Distinct roles of anterolateral system projection neuron populations in pain circuits

Chronic pain is a major clinical problem that affects ~30% of the population and results in significant societal and economic impact. Currently available treatments often provide inadequate pain relief, and a major reason for the limited success in design of new treatments is lack of knowledge about the neuronal circuits that underlie pain. Primary afferent neurons, many of which signal noxious stimuli, transmit sensory information to the spinal cord. Here it is processed through complex neuronal circuits before being transmitted to the brain through projection neurons. Many of these neurons belong to the anterolateral system (ALS), and this pathway is required for conscious perception of pain. However, pain is a multidimensional experience involving sensory-discriminative and motivational-affective components, and these may involve different spinal circuits. ALS neurons are located in both the superficial and deep grey matter of the spinal cord, and until now the cellular organisation of the ALS was poorly understood. We recently made use of a transgenic mouse (Phox2a::Cre), which labels a major developmental subset of ALS neurons, to define five transcriptomic populations based on single nucleus RNA sequencing (snRNAseq). Three of these populations (ALS1-3) were located in the upper laminae (I-IV), while the remaining two (ALS4-5) were in deep grey matter (laminae V-VII, X). Importantly, ALS1 cells include a well-characterised class of neurons known as antenna cells, as well as a transcriptomic counterpart in lamina I. Antenna cells represent a major postsynaptic target for nociceptive primary afferents, but at present nothing is known about the ALS1 lamina I cells. A major focus of this project will be to investigate the connectivity and function of ALS1 cells. Furthermore, there is considerable debate concerning the contribution of superficial and deep ALS projection neurons to the different pain dimensions. We will therefore investigate how the ALS1 cells (an exemplar of superficial cells) and ALS4-5 (deep) cells differ in their projection targets, and in how they drive the different pain dimensions in acute and inflammatory conditions. We will use a multidisciplinary approach involving 5 complementary workpackages (WPs). In WP1 we will refine our transcriptomic analysis to cover all types of ALS projection neuron. This will also form the basis for optimal targeting of ALS1 cells in subsequent WPs. In WP2 we will investigate the synaptic input to antenna and lamina I ALS1 cells, to determine whether these are likely to be functionally homogeneous. We will also use anterograde viral tracing to compare projection patterns of superficial (ALS1) and deep (ALS4/5) projection neurons, and test the prediction that these differentially innervate brain regions involved in pain mechanisms. In WP3, we will use electrophysiology and calcium imaging to further compare the properties of the two different types of ALS1 neuron. In WP4 we will activate cells belonging to ALS1 and ALS4/5 and test the hypothesis that ALS1 cells drive somatotopically appropriate behaviours, while activation of ALS4/5 cells is aversive. Finally, in WP5 we will inhibit neurons in each population in an inflammatory pain state and test the prediction that targeting ALS1 and ALS4/5 cells selectively reduces somatotopically-relevant signs (ALS1), or the aversiveness of the pain percept (ALS4/5). This work will provide important insights into pain neurobiology by identifying neuronal circuits that are involved in acute and inflammatory pain. It should also reveal potential targets for the development of new strategies for pain relief.