Genetic approaches to understanding pain mechanisms: Zfhx2 and peripheral sensory neuron ablation mouse transgenic models

Latest cutting-edge sequencing has allowed researchers to obtain a full array of differentially expressed neuronal genes within the peripheral nervous system. Understanding this heterogeneity and functional implication could unveil new therapeutic targets towards a more precise medicine. Combining a novel reporter mouse with Cre recombinase strategies, I examined the spatial and functional organization of transcriptomically different subpopulations of neurons in the mouse DRG in pathological and nonpathological states. Results herein include: confirmation of Cre activity and specificity in all lines studied by RNA scope when compared to previous reports and transcriptomic analysis; significant upregulation of DRG gal expression after Complete Freund's Adjuvant (CFA) induced inflamation; normal weight and exploratory behaviour for all lines tested; motor activity assessed by Rotarod not significant, but further motor coordination tests on animals missing Th DRG neurons showed significant impairment; noxious mechanosensation reduced in animals lacking SCN10aCre and Tmem45b DRG; confirmation of CGRP-positive neurons role in heat and cold perception as well as in the formalin inflammatory model; Von Frey hypersensitivity on animals lacking CGRP; and lastly TrkBpositive neurons responsible for significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model whilst no effect in cancer induced bone pain model. Parallelly, by reverse genetics approach, I explore the contribution of the Zfhx2 gene, whose mutation has been identified as responsible for the Marsili pain insensitivity syndrome, in two different animal models of nociception. Behavioural characterisation of bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine mutation, as well as Zfhx2 null mutant mice, shows significant deficits in pain sensitivity in thermal and mechanical tests respectively. In summary, as well as validating several new useful transgenic mouse lines, this thesis provides insights into genes and neuronal subpopulations important in pain pathways and provides potential platforms for translational studies of pain syndromes

Spinal cord plasticity in peripheral inflammatory pain

Inflammatory pain is a debilitating condition that can occur following tissue injury or inflammation and results in touch evoked pain (allodynia), exaggerated pain (hyperalgesia) and spontaneous pain, yet the neural plasticity underlying these symptoms is not fully understood. However, it is known that lamina I neurokinin 1 receptor expressing (NK1R+) spinal cord output neurons are crucial for the manifestation of inflammatory pain. There is also evidence that the afferent input to and the postsynaptic response of these neurons may be altered in inflammatory pain, which could be relevant for inflammatory pain hypersensitivity. Therefore, the aim of this thesis was to study inflammatory pain spinal plasticity mechanisms by investigating the synaptic input to lamina I NK1R+ neurons. In ex vivo spinal cord and dorsal root preparations from the rat, electrophysiological techniques were used to assess inflammation-induced changes in and pharmacological manipulation of the primary afferent drive to lamina I NK1R+ neurons. The excitatory input to lamina I NK1R+ neurons was examined and it was found that inflammation did not alter the relative distribution of the type of primary afferent input received and did not potentiate monosynaptic A δ or monosynaptic C-fibre input, the predominant input to these neurons. Spontaneous excitatory input was significantly elevated in the subset of neurons that received monosynaptic A δ-fibre input only, regardless of inflammation. It has recently been shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby ChemR23 agonists can decrease inflammatory pain hypersensitivity, by a mechanism that involves the attenuation of potentiated spinal cord responses. This study has found that the ChemR23 agonist, chemerin, attenuated capsaicin potentiation of excitatory input to lamina I NK1R+ neurons and significantly reduced monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. However, chemerin was without effect in non-potentiated conditions. In exploring potential inflammatory pain spinal plasticity mechanisms, I have investigated a phenomenon called activity-dependent slowing (ADS), whereby repetitive stimulation of C-fibres at frequencies of 1Hz or above results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. This is proposed to limit nociceptive input to the spinal cord, thus regulating plasticity. Results demonstrate that inflammation significantly attenuated C-fibre ADS in isolated dorsal roots. Furthermore, ADS in monosynaptic C-fibre input to lamina I NK1R+ neurons was significantly reduced in inflammatory pain, which could facilitate nociceptive drive to these key spinal cord output neurons and promote inflammatory pain spinal cord plasticity. In conclusion, the major novel findings of this thesis are firstly, that chemerin can attenuate primary afferent input to lamina I NK1R+ neurons in potentiated conditions, which supports recent studies that suggest ChemR23 is a potential target for the development of new analgesics. Secondly, it was discovered that ADS in monosynaptic C-fibre inputs to lamina I NK1R+ neurons is altered in inflammatory pain, which could be relevant for inflammatory pain spinal plasticity. The findings presented in this thesis could contribute to the development of novel inflammatory pain treatments

Serotonergic modulation of the ventral pallidum by 5HT1A, 5HT5A, 5HT7 AND 5HT2C receptors

Introduction: Serotonin's involvement in reward processing is controversial. The large number of serotonin receptor sub-types and their individual and unique contributions have been difficult to dissect out, yet understanding how specific serotonin receptor sub-types contribute to its effects on areas associated with reward processing is an essential step. Methods: The current study used multi-electrode arrays and acute slice preparations to examine the effects of serotonin on ventral pallidum (VP) neurons. Approach for statistical analysis: extracellular recordings were spike sorted using template matching and principal components analysis, Consecutive inter-spike intervals were then compared over periods of 1200 seconds for each treatment condition using a student’s t test. Results and conclusions: Our data suggests that excitatory responses to serotonin application are pre-synaptic in origin as blocking synaptic transmission with low-calcium aCSF abolished these responses. Our data also suggests that 5HT1a, 5HT5a and 5HT7 receptors contribute to this effect, potentially forming an oligomeric complex, as 5HT1a antagonists completely abolished excitatory responses to serotonin application, while 5HT5a and 5HT7 only reduced the magnitude of excitatory responses to serotonin. 5HT2c receptors were the only serotonin receptor sub-type tested that elicited inhibitory responses to serotonin application in the VP. These findings, combined with our previous data outlining the mechanisms underpinning dopamine's effects in the VP, provide key information, which will allow future research to fully examine the interplay between serotonin and dopamine in the VP. Investigation of dopamine and serotonins interaction may provide vital insights into our understanding of the VP's involvement in reward processing. It may also contribute to our understanding of how drugs of abuse, such as cocaine, may hijack these mechanisms in the VP resulting in sensitization to drugs of abuse