Neurons In Laminae Research Articles

High-frequency spinal cord stimulation treatment attenuates the increase in spinal glutamate release and spinal miniature excitatory postsynaptic currents in rats with spared nerve injury-induced neuropathic pain

High-frequency spinal cord stimulation (HFSCS) at 10 kHz provides paresthesia-free treatment for chronic pain. However, the underlying mechanisms of its action have not been fully elucidated. The aim of the present study was to investigate the effect of HFSCS treatment on spinal glutamate release and uptake in spared nerve injury (SNI) rats. HFSCS was applied to the T10/T11 spinal cord 3 days after SNI. The concentration of spinal glutamate, glutamate transporter activity and miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II were evaluated. HFSCS treatment alleviated SNI pain induced by mechanical and cold allodynia. HFSCS treatment also partially restored altered spinal glutamate uptake activity, the levels of spinal glutamate, and the frequency of mEPSCs following SNI. In conclusion, HFSCS treatment attenuated SNI-induced neuropathic pain and partially restored the altered glutamate uptake after SNI.

In-Depth Characterization of Layer 5 Output Neurons of the Primary Somatosensory Cortex Innervating the Mouse Dorsal Spinal Cord.

Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK+ S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing.

Intracellular Calcium Responses Encode Action Potential Firing in Spinal Cord Lamina I Neurons.

Maladaptive plasticity of neurons in lamina I of the spinal cord is a lynchpin for the development of chronic pain, and is critically dependent on intracellular calcium signaling. However, the relationship between neuronal activity and intracellular calcium in these neurons is unknown. Here we combined two-photon calcium imaging with whole-cell electrophysiology to determine how action potential firing drives calcium responses within subcellular compartments of male rat spinal cord lamina I neurons. We found that single action potentials generated at the soma increase calcium concentration in the somatic cytosol and nucleus, and these calcium responses invade dendrites and dendritic spines by active backpropagation. Calcium responses in each compartment were dependent on voltage-gated calcium channels, and somatic and nuclear calcium responses were amplified by release of calcium from ryanodine-sensitive intracellular stores. Grouping single action potential-evoked calcium responses by neuron type demonstrated their presence in all defined types, as well as a high degree of similarity in calcium responses between neuron types. With bursts of action potentials, we found that calcium responses have the capacity to encode action potential frequency and number in all compartments, with action potential number being preferentially encoded. Together, these findings indicate that intracellular calcium serves as a readout of neuronal activity within lamina I neurons, providing a unifying mechanism through which activity may regulate plasticity, including that seen in chronic pain.SIGNIFICANCE STATEMENT Despite their critical role in both acute pain sensation and chronic pain, little is known of the fundamental physiology of spinal cord lamina I neurons. This is especially the case with respect to calcium dynamics within these neurons, which could regulate maladaptive plasticity observed in chronic pain. By combining two-photon calcium imaging and patch-clamp electrophysiological recordings from lamina I neurons, we found that action potential firing induces calcium responses within the somatic cytosol, nucleus, dendrites, and dendritic spines of lamina I neurons. Our findings demonstrate the presence of actively backpropagating action potentials, shifting our understanding of how these neurons process information, such that calcium provides a mechanism for lamina I neurons to track their own activity.

Antagonistic regulation by insulin-like peptide and activin ensures the elaboration of appropriate dendritic field sizes of amacrine neurons.

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.

Blockade of BDNF signalling attenuates chronic visceral hypersensitivity in an IBS-like rat model.

