Molecular mechanisms of nociception and pain

My thesis uses in vivo calcium imaging to investigate the cell and molecular mechanisms of two unusual pain states: congenital analgesia and cold allodynia. Genetic deletion of voltage-gated sodium channel NaV1.7 in mice and humans leads to profound pain insensitivity. Paradoxically, peripherally-targeted pharmacological antagonists of NaV1.7 fail to relieve pain in the clinic. To determine the mechanism of analgesia in NaV1.7 null mutants, I used optical, electrophysiological and behavioural methods to investigate the effect of peripheral NaV1.7 deletion on nociceptor function. Surprisingly, both calcium imaging and extracellular recording of NaV1.7-deficient sensory neurons in vivo found limited deficits in the response to noxious stimuli. Synaptic transmission from nociceptor central terminals in the spinal cord was however compromised following NaV1.7 deletion. Importantly, both synaptic deficits and behavioural analgesia were reversed by blocking central opioid receptors. Collectively, these data account for the failure of peripherally-targeted NaV1.7 blockers and point to a central mechanism of analgesia in NaV1.7 null mutants that requires opioid receptors. Chronic pain patients suffering from cold allodynia experience normally innocuous cooling as excruciating pain, but the cells and molecules driving cold allodynia remain elusive. I used in vivo calcium imaging to investigate how the activity of cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, peripheral nerve injury and ciguatera poisoning. In neuropathic mice exhibiting cold allodynia, a subset of cold-insensitive, large-diameter, peptidergic nociceptors became responsive to cooling. Diptheria toxin-mediated ablation of these silent cold-sensing neurons decreased neuropathic cold hypersensitivity. Voltage-gated potassium channels KV1.1 and KV1.2 were highly expressed in silent cold-sensing neurons and pharmacological inhibition of these channels rapidly induced cold responsiveness in cold-insensitive neurons. Taken together, I reveal that silent-cold sensing neurons contribute to cold allodynia in neuropathic pain and identify KV1 channel downregulation as a driver of de novo cold sensitivity, in vivo

Peripheral Neuropathy

Understanding the rapid changes in the evaluation and management of peripheral neuropathies, as well as the complexity of their mechanism, is a mandatory requirement for the practitioner to optimize patient's care. The objective of this book is to update health care professionals on recent advances in the pathogenesis, diagnosis and treatment of peripheral neuropathy. This work was written by a group of clinicians and scientists with large expertise in the field

Exploring the use of synthetic delivery platforms and small molecule adjuvants to improve the efficacy of spinal interleukin-10 gene therapy for chronic neuropathic pain

The focus of the work in this dissertation is to improve the efficiency of a gene therapy for the treatment of chronic pain. The introduction, Chapter 1, is intended to orient the reader to the underlying physiological principles and anatomical structures involved in general sensory and pain transmission in the peripheral and central nervous systems. Pain modulatory systems are described in detail. Also included is a discussion of how peripheral nerve injury can provoke immune changes at the spinal level, including the activation of spinal macrophages and glial cells (microglia, astrocytes and oligodendrocytes) with the release of immune modulators, such as pro- and antiinflammatory cytokines, that can lead to the development of chronic pain. One of the most important of these is the anti-inflammatory cytokine, Interleukin-10 (IL-10). As a framework for the experiments described in the dissertation, earlier studies using spinal injections of IL-10 protein and DNA containing the gene for IL-10 for the treatment of neuropathic pain in a chronic constriction injury (CCI) rodent model are introduced. Also presented in this dissertation, are some of the key problems of delivering DNA to cells (tranfection). The studies in Chapter 2 explore the use of one novel non-viral synthetic platform, a silica/lipid nanoparticle or protocell,\u27 as a potential platform for IL-10 transgene delivery to the central nervous system (CNS). These particles had never before been examined in vivo in the CNS. The first objective was to determine if they simply would be tolerated by animals following peri-spinal injection. The second objective was to determine their biodistribution in the whole body following these injections and the cell type interacting with them near the spinal injection site. The final objective was to determine if the IL-10 transgene produced functional IL-10 protein following loading on protocells and if the gene loaded on protocells would produce a therapeutic pain reversal in neuropathic animals. The studies in Chapter 3 are based on previously published results of a critical interval following spinal injection of a transgene, the \u27sensitization period\u27, during which there is immune cell enrichment in the cerebral spinal fluid (CSF) in the subarachnoid matrix. This local enrichment of immune cells in the spinal CSF, is key to the development of the experimental approach used in Chapter 3, which is to prime improved cellular uptake of the IL-10 gene with small molecules as immune adjuvants. In Chapter 2 and Chapter 3, each experimental data set is presented in the form of the original manuscripts, submitted for external peer-review and publication. Chapter 4 includes a discussion of the gene therapy approaches used in this work and by other investigators. Also considered are some future directions, including the use of a different synthetic polymer, polylactic co-glycolic acid, PLGA, that is FDA approved and highly biodegradable in the body. A concluding statement completes the work of this dissertation

