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The meninges have long been accepted as purely structural, protecting the central nervous system (CNS). Similarly, the choroid plexus (CP) was thought to only produce cerebrospinal fluid. Advances have dispelled these limited views, and the meninges and CP are now known as critical sites of immune activity, housing various immune cells, including B and T cells, monocytes/macrophages, and dendritic cells, and are increasingly implicated in neuroinflammation. The broad aim of this thesis is to characterise the immune profile of the meninges, CP, and secondary lymphoid tissue in animal models of CNS and peripheral neuropathy. New evidence in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggests inflammation of the meninges and CP changes are crucial for disease induction, warranting further investigation. Firstly, we demonstrated significant changes in the meninges and lymphoid tissue in EAE. Adoptive transfer of immunosuppressive activated regulatory T cells (Tregs) resulted in their migration to the pia and CP, and drainage from the CNS back into lymph nodes 2-4 days following delivery. Treg treatment at the peak of EAE showed mild changes in meningeal/lymphatic immunity. Secondly, to study meningeal involvement in peripheral neuropathy, where literature is severely lacking, we utilised an animal model of peripheral nerve injury, sciatic nerve chronic constriction injury (CCI), and 2 animal models of chemotherapy-induced peripheral neuropathy (CIPN). The immune profile of the meninges, CP, and lymphoid tissue was compared across male and female mice in acute stages of CCI and in a chronic model. We found the immune profile of the meninges, CP, and lymphoid tissue at acute stage of injury is different for males and females, is altered at 7 days and 21 days post-injury and appears to resolve by a chronic time point. In CIPN animal models, we demonstrated the immune profile of the meninges and CP is minimally affected, however

The experiments reported in this thesis investigated arousal and motivational drive by chemogenetically evoking cardiovascular and locomotor responses from the tuberal hypothalamus. The first experiment showed that hSyn and CaMKIIα promoted hM3Dq adeno-associated viral vectors (AAVs) express well in the medial tuberal hypothalamus (MH) and the lateral tuberal hypothalamus (LH), and there were differences between the two AAVs in terms of their tropism. The hSyn AAV showed tropism towards GABAergic neurons over glutamatergic neurons, whereas the CaMKIIα AAV showed tropism towards glutamatergic over GABAergic neurons, for both regions. Experiment 2 confirmed that CNO injection (3 mg/kg i.p.) greatly increased neuron activity in a hM3Dq transduced hypothalamus hemisphere, as measured by cFos expression. cFos expressed strongly throughout the MH and LH, including orexin neurons. Experiment 3A demonstrated that chemogenetic activation of the tuberal hypothalamus elicited marked increases in locomotor activity, heart rate (HR), and mean arterial blood pressure (MAP) over vehicle, which persisted for the 2 hr measurement period (and beyond). Changes in locomotor activity depended on which region within the hypothalamus was activated as well as the promoter used. Strong locomotor responses were elicited from the hSyn and CaMKIIα AAVs in the MH, but only from the CaMKIIα AAV in the LH. In contrast, HR and MAP increases were stronger when evoked from the MH than the LH, and this was independent of the promoter used. Experiment 3B showed that the cardiovascular component of these responses was centrally mediated, rather than being caused by locomotor hyperactivity. Experiment 3C showed that orexin neurons are a key output neuron population that contributes to these responses, as blockade of orexin neurotransmission reduced the evoked responses by approximately half. Taken together, these results show that cardiovascular and locomotor responses, which facilitate a wide variety o

Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five G-protein coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by sphingosine kinase 1 (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases. This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic genes (BDNF, PDGFB, HBEGF, and LIF) in primary astrocytes, but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2. The clinical drug Fingolimod (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720-P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response. Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes. Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic gene expression than FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with