BackgroundIrritable bowel syndrome (IBS) is a common functional disease characterized by chronic abdominal pain and changes in bowel movements. Effective therapy for visceral hypersensitivity in IBS patients remains challenging. This study investigated the roles of brain‐derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) and the effect of ANA‐12 (a selective antagonist of TrkB) on chronic visceral hypersensitivity in an IBS‐like rat model.MethodsAn IBS‐like rat model was established through neonatal maternal separation (NMS), and visceral hypersensitivity was assessed by electromyographic (EMG) responses of the abdominal external oblique muscles to colorectal distention (CRD). Different doses of ANA‐12 were injected intrathecally to investigate the effect of that drug on visceral hypersensitivity, and the open field test was performed to determine whether ANA‐12 had side effects on movement. Thoracolumbar spinal BDNF, TrkB receptor and Protein kinase Mζ (PKMζ) expression were measured to investigate their roles in chronic visceral hypersensitivity. Whole‐cell recordings were made from thoracolumbar superficial dorsal horn (SDH) neurons of lamina II.ResultsThe expression of BDNF and TrkB was enhanced in the thoracolumbar spinal cord of the NMS animals. ANA‐12 attenuated visceral hypersensitivity without side effects on motricity in NMS rats. PKMζ expression significantly decreased after the administration of ANA‐12. The frequency of spontaneous excitatory postsynaptic currents (sEPSCs) increased in the thoracolumbar SDH neurons of lamina II in NMS rats. The amplitude and frequency of sEPSCs were reduced after perfusion with ANA‐12 in NMS rats.ConclusionsNeonatal maternal separation caused visceral hypersensitivity and increased synaptic activity by activating BDNF‐TrkB‐PKMζ signalling in the thoracolumbar spinal cord of adult rats. PKMζ was able to potentiate AMPA receptor (AMPAR)‐mediated sEPSCs in NMS rats. ANA‐12 attenuated visceral hypersensitivity and synaptic activity by blocking BDNF/TrkB signalling in NMS rats.SignificanceANA‐12 attenuates visceral hypersensitivity via BDNF‐TrkB‐PKMζ signalling and reduces synaptic activity through AMPARs in NMS rats. This knowledge suggests that ANA‐12 could represent an interesting novel therapeutic medicine for chronic visceral hypersensitivity.

Neural processing of linearly and circularly polarized light signal in a mantis shrimp Haptosquilla pulchella.

Stomatopods, or mantis shrimp, are the only animal group known to possess circular polarization vision along with linear polarization vision. By using the rhabdomere of a distally located photoreceptor as a wave retarder, the eyes of mantis shrimp are able to convert circularly polarized light into linearly polarized light. As a result, their circular polarization vision is based on the linearly polarized light-sensitive photoreceptors commonly found in many arthropods. To investigate how linearly and circularly polarized light signals might be processed, we presented a dynamic polarized light stimulus while recording from photoreceptors or lamina neurons in intact mantis shrimp Haptosquilla pulchella The results indicate that all the circularly polarized light-sensitive photoreceptors also showed differential responses to the changing e-vector angle of linearly polarized light. When stimulated with linearly polarized light of varying e-vector angle, most photoreceptors produced a concordant sinusoidal response. In contrast, some lamina neurons doubled the response frequency in reacting to linearly polarized light. These responses resembled a rectified sum of two-channel linear polarization-sensitive photoreceptors, indicating that polarization visual signals are processed at or before the first optic lobe. Noticeably, within the lamina, there was one type of neuron that showed a steady depolarization response to all stimuli except right-handed circularly polarized light. Together, our findings suggest that, between the photoreceptors and lamina neurons, linearly and circularly polarized light may be processed in parallel and differently from one another.

Distinct expression of potassium channels regulates visual response properties of lamina neurons in Drosophila melanogaster.

The computational organization of sensory systems depends on the diversification of individual cell types with distinct signal-processing capabilities. The Drosophila visual system, for instance, splits information into channels with different temporal properties directly downstream of photoreceptors in the first-order interneurons of the OFF pathway, L2 and L3. However, the biophysical mechanisms that determine this specialization are largely unknown. Here, we show that the voltage-gated Ka channels Shaker and Shal contribute to the response properties of the major OFF pathway input L2. L3 calcium response kinetics postsynaptic to photoreceptors resemble the sustained calcium signals of photoreceptors, whereas L2 neurons decay transiently. Based on a cell-type-specific RNA-seq data set and endogenous protein tagging, we identified Shaker and Shal as the primary candidates to shape L2 responses. Using in vivo two-photon imaging of L2 calcium signals in combination with pharmacological and genetic perturbations of these Ka channels, we show that the wild-type Shaker and Shal function is to enhance L2 responses and cell-autonomously sharpen L2 kinetics. Our results reveal a role for Ka channels in determining the signal-processing characteristics of a specific cell type in the visual system.

NMDA receptor activation induces long-term potentiation of glycine synapses.

