Shining light on the contribution of spinal projection neurons to the local spinal cord and wider nervous system

The spinal cord dorsal horn (DH) is the first destination for sensory signals arising from the body within the central nervous system (CNS). For the perception of pain, a stream of nociceptive signals are sent from the spinal cord to the brain governed by these sensory inputs, and local processing by a highly heterogeneous neuronal population in the DH. A better understanding this intrinsic spinal cord processing remains critical to improve strategies that treat short-term, or chronic pain. A prime treatment target within the spinal cord processing unit is the output neurons of the dorsal horn, the Projection Neurons (PNs), and this thesis focusses on the underappreciated fact that PNs give rise to local (spinal) axon collaterals. Chapter 1 discusses this long-studied neuron population emphasising ultimately that little is known about the function of PNs and even less about their local axon collaterals. Fortunately, genetic, and viral technologies are unlocking the ability to implement new approaches to interrogate the circuits and terminations PNs signal through. After discussing the potential ways that the new technologies can be used to study PNs, specifically those that project to the parabrachial nucleus (PBN) of the pons, referred to as Spinoparabrachial Neurons (SPBNs), this thesis work interrogates SPBNs, their presynaptic circuits, intrinsic functional characteristics, and post-synaptic circuits. Chapter 2 takes a viral approach to target SPBNs, showing virally-mediated robust labelling in spinal cord regions typically reported with injection to the PBN before electrophysiologically and morphologically characterising superficial and deep DH SPBNs. This work confirmed, for the first time, that mouse SPBNs have axons that give rise to local axon collaterals which included the pre- and post-synaptic machinery required for functional synapses. It also demonstrated marked heterogeneiety within the SPBN population, distinguishing four different functional classes of SPBNs. With the virus-mediated approach validated, Chapter 3 utilised viral SPBN labelling to assess the potential input of two interneuron populations to SPBNs, identified by calcium binding proteins calretinin (CRIN) and parvalbumin (PVIN). This work defined a strong direct and indirect pathway for CRINs to recruit SPBNs with the majority of SPBNs showing robust responses to CRIN activation. PVIN inputs were varied, indicating direct and indirect excitation and indirect inhibition of SPBNs with a primary afferent depolarisation mediated mechanism also contributing to indirect excitation. Chapter 4 exploited the discrete labelling of a deep DH (DDH)SPBN population that was selectively transduced by a particular viral serotype. Utilising Optogenetics in an in vivo photostimulation preparation combined with neuronal activation markers, and patch-clamp based circuit mapping, I provide the first clear evidence that DDH SPBNs influence local nociceptive circuitry through axon collateral signalling across a wide spinal cord territory. This work also provides evidence to suggest DDH SPBNs recruit inhibitory interneurons. Chapter 5 culminates in the development of an intersectional viral injection strategy to specifically label SPBNs,including those in superficial DH (Lamina I) and the DDH, before assessing local signalling from this population across the DH. Optogenetic activation and patch clamp recording from SPBNs revealed a recurrent, SPBN derived, inhibitory response with a likely role in regulating SPBN excitability. Optogenetic activation of SPBNs in anaesthetised mice produced Fos expression in activated DH neurons, with subsequent labelling identifying recruitment of both excitatory or inhibitory phenotypes. Together, this data shows a highly engaged SPBN axon collateral network, recruiting a range of excitatory and inhibitory circuits to substantially influence local spinal processing mechanisms. Overall, my thesis provides new knowledge on the presynaptic circuits, intrinsic functional characteristics, and post-synaptic circuits that DDH and SDH SPBNs interact with in the spinal DH. This has provided the first functional evidence of SPBN collateral signalling within the DH that will eventually require a paradigm shift to how we think about the roles of SPBNs and spinal circuits and the wider implications for a local efferent copy of output signals destined for the brain on sensory experiences including pain.