the present study (brainstem neurons) showed an increase in sIPSC frequency and attributed this effect to the activation of pre-synaptic

74 inhibitory interneurons (Ferrini et al., 2007). These results differ to our conclusions that capsaicin acts via a postsynaptic modulation of glycine channel activity or conductance, with little pre-synaptic effect, as observed from its effect on sIPSCs and mIPSC amplitude. Although not significant, capsaicin did tend to elicit an outward current during sIPSC recordings (Figure 4.1) and showed no effect in other conditions tested (Figure 4.2 and 4.3). This stands in contrast to the consistent and significant inward current elicited by capsaicin when recording sEPSCs and mEPSCs. As capsaicin was reducing inhibitory glycinergic membrane conductance, we would expect that this would produce a net inward current, so the absence of such an effect lends support to the hypothesis that the inward current seen during excitatory transmission experiments was primarily due to increased excitatory glutamatergic membrane conductance. However, further investigation of this effect is needed to underpin this hypothesis. Finally, we tested whether the effect of capsaicin can be blocked by pre-application of a competitive antagonist of TRPV1 receptors, capsazepine (Bevan et al., 1992). Our results show that pre-application of capsazepine did not block the effect of capsaicin on sIPSC amplitude, however, the effect on sIPSC half-width was blocked. It was also observed that sIPSC frequency was significantly reduced in presence of capsazepine. However, it was interesting to note that capsazepine alone, did not show any significant changes to any of the shape parameters tested, which shows that there is no tonic TRPV1 activity affecting inhibitory glycinergic transmission. This is contrary to the observation that capsazepine did exhibit a tonic effect on excitatory glutamatergic recordings, as shown by a change in sEPSC amplitude under capsazepine alone condition. A possible reason for this reduction in frequency can be attributed to the fact that at concentrations required to observe the antagonistic activity of capsazepine (~5-10µM), it can modulate several other ion channels and receptors, including voltage-activated calcium channels (Docherty et al., 1997) and nicotinic acetylcholine receptors (Liu & Simon, 1997). The inability of capsazepine to block all the effects of capsaicin emphasizes capsaicin’s off-target effects. Capsaicin is reported to show off-target effects on several voltage gated channels like sodium (Wang et al., 2007), calcium (Köfalvi et al., 2006) and potassium (Yang et al., 2014). As miniature recordings were performed in presence of TTX, we can safely conclude that capsaicin do not act on voltage gated sodium channels. However, a direct modulation of calcium and/or potassium channels remains a possibility. Furthermore, an inhibition of mitochondrial respiratory chain (Shimomura et al., 1989) and decrease in mitochondrial membrane potential (Dedov et al., 2001) are also reported. It is very well

75 possible that capsaicin in present study either acted on one or more of the above targets which were not specifically studied here. In conclusion, this study shows for the first time that capsaicin, a classic TRPV1 agonist, reduces inhibitory glycinergic synaptic transmission to rat HMNs. Since this effect was TTX-insensitive, we also conclude that this effect is independent of action potential generation and is possibly mediated via a post-synaptic mechanism. Finally, we have also show that this inhibitory effect of capsaicin is mediated via a mechanism other than activation of classic TRPV1 receptors.

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Chapter 5 Alfaxalone and capsaicin do not show any species-related difference in their effect on neonatal C57/BL6 WT mouse HMNs Abstract Our studies on rat hypoglossal motor neurons showed significant modulation of inhibitory glycinergic and excitatory glutamatergic synaptic transmission following application of alfaxalone and capsaicin respectively. However, both alfaxalone-like steroid molecules and several TRP channel agonists can have species, age and gender related differences in their effects. We conducted a study to gauge the possibility of species differences in the effects of alfaxalone and capsaicin on inhibitory and excitatory synaptic transmission to HMNs in brainstem slice preparations. Wild type C57/BL6 mice HMNs were studied using similar conditions used for rat HMN recordings. Our results show that alfaxalone significantly reduced sIPSC amplitude (-38%) and half-width (-19%) along with frequency (-20%) in mouse HMNs. It was interesting to note that there was no significant change in the baseline holding current (Iholding) upon alfaxalone treatment. On the other hand, capsaicin application showed a significant increase in amplitude (+25%) and frequency (+118%) of sEPSCs to mouse HMNs. Similar to the effect seen in our rat study, a significant negative shift in the baseline holding current (Iholding) (-90%) was observed after capsaicin application. Our results show that, in mouse HMNs, alfaxalone reduces spontaneous glycinergic neurotransmission and capsaicin enhances spontaneous glutamatergic neurotransmission. From these results, we conclude that there is unlikely to be a significant species difference in the effect of either alfaxalone or capsaicin on HMNs.

