Synaptic plasticity in medial vestibular nucleus neurons: comparison with computational requirements of VOR adaptation

Background: Vestibulo-ocular reflex (VOR) gain adaptation, a longstanding experimental model of cerebellar learning, utilizes sites of plasticity in both cerebellar cortex and brainstem. However, the mechanisms by which the activity of cortical Purkinje cells may guide synaptic plasticity in brainstem vestibular neurons are unclear. Theoretical analyses indicate that vestibular plasticity should depend upon the correlation between Purkinje cell and vestibular afferent inputs, so that, in gain-down learning for example, increased cortical activity should induce long-term depression (LTD) at vestibular synapses. Methodology/Principal Findings: Here we expressed this correlational learning rule in its simplest form, as an anti-Hebbian, heterosynaptic spike-timing dependent plasticity interaction between excitatory (vestibular) and inhibitory (floccular) inputs converging on medial vestibular nucleus (MVN) neurons (input-spike-timing dependent plasticity, iSTDP). To test this rule, we stimulated vestibular afferents to evoke EPSCs in rat MVN neurons in vitro. Control EPSC recordings were followed by an induction protocol where membrane hyperpolarizing pulses, mimicking IPSPs evoked by flocculus inputs, were paired with single vestibular nerve stimuli. A robust LTD developed at vestibular synapses when the afferent EPSPs coincided with membrane hyperpolarisation, while EPSPs occurring before or after the simulated IPSPs induced no lasting change. Furthermore, the iSTDP rule also successfully predicted the effects of a complex protocol using EPSP trains designed to mimic classical conditioning. Conclusions: These results, in strong support of theoretical predictions, suggest that the cerebellum alters the strength of vestibular synapses on MVN neurons through hetero-synaptic, anti-Hebbian iSTDP. Since the iSTDP rule does not depend on post-synaptic firing, it suggests a possible mechanism for VOR adaptation without compromising gaze-holding and VOR performance in vivo

