The spinal dorsal horn contains numerous excitatory interneurons, which can be assigned to functional classes based on morphological, electrophysiological and neurochemical criteria. One population consists of neurons that express neurotensin, and these belong to a larger group defined by the presence of protein kinase Cγ (PKCγ). It has been proposed that PKCγ neurons form part of a circuit that can convey low-threshold mechanoreceptive information to nociceptive projection neurons in lamina I, forming a pathway that could underlie mechanical allodynia in pathological pain states. However, despite their potential importance, relatively little is known about the properties of the neurotensin-expressing cells. Here we have used a neurotensin-Cre line, together with intraspinal injection of AAVs coding for Cre-dependent constructs, to characterise the morphological and electrophysiological properties of these cells. Reconstruction of their dendritic trees revealed that they were morphologically diverse, although many could be assigned to a class known as central cells. All cells examined received synaptic contacts from putative A- and C-low-threshold mechanoreceptors (identified by expression of VGLUT1 and VGLUT3, respectively). However, these only accounted for a minority of their excitatory synapses. Around 40% of their synapses were from VGLUT2-immunoreactive boutons, which are likely to have originated mainly from local excitatory interneurons. Electrophysiological analysis revealed similarities to, and differences, from other neurochemically-defined excitatory interneuron populations. Our findings are compatible with the proposed role of neurotensin cells in mechanical allodynia, but suggest additional functions for these cells.

Primary afferent neurons detect sensory stimuli in the periphery and transmit this information to the dorsal horn of the spinal cord, where it is processed by excitatory and inhibitory controls before being sent to the brain. Our understanding of the synaptic architecture of these spinal circuits in the rodent has been massively advanced using antibodies raised against scaffolding proteins Homer1 and gephyrin, which anchor glutamate and GABA receptors to the membrane, respectively. Few studies have attempted to visualize spinal cord synapses in human tissue, partly due to a lack of high-quality tissue with low postmortem intervals. In this study, we reveal both excitatory and inhibitory synapses at a high resolution in human lumbar spinal cord tissue using Homer1 and gephyrin immunolabeling and show that the basic organization of these proteins within the dorsal horn is similar to that in the rodent. Homer1+ puncta are highly colocalized with ionotropic glutamate receptors, and over 75% are in contact with a presynaptic axon terminal containing the vesicular glutamate transporter 2 (VGluT2). Similarly, most gephyrin+ profiles are coextensive with the GABA A -α 2 subunit but fewer than 10% colocalize with Homer1+ puncta, confirming the specificity of these markers. Finally, we use Homer1 immunolabeling to demonstrate that primary afferents can form complex synaptic arrangements in human spinal cord. We conclude that these antibodies can be used as reliable tools for the study of human synaptic circuitry, and we have used them to reveal insight into the spinal connections underlying somatosensation that can be expanded upon in future studies.

Anterolateral system (ALS) spinal projection neurons are essential for pain perception. However, these cells are heterogeneous, and there has been extensive debate about the roles of ALS populations in the different pain dimensions. We recently performed single-nucleus RNA sequencing on a developmentally-defined subset of ALS neurons, and identified 5 transcriptomic populations. One of these, ALS4, consists of cells that express Sst, the gene coding for somatostatin, and we reported that these were located in the lateral part of lamina V. Here we use a SstCre mouse line to characterise these cells and define their axonal projections. We find that their axons ascend mainly on the ipsilateral side, giving off collaterals throughout their course in the spinal cord. They target various brainstem nuclei, including the parabrachial internal lateral nucleus, and the posterior triangular and medial dorsal thalamic nuclei. We also show that in the L4 segment Sst is expressed by ~ 75% of ALS neurons in lateral lamina V and that there are around 120 Sst-positive lateral lamina V cells on each side. Our findings indicate that this is a relatively large population, and based on projection targets we conclude that they are likely to contribute to the affective-motivational dimension of pain.

Anterolateral system (ALS) projection neurons underlie perception of pain, itch and skin temperature. These cells are heterogeneous, and there have therefore been many attempts to define functional populations. A recent study identified two classes of ALS neuron in mouse superficial dorsal horn (SDH) based on expression of the G protein-coupled receptors Tacr1 or Gpr83. It was reported that cells expressing these receptors formed largely non-overlapping populations, and that ~60% of ALS cells in SDH expressed Tacr1. An additional finding was that while Tacr1- and Gpr83-expressing ALS cells projected to several brain nuclei, their axons did not reach the ventral posterolateral (VPL) thalamic nucleus, which is reciprocally connected to the primary somatosensory cortex. These results were surprising, because we had reported that ~90% of SDH ALS neurons in the mouse possess the neurokinin 1 receptor (NK1r), which is encoded by Tacr1, and in addition the VPL is thought to receive input from lamina I ALS cells. Here we use retrograde and anterograde labelling in Tacr1CreERT2 and Gpr83CreERT2 mice to reinvestigate the expression of the receptors by ALS neurons and to reassess their projection patterns. We find that ~90% of ALS neurons in SDH express Tacr1, with 40%-50% expressing Gpr83. We also show that axons of both Tacr1- and Gpr83-expressing ALS neurons reach the VPL. These results suggest that ALS neurons in the SDH that express these GPCRs show considerable overlap, and that they are likely to contribute to the sensory-discriminative dimension of pain through their projections to VPL.

