Biology subjects

Kucharczyk, M. W.

Publications and source records attributed to Kucharczyk, M. W..

5 recordsLinked to original sources

bioRxiv2023

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify 3 clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 & ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons. Significance StatementThe anterolateral system (ALS) is a major ascending pathway from the spinal cord that underlies perception of pain, itch and skin temperature. It is therefore an important target for the development of new treatments for chronic pain. Our understanding of this system has been hampered by the considerable diversity of its constituent cells. Here we dissect the complex heterogeneity of these cells by using high-resolution RNA sequencing. We reveal five distinct types of ALS neurons, which are differentially distributed within the spinal cord, and probably represent functional populations. Our data provide novel insights into the molecular architecture of the ALS, and will be important for future studies to define the roles of different ALS cell types in sensory processing.

neuroscience↗

bioRxiv2022

The origin of diffuse noxious inhibitory controls

Pain alerts us to actual or potential tissue damage. During acute pain, our central nervous system acts endogenously to modulate pain processing, thus reducing or enhancing pain perception. However, during chronic pain, the balance between inhibitory and facilitatory processes are tipped in favour of pro-pain modulation. Diffuse noxious inhibitory controls (DNIC) is a naturally occurring pain inhibitory pathway that projects from the brainstem to the spinal cord to inhibit neuronal activity therein in a manner that is 1) subserved by noradrenaline, and 2) dysfunctional in chronicity. To harness its high therapeutic potential, we aimed to anatomically and functionally define DNIC. Through employing an intersectional opto- and chemogenetic approach to modulate activity in brainstem noradrenergic nuclei, here we show that spinal neuronal firing observed upon DNIC activation during electrophysiological experiments, and animal pain thresholds observed during behavioural experiments, are modulated in a pro-pain manner upon opto-manipulation of A5 spinally projecting noradrenergic neurons, thus evidencing the DNIC origin. Given the plasticity of the functional expression of DNIC in disease, and the success of back and forward translation of paradigms that evoke DNIC in pre-clinical and clinical models, our findings offer an attractive avenue of studies for disease specific analgesic interventions.

neuroscience↗

bioRxiv2021

Distinct brainstem to spinal cord noradrenergic pathways differentially regulate spinal neuronal activity

Brainstem to spinal cord pathways modulate spinal neuronal activity. We implemented locus coeruleus (LC) targeting strategies by microinjecting CAV-PRS-ChR2 virus in the spinal cord (LC:SC module) or LC (LC:LC module). While activation of both modules inhibited evoked spinal neuronal firing via 1-adrenoceptor-mediated actions, LC:SC opto-activation abolished diffuse noxious inhibitory controls. The LC as a pain generator is likely mechanistically underpinned by maladaptive communication with discrete descending modulatory pathways.

neuroscience↗

bioRxiv2020

The stage specific plasticity of descending modulatory controls in a rodent model of cancer induced bone pain

Pain resulting from metastatic bone disease is a major unmet clinical need. Studying spinal processing in rodent models of cancer pain is desirable since the percept of pain is influenced in part by modulation at the level of the transmission system in the dorsal horn of the spinal cord. Here a rodent model of cancer induced bone pain (CIBP) was generated following syngenic rat mammary gland adenocarcinoma cell injection in the tibia of male Sprague Dawley rats. Disease progression was classified as early or late stage according to bone destruction. Even though wakeful CIBP rats showed progressive mechanical hypersensitivity, subsequent in vivo electrophysiological measurement of mechanically evoked deep dorsal horn spinal neuronal responses revealed no change. Rather, a dynamic reorganization of spinal neuronal modulation by descending controls was observed, and this was maladaptive only in the early stage of CIBP. Interestingly, this latter observation corresponded with the degree of damage to the primary afferents innervating the cancerous tissue. Plasticity in the modulation of spinal neuronal activity by descending control pathways reveals a novel opportunity for targeting CIBP in a stage-specific manner. Finally, the data herein has translational potential since the descending control pathways measured are present also in man. Simple SummaryThe mechanisms that underlie pain resulting from metastatic bone disease remain elusive. This translates to a clinical and socioeconomic burden; targeted therapy is not possible, and patients do not receive adequate analgesic relief. Complicating matters is the heterogeneous nature of metastatic bone disease. Early stage cancers are molecularly very different to their late stage counterparts and so too is the pain associated with infant and advanced tumours. Thus, analgesic approaches should differ according to disease stage. In this article we demonstrate that a unique form of brain inhibitory control responsible for modulation of incoming pain signals at the level of the spinal cord changes with the progression of bone tumours, This corresponds with the degree of damage to the primary afferents innervating the cancerous tissue. Plasticity in the modulation of spinal neuronal activity by descending control pathways reveals a novel opportunity for targeting bone cancer pain in a stage-specific manner.

neuroscience↗

bioRxiv2018

Novel mechanisms of bone cancer pain revealed with in vivo GCaMP6s imaging

Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature and not easily managed by available therapies. The peripheral sensory profile of primary afferents responsible for transmitting the pain-related messages from cancerous bone to central sites is investigated here. We imaged thousands of primary sensory dorsal root ganglion neurons in vivo in healthy (sham-operated) and cancer-induced bone pain (CIBP) rats in order to analyse and compare their function. Utilising Markov Cluster Analysis we identified distinct clusters of primary afferent responses to limb compression and position. In CIBP rats, three times as many sensory afferents responded to knee compression in the leg ipsilateral to the tumour compared to sham-operated rats. We present evidence that the observed increase in sensory afferent response was not due to increased individual afferent activity but rather represents activation of silent nociceptors, whose origin we propose is largely from outside of the bone.

neuroscience↗