Obesity is a health problem affecting more than 40% of US adults and 13% of the global population. Anti-obesity treatments including diet, exercise, surgery and pharmacotherapies have so far failed to reverse obesity incidence. Herein, we target obesity with a pharmacotherapeutic approach that decreases caloric efficiency by mitochondrial uncoupling. We show that a recently identified mitochondrial uncoupler BAM15 is orally bioavailable, increases nutrient oxidation, and decreases body fat mass without altering food intake, lean body mass, body temperature, or biochemical and haematological markers of toxicity. BAM15 decreases hepatic fat, decreases inflammatory lipids, and has strong antioxidant effects. Hyperinsulinemic-euglycemic clamp studies show that BAM15 improves insulin sensitivity in multiple tissue types. Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Of the multitude of motor, sensory and cognitive complaints reported by sufferers, pain represents an extremely common and difficult to treat symptom in MS. This is an issue that, at least in part, stems from a lack of reliable and translatable pain outcome measures in animal models of MS. Experimental autoimmune encephalomyelitis (EAE) shares many key pathological characteristics with MS, namely neuroinflammation, demyelination and axonal damage. The broad aim of this thesis was to investigate the effects of immunoregulatory approaches on pain associated with EAE. We show here that in addition to stimulus-evoked pain, mice with EAE also develop spontaneous pain, an important and often overlooked component of MS-associated pain. Comprehensive analysis of immune, glial and neural changes in correspondence with observable pain behaviours showed that distinct stages of EAE are associated with specific pain phenotypes and neuroinflammation in both the peripheral and central nervous systems. We next aimed to test the effects of modulation of regulatory T (Treg) cells, potent suppressors of the immune system, on EAE progression and pain behaviours. We found that both systemic and centrally-delivered Treg cells were effective in reducing pain behaviours in EAE mice, and centrally-delivered Treg cells appeared associated with a reduction in astrogliosis and increased myelination in key regions comprising the facial nociceptive pathway. Similarly, intrathecal injection of interleukin (IL)-35, a novel anti-inflammatory cytokine produced by Treg cells, significantly reduced stimulus-evoked and spontaneous pain behaviours in EAE mice. This effect was Treg cell-dependent and appeared associated with an upregulation of IL-10 expression in CNS-infiltrating T and B cells, a reduction in the number of monocytes accumulated within the brain stem trigeminal nuclei, and the inhi

This thesis sought to investigate the role of orexin receptors in cardiovascular control, and to determine the role ofthe orexin system in essential hypertension.Immunolabelling revealed that most orexin neurons express orexin receptors (OxR1 and OxR2) themselves, with65% co-expressing both receptors, whereas sympathetic preganglionic neurons of the intermediolateral columnwere only seen to express OxR1. This suggests that much of the actions of orexin receptor antagonists may be on theorexin neurons themselves, which would potentially reduce all the orexin mediated components of a response atthe same time.Orexin levels in the CSF were shown to be tonically upregulated in the adult spontaneously hypertensive rat (SHR)model of essential hypertension, as compared to normotensive Wistar Kyoto (WKY) and Wistar rats. Using orexinimmunolabelling, we determined that the previously reported increased expression of orexin neurons in the medialhypothalamus is present in pre-hypertensive juvenile SHRs, and therefore precedes the hypertensive state in theseanimals. We also discovered a significantly increased orexin neuron population in brains of the BPH/2J Schlagermouse, another animal model of essential hypertension. This suggests that orexin upregulation may be an indicator,if not a precursor to essential hypertension across multiple mammalian species.Both destruction of medial hypothalamic neurons through vivo-morpholinos and targeted rAAV-delivered shRNAknockdown of orexin within this region were successful in significantly and permanently attenuating hypertensionin the adult SHR. Similar injections had no effect on resting blood pressure in normotensive WKY rats. The consistencyof the reduction seen in both experiments suggests that orexin is the primary source of the hypertensive toneproduced from this region in the SHR.Thus, this thesis has identified potential pathways for the cardiovascular actions of orexin receptor antagonists,characterised an overactive and upregul

