NaV1.7 mRNA and protein expression in putative projection neurons of the human spinal dorsal horn

NaV1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding NaV1.7, SCN9A, are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting NaV1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of NaV1.7. We report for the first time that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. NaV1.7 protein was found in the central axons of sensory neurons terminating in lamina 1–2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic SCN9A mutations, and in achieving analgesic efficacy in clinical trials.


Introduction
In the mid-2000s, it was found that loss-and-gain-of-function mutations in the gene encoding voltage-gated sodium channel (Na V ) 1.7 (Baker and Nassar, 2020), SCN9A, resulted in congenital pain insensitivity (Cox et al., 2006) or in extreme pain disorders like primary erythromelalgia (Yang et al., 2004;Mann et al., 2019), paroxysmal extreme pain disorder (Fertleman et al., 2006;Hua et al., 2022), and idiopathic small fiber neuropathy (Faber et al., 2012), respectively. The rarity of finding a gene that is critical for sensory pathophysiology and that is backed by human genetics framed Na V 1.7 as a prime therapeutic target for pain treatment. Several Na V 1.7 inhibitors have been developed, and a series of clinical trials have been conducted with mixed reports of success (Price et al., 2017;McDonnell et al., 2018;Kingwell, 2019;Alles and Smith, 2021;Biogen, 2021;Eagles et al., 2022).
While these outcomes could be due to poor drug pharmacokinetics and/or limited selectivity towards other sodium channels, one hypothesis is that peripherally restricted drugs lack significant efficacy because spinal Na V 1.7 must be targeted to achieve analgesia. This idea has been supported by mouse studies which suggest that Na V 1.7 in the central terminals of nociceptors is critical for nociceptive neurotransmission in the dorsal horn (MacDonald et al., 2021). However, it is also notable that mouse genetic studies generating gain-of-function mutations in Scn9a have failed to recapitulate human phenotypes (Chen et al., 2021). Na V 1.7 is predominantly expressed in the peripheral nervous system in sympathetic neurons and nociceptive sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (Hameed, 2019a). Subcellularly, it is localized to the membrane of sensory neurons including their soma, peripheral axons that innervate the skin, muscle and other organs, and central axons that cross the blood brain barrier and terminate in the dorsal horn of the spinal cord (Black et al., 2012;Shiers et al., 2020;Shiers et al., 2021). It is a key regulator of neuronal excitability as it mediates Na currents during membrane depolarization and action potential firing (McDermott et al., 2019;Middleton et al., 2022) and is dysregulated in pathological pain conditions in both rodents and humans (Black et al., localized to presynaptic axons and fibers in both the rodent and human spinal dorsal horn (Black et al., 2012;Shiers et al., 2021). A central analgesic mechanism for Na V 1.7 was recently proposed wherein Na V 1.7 -null mice that were pain insensitive, retained nociceptor firing properties but displayed opioid-dependent impaired synaptic transmission and neurotransmitter release at sensory neuron presynaptic terminals in the spinal dorsal horn (MacDonald et al., 2021). Indeed, this could be an important mechanism of action for Na V 1.7's key role in nociception but there is some evidence, although inconsistent, for Na V 1.7 expression post-synaptically within the spinal cord. Na V 1.7 mRNA has been identified in several subsets of mouse spinal cord neurons using single-cell RNA sequencing (Russ et al., 2021), but only its motor neuron expression could be confirmed with in situ hybridization (Alles et al., 2020; Allen Institute for Brain Science, 2022a). However, electron microscopy revealed a substantial proportion of Na V 1.7 immunoreactivity was localized to dendrites of mouse spinal dorsal horn neurons, but these authors proposed that the protein originated from presynaptic fibers and was transferred to post-synaptic sites through a mechanism that remains to be elucidated (Alles et al., 2020).