Introduction Steroids or steroid-like molecules and several TRP channel modulators are implicated in modulating various neuronal functions (Zakon, 1998; Moran et al., 2004). In particular, steroids can regulate a variety of functions in central neurons by acting either on membrane or intracellular steroid receptors to bring about both fast and delayed effects (Joëls, 1997). Electrophysiological studies show that steroids can potentially alter the electrical excitability of neuronal membranes and thereby cause changes in firing properties (Okuhara & Beck, 1998; Pielecka et al., 2006). These effects are not limited to endogenously synthetised steroids (neurosteroids), such as progesterone and its metabolites allopregnanolone (Robichaud & Debonnel, 2004), but have also exhibited by synthetic steroid molecules like (Robichaud & Debonnel, 2006), alfaxalone (Ahrens et al., 2008). 77

However, an interesting phenomenon is the variation in steroid effects based on sex or species of animals tested. Secretion of testosterone in male rodents is a major differentiation factor resulting in differences in physiology and behaviour between male and female rodents (Bonthuis et al., 2010). Significant sex differences in responses to steroid treatments are reported (Alele & Devaud, 2007), most likely due to the variation in the basal levels of endogenous steroids between the two groups. For example, differences in the sedative effects of allopregnanolone were observed between male and female rats (Irwin et al., 2014). An important determinant in the variability of effects is attributed to differences in the steroid metabolising enzymes in the . CYP2C12 is one of the isoforms of the cytochrome P450 (CYP2) enzyme involved in hydroxylation of steroids and is highly expressed in the of adult female rats compared to males (Martignoni et al., 2006). Similarly, CYP2C11 levels are dramatically induced at in male, but not female, rats (Martignoni et al., 2006). On the other hand, there is considerable support in the literature for significant species-related differences between rats and mice. These differences can either be generated within the species e.g. genetic mutation within a closed colony (Egan et al., 2007) or can also be due to variation in breeding protocols (Bonthuis et al., 2010). Species-related differences can also be observed because of naturally occurring anatomical variations which can lead to striking differences in adult life. For example, in the preoptic area, steroidal modulation of neuronal nitric oxide synthases (nNOS) expression is opposite in rats and mice. In rats, acting via receptors bring about an increase in nNOS levels. However, in mice, an opposite effect is observed where acting via receptors increase nNOS expression (Bonthuis et al.,

2010). Another prominent difference between rats and mice is in the expression of the protein calbindin in the sexually dimorphic nucleus (SDN). In rats, a lower immunoreactivity is observed at birth and further increases post-natally. However, in mice, the expression is high pre-natally (Bonthuis et al., 2010). Studies on neuroanatomical markers in the brains of rats and mice have also demonstrated species differences (Bonthuis et al., 2010). As for sex-related differences, the majority of species-related differences between rats and mice arise because of differences in enzyme catalytic activity and metabolic clearance. Particularly with steroids, species differences are reported owing to a significant difference in metabolising enzymes in liver (Busso & Ruiz, 2011).