Of the fast ionotropic synapses, glycinergic synapses are the least well understood, but are vital for the maintenance of inhibitory signaling in the brain and spinal cord. Glycinergic signaling comprises half of the inhibitory signaling in the spinal cord, and glycinergic synapses are likely to regulate local nociceptive processing as well as the transmission to the brain of peripheral nociceptive information. Here we have investigated the rapid and prolonged potentiation of glycinergic synapses in the superficial dorsal horn of young male and female mice after brief activation of NMDA receptors (NMDARs). Glycinergic inhibitory postsynaptic currents (IPSCs) evoked with lamina II-III stimulation in identified GABAergic neurons in lamina II were potentiated by bath-applied Zn2+ and were depressed by the prostaglandin PGE2, consistent with the presence of both GlyRα1- and GlyRα3-containing receptors. NMDA application rapidly potentiated synaptic glycinergic currents. Whole-cell currents evoked by exogenous glycine were also readily potentiated by NMDA, indicating that the potentiation results from altered numbers or conductance of postsynaptic glycine receptors. Repetitive depolarization alone of the postsynaptic GABAergic neuron also potentiated glycinergic synapses, and intracellular EGTA prevented both NMDA-induced and depolarization-induced potentiation of glycinergic IPSCs. Optogenetic activation of trpv1 lineage afferents also triggered NMDAR-dependent potentiation of glycinergic synapses. Our results suggest that during peripheral injury or inflammation, nociceptor firing during injury is likely to potentiate glycinergic synapses on GABAergic neurons. This disinhibition mechanism may be engaged rapidly, altering dorsal horn circuitry to promote the transmission of nociceptive information to the brain.

Chronic pain models amplify transient receptor potential vanilloid 1 (TRPV1) receptor responses in adult rat spinal dorsal horn

Persistent pain is associated with negative affect originating from hypersensitivity and/or allodynia. The spinal cord is a key area for nociception as well as chronic pain processing. Specifically, the dorsal horn neurons in lamina II (substantia gelatinosa: SG) receive nociceptive inputs from primary afferents such as C fibers and/or Aδ fibers. Transient receptor potential vanilloid 1 (TRPV1) is a major receptor to sense heat as well as nociception. TRPV1 are expressed in the periphery and the central axon terminals of C fibers and/or Aδ fibers in the spinal cord. Activating TRPV1 enhances the release of glutamate in the spinal cord from naïve rodents.Here, we studied whether or not chronic pain could alter the response of TRPV1 channels to exogenous, capsaicin through study of synaptic transmission and neural activity in rat SG neurons. Using in vitro whole-cell patch-clamp recording, we found that bath application of capsaicin facilitated both the frequency and amplitude of miniature and spontaneous excitatory postsynaptic currents beyond a nerve injury and a complete Freund's adjuvant injection observed in the naïve group. Strikingly, capsaicin produced larger amplitudes of inward currents in pain models than compared to the naïve group. By contrast, the proportions of neurons that show capsaicin-induced inward currents were similar among naïve and pain groups. Importantly, the capsaicin-induced inward currents were conducted by TRPV1 and required calcium influx that was independent of voltage-gated calcium channels.Our study provides fundamental evidence that chronic inflammation and neuropathic pain models amplify the release of glutamate through the activation of TRPV1 in central axon terminals, and that facilitation of TRPV1 function in rat spinal SG neurons may contribute to enhanced capsaicin-induced inward currents.

Expression of Neuropeptide FF Defines a Population of Excitatory Interneurons in the Superficial Dorsal Horn of the Mouse Spinal Cord that Respond to Noxious and Pruritic Stimuli

The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch.

Recruitment of Spinoparabrachial Neurons by Dorsal Horn Calretinin Neurons

The dorsal horn of the spinal cord is the first integration site of somatosensory inputs from the periphery. In the superficial layers of the dorsal horn, nociceptive inputs are processed by a complex network of excitatory and inhibitory interneurons whose function and connectivity remain poorly understood. We examined the role of calretinin-expressing interneurons (CR neurons) in such processing and show that they receive direct inputs from nociceptive fibers and polysynaptic inputs from touch-sensitive Aβ fibers. Their activation by chemogenetic or optogenetic stimulation produces mechanical allodynia and nocifensive responses. Furthermore, they monosynaptically engage spinoparabrachial (SPb) neurons in lamina I, suggesting CR neurons modulate one of the major ascending pain pathways of the dorsal horn. In conclusion, we propose a neuronal pathway in which CR neurons are positioned at the junction between nociceptive and innocuous circuits and directly control SPb neurons in lamina I.

Properties of neurons in the superficial laminae of trigeminal nucleus caudalis.