We hypothesized that SCN9A mRNA and Na V 1.7 protein might be expressed by human dorsal horn projection neurons. If post-synaptic Na V 1.7 exists, especially within the pain neurocircuitry in the spinal dorsal horn such as projection neurons, this would provide important evidence to explain the lack of efficacy of peripherally restricted Na V 1.7 inhibitors and invigorate research towards investigating how Na V 1.7 expression in intrinsic dorsal horn neurons regulates nociception in addition to its well-described role in nociceptors.

Tissue preparation
All human tissue procurement procedures were approved by the Institutional Review Boards at the University of Texas at Dallas. Human lumbar spinal cords were surgically extracted using a ventral approach (Valtcheva et al., 2016) from organ donors within 4 hours of cross-clamp, frozen immediately in dry ice, and stored in a -80°C freezer. All tissues were recovered in the Dallas area via a collaboration with the Southwest Transplant Alliance. Donor information is provided in Table 1. All tissues were collected from neurologic determination of death donors. Human spinal cords were gradually embedded in OCT in a cryomold by adding small volumes of OCT over dry ice to avoid thawing. All tissues were cryostat sectioned at 20 µm onto SuperFrost Plus charged slides.

Immunohistochemistry (IHC)
Slides were removed from the cryostat and immediately transferred to cold 10% formalin (4°C; pH 7.4) for 15 minutes. The tissues were then dehydrated in 50% ethanol (5 min), 70% ethanol (5 min), 100% ethanol (5 min), 100% ethanol (5 min) at room temperature. The slides were air dried briefly and then boundaries were drawn around each section using a hydrophobic pen (ImmEdge PAP pen, Vector Labs). When hydrophobic boundaries had dried, the slides were submerged in blocking buffer (10% Normal Goat Serum, 0.3% Triton-X 100 in 0.1M Phosphate Buffer (PB) for 1 hour at room temperature. Slides were then rinsed in 0.1M PB, placed in a light-protected humidity-. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint 6 controlled tray and incubated in primary antibodies diluted in blocking buffer overnight at 4°C. The next day, slides were washed in 0.1M PB and then incubated in their respective secondary antibody diluted at 1:2000 with DAPI (1:5000; Cayman Chemical; Cat 14285) in blocking buffer for 1 hour at room temperature. The antibodies used are provided in Table 2. The sections were washed in 0.1M PB and then covered with True Black (20% diluted in 70% Ethanol), a blocker of lipofuscin, for 1 minute. Sections were then washed in water, air dried and coverslipped with Prolong Gold Antifade reagent (Cat P36930, Fisher Scientific).

antibody information and validation
The Na V 1.7 mouse monoclonal antibody has been knockout validated using immunocytochemistry on mouse cultured DRG neurons and using IHC on rat brain (Grubinska et al., 2019). We have also recently shown that this antibody robustly stains human DRG and shows a specific and similar expression pattern to its mRNA (Shiers et al., 2020;Shiers et al., 2021;Tavares-Ferreira et al., 2022).

Imaging
All sections were imaged on an Olympus FV3000 confocal microscope. Acquisition parameters were set based on guidelines for the FV3000 provided by Olympus. In particular, the gain was kept at the default setting 1, HV ≤ 600, offset = 4, and laser power ≤ 10%. For the MAP2/Na V 1.7 experiment, an epifluorescent mosaic image of the entire spinal cord section was captured on an Olympus vs120 slide scanner as a means to visualize the precise anatomical location of the confocal images.
For human spinal cord RNAscope, multiple 20X confocal images with overlapping lipofuscin signal were acquired of adjacent regions of the spinal dorsal horn (mosaic imaging). The images were manually stitched together in Adobe Photoshop (v21.2.12, 2020) by overlaying the overlapping . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint lipofuscin in each image. Once the entire spinal dorsal horn was visualized, laminar boundaries were drawn in Adobe Illustrator using a reference atlas (Sengul et al., 2013;Shiers et al., 2021). This imaging was performed on one section from 3 donors.

Images Analysis
Raw images were brightened and contrasted in Olympus CellSens (v1.18). Images were pseudocolored for visualization purposes.
