Abstract
The detection of stimuli that produce painful behaviors occurs within a subset of sensory neurons, namely nociceptors. The signals elicited by nociceptors are transmitted to the spinal cord through synaptic relay points in the dorsal horn. Nociceptors undergo significant plastic changes upon nerve injury and inflammation, where they augment painful signaling and can further modulate the excitability of central neurons, resulting in persistent pain. Elucidating the cellular and molecular properties of the somatosensory apparatus provides a deeper understanding of the physiology of painful signaling and opens therapeutic contingencies for the treatment of painful conditions. However, the plasticity of nociceptors and their contribution to painful pathologies has not been fully captured by existing methodologies. The current in vivo behavioral and electrophysiological techniques lack the spatial resolution to detect molecular contributions to nociceptor excitability, while in vitro studies have focused on the cell bodies of nociceptors and not the axons, that often represent the actual sites of injury.To address this problem, we have employed a novel cell culture model which exploits advances in microfluidics. Microfluidic cultures allow the investigation of the functional properties of nociceptor axons separately from their respective cell bodies. Three problems are addressed using variations of this technique in my thesis. First, the properties that the inflammatory mediator prostaglandin E2 (PGE2) exerts on sensory axons are elucidated. Second, the periphery-to-center circuit between DRG and DH neurons is recapitulated and the formation of functional synapses between them is reported. Third, the pre-synaptic properties of transient receptor potential (TRP) and voltage-gated sodium (Nav) channels are examined and subsequently their contribution to DH excitability between axotomized and non-axotomized cultures is compared.
The results from the first investigation reveal a direct and persistent depolarization of sensory axons by PGE2. It is shown that the PGE2-evoked activity is dependent on the EP4/cAMP/PKA pathway. Evidence is provided for the mediation of this activity by the TTX-resistant sodium channel Nav1.8 and by the calcium activated chloride channel ANO1. Hence, it is proposed that PGE2 possesses a dual role as a sensitizer, but also as direct activator of sensory axons. The molecular mechanisms that underpin the latter phenomenon are delineated.
In the second investigation, mouse postnatal DRG and DH neurons are co-cultured in microfluidic devices. The co-culture model recapitulates salient features of synaptic transmission between DRG and DH, allowing the recording of DH neuron activity following DRG neuron stimulation. The model is important because it allows a spatially resolved interrogation of the molecular properties of nociceptors and the extent to which they contribute to synaptic transmission.
Last, the co-culture model is employed to investigate the contribution of pre-synaptic TRP and Nav channels to synaptic transmission. As a surrogate model of nerve transection, an axotomy is introduced and compared with the contribution of these channels under intact conditions. A role for Nav1.7 channels in synaptic transmission under intact conditions is reported, however it is lost following axotomy. Surprisingly, Nav1.6 channels emerge as principal contributors to synaptic transmission only following axotomy. A novel finding of this thesis regards the pre-synaptic role of TRPV1 and TRPA1 channels. Traditionally considered as transducer channels of thermal and chemical noxious stimuli, these channels show significant pre-synaptic activity where they dynamically contribute to synaptic transmission, a property which is lost following axotomy.
Overall, this thesis demonstrates the versatility of the microfluidic platform and unravels novel properties of a neuroimmune interaction relevant to inflammatory pain, synaptic transmission between DRG and DH neurons and finally, the pre-synaptic contribution of TRP and Nav channels in normal and axotomized conditions. The delineation of the molecular properties of nociceptors using pharmacological or genetic manipulations at distinct anatomical parts may further reveal unknown aspects about the physiology and pathophysiology of these neurons. The platform possesses an industrial utility too, as it can be used for the screening of novel analgesic molecules and the determination of how they affect the excitability of nociceptors or DH neurons.
| Date of Award | 1 Jan 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Ramin Raouf (Supervisor) & Marzia Malcangio (Supervisor) |