The trigeminal nucleus caudalis (TNc) receives extensive afferent innervation from peripheral sensory neurons of the trigeminal ganglion (TG), and is the first central relay in the circuitry underpinning orofacial pain. Despite the initial characterization of the neurons in the superficial laminae, many questions remain. Here we report on electrophysiological properties of 535 superficial lamina I/II TNc neurons. Based on their firing pattern, we assigned these cells to five main groups, including (1) tonic, (2) phasic, (3) delayed, (4) H‐current, and (5) tonic‐phasic neurons, groups that exhibit distinct intrinsic properties and share some similarity with groups identified in the spinal dorsal horn. Driving predominantly nociceptive TG primary afferents using optogenetic stimulation in TRPV1/ChR2 animals, we found that tonic and H‐current cells are most likely to receive pure monosynaptic input, whereas delayed neurons are more likely to exhibit inputs that appear polysynaptic. Finally, for the first time in TNc neurons, we used unsupervised clustering analysis methods and found that the kinetics of the action potentials and other intrinsic properties of these groups differ significantly from one another. Unsupervised spectral clustering based solely on a single voltage response to rheobase current was sufficient to group cells with shared properties independent of action potential discharge pattern, indicating that this approach can be effectively applied to identify functional neuronal subclasses. Together, our data illustrate that cells in the TNc with distinct patterns of TRPV1/ChR2 afferent innervation are physiologically diverse, but can be understood as a few major groups of cells having shared functional properties.

Acute spatial spread of NO-mediated potentiation during hindpaw ischaemia in mice.

Key points Neuropathic pain spreads spatially beyond the injured sites, and the mechanism underlying the spread has been attributed to inflammation occurring in the spinal cord.However, the spatial spread of spinal/cortical potentiation induced by conduction block of the peripheral nerves can be observed prior to inflammation.In the present study, we found that spreading potentiation and hypersensitivity acutely induced by unilateral hindpaw ischaemia are nitric oxide (NO)‐dependent and that NO is produced by ischaemia and quickly diffuses within the spinal cord.We also found that NO production induced by ischaemia is not observed in the presence of an antagonist for group II metabotropic glutamate receptors (mGluRs) and that neuronal NO synthase‐positive dorsal horn neurons express group II mGluRs.These results suggest strongly that NO‐mediated spreading potentiation in the spinal cord is one of the trigger mechanisms for neuropathic pain. Cortical/spinal responses to hindpaw stimulation are bilaterally potentiated by unilateral hindpaw ischaemia in mice. We tested the hypothesis that hindpaw ischaemia produces nitric oxide (NO), which diffuses in the spinal cord to induce spatially spreading potentiation. Using flavoprotein fluorescence imaging, we confirmed that the spreading potentiation in hindpaw responses was induced during ischaemia in the non‐stimulated hindpaw. This spreading potentiation was blocked by spinal application of l‐NAME, an inhibitor of NO synthase (NOS). Furthermore, no spreading potentiation was observed in neural NOS (nNOS) knockout mice. Spinal application of an NO donor was enough to induce cortical potentiation and mechanical hypersensitivity. The spatial distribution of NO during unilateral hindpaw ischaemia was visualized using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein (DAF‐FM). An increase in fluorescence derived from the complex of DAF‐FM with NO was observed on the ischaemic side of the spinal cord. A similar but smaller increase was also observed on the contralateral side. Somatosensory potentiation after hindpaw ischaemia is known to be inhibited by spinal application of LY354740, an agonist of group II metabotropic glutamate receptors (mGluRs). We confirmed that the spinal DAF‐FM fluorescence increases during hindpaw ischaemia were not observed in the presence of LY354740. We also confirmed that approximately half of the nNOS‐positive neurons in the superficial laminae of the dorsal horn expressed mGluR2 mRNA. These results suggest that disinhibition of mGluR2 produces NO which in turn induces a spreading potentiation in a wide area of the spinal cord. Such spreading, along with the consequent non‐specific potentiation in the spinal cord, may trigger neuropathic pain.

Distribution of brain derived neurotrophic factor (BDNF) immunostaining along the C2–C5 spinal cord in adult rats