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Taken together, prior studies have reported sex and species differences in the effects of steroids and it thus becomes important to investigate if alfaxalone, a synthetic neuroactive steroid, shows any species-related variation in its effects on mouse HMNs. It is interesting to note that considerable species-related differences exist for the effect of capsaicin (Szallasi & Blumberg, 1999). Age-, strain- and species-related differences in sensitivity to capsaicin is extensively reported in previous studies. For example, cell bodies of primary sensory neurons are more susceptible to capsaicin in newborn than in adult rats, as concluded from the study that showed complete impairment versus partial recovery after capsaicin treatment in newborn and adult rats respectively (Jancso et al., 1977). Amphibians (toads) show low sensitivity to capsaicin (Hawkins et al., 1991) when compared to rats. Similarly, a significant difference in the amino acid sequence homology is observed between avian and mammalian capsaicin receptors (Jordt & Julius, 2002). The human hTRPV1 channel is harder to open and hence, the efficacy of capsaicin as a TRPV1 agonist is lower for the human receptor than the rat receptor (Wang et al., 2010). Measurement of compound action potentials in the saphenous nerve show low sensitivity to capsaicin in guinea-pig and when compared to rats (Baranowski et al., 1986). Considering this variability in the effects of capsaicin, we became interested in investigating if there are any species-related differences between rat and mice, in comparing the effects of capsaicin on spontaneous excitatory currents in mouse HMNs.

Results Alfaxalone (25µM) reduces spontaneous glycinergic IPSC amplitude, half-width and frequency in mouse hypoglossal motor neurons (HMNs) Our earlier investigation on the effect of alfaxalone on IPSCs (Chapter 2) to rat HMNs had shown a significant reduction in amplitude and frequency of glycinergic inhibitory currents, which was accompanied by a positive outward shift in the baseline holding current (Iholding). However, no study has investigate if alfaxalone affects glycinergic inhibitory synaptic transmission to mouse HMNs. To check if there is any species related variation in the effect of alfaxalone when tested on C57/BL6 mice, a similar experiment was designed with same internal solutions and pharmacological blockers of GABAergic and glutamatergic synaptic transmission, NBQX (10µM), APV (50µM) and bicuculline (5µM). Mean sIPSC amplitude was significantly reduced from -62.7 to -39.2pA (-38% change from control, p=0.003, n=12, Figure 5.1E, Table 5.1). Representative amplitude 79 traces (Figure 5.1 D) shows a reduction with alfaxalone. There was also a significant reduction in the half-width of sIPSCs from 9.6 to 7.8ms (-19% change from control, p=0.001, n=12, Figure 5.1H, Table 5.1). Similarly, a significant decrease was also observed in sIPSC frequency from 1.98 to 1.59Hz (-20% change from control, p=0.041, n=12, Figure 5.1C, Table 5.1). A representative frequency trace under control and alfaxalone conditions (Figure 5.1B) show the reduction in sIPSC frequency. The distribution of sIPSC amplitudes and inter-event intervals in control and alfaxalone conditions are shown in Figure 5.1F, G. A Kolmogorov-Smirnov test applied to the amplitude and inter-event interval distribution shows a significant shift toward low amplitude and high frequency events respectively. Figure 2.1I show no change in sIPSC 10–90% rise time after alfaxalone application. It was interesting to note that baseline holding current (Iholding) showed no significant change in either direction in presence of alfaxalone which differs from our results in rat HMNs. These results suggest that alfaxalone causes a reduction in inhibitory glycinergic synaptic transmission to mouse HMNs. Similarly, a robust decrease in both frequency and amplitude of sIPSCs show a possible pre- and post-synaptic mechanism similar to observations in rat HMNs.

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Figure 5.1 Alfaxalone (Alfa) reduces spontaneous inhibitory post-synaptic transmission to WT C57BL/6 mouse HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with alfaxalone (25µM) dissolved in aCSF for 2-4min before recording the effect of alfaxalone on spontaneous inhibitory post-synaptic currents (sIPSCs). (B) Representative sIPSC frequency trace recorded under control and in presence of 25µM alfaxalone from HMNs voltage-clamped at -60mV. (C) Mean sIPSC frequency under control and alfaxalone conditions showing a significant decrease in frequency with alfaxalone. (D) Representative sIPSC amplitude showing a significant decrease after alfaxalone application. (E) Mean sIPSC amplitude significantly reduced after alfaxalone. Averaged cumulative frequency distribution of sIPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test to amplitude and inter-event interval showed a significant difference between control and alfaxalone conditions. Mean sIPSC half-width (H) rise time

(I) and baseline holding current (Iholding) (J) remain unchanged after alfaxalone application. All recordings were made with NBQX (10µM), APV (50µM) and bicuculline (5µM). Data represented as mean ± S.D. for n=12 cells, each from separate brain slice. Significance is shown as *p<0.05, **p<0.01. A paired two-tailed t-test was used to determine whether the difference between data points under control and alfaxalone conditions was statistically significant.

