Recent work demonstrated that activation of spinal D1 and D5 dopamine receptors (D1/D5Rs) facilitates non-Hebbian long-term potentiation (LTP) at primary afferent synapses onto spinal projection neurons. However, the cellular localization of the D1/D5Rs driving non-Hebbian LTP in spinal nociceptive circuits remains unknown, and it is also unclear whether D1/D5R signaling must occur concurrently with sensory input in order to promote non-Hebbian LTP at these synapses. Here we investigate these issues using cell-type-selective knockdown of D1Rs or D5Rs from lamina I spinoparabrachial neurons, dorsal root ganglion (DRG) neurons, or astrocytes in adult mice of either sex using Cre recombinase-based genetic strategies. The LTP evoked by low-frequency stimulation of primary afferents in the presence of the selective D1/D5R agonist SKF82958 persisted following the knockdown of D1R or D5R in spinoparabrachial neurons, suggesting that postsynaptic D1/D5R signaling was dispensable for non-Hebbian plasticity at sensory synapses onto these key output neurons of the superficial dorsal horn (SDH). Similarly, the knockdown of D1Rs or D5Rs in DRG neurons failed to influence SKF82958-enabled LTP in lamina I projection neurons. In contrast, SKF82958-induced LTP was suppressed by the knockdown of D1R or D5R in spinal astrocytes. Furthermore, the data indicate that the activation of D1R/D5Rs in spinal astrocytes can either retroactively or proactively drive non-Hebbian LTP in spinoparabrachial neurons. Collectively, these results suggest that dopaminergic signaling in astrocytes can strongly promote activity-dependent LTP in the SDH, which is predicted to significantly enhance the amplification of ascending nociceptive transmission from the spinal cord to the brain.

Spinal cord dorsal horn inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain are poorly understood. Here, we show that a critical step in the loss of inhibitory tone is the change in the firing pattern of inhibitory parvalbumin (PV)-expressing neurons (PVNs). Our results show that PV, a calcium-binding protein, controls the firing activity of PVNs by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient for the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of the firing pattern is due to the recruitment of calcium-activated potassium (SK) channels, and blocking them during chronic pain restores normal tonic firing and alleviates chronic pain. Our findings indicate that PV is essential for controlling the firing pattern of PVNs and for preventing allodynia. Developing approaches to manipulate these mechanisms may lead to different strategies for chronic pain relief.

UNASSIGNED: Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species.
UNASSIGNED: We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans.
UNASSIGNED: To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets.
UNASSIGNED: We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans.
UNASSIGNED: The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.
Contexte: Les données probantes précliniques et cliniques indiquent que le cannabis possède des propriétés analgésiques potentielles. Cependant, l’expression et la localisation des récepteurs cannabinoïdes au sein des circuits de traitement de la douleur de la moelle épinière restent à caractériser selon le sexe et les espèces.Objectifs: Nous avons cherché à étudier l’expression différenciée du récepteur cannabinoïde de type 1 (CB1) dans les différentes couches de la corne dorsale et les populations cellulaires chez des rats et des êtres humains adultes de sexe masculin et féminin.Méthodes: Pour étudier et quantifier l’expression des récepteurs CB1 dans la corne dorsale de la moelle épinière chez différentes espèces, nous avons perfectionné les procédures d’immunohistochimie pour obtenir des résultats de coloration réussis sur des échantillons de tissus provenant de rats et d’êtres humains, ainsi que des images confocales. Les résultats immunohistochimiques ont été complétés par l’analyse de l’expression du gène CB1 (CNR1) dans la corne dorsale des rongeurs et des humains en utilisant des ensembles de données de séquençage d’ARN au niveau des cellules uniques et des noyaux.Résultats: Nous avons constaté que le CB1 était principalement localisé dans le neuropile au sein de la corne dorsale superficielle chez les rats et les humains, avec une coloration somatique du CB1 dans les différentes couches de la corne dorsale. Chez les deux espèces, l’immunoréactivité du récepteur CB1 était significativement plus élevée dans la couche superficielle de la corne dorsale par rapport aux couches plus profondes, indépendamment du sexe. De manière intéressante, nous avons constaté que l’immunoréactivité du CB1 n’était pas principalement localisée dans les afférences peptidergiques chez les rats et les humains. De plus, nous avons observé une forte expression du gène CNR1 (CB1), mais pas du CNR2 (CB2), au sein de sous-populations de neurones de la corne dorsale chez les rongeurs et les êtres humains.Conclusions: La localisation privilégiée et constante des récepteurs CB1 dans la couche superficielle de la corne dorsale chez les rongeurs et les humains, quel que soit leur sexe, revêt une importance majeure pour la compréhension des fonctions de ce récepteur des cannabinoïdes dans les mécanismes médullaires de la nociception et de l’analgésie.