Electrophysiological profile and monosynaptic circuitry of efferent vestibular nucleus neurons

As with other sensory modalities, the vestibular system recruits efferent circuitry to transport information from the central nervous system (CNS) to the sensory periphery. This efferent vestibular system (EVS) originates in the brainstem and terminates on vestibular hair cells and afferent fibres in the semicircular canals and otolith organs. Understanding how this central component outputs to the vestibular organs, and mediates motor and vestibular coordination, could potentially impact clinical treatment of vestibular disorders. Previous EVS work has primarily focused on the anatomy, pharmacology, synaptic mechanisms, and peripheral effects of efferent vestibular nucleus (EVN) activation. Although this work is fundamental to understanding this system and its mechanism of action, the behavioural function of the EVS is yet to be ascribed. For this, we need to appreciate the physiology of EVN neurons, and their context of activation within the CNS. In this thesis, I characterise the electrophysiological profile of EVN neurons, and trace their direct monosynaptic circuitry. My methodology includes whole-cell current- and voltage- clamp electrophysiology, and glycoproteindeficient rabies virus tracing techniques. Using these, I enrich understanding of EVN action, and hint at potential functional roles from their CNS partners. The data presented in this thesis provides novel insights into the EVS. EVN neurons are characterised with a homogeneous output, but a heterogeneous synaptic input profile. Inputs to the EVN originate from diverse areas in the brainstem and cortex. These findings suggest that the EVN modulates vestibular end organs in multiple different behavioural contexts. This work forms the basis of subsequent EVS behavioural investigations such as loss of function experiments targeting input regions via optogentic means and subsequent EVS recordings, or silencing of EVN activity and subsequent behavioural testing. Collectively, my results, these future directions, and the existing body of EVS literature, brings us closer than ever to understanding and ascribing a functional role for the EVS

Association of NaV1.8 with lipid rafts in DRG sensory neurons

Voltage gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neuronal cells. NaV1.8 is a Tetrodotoxin resistant VGSC expressed in nociceptors and underlies the majority of sodium currents during action potentials. Many studies have highlighted a key role of NaV1.8 in different pain pathways. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids characterised by unique physical features: a liquid ordered phase and the resistance to nonionic detergent at 4°C. Lipid rafts are thought to act as platforms on the membrane where proteins and lipids can be compartmentalised and functionally clustered. In the present study we investigated NaV1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We hypothesised that lipid rafts on primary sensory neurons act as a platform on the membrane where NaV1.8 can be trafficked and underlie action potentials generation. We demonstrated that NaV1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. We also found that NaV1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We investigated the functional meaning of NaV1.8-raft association by studying action potential propagation in sensory neurons, in response to mechanical and chemical stimulation, by calcium imaging. Disruption of the association between NaV1.8 and lipid rafts in cultured sensory neurons, by methyl-betacyclodextrin and 7-ketocholesterol, caused a reduction in the number of cells able to propagate action potentials. In addition, lipid raft depletion caused a remarkable reduction in the conduction velocity upon mechanical stimulation. These findings highlight the importance of the association between NaV1.8 and lipid rafts in the conduction of action potentials and could lead to new perspectives in the study of NaV1.8 trafficking and nociceptor excitability

Lycium barbarum (wolfberry) polysaccharide facilitates ejaculatory behaviour in male rats

Poster Session AOBJECTIVE: Lycium barbarum (wolfberry) is a traditional Chinese medicine, which has been considered to have therapeutic effect on male infertility. However, there is a lack of studies support the claims. We thus investigated the effect of Lycium barbarum polysaccharide (LBP), a major component of wolfberry, on male rat copulatory behavior. METHOD: Sprague-Dawley rats were divided into two groups (n=8 for each group). The first group received oral feeding of LBP at dosage of 1mg/kg daily. The control group received vehicle (0.01M phosphate-buffered saline, served as control) feeding daily for 21 days. Copulatory tests were conducted at 7, 14 and 21 days after initiation of treatment. RESULTS: Compared to control animals, animals fed with 1mg/kg LBP showed improved copulatory behavior in terms of: 1. Higher copulatory efficiency (i.e. higher frequency to show intromission rather than mounting during the test), 2. higher ejaculation frequency and 3. Shorter ejaculation latency. The differences were found at all time points (Analyzed with two-tailed student’s t-test, p<0.05). There is no significant difference found between the two groups in terms of mount/intromission latency, which indicates no difference in time required for initiation of sexual activity. Additionally, no difference in mount frequency and intromission frequency was found. CONCLUSION: The present study provides scientific evidence for the traditional use of Lycium barbarum on male sexual behavior. The result provides basis for further study of wolfberry on sexual functioning and its use as an alternative treatment in reproductive medicine.postprintThe 30th Annual Meeting of the Australian Neuroscience Society, in conjunction with the 50th Anniversary Meeting of the Australian Physiological Society (ANS/AuPS 2010), Sydney, Australia, 31 January-3 February 2010. In Abstract Book of ANS/AuPS, 2010, p. 177, abstract no. POS-TUE-19