Table 5.1 Spontaneous IPSC parameters of neonatal C57BL/6 WT mice HMNs upon application of 25µM alfaxalone. Control 25µM Alfa Statistical Parameter Mean ±SD n Mean ±SD n Significance Amplitude (pA) -62.7 37.08 12 -39.2 23.4 12 ** p=0.003 Half-width (ms) 9.6 4.5 12 7.8 3.5 12 ** p=0.001 Rise time (ms) 2.7 1.7 12 3.5 2.2 12 ns, p=0.064 Frequency (Hz) 1.98 0.76 12 1.59 0.91 12 * p=0.041

Iholding (pA) -173 159.7 12 -171.3 192.6 12 ns, p=0.93

Capsaicin (10µM) increases spontaneous excitatory post-synaptic current (sEPSC) frequency and amplitude to mouse hypoglossal motor neurons (HMNs) Our results in Chapter 3 had clearly shown a significant positive modulation of sEPSCs in rat HMNs at two different concentrations of capsaicin (1 and 10µM). However,

82 to our knowledge, the effect of capsaicin on spontaneous excitatory currents in mouse HMNs are yet to be reported. Similarly, considering the fact that there is significant species related variation in sensitivity of capsaicin, we decided to investigate if capsaicin shows any species difference when tested on C57/BL6 mice. As previous studies on capsaicin effects in mice (Güler et al., 2012) show robust effects in slice electrophysiology when capsaicin was used at 10µM, we decided to use this higher dose for our studies on spontaneous glutamatergic excitatory currents. After capsaicin application (7-11 min, 10µM), it was noted that there is a significant increase in sEPSC frequency and amplitude (Figure 5.2 B-G). An increase in frequency is represented in Figure 5.2B where an increase in the number of negative going excitatory events is observed under capsaicin condition. Figure 5.2C shows an increase in mean sEPSC frequency from 2.02 to 4.39Hz (+118% of control, p=0.03; n=8; Table 5.2). Figure 5.2D represents sEPSC amplitude under both conditions. An increase in mean sEPSC amplitude from -18.1 to -22.6pA is shown in Figure 5.2E (+25% increase from control, p=0.021, n=8, Table 5.2). Figure 5.2F and G showing distribution of amplitude and inter- event interval under control and capsaicin conditions. A Kolmogorov-Smirnov test applied to the inter-event interval distribution shows a significant shift high frequency events. Other sEPSC shape parameters, such as half-width (Fig 5.2H) and 10-90% rise time (Fig 5.2I) remained unchanged during capsaicin application. However, baseline holding current

(Iholding), showed a significant inward shift from -60.4 to -114.9pA (+90% of control, p=0.011, n=8, Fig 5.2J).

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Figure 5.2 Capsaicin (Caps) increases glutamatergic excitatory post-synaptic current frequency and amplitude to mouse HMNs. (A) A time line of the experimental protocol. After baseline control recording, slices were superfused with capsaicin (10µM) dissolved in aCSF for 7-11min before recording the effect of capsaicin (Caps) on spontaneous excitatory post-synaptic currents (sEPSCs). (B) Representative sEPSC traces recorded in the absence (control) and presence of 10µM capsaicin from a HMN voltage-clamped at -60mV. (C) Mean sEPSC frequency showing a significant increase during control vs capsaicin conditions. (D) Representative averaged amplitude traces under control and capsaicin conditions. (E) Mean sEPSC amplitude plot showing a significant increase in sEPSC amplitude with capsaicin. Averaged, cumulative frequency distribution plots of sEPSC amplitude (F) and inter-event interval (G). Kolmogorov-Smirnov test of inter-event interval shows a significant difference between control and capsaicin conditions. Mean sEPSC half-width (H) and rise time (I) did not change after capsaicin application. (J) Baseline holding current (Iholding) was significantly altered in inward direction by capsaicin. All recordings were made in the presence of 20µM strychnine HCl. Data represented as mean ± S.D. for n=8 cells, each from a separate brain slice. Significance is shown as *p<0.05. A paired two-tailed t-test was used to determine whether the difference between data points under control and capsaicin conditions was statistically significant.