Neuropathic pain occurring as a consequence of a disease or lesion in the somatosensory nervous system is devastating and refractory to current treatments. Neuropathic pain negatively affects the quality of life of patients and causes an economic burden to the sufferers and the global economy. Thus, there is an overwhelming need to investigate the underlying mechanisms and develop novel treatment approaches. Over the past decade, it has become clear that neuroinflammation plays a critical role in the pathogenesis of neuropathic pain. Indeed, the contribution of leukocytes such as T cells and macrophages, immune-like glial cells such as microglia and astrocytes, and inflammatory cytokines have been implicated in neuropathic pain caused by nervous system injury. Modification of such neuroinflammatory responses via immunomodulation is a potential novel therapeutic approach for the treatment of neuropathic pain.Myelin-derived altered peptide ligands (APLs) generated by transforming the amino acid sequence of native myelin peptides are known to modify immune and inflammatory responses, and are able to inhibit antigen-specific T cell function. APLs have demonstrated promising therapeutic effects in several in vivo and in vitro assays by changing the activation and functions of immune cells, and altering their cytokine profiles. In this thesis, we found that immunisation with the native myelin basic protein (MBP) peptide produced pain hypersensitivity and neuroinflammation in uninjured animals and that co-immunisation with the relevant APL reduced these effects. Next, we showed that immunisation with MBP-derived APL in animals with peripheral nerve injury significantly attenuated pain hypersensitivity and suppressed neuroinflammation, as well as altered some systemic immune responses. However, the same immunisation strategy failed to exert an analgesic effect following spinal cord injury. Lastly, the effects of gene therapy to introduce the anti-inflammatory cytokine inter

In recent times, oxidative stress has been identified as a major instigator of numerous conditions of aging and inflammation. The retina is subject to high levels of oxidative stress due to its high oxygen per weight requirements and perpetual exposure to light stress. Several diseases, such as macular degeneration and diabetic retinopathy are positively identified as having an oxidative stress pathophysiological component. These conditions are the leading causes of blindness in the first world adult population, thus representing significant morbidity to both economy and society. The retina has endogenous protection mechanisms that provide a degree of protection against common insults. One such mechanism involves induction of changes to the trans-retinal Müller cell. In response to certain physical or biological stimuli, the Müller cell becomes reactive, resulting in phenotypic changes such as increased release of growth factors, changes in extracellular fluid and ion uptake or proliferation that in certain contexts may limit retinal damage. However, in other contexts, such a response may be inappropriate, exacerbating retinal damage or disease. In order to understand and perhaps control Müller cell reactive expression, an unprecedented look at the early signalling cascades in response to prototypical oxidative stress agent, hydrogen peroxide, was undertaken. Conditions whereby hydrogen peroxide induced reactive Müller cell migration, proliferation and hypertrophy were optimized. Using these conditions, assay of the expression of select major signal transducing entities was performed in the presence and absence of inhibitors. This method identified hydrogen peroxide as enhancing ADAM-17 activity and tyrosine kinase receptors such as the epidermal growth factor receptor. Transduction of the hydrogen peroxide stimulated signal caused activation of ERK/MAPK and PI3K/Akt signalling pathways with subsequent induction of Egr1 expression and activity. Concomitantly, the ER

Conditioned fear to a tone is associated with disinhibition of brainstem-projecting neurons in the central nucleus of the amygdala (CeA). This is thought to trigger freezing via projections to the ventrolateral periaqueductal gray (VLPAG). However, the proportion of these projections containing GABA and glutamate is unknown. Using in situ hybridisation for glutamic acid decarboxylase (GAD67) and vesicular glutamate transporters (VGLUT1, VGLUT2) combined with retrograde labelling from the VLPAG, we showed that 90% of VLPAG-projecting CeA neurons contained GAD67, and none contained VGLUT1 or VGLUT2. Next, by combining Fos immunohistochemistry with retrograde labelling from the VLPAG, we found that sustained re-exposure to a feared context induced Fos expression in only 0.7% of VLPAG-projecting CeA neurons. We then attempted to selectively activate neurons in the medial CeA with orexin-A in freely moving rats, whilst recording cardiovascular and behavioural response. Orexin was associated with enhanced heart rate (HR), mean arterial pressure (MAP), activity, tail temperature and arousal. Next, we administered the same treatment to rats before sustained re-exposure to a fear-conditioned context. We showed that orexin prevented normal within-trial reductions in freezing, but had no effect on HR or MAP responses, activity or total time spent freezing. In a follow up re-exposure, rats previously injected with orexin were treated with saline. These rats did not show any sign of previous extinction learning, and spent more time freezing than rats receiving saline in the first re-exposure. Conversely, rats injected with orexin which were previously injected with saline displayed low levels of freezing. Finally, we sought to determine the behavioural and cardiovascular effects of microinjection of orexin-A in the VLPAG, an important structure in passive coping responses. We showed that orexin enhanced arousal, activity, HR, MAP and tail temperature. These findings support the