Developments in functional magnetic resonance imaging (fMRI) at ultra-high field (UHF) now allow for insights into human brain function at mesoscopic scale. However, similar progress has not been achieved for the human spinal cord, despite its importance for reciprocal brain-body communication. Here, we therefore integrate substantial methodological improvements that enable previously unattainable high-resolution UHF spinal fMRI. Using a heat-pain paradigm in healthy volunteers, we investigate fMRI responses in the dorsal horn: we characterize their spatial pattern, establish their robustness and demonstrate that they can be reliably observed even for individual participants. Most importantly, we uncover two physiologically relevant spinal response components – phasic and tonic heat responses – that map differentially onto superficial and deep layers of the dorsal horn. Taken together, our novel UHF fMRI approach allows for differentiating responses across fundamental spinal processing units and will enable insights into human spinal cord function at mesoscopic scale in health and disease. ### Competing Interest Statement The Max Planck Institute for Human Cognitive and Brain Sciences and Wellcome Centre for Human Neuroimaging have institutional research agreements with Siemens Healthineers. N.W. holds a patent on acquisition of MRI data during spoiler gradients (US 10,401,453 B2). Max Planck Society, https://ror.org/01hhn8329 European Research Council, https://ror.org/0472cxd90, 758974

Significance The 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 development of 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 a major subset of these cells, 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 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.

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 three 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 and 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.

Unmyelinated C-fibres have been classically defined as peptidergic or non-peptidergic based on the presence or absence of calcitonin gene-related peptide and/or substance P. Peptidergic primary afferents terminate in lamina I of the rodent spinal dorsal horn, where they form simple synaptic arrangements. In contrast, the axonal boutons of non-peptidergic afferents make multiple output synapses and have been shown to form the central axon of type I glomeruli. There is an expansion in the expression of these peptides in humans as a greater proportion of primary afferents contain the mRNA for the peptide precursor proteins than in the rodent. The dorsal horn plexus of these afferents also extends more ventrally into lamina II in humans. Here, we have used confocal microscopy on lumbar spinal cord sections from human organ donors (18 – 73 years) to show that the axonal boutons of peptide-containing primary afferents form much more complex synaptic arrangements in humans, with many output synapses. Using transmission electron microscopy, we have also identified many profiles in the substantia gelatinosa of the human spinal cord with defined characteristics of type I glomeruli. Whilst these profiles rarely contain large dense-core vesicles (LDCVs) responsible for peptide release in the rodent, we frequently found they contain many LDCVs in the human. Together, these data suggest that peptidergic primary afferents engage much more complex circuitry in the human spinal cord, although further research is required to unravel these circuits and how they may change with age and in pain states.

Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75-80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here, we investigate the role of interneurons that continue to express NPY (NPY-INs) in the adult mouse spinal cord. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY-IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.

Neuropeptide Y targets the Y1 receptor (Y1) in the spinal dorsal horn (DH) to produce endogenous and exogenous analgesia. DH interneurons that express Y1 (Y1-INs; encoded by Npy1r) are necessary and sufficient for neuropathic hypersensitivity after peripheral nerve injury. However, as Y1-INs are heterogenous in composition in terms of morphology, neurophysiological characteristics, and gene expression, we hypothesized that a more precisely defined subpopulation mediates neuropathic hypersensitivity. Using fluorescence in situ hybridization, we found that Y1-INs segregate into 3 largely nonoverlapping subpopulations defined by the coexpression of Npy1r with gastrin releasing peptide (Grp/Npy1r), neuropeptide FF (Npff/Npy1r), and cholecystokinin (Cck/Npy1r) in the superficial DH of mice, nonhuman primates, and humans. Next, we analyzed the functional significance of Grp/Npy1r, Npff/Npy1r, and Cck/Npy1r INs to neuropathic pain using a mouse model of peripheral nerve injury. We found that chemogenetic inhibition of Npff/Npy1r-INs did not change the behavioral signs of neuropathic pain. Further, inhibition of Y1-INs with an intrathecal Y1 agonist, [Leu31, Pro34]-NPY, reduced neuropathic hypersensitivity in mice with conditional deletion of Npy1r from CCK-INs and NPFF-INs but not from GRP-INs. We conclude that Grp/Npy1r-INs are conserved in higher order mammalian species and represent a promising and precise pharmacotherapeutic target for the treatment of neuropathic pain.