Neuropathic pain can result from injury to, or disease of the nervous system. It is notoriously difficult to treat. Peripheral nerve injury promotes Schwann cell activation and invasion of immunocompetent cells into the site of injury, spinal cord and higher sensory structures such as thalamus and cingulate and sensory cortices. Various cytokines, chemokines, growth factors, monoamines and neuropeptides effect two-way signalling between neurons, glia and immune cells. This promotes sustained hyperexcitability and spontaneous activity in primary afferents that is crucial for onset and persistence of pain as well as misprocessing of sensory information in the spinal cord and supraspinal structures. Much of the current understanding of pain aetiology and identification of drug targets derives from studies of the consequences of peripheral nerve injury in rodent models. Although a vast amount of information has been forthcoming, the translation of this information into the clinical arena has been minimal. Few, if any, major therapeutic approaches have appeared since the mid 1990\'s. This may reflect failure to recognise differences in pain processing in males vs. females, differences in cellular responses to different types of injury and differences in pain processing in humans vs. animals. Basic science and clinical approaches which seek to bridge this knowledge gap include better assessment of pain in animal models, use of pain models which better emulate human disease, and stratification of human pain phenotypes according to quantitative assessment of signs and symptoms of disease. This can lead to more personalized and effective treatments for individual patients. Significance statement: There is an urgent need to find new treatments for neuropathic pain. Although classical animal models have revealed essential features of pain aetiology such as peripheral and central sensitization and some of the molecular and cellular mechanisms involved, they do not adequately model the multiplicity of disease states or injuries that may bring forth neuropathic pain in the clinic. This review seeks to integrate information from the multiplicity of disciplines that seek to understand neuropathic pain; including immunology, cell biology, electrophysiology and biophysics, anatomy, cell biology, neurology, molecular biology, pharmacology and behavioral science. Beyond this, it underlines ongoing refinements in basic science and clinical practice that will engender improved approaches to pain management.

Photobiomodulation is an effective treatment for pain. We previously reported that the direct laser irradiation of the exposed sciatic nerve inhibited firing in the rat spinal dorsal horn evoked by mechanical stimulation, corresponding to the noxious stimulus. However, percutaneous laser irradiation is used in clinical practice, and it is unclear whether it can inhibit the firing of the dorsal horn. In this study, we investigated whether the percutaneous laser irradiation of the sciatic nerve inhibits firing. Electrodes were inserted into the lamina II of the dorsal horn, and mechanical stimulation was applied using von Frey filaments (vFFs) with both pre and post laser irradiation. Our findings show that percutaneous laser irradiation inhibited 26.0 g vFF-evoked firing, which corresponded to the noxious stimulus, but did not inhibit 0.6 g and 8.0 g vFF-evoked firing. The post- (15 min after) and pre-irradiation firing ratios were almost the same as those for direct and percutaneous irradiation. A photodiode sensor implanted in the sciatic nerve showed that the power density reaching the sciatic nerve percutaneously was attenuated to approximately 10% of that on the skin. The relationship between the laser intensity reaching the nerve and its effect could be potentially useful for a more appropriate setting of laser conditions in clinical practice.

Mirror-image pain arises from pathologic alterations in the nociceptive processing network that controls functional lateralization of the primary afferent input. Although a number of clinical syndromes related to dysfunction of the lumbar afferent system are associated with the mirror-image pain, its morphophysiological substrate and mechanism of induction remain poorly understood. Therefore, we used ex vivo spinal cord preparation of young rats of both sexes to study organization and processing of the contralateral afferent input to the neurons in the major spinal nociceptive projection area Lamina I. We show that decussating primary afferent branches reach contralateral Lamina I, where 27% of neurons, including projection neurons, receive monosynaptic and/or polysynaptic excitatory drive from the contralateral Aδ-fibers and C-fibers. All these neurons also received ipsilateral input, implying their involvement in the bilateral information processing. Our data further show that the contralateral Aδ-fiber and C-fiber input is under diverse forms of inhibitory control. Attenuation of the afferent-driven presynaptic inhibition and/or disinhibition of the dorsal horn network increased the contralateral excitatory drive to Lamina I neurons and its ability to evoke action potentials. Furthermore, the contralateral Aβδ-fibers presynaptically control ipsilateral C-fiber input to Lamina I neurons. Thus, these results show that some lumbar Lamina I neurons are wired to the contralateral afferent system whose input, under normal conditions, is subject to inhibitory control. A pathologic disinhibition of the decussating pathways can open a gate controlling contralateral information flow to the nociceptive projection neurons and, thus, contribute to induction of hypersensitivity and mirror-image pain.SIGNIFICANCE STATEMENT We show that contralateral Aδ-afferents and C-afferents supply lumbar Lamina I neurons. The contralateral input is under diverse forms of inhibitory control and itself controls the ipsilateral input. Disinhibition of decussating pathways increases nociceptive drive to Lamina I neurons and may cause induction of contralateral hypersensitivity and mirror-image pain.