This thesis attempted to identify the site(s) of action of orexin and 5HT1A receptor agonists in the central network thatmediate/modulate the cardiovascular and behavioural changes evoked by psychological and physical stress.Central blockade of orexin receptors with Almorexant in the lateral ventricle significantly reduced the locomotor response toNovelty stress but did not significantly reduce the associated cardiovascular response. Comparison of the effect of orexin-A inthe lateral ventricle, fourth ventricle and thoracic cord showed that orexin injections in the upper thoracic cord could evokesubstantial cardiovascular effects, comparable to those evoked from the fourth and lateral ventricle, suggesting that orexin couldact at spinal level to mediate the cardiovascular effects of stress. This was confirmed by showing high levels of Fos expression inorexin neurons retrogradely labelled from the upper thoracic cord after Fear, Novelty and Exercise. Finally, orexin-A in themedullary raphe evoked clear cardiovascular effects, indicating that the medullary raphe could also be an important supraspinalsite of action of orexin in the cardiovascular response to stress.The 5HT1A receptor agonist 8-OH-DPAT evoked greater reduction in the cardiovascular response to Novelty, Restraint, Fear andCold after injection in the fourth ventricle than after injections in the lateral ventricle. This indicates that the lower brainstem islikely the main site of action of 8-OH-DPAT for the cardiovascular component of these stressors, suggesting a directsympathoinhibitory effect. However, the reduction of the freezing response evoked by Fear was greater after lateral ventricle thanfourth ventricle injections, suggesting an anxiolytic effect in the forebrain and/or midbrain region.Thus, this thesis has identified the most probable site(s) of action of orexin and 5HT1A agonists for the cardiovascular responseto stress. Orexin is likely to have a direct action in the upper thoracic cord toget

Disorders of the autonomic nervous system constitute serious complications of spinal cord injury (SCI) and their treatment is usually highly prioritised by spinal patients. Among these, autonomic dysreflexia and impaired thermoregulation are potentially life threatening conditions and require effective management. Olfactory ensheathing cells (OECs), progenitor cells and polymeric scaffolds have been tested in animal models of SCI and some of them have been considered for clinical trials. However, evaluation of the effect of such interventions on autonomic functions has received only rudimentary attention and would require a more thorough experimental assessment before the methods are utilised in human patients. This thesis tested two potential therapeutic strategies for autonomic dysreflexia and examined disorders of thermoregulatory functions in a rat model of spinal cord transection. Magnitude and duration of autonomic dysreflexia were evaluated with radio telemetry in spinalised animals treated with (i) implants of OECs and olfactory neurosphere-derived cells seeded in poly(lactic co glycolic) porous scaffolds or with (ii) transplants of OECs alone. (iii) Effects of SCI and of OECs on the morphology of sympathetic preganglionic neurons (SPNs; which are involved in pathogenesis of autonomic dysreflexia) stained for NADPH diaphorase were examined. (iv) Doppler ultrasonography and infrared thermography were used to assess responses of tail blood flow and surface temperature to cold.Transplants of OECs alone, but not in combination with olfactory neurosphere-derived cells and polymeric scaffolds, resulted in significantly shortened episodes of autonomic dysreflexia. This may be attributed to the alterations to the morphology of SPNs adjacent to the lesion: a transient increase in the morphometric features of the SPNs was evoked by spinal cord transection and this was further altered by transplantation of OECs. The thesis also showed that local responses of tail blood

As a street directory is essential to a traveller, so a map of the human brain is the most fundamental guide for anatomists, pathologists, neurologists, radiologists and psychologists. Neuroscience requires standard maps, terminology and coordinate systems for the effective study of the normal and diseased brain (Mazziotta, submitted for publication). In geographic atlases on a single physical reality a myriad of abstract representations can be superimposed, such as climate, religious affiliations and crime statistics. In an analogous fashion to mapping roads on LANDS AT images, the connections, neurochemistry, cell types and other brain imaging data can be superimposed on anatomical data, the "backbone " of the wider neuroscience research activity (Perchura and Martin, 1991).