Table 5.2 Spontaneous EPSC parameters of neonatal C57BL/6 WT mice HMNs upon application of 10µM capsaicin. Control 10µM Caps Statistical Parameter Mean ±SD n Mean ±SD n Significance Amplitude (pA) -18.1 4.5 8 -22.6 3.9 8 * p=0.021 Half-width (ms) 2.2 0.5 8 2.0 0.5 8 ns, p=0.51 Rise time (ms) 1.6 0.4 8 1.2 0.4 8 ns, p=0.12 Frequency (Hz) 2.02 0.9 8 4.39 2.79 8 * p=0.03

Iholding (pA) -60.4 48 8 -114.9 40.2 8 * p=0.011

Discussion Our present study was aimed at investigating if any species-related difference in the effects of alfaxalone and capsaicin on mouse HMNs exists. Although there is evidence of species variation in effects of several steroids and TRP channel modulators, our study did

85 not show any such variation in alfaxalone and capsaicin effects on inhibitory and excitatory currents respectively. Previously, Lau and colleagues (Lau, 2013) showed that alfaxalone, a synthetic neuroactive steroid molecule, reduces spontaneous glycinergic synaptic transmission to rat HMNs. We show (Chapter 2) that alfaxalone also significantly reduced miniature glycinergic inhibitory synaptic transmission in the same species (Wistar rat). However, considering the steroid nature of alfaxalone, it became desirable to check if there is any species-related difference in effects of alfaxalone between rats and mice. Our results for spontaneous glycinergic currents on mouse HMNs (present work) show that, along with reduction in amplitude and frequency, there was also a significant decrease in half-width, which is similar to that observed by Lau and colleagues (Lau, 2013) in their study of rat HMNs. The same study also showed a significant negative shift in

Iholding, which was not observed in the present study on mice. The cause for this discrepancy remains unidentified and could be attributed to species difference between neonatal rats and mice. On the other hand, in our study of the effects of alfaxalone on miniature glycinergic currents in rat HMNs, modulation in half-width was not noted, which contradicts the effect observed in sIPSCs in mouse study, where a significant decrease in half-width was observed. This difference can be attributed to the fact that we only tested miniature glycinergic synaptic transmission in rats, which were performed in presence of TTX; however, no miniature recordings were performed the mice study. To explore this phenomenon further, miniature IPSC recording could be performed on mouse HMNs in the presence of alfaxalone. Capsaicin, a classical TRPV1 receptor agonist, caused a significant increase in glutamatergic synaptic transmission to mouse HMNs, which was evident as an increase in both frequency and amplitude of spontaneous synaptic currents. Modulation of both frequency and amplitude indicate a possibility of dual pre- and post-synaptic mechanisms causing increased glutamate release and enhanced postsynaptic glutamate receptor activation. It is also important to note that this effect was also accompanied by a negative shift in the baseline holding current (Iholding), which might contribute to modulation of motor neuron excitability. Comparing the effects of 10µM capsaicin on rat and mouse HMNs show a close similarity in the relative effects from control. Increases in amplitude of sEPSCs in rat and mouse from control were very similar, +26% and +25% respectively (Table 3.2 and 5.2). Similarly, the increase in sEPSC frequency when compared to control was very similar in both rat and mouse study (+116% and +118% respectively). This emphasizes the fact that 86 there is unlikely to be significant species related variation in the sensitivity of rat and mouse HMNs to capsaicin. In conclusion, we report that alfaxalone and capsaicin effects on spontaneous inhibitory and excitatory currents respectively did not show significant species-related difference between neonatal Wistar rats and C57/BL6 mice.