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.

There is growing literature supporting cannabinoids as a potential therapeutic for pain conditions. The development of chronic pain has been associated with reduced concentrations of the endogenous cannabinoid anandamide (AEA) in the midbrain dorsal periaqueductal gray (dPAG), and microinjections of synthetic cannabinoids into the dPAG are antinociceptive. Therefore, the goal of this study was to examine the role of the dPAG in cannabinoid-mediated sensory inhibition. Given that cannabinoids in the dPAG also elicit sympathoexcitation, a secondary goal was to assess coordination between sympathetic and antinociceptive responses. AEA was microinjected into the dPAG while recording single unit activity of wide dynamic range (WDR) dorsal horn neurons (DHNs) evoked by high intensity mechanical stimulation of the hindpaw, concurrently with renal sympathetic nerve activity (RSNA), in anesthetized male rats. AEA microinjected into the dPAG decreased evoked DHN activity (n = 24 units), for half of which AEA also elicited sympathoexcitation. AEA actions were mediated by cannabinoid 1 receptors as confirmed by local pretreatment with the cannabinoid receptor antagonist AM281. dPAG microinjection of the synaptic excitant DL-homocysteic acid (DLH) also decreased evoked DHN activity (n = 27 units), but in all cases this was accompanied by sympathoexcitation. Thus, sensory inhibition elicited from the dPAG is not exclusively linked with sympathoexcitation, suggesting discrete neuronal circuits. The rostrocaudal location of sites may affect evoked responses as AEA produced sensory inhibition without sympathetic effects at 86 % of caudal compared to 25 % of rostral sites, supporting anatomically distinct neurocircuits. These data indicate that spatially selective manipulation of cannabinoid signaling could provide analgesia without potentially harmful autonomic activation.

Spinal interneurons located in the dorsal horn induce primary afferent depolarization (PAD) controlling the excitability of the afferent\'s terminals. Following inflammation, PAD may reach firing threshold contributing to maintain inflammation and pain. Our aim was to study the collective behavior of dorsal horn neurons, its relation to backfiring of primary afferents and the effects of a peripheral inflammation in this system. Experiments were performed on slices of spinal cord obtained from naïve adult mice or mice that had suffered an inflammatory pretreatment. Simultaneous recordings from groups of dorsal horn neurons and primary afferents were obtained and machine-learning methodology was used to analyze effective connectivity between them. Dorsal horn recordings showed grouping of spontaneous action potentials from different neurons in \"population bursts.\" These occurred at irregular intervals and were formed by action potentials from all classes of neurons recorded. Compared to naïve, population bursts from treated animals concentrated more action potentials, had a faster onset and a slower decay. Population bursts were disrupted by perfusion of picrotoxin and held a strong temporal correlation with backfiring of afferents. Effective connectivity analysis allowed pinpointing specific neurons holding pre- or post-synaptic relation to the afferents. Many of these neurons had an irregular fast bursting pattern of spontaneous firing. We conclude that population bursts contain action potentials from neurons presynaptic to the afferents which are likely to control their excitability. Peripheral inflammation may enhance synchrony in these neurons, increasing the chance of triggering action potentials in primary afferents and contributing toward central sensitization.

A central principle of synaptic transmission is that action potential-induced presynaptic neurotransmitter release occurs exclusively via Ca2+ -dependent secretion (CDS). The discovery and mechanistic investigations of Ca2+ -independent but voltage-dependent secretion (CiVDS) have demonstrated that the action potential per se is sufficient to trigger neurotransmission in the somata of primary sensory and sympathetic neurons in mammals. One key question remains, however, whether CiVDS contributes to central synaptic transmission. Here, we report, in the central transmission from presynaptic (dorsal root ganglion) to postsynaptic (spinal dorsal horn) neurons in vitro, (i) excitatory postsynaptic currents (EPSCs) are mediated by glutamate transmission through both CiVDS (up to 87%) and CDS; (ii) CiVDS-mediated EPSCs are independent of extracellular and intracellular Ca2+ ; (iii) CiVDS is faster than CDS in vesicle recycling with much less short-term depression; (iv) the fusion machinery of CiVDS includes Cav2.2 (voltage sensor) and SNARE (fusion pore). Together, an essential component of activity-induced EPSCs is mediated by CiVDS in a central synapse.