Huma, Zilli (2014) Spinoreticular tract neurons: the spinoreticular tract as a component of an ascending descending loop. PhD thesis. http://theses.gla.ac.uk/5628 Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] SPINORETICULAR TRACT NEURONS: THE SPINORETICULAR TRACT AS A COMPONENT OF AN ASCENDING DESCENDING LOOP Dr. Zilli Huma MBBS, FCPS in General surgery (College of Physicians and Surgeons, Pakistan) Thesis submitted in fulfilment for the degree of Doctor of Philosophy Institute of Neuroscience and Psychology College of Medical, Veterinary and Life Sciences University of Glasgow Glasgow, Scotland October 2014 "In the name of Allah, most Gracious, most Compassionate" Dedication In loving memory of my dear brother Syed Wasif Ali Shah and beloved father-in–law Syed Manzar Hussain Summary The lateral reticular nucleus (LRN) is a component of the indirect spino-reticulo- cerebellar pathway that conveys sensorimotor information to the cerebellum. Although extensive work has been done on this pathway using electrophysiological techniques in cat, little is known about its infrastructure or neurochemistry in both cat and rat. Thus defining the morphology of this spinoreticular pathway would provide a better understanding of its intricate connections and the role of various neurotransmitters involved, which in turn would provide insight into the process by which these neurons carry out, for example, reflex modulation. We thus became interested in finding out more about the role of the spinoreticular neurons (SRT) in this pathway, what and how these cells receive inputs, their role within the spinal circuitry and how they modulate sensorimotor output. Thus, in view of these limitations, we formulated a hypothesis: ‘That spinoreticular neurons form a component of a feedback loop which influences activity of medullary descending control systems’. To test this hypothesis we developed four main aims: (1) to find out the distribution pattern of spinoreticular tract (SRT) neurons and their axonal projections to the LRN; (2) to examine the origins of two bulbospinal pathways projecting to the rat lumbar spinal cord via the medial longitudinal fasciculus (MLF) and caudal ventrolateral medulla (CVLM); (3) to determine the origin of excitatory and inhibitory contacts on SRT neurons in rat and cat lumbar spinal cord; and (4) to analyse some of the neurochemical phenotypes of SRT neurons and their response to noxious stimulus. In order to fulfil these aims, we combined tract tracing by retrograde and in some cases anterograde transport of the b subunit of cholera toxin (CTb) and retrograde transport of fluorogold (FG) along with immunohistochemistry in rats. In addition to this, SRT cells in cat were identified electrophysiologically and intracellularly labelled with Neurobiotin (NB), in vivo which were further investigated by using immunohistochemistry. As most of the electrophysiological data available to date is from cat studies so in this study we wanted to see how well this correlated to the anatomical results obtained from both cat and rat experiments. I Results from Aim 1 demonstrated that, although there was extensive bilateral labelling of spinoreticular neurons in rat on both sides of the lumbar spinal cord, ~ 70% were contralateral, to the LRN injection site, in the ventromedial Lamina V to VIII. There were also some SRT cells that project ipsilaterally (31-35%) in addition to ~8% projecting bilaterally to both lateral reticular nuclei. Further experiments showed that the majority of SRT axons ascending via the ventrolateral funiculus terminate within the ipsilateral LRN with fewer projections to the contralateral LRN (2.6:1 ratio). These projections are predominantly excitatory (~80% both vesicular glutamate transporter 1 and 2; VGLUT-1, VGLUT-2) in addition to a significant inhibitory component (~15%, vesicular GABA transporter; VGAT), that consists of three subtypes of axons containing GABA, glycine or a mixture of GABA and glycine. LRN pre-cerebellar neurons receive convergent connections from excitatory (~13%) and inhibitory (~2%), SRT axons. Experiments undertaken to meet the second aim of this thesis revealed that, in rat, bulbar cells projecting via the MLF (medial longitudinal fasciculus) or the CVLM (caudal ventrolateral medulla) to the lumbar spinal cord have mostly overlapping spatial distributions. The vast majority of cells in both pathways are located in identical reticular areas of the brainstem. Furthermore, both