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General discussion Rational development of a safe and reliable anaesthetic regime for laboratory rodents was the over-arching aim of this study. As anaesthetic protocols are widely used in biomedical research laboratories, effort in understanding and developing better anaesthetics is highly desirable. The present study was aimed at furthering our understanding of a well-known anaesthetic agent that is widely used in several large animal species, but not in laboratory rodents. We focused on alfaxalone, which is successfully used in cats, dogs, horses, etc, with minimal side effects. As alfaxalone is associated with a high margin of safety in larger animals, it became important to understand if it can provide an effective and safe alternative to current anaesthetics in regular laboratory practices for rats and mice. However, severe muscular twitching is observed when the same alfaxalone formulation is administered to laboratory rodents. Hence, understanding the mechanism underpinning twitching behaviour in rats was the primary focus of our study. It was assumed that the severe twitching observed in rats could be brought about by modulation of inhibitory as well as excitatory synaptic transmission to brain stem motor neurons. As the anaesthetic activity of alfaxalone is attributed to its well documented modulation of GABAergic transmission, we focused on glycinergic inhibitory and glutamatergic excitatory synaptic transmissions in our study. As muscle twitching is most likely driven by motor neuron activity, it was imperative to investigate the modulation brought about by alfaxalone at synaptic levels to motor neurons. A specialized group of cells in the brain stem, viz. hypoglossal motor neurons (HMNs) were selected for this investigation. Electrophysiological recordings were obtained from individual motor neurons to study inhibitory and excitatory synaptic transmission to HMNs in slice preparations after alfaxalone application. First, modulation of miniature inhibitory glycinergic transmission was investigated which showed that alfaxalone robustly decreases amplitude and frequency of inhibitory currents. We also showed that activity-dependent and -independent excitatory glutamatergic transmission and repetitive action potential firing in HMNs is not affected by alfaxalone. Hence, our data confirms that alfaxalone selectively reduces glycinergic inhibition to rat HMNs, which we believe causes a severe disruption of excitatory-inhibitory balance and leads to motor neuron activity and muscular twitching. A regular observation after alfaxalone application was the modulation of baseline holding current (Iholding), therefore, we investigated the possibility that alfaxalone activated either selective or multiple TRP channels that might produce HMN depolarization and an increase in intracellular Ca2+ concentration. It can be argued that upon this increase in 88 intracellular Ca2+ from several, yet unidentified secondary signaling pathways, causes release of endocannabinoids and/or endovanilloids. This led us to investigate the possible modulation of synaptic activity by capsaicin, a TRPV1 agonist. Interestingly, capsaicin itself had a strong modulatory role in the synaptic neurotransmission to HMNs. We have for the first time shown that capsaicin increases excitatory glutamatergic and reduces inhibitory glycinergic synaptic transmission to HMNs without affecting the intrinsic excitability. We also show that these effects are not mediated by typical TRPV1 activation.

Although capsaicin did change Iholding, it does not support our hypothesis that alfaxalone might bring about this selective modulation of TRPV1 receptors, as we believe that modulation of Iholding after capsaicin was an indirect effect of increased glutamate release in the synaptic cleft. This idea is also supported by the fact that capsazepine, a TRPV1 antagonist, was unable to block the effects of capsaicin. Although our hypothesis was not supported by our data, it has led us to a new line of investigation and has provided novel insights into the modulation of synaptic transmission by capsaicin. Finally, as multiple reports suggest species-related differences in the effects of steroid-like molecules and TRP channels modulators, we decided to investigate if the effects of alfaxalone and capsaicin on inhibitory and excitatory synaptic transmission to HMNs in rats are also replicated in wild type C57/BL6 mice. We show that there is no species-related variation in the effect of either alfaxalone or capsaicin. This supports the use of alfaxalone anaesthesia in the two most commonly used species in biomedical research, provided that neuromotor excitation can be prevented by the use of suitable pre- medications. Our present work on alfaxalone has provided the first evidence that this neuroactive steroid selectively reduces inhibitory synaptic transmission to motor neurons. This data can be used to design better anaesthetic regimes using alfaxalone, and will particularly help in selecting appropriate premedications before alfaxalone anaesthesia in laboratory rodents. Similarly, our work on capsaicin has shown for the first time that capsaicin significantly modulates synaptic transmission to hypoglossal motor neurons, independent from TRPV1 activation, thus broadening our understanding about the pharmacology of capsaicin.

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Appendix 1. Link for the publication included in thesis: https://www.ncbi.nlm.nih.gov/pubmed/29259542

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2. Animal ethics approval letter

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