For RNAscope, the number of nuclei expressing SCN9A-only, TACR1-only, and SCN9A-and-TACR1 were counted in each laminar region. For density analysis, the number of RNAscope positive nuclei (neurons) was divided by the area of each laminar subregion (neuron / μ m 2 ). One section from three donors was analyzed. Graphs were generated using GraphPad Prism version 8.4.3 (GraphPad Software, Inc. San Diego, CA USA).

SCN9A mRNA is expressed in TACR1+ putative projection neurons in the human spinal dorsal horn
The substance P receptor (protein: Nk1r, mRNA: Tacr1) labels a subset of projection neurons in the rodent spinal dorsal horn that transmit pain and itch information to the parabrachial nucleus in the brainstem (Blomqvist and Mackerlova, 1995;Mantyh et al., 1997;Carstens et al., 2010). These neurons are localized to the superficial laminae, primarily lamina 1 (Mantyh et al., 1997;Carstens et al., 2010), but they are also found in the deep dorsal horn, mostly in lamina V (Brown et al., 1995).
We assessed the distribution of SCN9A (Na V 1.7) and TACR1 (Nk1r) mRNA in the human spinal dorsal horn using RNAscope. We found that SCN9A and TACR1 mRNAs were detected in cells throughout all laminae in the human spinal dorsal horn (FIG 1A-E), and in motor neurons in the ventral horn (FIG 1F). Large SCN9A/TACR1 co-expressing neurons were prevalent in lamina 1 and were ~30-40μm in diameter (FIG 1B). In the dorsal horn (laminae 1-7), virtually all of the TACR1+ cells were SCN9A+ (95%), but only 36% of SCN9A+ cells were TACR1+ (FIG 1G-H). SCN9A+ and TACR1+ co-expressing neurons were more densely populated in laminae 1-2 (FIG 1I). This distribution closely resembles recently published human spinal cord spatial transcriptomic data (Yadav et al., 2023) in which both SCN9A and TACR1 mRNAs were found throughout the dorsal and ventral horns (FIG 2A). SCN9A was detected in a broad population of excitatory and inhibitory dorsal horn neurons using single-nucleus RNA sequencing of human spinal cord (Yadav et al., 2023), many of which co-expressed TACR1 (FIG 2B).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint Figure 1. Distribution of SCN9A (Na V 1.7) and TACR1 (NK1R) mRNAs in the human spinal dorsal horn using RNAscope. A) Representative stitched mosaic image of human lumbar spinal cord labeled with RNAscope in situ hybridization for SCN9A (red) and TACR1 (green) mRNAs and co-stained with DAPI (cyan). The 488 channel was left unstained (pseudocolored to cyan) to reveal background autofluorescence and lipofuscin which is present in all human neurons. 20X images for each channel are shown for B) laminae 1-2, C) laminae 3-4, D) lamina 5, and E) laminae 6-7. The inset images are a zoomed-in, cropped image of a single SCN9A/TACR1 co-positive cell. F) SCN9A and TACR1 mRNAs were coexpressed by motor neurons in the ventral horn. G) Percentage of SCN9A+ neurons in the dorsal horn that coexpressed TACR1 (purple bar), and the percentage of TACR1+ neurons that coexpressed SCN9A (blue bar). H) Percentage of TACR1+ neurons that were copositive for SCN9A, and the percentage of SCN9A+ neurons that were copositive for TACR1 for each lamina (L1-L7) of the spinal dorsal horn. I) Density of each neuronal subpopulation within each 1 1 Figure 2. Spatial and single-nuclear RNA-sequencing detection of SCN9A and TACR1 in the human spinal dorsal horn. A) Normalized spatial transcriptomic gene expression for SCN9A and TACR1 per barcoded spot on aggregated human lumbar spinal cord sections (Yadav et al., 2023). Solid lines mark grey matter boundaries. B) Dot plot showing the average gene expression for SCN9A and TACR1 for each human spinal cord cluster identified using single-nucleus RNA sequencing. Data found at https://vmenon.shinyapps.io/humanspinalcord/ Na V 1.7 protein is localized pre-synaptically in the human spinal cord.