pathways have a mixture of crossed and uncrossed axonal fibres, as double labelled cells were located both ipsi and contralateral to unilateral spinal injection sites. Bulbospinal (BS) cells that project via CVLM, form predominantly excitatory contacts with spinoreticular cells but there is also an inhibitory component targeting these cells; ~56% and ~45% of the BS contacts, respectively, In investigating the third aim to provide insight into the inputs to spinoreticular cells in two species, rat and cat we observed that; in both species these cells receive predominantly inhibitory inputs (VGAT) in addition to excitatory glutamatergic contacts that are overwhelmingly VGLUT-2 positive (88% to 90%). Thus, it appears that most inputs to these cells are from putative interneuronal populations of cells, for example PV (parvalbumin) and ChAT cells (choline acetyl transferase). SRT neurons in the rat receive a significant proportion of contacts from proprioceptors (~17%) but in the cat these cells do not seem to II respond monosynaptically to inputs from somatic nerves. Furthermore, a significant proportion of contacts on rat SRT cells originate from myelinated cutaneous afferents (~68%). Data from the final series of experiments demonstrate the heterogeneity of spinoreticular neurons in terms of immunolabelling by neurochemical markers as well as their varied responses to noxious stimulation. Many SRT neurons express NK-1 receptors (~27%, neurokinin 1) and approximately 20% of SRT neurons were immunoreactive for calcium binding proteins, CB, CR (calretinin) or both CB & CR and hardly any cells labelled for ChAT. While a smaller proportion immunolabelled for neuronal nitric oxide synthase (nNOS). Nine percent of SRT cells responded to mechanical noxious stimulation as demonstrated by phosphorylation of extracellular signal regulated kinase (ERK). The present findings provide a new basis for understanding the organisation and functional connectivity of spinoreticular tract neurons which convey information from peripheral and spinal inputs to the LRN where it is integrated with information from the brain and conveyed to the cerebellum and their role in a spino-bulbo-spinal loop that is responsible for modulating activity of pre-motor networks to ensure co-ordinated motor output. III Acknowledgement I would like to begin by thanking Almighty Allah for helping me finish this project in time and for all the amenities He has provided to make it possible. My sincere gratitude goes to my supervisor, Professor David J Maxwell for accepting me as a PhD student when all seemed lost and for his guidance and support throughout my research and write up, for always being there. A heartfelt thank you, to Dr Ingela Hammar from the Department of Physiology, University of Gothenburg, Sweden, for her continuous support, being my mentor and wonderful host. Thank you to my advisors, Professor Andrew J Todd for his invaluable comments and observations and Professor Mhairi McRae for her expert advice especially in all matters statistical. There is a long list of people within the spinal cord group who have helped, advised or just been there for me throughout this PhD, in particular, Robert Kerr and Christine Watt for not only their expert technical assistance but also for all the tit bits of information about Scottish life. Special thanks to two wonderful friends and colleagues Sony and Anne for all your help and being a shoulder to cry on in dire times. I am greatly indebted to my family for all their sacrifices and allowances on my behalf, my parents and brothers, in helping me fulfil a lifelong dream. A special thank you to my loving husband Masud for without you this PhD would not have even been conceivable, for your belief, love and endless patience. Thank you to my awesome kids Hasan, Fatima and Haris for just being there and for putting up with my absences, even when I am physically present. Last but not least I would like to thank my funding body, Higher Education Commission and Khyber Medical University, Pakistan, for providing me this unique opportunity of pursuing higher studies in this beautiful and friendly city, Glasgow. IV Author’s declaration All work in this thesis was carried out solely by me, apart from some of the surgical procedures and electrophysiology. Professor David Maxwell contributed to this work by performing surgical procedures on rats. Dr Ingela Hammar contributed by performing surgical procedures and electrophysiological