Gastrin-releasing peptide (GRP) in the spinal dorsal horn acts on the GRP receptor, and this signalling mechanism has been strongly implicated in itch. However, the source of GRP in the dorsal horn is not fully understood. For example, the BAC transgenic mouse line GRP::GFP only captures around 25% of GRP-expressing cells, and Grp mRNA is found in several types of excitatory interneuron. A major limitation in attempts to identify GRP-expressing neurons has been that antibodies against GRP cross-react with other neuropeptides, including some that are expressed by primary afferents. Here we have developed two antibodies raised against different parts of the precursor protein, pro-GRP. We show that labelling is specific, and that the antibodies do not cross-react with neuropeptides in primary afferents. Immunoreactivity was strongest in the superficial laminae, and the two antibodies labelled identical structures, including glutamatergic axons and cell bodies. The pattern of pro-GRP-immunoreactivity varied among different neurochemical classes of excitatory interneuron. Cell bodies and axons of all GRP-GFP cells were labelled, confirming reliability of the antibodies. Among the other populations, we found the highest degree of co-expression (>50%) in axons of NPFF-expressing cells, while this was somewhat lower (10-20%) in cells that expressed substance P and NKB, and much lower (<10%) in other classes. Our findings show that these antibodies reliably detect GRP-expressing neurons and axons, and that in addition to the GRP-GFP cells, excitatory interneurons expressing NPFF or substance P are likely to be the main source of GRP in the spinal dorsal horn.

The superficial dorsal horn (SDH) of the spinal cord contains a diverse array of neurons. The vast majority of these are interneurons, most of which are glutamatergic. These can be assigned to several populations, one of which is defined by expression of gastrin-releasing peptide receptor (GRPR). The GRPR cells are thought to be “tertiary pruritoceptors,” conveying itch information to lamina I projection neurons of the anterolateral system (ALS). Surprisingly, we recently found that GRPR-expressing neurons belong to a morphological class known as vertical cells, which are believed to transmit nociceptive information to lamina I ALS cells. Little is currently known about synaptic circuits engaged by the GRPR cells. Here we combine viral-mediated expression of PSD95-tagRFP fusion protein with super-resolution microscopy to reveal sources of excitatory input to GRPR cells. We find that they receive a relatively sparse input from peptidergic and non-peptidergic nociceptors in SDH, and a limited input from A- and C-low threshold mechanoreceptors on their ventral dendrites. They receive synapses from several excitatory interneuron populations, including those defined by expression of substance P, neuropeptide FF, cholecystokinin, neurokinin B, and neurotensin. We investigated downstream targets of GRPR cells by chemogenetically exciting them and identifying Fos-positive (activated) cells. In addition to lamina I projection neurons, many ALS cells in lateral lamina V and the lateral spinal nucleus were Fos-positive, suggesting that GRPR-expressing cells target a broader population of projection neurons than was previously recognised. Our findings indicate that GRPR cells receive a diverse synaptic input from various types of primary afferent and excitatory interneuron, and that they can activate ALS cells in both superficial and deep regions of the dorsal horn.

Objective:Attention Deficit Hyperactivity Disorder (ADHD) is associated with a range of adverse outcomes in adult life. However it is unclear whether the risk pathways to adverse adult outcomes are established during childhood or whether associations are driven by concurrent ADHD symptoms that have persisted to adulthood.Methods:We examined associations between broadly defined child-limited (remitted) and persistent ADHD (assessed using the ADHD subscale of the Strengths and Difficulties Questionnaire) with social outcomes (low emotional and instrumental support, antisocial behaviour, employment, receipt of state benefits as an indicator of socio-economic disadvantage, homelessness) at age 25 years in a UK longitudinal population sample ALPSAC (the Avon Longitudinal Study of Parents and Children, age 25 data collected between years 2017 and 2018): total N=6439.Results:Up to 20% of young-people with less favourable social outcomes at age 25 had persistent ADHD. Persistent ADHD was associated with an increased likelihood of being not in education, employment or training (NEET: OR=3.71, 95% CI=2.06 to 6.67, p=1x10-05) and receiving state benefits (OR=2.72, 95% CI=1.62 to 4.57, p=2x10-04) at age 25 years compared to those without ADHD. We did not find strong evidence of associations between child-limited ADHD and social outcomes (NEET OR=1.20, 95% CI=0.54 to 2.69, p=0.65; state benefits OR=1.38, 95% CI=0.76 to 2.51, p=0.29). Persistent ADHD associations with negative social outcomes were observed across family-of-origin income groups, sex and were not explained by comorbidity.Conclusion:Our findings highlight the importance of continued monitoring and management of ADHD symptoms and related social as well as clinical outcomes across development into adulthood. Future research is needed to identify what factors promote positive social outcomes, including effective treatment of adult ADHD symptoms.

Abstract Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are believed to transmit nociceptive information. In this study, we have used a GRPRCreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus, and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain-related and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.