Na V 1.7 protein staining in the human spinal cord was robustly found in the dorsal rootlet and substantia gelatinosa which comprises lamina 1 and 2, as we have previously reported (Shiers et al., 2021) (FIG 3A-3B).
Na V 1.7 staining gave a neuropil-like pattern, potentially indicative of synaptic staining, throughout laminae 1-2 (FIG 3B). When colabeled with the nociceptive presynaptic marker, CGRP, and the presynaptic active-zone protein, Bassoon (FIG 3C), we observed Na V 1.7 signal that co-localized or was in close proximity to these presynaptic markers (Fig 3D). However, not all of the Na V 1.7 signal in the human spinal cord appeared to be localized to the presynaptic compartment.  et al., 1979;Jones et al., 1987;Craig, 2006), a hypothesis we explored with additional experiments. Additionally, we observed Na V 1.7 protein staining in the cytoplasm of motor neurons in the ventral horn (FIG 3G).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint Figure 3. Na V 1.7 protein expression in the human lumbar spinal cord. A) Mosaic image of Na V 1.7 (red) protein staining in the human lumbar spinal cord, costained with DAPI (blue). The green box represents B) the dorsal horn from L1-L6 where robust Na V 1.7 neuropil staining was observed in lamina 1-lamina 2. C) Representative 100X image of Na V 1.7 (red) protein .
CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint co-stained with the nociceptive presynaptic marker, CGRP (blue), the presynaptic active zone protein, Bassoon (green), and DAPI (cyan) in lamina 2 of the spinal dorsal horn. A cropped, zoomed in area outlined in white is shown in D) where Na V 1.7 signal is colocalized with CGRP and/or Bassoon (white arrows) or is in close proximity to these proteins. However, not all Na V 1.7 signal appeared to be localized to the presynaptic compartment. E) Axonal Na V 1.7 was observed in the deeper laminae, most prominently in lamina 5. The magenta box in panel A represents F) the anterior commissure where Na V 1.7 was also detected in axons. The white box in panel A represents G) the ventral horn where Na V 1.7 was localized to the cytoplasm of large motor neurons. Scales bar = panel Evidence for post-synaptic Na V 1.7 protein in the human spinal cord.
In an effort to identify post-synaptic Na V 1.7 immunoreactivity in the spinal dorsal horn, we costained Na V 1.7 with a variety of subcellular protein markers. First, we assessed Na V 1.7 with the cytoskeletal marker, MAP2, which is primarily localized to dendrites (FIG 4A). We assessed the dorsal horn from 3 organ donors but observed no convincing evidence for any Na V 1.7 signal that was localized to the dendritic compartment of resident dorsal horn neurons, nor their soma in neither lamina 1 (FIG 4B), nor lamina 2 (FIG 4C), nor lamina 4-5 (FIG 4D). In the anterior commissure, intensely labeled Na V 1.7 axonal fibers could be observed crossing hemispheres (FIG 4E), but we could not determine what neurons these axons originated from; however, it is likely they are projection neurons as this is a major hub for cross-hemispheric spinal neurotransmission.