BDNF is a powerful modulator of neuroplasticity and can be found across the rostral‐caudal extent of the spinal neuroaxis in neurons, astrocytes and microglia. BDNF is necessary and sufficient to drive plasticity in the phrenic motor system, likely through either pre‐ and/or post‐synaptic actions on phrenic motoneurons. To our knowledge, the relative distribution of BDNF expression across this region of the mid‐cervical spinal cord has not been systematically “mapped”. Accordingly, in ongoing experiments, we are quantitatively evaluating the distribution of BDNF positive neurons in the C2–C5 spinal cord in adult Sprague‐Dawley rats. The guiding hypothesis is that BDNF will be most prevalent in the C3‐5 ventral horn in the region of the phrenic motor nucleus. Tissues were harvested from adult male rats and processed with standard immunochemistry methods using a BDNF antibody (Alomone, 1:500 dilution). Control experiments showed an absence of spinal cord staining when tissues were incubated without the primary antibody and while keeping all other aspects constant. A custom MATLAB code was used to quantify the number of BDNF positive cells throughout C2–C5. Specifically, BDNF expressing soma were identified as areas with fluorescent intensity above a common threshold. To separate putative neurons, identified regions were sorted by size and only regions with an area of 250–1500 μm2 were included for further analysis. We observed BDNF expression within neurons in laminae I, II, VII, VIII, and IX across the mid‐cervical spinal cord. Further, the number of BDNF positive neurons increased in a rostral to caudal manner from C2 to C5 (P<0.05). There were more BDNF expressing neurons located in the ventral horn when compared to the dorsal horn across C2–C5 (p< 0.01). Finally, smaller BDNF positive cells were evident throughout the gray and white matter which is consistent with microglial distribution in the spinal cord, however, this staining has not been verified at this time. These initial results suggest that naturally occurring levels of BDNF vary across the cervical spinal cord, and are greatest in the region of the phrenic motor nucleus.Support or Funding Information1R01HL139708‐01A1 (DDF), T32‐HD043730 (MDS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem.The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood.Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group.Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

The glial sodium-potassium-2-chloride cotransporter is required for synaptic transmission in the Drosophila visual system

The Drosophila Ncc69 gene encodes a Na+-K+-2Cl−-cotransporter (NKCC) that is critical for regulating intra- and extracellular ionic conditions in different tissues. Here, we show that the Ncc69 transporter is necessary for fly vision and that its expression is required non-autonomously in glia to maintain visual synaptic transmission. Flies mutant for Ncc69 exhibit normal photoreceptor depolarization in response to a light pulse but lack the ON and OFF-transients characteristic of postsynaptic responses of lamina neurons, indicating a failure in synaptic transmission. We also find that synaptic transmission requires the Ncc69 regulatory kinases WNK and Fray in glia. The ERG phenotype is associated with a defect in the recycling of the histamine neurotransmitter. Ncc69 mutants exhibit higher levels of the transport metabolite carcinine in lamina cartridges, with its accumulation most intense in the extracellular space. Our work reveals a novel role of glial NKCC transporters in synaptic transmission, possibly through regulating extracellular ionic conditions.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn.

Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non‐overlapping populations in laminae I‐III, based on expression of substance P, gastrin‐releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I‐II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin‐releasing peptide populations, but show limited overlap with the substance P cells. Laminae II‐III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ‐immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin‐releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.

The Neural Pathways for the Hypothetic Nociceptive-Sympathetic Coupling of NBA for Pain Sensation

In May 2016, it was suggested by the US athletes playing games of National Basketball Association (NBA) in television that the pain sensation might result from the post-sensory nociceptive-sympathetic coupling. In this re-view, it is attempted to newly delineate the underlying neural pathways for this post-sensory nociceptive-sympathetic coupling. Based on the con-temporary feedback neural circuits of pain transmission in spinal cord, it is summarized that the Aδ- and C-fibers relay the sensory noxious signals to the laminae I, II and V of dorsal spinal horn, and in turn activate the peria-queductal grey (PAG) in midbrain, which feedbacks via raphe to inhibit the nociceptive transmission of spinal cord. Herein it is newly added that, as extension of the contemporary feedback pain circuits, the neurons in laminae I and V of dorsal spinal horn as well as those in PAG can additionally activate the sympathetic outputs, completing the post-sensory nocicep-tive-sympathetic coupling. It is emphasized that such nocicep-tive-sympathetic coupling would contribute at least partially to the pain sensation because of sympathetically maintained pain. It is also pointed out that the neurons in parabrachial area (PB) can concurrently relay the nociceptive inputs from spinal cord while regulating the respiration, coor-dinating the nociceptive and respiratory activities during pain sensation. Because both PAG and PB are below thalamus, it is speculated that these sympathetic and respiratory associations with pain are primitive and uni-versal in evolution in vertebrates. It is expected to promote this hypothesis to revise the pain mechanisms in vertebrates and the relevant investigations in the future.

Expression of Calretinin Among Different Neurochemical Classes of Interneuron in the Superficial Dorsal Horn of the Mouse Spinal Cord

Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.

Neuron-Subtype-Specific Expression, Interaction Affinities, and Specificity Determinants of DIP/Dpr Cell Recognition Proteins

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies invivo.