Hypothalamic Melanocortin Regulation of Energy Balance and Metabolism

Genetic and environmental factors both contribute to obesity, however studies in twins and adoptees demonstrate that genetic predisposition and susceptibility are driving factors in the development of this disease. Although numerous human mutations are associated with an increase in obesity prevalence, melanocortin-4 receptor (MC4-R) mutations are the most common monogenic form of severe obesity and genetic deletion of this receptor in rodents also leads to an obese phenotype. The G-protein coupled MC4-R is a target for the peptide products of Proopiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons residing in the arcuate nucleus of the hypothalamus. POMC-derived alpha-melanocyte-stimulating hormone (MSH) is an agonist for the MC4-R and promotes negative energy balance, while the melanocortin-receptor antagonist AgRP promotes positive energy balance. Given the strong influence of the hypothalamic melanocortin system on energy balance, this thesis sought to investigate unexplored aspects of this system in relation to obesity. POMC is post-translationally processed to biologically active peptides with opposing actions. Alpha-MSH is well established to decrease food intake and increase energy expenditure, however POMC-derived beta-endorphin (beta-EP) has been shown in limited studies to increase food intake. Our experiments in intracerebroventricular (icv) cannulated rats demonstrate that the effects of beta-EP alone on feeding are complex. Beta-EP acutely stimulated food intake during both the light and dark cycle, however orexigenic effects were not sustained in a chronic model; in fact, higher doses of chronic beta-EP decreased food intake. Subthreshold doses of beta-EP also reversed alpha-MSH-induced suppression in feeding and weight gain in an acute fasting and refeeding model as well as a chronic infusion model. Beta-EP 1-27, a product of C-terminal beta-EP cleavage reported to have reduced opioid activity, did not stimulate food intake alone, nor could it reverse alpha-MSH-induced suppression in feeding. These studies show that POMC-derived peptides alpha-MSH and beta-EP can interact to regulate food intake and body weight and highlight the importance of understanding how the balance between these peptides is maintained, as well as the potential role of differential POMC processing in regulating energy balance. AgRP is also a critical component of the melanocortin system; however, studies in which the AgRP peptide was deleted show only a mild phenotype suggesting that developmental compensation exists in this model. Consequently the role of the AgRP peptide was investigated using a novel AgRP inhibitor developed by TransTech Pharma, Inc. Results show that this inhibitor was extremely effective in reversing exogenous icv AgRP-induced metabolic and neuroendocrine parameters in rats, and these parameters were unaffected in saline infused rats receiving this drug. This AgRP inhibitor also reduced food intake, weight gain and adiposity in diet-induced obese (DIO) and ob/ob mice and increased thyroxine (T4) levels in DIO mice, consistent with AgRP's reported effects; however this drug did not affect food intake or weight gain in lean chow fed mice. The AgRP inhibitor also suppressed rebound feeding and potently reduced food intake in mice immediately upon initiation of a high fat diet (HFD). As some of these effects were also observed in AgRP knockout (KO) mice, this indicates that there are clear off-target effects that are not due to AgRP antagonism. Although there are many potential reasons why a drug may yield anorexia and weight loss, the fact that these effects were only observed in obese models or in the presence of increased dietary fat, suggests the possibility that another molecule that promotes positive energy balance and fat intake is also being targeted. As the melanocortin system can also regulate pituitary function, this thesis investigated circulating and pituitary prolactin levels in models with genetic manipulation of this system. Previous studies have shown that acute stimulation of the melanocortin system by alpha-MSH or antagonism by AgRP decreases and increases prolactin levels, respectively. However, the effects of chronic melanocortin manipulation were unknown. Male mice with selective MSH overexpression and AgRP deletion were found to have decreased blood prolactin levels under both stressed and unstressed conditions, and mice with AgRP deletion also had lower prolactin content in the pituitary. As prolactin is tonically inhibited by dopamine and acute melanocortin regulation of prolactin release has been shown to be dopamine-mediated, we sought to show this mechanism in these genetic models. Measurements of mediobasal hypothalamic (MBH) tyrosine hydroxylase mRNA levels as well as dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content did not differ between groups, nor did prolactin measurements after functional dopamine receptor antagonism. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first report of reduced baseline and stress-induced prolactin release and pituitary prolactin content in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels. The melanocortin system has been shown to mediate some of the adaptive responses after introduction to the HFD including attenuation of hyperphagia and weight gain; thus, preliminary studies sought to investigate the effects of the HFD on hypothalamic melanocortin gene and peptide expression, as well as expression of enzymes responsible for POMC post-translational processing. Mice administered a 60% HFD were found to have increased levels of POMC precursor peptide after both acute (3 and 7 days) and chronic (8 week) HFD administration. After 3 days of HFD feeding, increased POMC peptide levels were accompanied by increased Pomc mRNA and alpha-MSH and beta-EP, however after chronic HFD feeding POMC peptide levels were elevated without an increase in these parameters. Additionally, chronic HFD feeding suppressed AgRP mRNA and peptide levels and tended to increase the alpha-MSH/beta-EP ratio and suppress mRNA levels of prolylcarboxypeptidase (Prcp), an enzyme responsible for inactivating alpha-MSH. These data suggest that the HFD can modulate melanocortin gene and peptide expression with distinct time-related changes and are consistent with the hypothesis that POMC-derived peptides act in concert to regulate energy balance and metabolism. However, these results are preliminary and further investigation of the effects of HFD feeding on peptide and processing enzyme expression in more anatomically discrete hypothalamic nuclei as well as dynamic studies of peptide release are required. These data provide a contribution to the field of melanocortin regulation of energy balance by further elucidating the effects of POMC processed peptides both alone and in interaction, by providing insight into the effects of a novel AgRP inhibitor on energy balance and metabolism, by investigating the effects of genetic models of chronic melanocortin manipulation on the pituitary hormone prolactin, as well as by examining the effects of HFD feeding on melanocortin gene and peptide expression

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus:a role for CD8 T cells

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed

Time cost associated with sports participation for athletes with high support needs : A time-motion analysis of tasks required for para swimming

Objectives People with cerebral palsy and high support needs (CP&HSN) are profoundly inactive but also under-represented in studies evaluating physical activity interventions. Reasons for their exclusion have not been evaluated. We hypothesised that CP&HSN would be associated with high time costs of preparatory activities (eg, getting dressed/undressed), possibly contributing to low participation and under-representation. Accordingly, this pilot study aimed to: (1) evaluate whether the time required for preparatory activities was extremely different (≥3 SD) between swimmers with and without CP&HSN; and (2) provide a qualitative indication of the preparatory tasks undertaken by swimmers with CP&HSN. Methods Each of three experienced (5 years) para swimmers with CP&HSN and 20 non-disabled swimmers were timed entering and then exiting the pool on three occasions. Mean entry and exit time for each para swimmer was compared with the group mean for non-disabled swimmers, and differences of greater than 3.0 SD were considered extreme. A qualitative description of the tasks completed by the para swimmers was recorded. Results The differences in time costs between para and non-disabled swimmers met the criterion of extreme. Pool entry times for para swimmers were 8–13 times greater (Effect size = 4.1–8.7). Pool exit times were 6–10 times greater (ES=7.0–9.5). 90% of tasks completed by para swimmers required personal assistance or wheeled mobility. Conclusions This pilot study suggests that, compared with non-disabled swimmers, time costs for preparation to commence or depart training are extremely high for swimmers with CP&HSN. Further research is required to evaluate the veracity of these findings