Interestingly, we noted that Na V 1.7 appeared to be localized to the cytoplasm of motor neurons, including their soma and what appeared to be their axon initial segment (AIS). Recent work with Na V 1.7 in the rodent DRG found that sensory neurons contain an AIS that it is enriched with Na V 1.7 and that its localization there is critical for spontaneous activity in neuropathic pain (Nascimento et al., 2022). Other Navs are also known to be enriched in the AIS in the CNS (Leterrier, 2018). To confirm our observation in motor neurons, we co-stained Na V 1.7 with the AIS marker, Ankyrin-G, and found that, indeed, Na V 1.7 colocalized with Ankyrin-G at the AIS of motor neurons in the human ventral horn (Fig 4D). In the dorsal horn, we also found evidence for Na V 1.7 localized to the AIS of resident neurons (Fig 4E), but this was sparse, and we only observed this pattern in a few neurons across 3 donors. As we see robust Na V 1.7 mRNA expression in virtually all of the putative (TACR1+) projection neurons, its low prevalence in the AIS and absence in the soma and cytoplasm of resident neurons suggests that either Na V 1.7 is not translated into protein in these neurons, or which is more likely, that it is localized to the membrane of post-synaptic axons. This is further supported by Na V 1.7 labeling in the anterior commissure.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint Figure 4. Evidence for post-synaptic Na V 1.7 expression in the human spinal cord. A) Representative 10X image of Na V 1.7 (red), MAP2 (green), and DAPI (blue) staining in the human lumbar dorsal horn. The white outline demarcates the substantia gelatinosa which comprises lamina 1 and lamina 2. B) Representative 40X images of Na V 1.7 (red), MAP2 (green), and DAPI (blue) in lamina 1, and C) lamina 2. White arrows point to MAP2 signal that is localized around resident . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint neurons (appears to be the plasma membrane) but are absent of Na V 1.7 signal. The image inset in panel C shows a 100X image of a large, L2 neuron with a large apical dendrite that is devoid of Na V 1.7 signal. D) 20X image of Na V 1.7-positive axonal fibers in the deeper lamina around L4-L5. The white arrow points to Na V 1.7 and MAP2 copositive signal (yellow in overlay) that does not have a nucleus and is not a cell body. E) 20X image of Na V 1.7 staining in the anterior commissure (ac) where intensely labeled Na V 1.7-positive axons are highlighted (white arrow). F) A 100X image of Na V 1.7 (red), Ankyrin-G (green), and DAPI (blue) staining in a motor neuron in the ventral horn. G) A cropped, zoomed-in image of Na V 1.7 (red), Ankyrin-G (green), and DAPI (blue)

Discussion
In the present study, we found evidence for the presence of Na V 1.7 expression in intrinsic neurons of the human spinal cord, including in the dorsal and ventral horns. First, we identified that SCN9A (protein: Na V 1.7) mRNA was detected in virtually all TACR1+ (protein: Nk1r) neurons and that these neurons were more densely populated in lamina 1, 2, 4 and 5, with their lowest density in lamina 3, 6, and 7. In rodents, the Nk1r is expressed by a subset of large-diameter projection neurons that send projections to higher brain regions like the parabrachial nucleus, and are found predominantly in lamina 1 and in the deeper dorsal horn around lamina 5 (Brown et al., 1995;Marshall et al., 1996;Todd et al., 2000). It is important to note that not all rodent projection neurons are Tacr1+  Similarly, Scn9a mRNA was also detected in a variety of Tacr1 co-expressing neuron populations in mouse spinal cord using single cell sequencing (Russ et al., 2021). However, only its motor neuron expression could be validated with in situ hybridization (Alles et al., 2020; Allen Institute for Brain Science, 2022a). While sensitivity issues could underlie these technical differences, detection of Na V 1.7 mRNA and protein in rodent spinal dorsal horn neurons has also been inconclusive likely due to its unique subcellular localization to the neuronal membrane which could comprise the soma, dendrites, axon initial segment, nodes of Ranvier, and/or synapse. Indeed, most reports suggest that spinal Na V 1.7 protein expression is entirely presynaptic due to its robust neuropil staining pattern within lamina 1-2 (Black et al., 2012;Shiers et al., 2021). However, immuno-electron microscopy detected Na V 1.7 protein localized to dendrites of mouse dorsal horn neurons, but it was . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint hypothesized that these proteins originated in the presynaptic compartment and were transferred to post-synaptic sites through an unknown mechanism (Alles et al., 2020).
