Using chemical and light activation of neurons to study pain mechanisms and screen analgesic compounds

Iredale, Jacqueline A.

Title: Using chemical and light activation of neurons to study pain mechanisms and screen analgesic compounds
Creator: Iredale, Jacqueline A.
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2023
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: The limited efficacy and/or often complicated side effect profiles of existing analgesic compounds, coupled with the lack of successful translation of preclinically promising novel analgesics has underscored a need for the existing paradigm to change. This includes scrutinising all phases from early discovery, through preclinical testing, clinical validation, and ultimately translation to practice. One barrier to improve the performance of this process is our limited understanding of the cellular mechanisms of analgesia within the pathway that transmits pain signals. Although the currently available tools for pain pathway investigation and analgesic screening have facilitated numerous important discoveries, one proposed roadblock for successful preclinical translation is limitations in these tools and assays. This thesis aims to address some of these gaps, with the goal of expanding the preclinical toolkit available to researchers studying the pain pathway and establish evidence for the likely efficacy of novel analgesic compounds. Chapter 1 introduces the background to this thesis, providing an overview of the existing literature on pain and analgesia. First, background information on the pain pathway and mechanisms are covered including pain transduction, transmission, and modulation, as well as an introduction of specific pain subtypes and classifications. Second, current analgesic options are presented, including both pharmacological and non-pharmacological approaches, as well as emerging analgesics. Finally, the case for new treatments is canvased and the current preclinical approaches to pain pathway investigation and analgesic screening is summarised. Based on review of these topics, I propose the development of two new approaches to pain pathway research and analgesic screening and outline how these approaches enhance the existing paradigm. Chapter 2 presents the development of the first model, a microelectrode array (MEA) recording preparation. One gap identified in the current range of preclinical pain and analgesic screening tools is an assay capable of investigating network level activity spinal cord while under a hyperexcited state, like that hypothesised to exist in chronic pain. MEAs are capable of monitoring electrical activity across numerous cells and circuits, providing high spatial and temporal resolution of neural signalling. Consequently, the MEA model is an in vitro platform to investigating spinal cord dorsal horn (DH) function at a macro-circuit level. The spinal DH is a known region of importance for pain processing and modulation prior to these signals producing perception. Using 4-aminopyridine (4-AP) to chemically produce hyperexcitability in DH neuronal networks, thought to mimic a chronic pain like state, the MEA model allows pain mechanisms to be assessed at the level of the DH as well as screening analgesics for spinal effects. The work of this chapter focusses on establishing the MEA model, showing stability of 4-AP induced activity, and localisation of this restricted to the DH. Two types of activity can be resolved under these conditions: extracellular action potential (EAPs); and local field potentials (LFPs), reflective of neuronal and circuit level activity respectively. Last, the pharmacological sensitivity of 4-AP induced activity is demonstrated, confirming that action potential discharge and synaptic transmission are necessary for the 4-AP response and that the preparation is amenable to drug action studies. The results provide evidence for the effectiveness of the MEA assay to model pain circuitry in a hyperexcited state. Chapter 3 builds on the established MEA model from Chapter 2, applying known analgesic compounds to the MEA model and assessing the sensitivity of 4-AP induced activity to these compounds. Morphine, as a gold standard analgesic, and the first line neuropathic pain treatment gabapentin, were applied to the MEA model. Importantly, both are established analgesics but do not have completely clarified mechanisms of action, particularly within the spinal cord. Morphine significantly attenuated several aspects of LFP activity, adding to evidence that its analgesics actions include effects on spinal signalling. Yet despite these results, morphine did not fully abolish 4-AP induced activity, consistent with the widespread actions of this drug within spinal and supraspinal pain pathways. Contrasting morphine, gabapentin had little effect on 4-AP induced activity in the MEA model, despite its principle mode of action thought to supress nociceptive afferent signalling to neurons in the DH. As canvased in discussion of this result, several factors including a delayed time course for gabapentin action, efficacy in neuropathic but not nociceptive pain, and potential for supraspinal mechanisms may have contributed to this outcome. Regardless, the application of both compounds on the MEA model highlights its ability to detect and differentiate spinal analgesia, with the contrasting results confirming that 4-AP induced hyperexcitability does not respond in a generic way to all classes of analgesics. Chapter 4 sought to extend the MEA 4-AP model’s utility by testing a series of novel analgesic compounds. Specifically, cannabinoid compounds were chosen as there is a long anecdotal history suggesting they have analgesic properties, however, more recent studies have provided conflicting evidence. Two specific cannabinoid isolates were selected for assessment in the MEA model, cannabidiol (CBD), with mounting evidence of its analgesia potential, and 8-tetrahydrocannabivarin (8-THCV), with less evidence for an analgesic potential. Surprisingly, both compounds produced similar effects on 4-AP induced DH hyperexcitation, increasing several EAP parameters, while also decreasing multiple LFP parameters. This cannabinoid mediated increase in EAPs lends itself to the hypothesis that this may reflect increased inhibitory neuron activity, which may be in turn important for producing a reduction to LFP signals. Importantly, this signature was distinct from the morphine and gabapentin results in Chapter 3, reinforcing that drug application does not cause a generalised effect in MEA recordings of 4-AP stimulation, which would be of little value. Ultimately, the significant reduction in LFP network activity, supports CBD and 8-THCV as having spinal analgesic actions. Chapter 5 describes development of the second ‘peripheral photostimulation’ model, its utility for investigating the pain pathway and a preliminary validation as an analgesic screening assay. Utilising transgenic and optogenetics techniques, the in vivo peripheral photostimulation model achieves selective expression of channelrhodopsin-2 (ChR2) in a population of nociceptors that can be identified by transient receptor potential cation channel subfamily V member 1 (TRPV1) expression, rendering this population sensitive to activation by exposure to 470 nm light. Nociceptive responses can then be studied by applying increasing intensities of light to the hindpaw, establishing an optical withdrawal threshold produced by pure activation of nociceptive circuitry. This has the distinct advantage of avoiding the confounds of mixed sensory activation necessary in existing sensory thresholding models such as von Frey testing. In establishing the model, baseline stability of optical threshold responses was assessed through repeated testing on subsequent days and following one week. Specificity of optically evoked responses was also confirmed in control experiments that applied photostimulation to animals expressing GFP rather than ChR2 in nociceptive afferents, and applying photostimulation outside the excitation wavelength for ChR2 in TRPV1::ChR2 animals. Next, mirroring the analgesic screening of the MEA model in Chapters 3 and 4, known analgesics, morphine and gabapentin, plus novel analgesics, CBD and 8-THCV, were administered before peripheral photostimulation was applied. Morphine fully abolished peripheral photostimulation across a range of light intensities confirming its sensitivity as a model for analgesic screening. Screening of other compounds did not progress past the preliminary stage, with gabapentin, CBD, and 8-THCV all requiring an increased sample size to determine their actions during peripheral photostimulation. Chapter 6 presents broad conclusions and future directions summarising the above findings and proposing further work to progress. Given the successful development of the two new models to investigate pain pathway function and analgesic screening, the case has been made for both approaches filling areas of need in the experimental spectrum that spans from single cell assays through to complex behavioural paradigms.
Subject: pain; cannabinoids; TRPV1; microelectrode array; analgesia
Identifier: http://hdl.handle.net/1959.13/1495870
Identifier: uon:54079
Language: eng
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