Concurrent infection with the filarial helminth Litomosoides sigmodontis attenuates or worsens Influenza A virus pathogenesis in a stage-dependent manner

Filarial helminths infect approximately 120 million people worldwide initiating a type 2 immune response in the host. Influenza A viruses stimulate a virulent type 1 pro-inflammatory immune response that in some individuals can cause uncontrolled immunopathology and fatality. Although coinfection with filariasis and influenza is a common occurrence, the impact of filarial infection on respiratory viral infection is unknown. The aim of this study was to determine the impact of pre-existing filarial infection on concurrent infection with influenza A virus. A murine model of co-infection was established using the filarial helminth Litomosoides sigmodontis and the H1N1 (A/WSN/33) influenza A virus (IAV). Co-infection was performed at 3 different stages of L. sigmodontis infection (larval, juvenile adult, and patency), and the impact of co-infection was determined by IAV induced weight loss and clinical signs, quantification of viral titres, and helminth counts. Significant alterations of IAV pathogenesis, dependent upon stage of infection, was observed on co-infection with L. sigmodontis. Larval stage L. sigmodontis infection alleviated clinical signs of IAV co-infection, whilst more established juvenile adult infection also significantly delayed weight loss. Viral titres remained unaltered at either infection stage. In contrast, patent L. sigmdodontis infection led to a reversal of age-related resistance to IAV infection, significantly increasing weight loss and clinical signs of infection as well as increasing IAV titre. These data demonstrate that the progression of influenza infection can be ameliorated or worsened by pre-existing filarial infection, with the outcome dependent upon the stage of filarial infection

An Influenza Virus M2 Protein Specific Chimeric Antigen Receptor Modulates Influenza A/WSN/33 H1N1 Infection In Vivo

A potential target for the development of universal vaccine strategies against Influenza A is the M2 protein – a membrane protein with a highly conserved extracellular domain. In this study we developed engineered T-cell receptors, by fusing M2-specific antibody sequences with T-cell receptor transmembrane and signaling domains to target influenza infected cells. When expressed on T-cells, these novel T-cell receptors (chimeric antigen receptors - CARs) are able to recognize specific antigens on the surface of target cells via an MHC-independent mechanism. Using an existing monoclonal antibody (14C2) specific for the M2 ectodomain (M2e), we generated an M2-specific CAR. We tested the specificity of this M2 CAR in vitro by measuring the activation of T-cells in response to M2-specific peptides or M2-expressing cell lines. Both Jurkat T-cells and peripheral blood mononuclear cells expressing the M2-specific CAR responded to specific antigen stimulation by upregulating NFAT and producing γ-interferon. To test whether the M2-specific CAR are effective at recognizing influenza infected cells in vivo we used an established BALB/c murine infection model. At day 4 post-infection, when M2 CAR expressing splenocytes could be detected in the lung, the Influenza A/WSN/33 virus titre was around 50% of that in control mice. Although the lung virus titre later increased in the treated group, virus was cleared in both groups of mice by day 8. The results provide support for the development of M2e as a target for cell mediated immunotherapy

The Role of Dendritic Cells in the Host Response to Marek’s Disease Virus (MDV) as Shown by Transcriptomic Analysis of Susceptible and Resistant Birds

Despite the successful control of highly contagious tumorigenic Marek’s disease (MD) by vaccination, a continuous increase in MD virus (MDV) virulence over recent decades has put emphasis on the development of more MD-resistant chickens. The cell types and genes involved in resistance therefore need to be recognized. The virus is primarily lymphotropic, but research should also focus on innate immunity, as innate immune cells are among the first to encounter MDV. Our previous study on MDV–macrophage interaction revealed significant differences between MHC-congenic lines 6(1) (MD-resistant) and 7(2) (MD-susceptible). To investigate the role of dendritic cells (DCs) in MD resistance, bone-marrow-derived DCs from these lines were infected with MDV in vitro. They were then characterized by cell sorting, and the respective transcriptomes analysed by RNA-seq. The differential expression (DE) of genes revealed a strong immune activation in DCs of the susceptible line, although an inherent immune supremacy was shown by the resistant line, including a significant expression of tumour-suppressor miRNA, gga-mir-124a, in line 6(1) control birds. Enrichment analysis of DE genes revealed high expression of an oncogenic transcription factor, AP-1, in the susceptible line following MDV challenge. This research highlights genes and pathways that may play a role in DCs in determining resistance or susceptibility to MDV infection