While we also detected intense presynaptic Na V 1.7 labeling in lamina 1-2 of the human spinal cord, not all of its expression was colocalized with presynaptic markers like CGRP and Bassoon. Na V 1.7 protein was also localized to axons in the deeper lamina, particularly lamina 4 and 5, and to axons in the anterior commissure, the white matter tract connecting the two spinal hemispheres and an important relay for dorsal horn projection neurons transmitting nociceptive information to the contralateral spinothalamic tract (Ku and Morrison, 2022). Because axon tracing methods cannot be employed in the human spinal cord, we could not identify if these were primary afferents or the axons of intrinsic dorsal horn neurons; however, ascending projections of primary afferents ascend in the dorsal columns and do not cross the midline, so it is exceedingly unlikely that these commissural axons are contributed by sensory neurons.
Interestingly, we did not detect Na V 1.7 localized to dendrites, but instead identified its expression in the AISs of some dorsal horn neurons in lamina1-2 and also in motor neurons. Other Nav family members are highly concentrated at the AIS and their localization there is critical for integration of synaptic currents into action potential generation (Grubb and Burrone, 2010;Leterrier, 2018). Importantly, neurons are known to increase or decrease the size of their AIS as a means to augment or depress their neuronal excitability in response to changing presynaptic input (Grubb and Burrone, 2010;Kuba et al., 2010;Grubb et al., 2011). As sensory neuron hyperexcitability and/or spontaneous activity is a major driver of abnormal nociceptive signaling into the dorsal horn in chronic pain conditions, it is possible that Na V 1.7 regulation at the AIS in resident dorsal horn neurons is critical for nociceptive processing in the dorsal horn.
While loss-of-function mutations in SCN9A result in pain insensitivity in both rodents and humans (Gingras et al., 2014;Shields et al., 2018;Grubinska et al., 2019), gain-of-function mutations engineered in mice to match human mutations that cause pain disorders do not recapitulate the human pain phenotype (Chen et al., 2021). One potential explanation for this is that the loss-offunction phenotype is dependent upon sensory neuron expression of Na V 1.7 which is conserved across species and the loss of function leads to a conserved loss of action potential generation in nociceptors. On the other hand, the human gain-of-function pain phenotype may require both peripheral sensitization and spinal amplification. As there has been little convincing evidence for Nav1.7 expression in intrinsic neurons of the rodent spinal cord, it is probable that spinal amplification of nociceptive signals does not occur in rodents but may occur in humans with gain-of-function mutations given the broad expression of SCN9A in dorsal horn neurons, including putative projection neurons. Gain-of-function mutations in SCN9A could lead to increased excitability of projection . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint neurons, which would be further amplified by increased nociceptive input from hyperexcitable nociceptors in the periphery. However, this mechanistic hypothesis may not be as straightforward as this because SCN9A mRNA was found in many neurons in the dorsal horn, including inhibitory interneurons by single-nuclear sequencing (Yadav et al., 2023). Nevertheless, dysregulated circuit dynamics in the dorsal horn caused by gain-of-function mutations in Nav1.7 potentially explain the difference in pain phenotype between humans and rodents.
In summary, we offer several compelling pieces of evidence to support the existence of Na V 1.7 mRNA and protein expression by intrinsic neurons of the human spinal dorsal horn: 1) SCN9A mRNA is expressed by human resident dorsal horn neurons detected by both RNAscope in situ hybridization, spatial sequencing, and single-nucleus sequencing; 2) all TACR1+ human resident dorsal horn neurons express SCN9A, a subset of which are likely projection neurons; 3) many SCN9A+ and TACR1+ co-expressing neurons were large diameter and were most abundant in laminar regions (lamina 1 and lamina 5) that are known to be enriched with projection neurons; 4) not all Na V 1.7 protein signal was limited to the presynaptic compartment as demonstrated with colabeling with CGRP and Bassoon; 5) Na V 1.7 was detected in the axon initial segment of some resident dorsal horn neurons; 6) Na V 1.7+ axons were detected in the anterior commissure. The existence of Na V 1.7 in dorsal horn neurons could explain the lack of analgesic efficacy of peripherally restricted Na V 1.7 inhibitors and offer new insight into a centrally mediated Na V 1.7 regulatory mechanism for nociceptive processing in the dorsal horn.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint  R  o  l  e  i  n  t  h  e  p  a  t  h  o  p  h  y  s  i  o  l  o  g  y  o  f  p  a  i  n  .  M  o  l  P  a  i  n  1  5  :  1  7  4  4  8  0  6  9  1  9  8  5  8  8  0  1  .   H  a  m  e  e  d  S  (  2  0  1  9  b  )  N  a  v  1  .  7  a  n  d  N  a  v  1  .  8  :  R  o  l  e  i  n  t  h  e  p  a  t  h  o  p  h  y  s  i  o  l  o  g  y  o  f  p  a  i  n  .  M  o  l  P  a  i  n  1  5  :  1  7  4  4  8  0  6  9  1  9  8  5  8  8  0  1  .   H  u  a  Y  ,  C  u  i  D  ,  H  a  n  L  ,  X  u  L  ,  M  a  o  S  ,  Y  a  n  g  C  ,  G  a  o  F  ,  Y  u  a  n  Z  (  2  0  2  2  )  A  n  o  v  e  l  S  C  N  9  A  g  e  n  e  v  a  r  i  a  n  t  i  d  e  n  t  i  f  i  e  d  i  n  a  C  h  i  n  e  s  e  g  i  r  l   w  i  t  h  p  a  r  o  x  y  s  m  a  l  e  x  t  r  e  m  e  p  a  i  n  d  i  s  o  r  d  e  r  (  P  E  P  D  )  :  a  r  a  r  e  c  a  s  e  r  e  p  o  r  t  .  B  M  C  M  e  d  G  e  n  o  m  i  c  s  1  5  :  1  5  9  .   J  o  n  e  s  M  W  ,  A  p  k  a  r  i  a  n  A  V  ,  S  t  e  v  e  n  s  R  T  ,  H  o  d  g  e  C  J  ,  J  r  .  (  1  9  8  7  )  T  h  e  s  p  i  n  o  t  h  a  l  a  m  i  c  t  r  a  c  t  :  a  n  e  x  a  m  i  n  a  t  i  o  n  o  f  t  h  e  c  e  l  l  s  o  f  o  r  i  g  i  n  o   . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint 0 S h i e r s S I , S a n k a r a n a r a y a n a n I  D  u  a  n  G  ,  L  i  u  Y  ,  G  u  o  S  ,  W  a  n  g  C  ,  Z  h  u  C  ,  Z  h  a  n  g  X  (  2  0  1  8  )  I  n  c  r  e  a  s  e  d  N  a  (  v  )  1  .  7  e  x  p  r  e  s  s  i  o  n  i  n  t  h  e  d  o  r  s  a  l  r  o  o  t  g  a  n  g  l  i  o  n   c  o  n  t  r  i  b  u  t  e  s  t  o  p  a  i  n  h  y  p  e  r  s  e  n  s  i  t  i  v  i  t  y  a  f  t  e  r  p  l  a  n  t  a  r  i  n  c  i  s  i  o  n  i  n  r  a  t  s  .  M  o  l  P  a  i  n  1  4  :  1  7  4  4  8  0  6  9  1  8  7  8  2  3  2  3  .   T  a  v  a  r  e  s  -F  e  r  r  e  i  r  a  D  ,  S  h  i  e  r  s  S  ,  R  a  y  P  R  ,  W  a  n  g  z  h  o  u  A  ,  J  e  e  v  a  k  u  m  a  r  V  ,  S  a  n  k  a  r  a  n  a  r  a  y  a  n  a  n  I  ,  C  e  r  v  a  n  t  e  s  A  M  ,  R  e  e  s  e  J  C  ,   C  h  a  m  e  s  s  i  a  n  A  ,  C  o  p  i  t  s  B  A  ,  D  o  u  g  h  e  r  t  y  P  M  ,  G  e  r  e  a  u  R  W  t  ,  B  u  r  t  o  n  M  D  ,  D  u  s  s  o  r  G  ,  P  r  i  c  e  T  J  (  2  0  2  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 5, 2023. ; https://doi.org/10.1101/2023.02.04.527110 doi: bioRxiv preprint