Multimodal sensory control of motor performance by glycinergic interneurons of the mouse spinal cord deep dorsal horn.

Sensory feedback is integral for contextually appropriate motor output, yet the neural circuits responsible remain elusive. Here, we pinpoint the medial deep dorsal horn of the mouse spinal cord as a convergence point for proprioceptive and cutaneous input. Within this region, we identify a population of tonically active glycinergic inhibitory neurons expressing parvalbumin. Using anatomy and electrophysiology, we demonstrate that deep dorsal horn parvalbumin-expressing interneuron (dPV) activity is shaped by convergent proprioceptive, cutaneous, and descending input. Selectively targeting spinal dPVs, we reveal their widespread ipsilateral inhibition onto pre-motor and motor networks and demonstrate their role in gating sensory-evoked muscle activity using electromyography (EMG) recordings. dPV ablation altered limb kinematics and step-cycle timing during treadmill locomotion and reduced the transitions between sub-movements during spontaneous behavior. These findings reveal a circuit basis by which sensory convergence onto dorsal horn inhibitory neurons modulates motor output to facilitate smooth movement and context-appropriate transitions.

Acute inhibition of acid sensing ion channel 1a after spinal cord injury selectively affects excitatory synaptic transmission, but not intrinsic membrane properties, in deep dorsal horn interneurons.

Following a spinal cord injury (SCI), secondary damage mechanisms are triggered that cause inflammation and cell death. A key component of this secondary damage is a reduction in local blood flow that initiates a well-characterised ischemic cascade. Downstream hypoxia and acidosis activate acid sensing ion channel 1a (ASIC1a) to trigger cell death. We recently showed that administration of a potent venom-derived inhibitor of ASIC1a, Hi1a, leads to tissue sparing and improved functional recovery when delivered up to 8 h after ischemic stroke. Here, we use whole-cell patch-clamp electrophysiology in a spinal cord slice preparation to assess the effect of acute ASIC1a inhibition, via a single dose of Hi1a, on intrinsic membrane properties and excitatory synaptic transmission long-term after a spinal cord hemisection injury. We focus on a population of interneurons (INs) in the deep dorsal horn (DDH) that play a key role in relaying sensory information to downstream motoneurons. DDH INs in mice treated with Hi1a 1 h after a spinal cord hemisection showed no change in active or passive intrinsic membrane properties measured 4 weeks after SCI. DDH INs, however, exhibit significant changes in the kinetics of spontaneous excitatory postsynaptic currents after a single dose of Hi1a, when compared to naive animals (unlike SCI mice). Our data suggest that acute ASIC1a inhibition exerts selective effects on excitatory synaptic transmission in DDH INs after SCI via specific ligand-gated receptor channels, and has no effect on other voltage-activated channels long-term after SCI.

Network model of nociceptive processing in the superficial spinal dorsal horn reveals mechanisms of hyperalgesia, allodynia, and spinal cord stimulation.

The spinal dorsal horn (DH) processes sensory information and plays a key role in transmitting nociception to supraspinal centers. Loss of DH inhibition during neuropathic pain unmasks a pathway from nonnociceptive Aß-afferent inputs to superficial dorsal horn (SDH) nociceptive-specific (NS) projection neurons, and this change may contribute to hyperalgesia and allodynia. We developed and validated a computational model of SDH neuronal circuitry that links nonnociceptive Aß-afferent inputs in lamina II/III to a NS projection neuron in lamina I via a network of excitatory interneurons. The excitatory pathway and the NS projection neuron were in turn gated by inhibitory interneurons with connections based on prior patch-clamp recordings. Changing synaptic weights in the computational model to replicate neuropathic pain states unmasked a low-threshold excitatory pathway to NS neurons similar to experimental recordings. Spinal cord stimulation (SCS) is an effective therapy for neuropathic pain, and accumulating experimental evidence indicates that NS neurons in the SDH also respond to SCS. Accounting for these responses may inform therapeutic improvements, and we quantified responses to SCS in the SDH network model and examined the role of different modes of inhibitory control in modulating NS neuron responses to SCS. We combined the SDH network model with a previously published model of the deep dorsal horn (DDH) and identified optimal stimulation frequencies across different neuropathic pain conditions. Finally, we found that SCS-generated inhibition did not completely suppress model NS activity during simulated pinch inputs, providing an explanation of why SCS does not eliminate acute pain.NEW & NOTEWORTHY Chronic pain is a severe public health problem that reduces the quality of life for those affected and exacts an enormous socio-economic burden worldwide. Spinal cord stimulation (SCS) is an effective treatment for chronic pain, but SCS efficacy has not significantly improved over time, in part because the mechanisms of action remain unclear. Most preclinical studies investigating pain and SCS mechanisms have focused on the responses of deep dorsal horn (DDH) neurons, but neural networks in the superficial dorsal horn (SDH) are also important for processing nociceptive information. This work synthesizes heterogeneous experimental recordings from the SDH into a computational model that replicates experimental responses and that can be used to quantify neuronal responses to SCS under neuropathic pain conditions.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Inhibitory Kcnip2 neurons of the spinal dorsal horn control behavioral sensitivity to environmental cold.

Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2GlyT2) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity.

Ca2+ -independent transmission at the central synapse formed between dorsal root ganglion and dorsal horn neurons.

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.

Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line.

Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as "antenna cells", which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only ∼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.

ErbB4+ spinal cord dorsal horn neurons process heat pain.

How the spinal cord transmits heat signals from the periphery to the brain remains unclear. In this issue of Neuron, Wang et al. (2022) identify a population of spinal cord neurons functioning in this pathway.

GABAB receptors constrain glutamate presynaptic release and postsynaptic actions in substantia gelatinosa of rat spinal cord.

The substantia gelatinosa (SG, lamina II of spinal cord gray matter) is pivotal for modulating nociceptive information from the peripheral to the central nervous system. γ-Aminobutyric acid type B receptors (GABABRs), the metabotropic GABA receptor subtype, are widely expressed in pre- and postsynaptic structures of the SG. Activation of GABABRs by exogenous agonists induces both pre- and postsynaptic inhibition. However, the actions of endogenous GABA via presynaptic GABABRs on glutamatergic synapses, and the postsynaptic GABABRs interaction with glutamate, remain elusive. In the present study, first, using in vitro whole-cell recordings and taking minimal stimulation strategies, we found that in rat spinal cord glutamatergic synapses, blockade of presynaptic GABABRs switched "silent" synapses into active ones and increased the probability of glutamate release onto SG neurons; increasing ambient GABA concentration mimicked GABABRs activation on glutamatergic terminals. Next, using holographic photostimulation to uncage glutamate on postsynaptic SG neurons, we found that postsynaptic GABABRs modified glutamate-induced postsynaptic potentials. Taken together, our data identify that endogenous GABA heterosynaptically constrains glutamate release via persistently activating presynaptic GABABRs; and postsynaptically, GABABRs modulate glutamate responses. The results give new clues for endogenous GABA in modulating the nociception circuit of the spinal dorsal horn and shed fresh light on the postsynaptic interaction of glutamate and GABA.

Discrete field potentials produced by coherent activation of spinal dorsal horn neurons.

In addition to the action potentials generated by the ongoing activation of single dorsal horn neurons in the anesthetized cat, we often recorded small negative field potentials with a fast-rising phase and a slow decay (dIFPs). These potentials could be separated in different classes, each with a specific and rather constant shape and amplitude. They were largest in spinal laminae III-V and gradually faded at deeper locations, without showing the polarity reversal displayed at these depths by the focal potentials produced by stimulation of muscle and cutaneous afferents. We propose that the dIFPs are postsynaptic field potentials generated by strongly coupled sets of dorsal horn neurons displaying a spatial orientation that generates closed field potentials in response to stimulation of high-threshold cutaneous and muscle afferents. These neuronal sets could form part of the spinal inhibitory circuitry that mediates presynaptic inhibition and Ib non-reciprocal postsynaptic inhibition and could be involved in the sensory-motor transformations activated by stimulation of high-threshold cutaneous afferents.

Dorsal column and root stimulation at Aß-fiber intensity activate superficial dorsal horn glutamatergic and GABAergic populations.

Neurostimulation therapies are frequently used in patients with chronic pain conditions. They emerged from Gate Control Theory (GCT), which posits that Aß-fiber activation recruits superficial dorsal horn (SDH) inhibitory networks to "close the gate" on nociceptive transmission, resulting in pain relief. However, the efficacy of current therapies is limited, and the underlying circuits remain poorly understood. For example, it remains unknown whether ongoing stimulation of Aß-fibers is sufficient to drive activity in SDH neurons. We used multiphoton microscopy in spinal cords extracted from mice expressing the genetically encoded calcium indicator GCaMP6s in glutamatergic and GABAergic populations; activity levels were inferred from deconvolved calcium signals using CaImAn software. Sustained Aß-fiber stimulation at the dorsal columns or dorsal roots drove robust yet transient activation of both SDH populations. Following the initial increase, activity levels decreased below baseline in glutamatergic neurons and were depressed after stimulation ceased in both populations. Surprisingly, only about half of GABAergic neurons responded to Aß-fiber stimulation. This subset showed elevated activity for the entire duration of stimulation, while non-responders decreased with time. Our findings suggest that Aß-fiber stimulation initially recruits both excitatory and inhibitory populations but has divergent effects on their activity, providing a foundation for understanding the analgesic effects of neurostimulation devices.Perspective: This article used microscopy to characterize the responses of mouse spinal cord cells to stimulation of non-painful nerve fibers. These findings deepen our understanding of how the spinal cord processes information and provide a foundation for improving pain-relieving therapies.

Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

Diversity of inhibitory and excitatory parvalbumin interneuron circuits in the dorsal horn.

ABSTRACT: Parvalbumin-expressing interneurons (PVINs) in the spinal dorsal horn are found primarily in laminae II inner and III. Inhibitory PVINs play an important role in segregating innocuous tactile input from pain-processing circuits through presynaptic inhibition of myelinated low-threshold mechanoreceptors and postsynaptic inhibition of distinct spinal circuits. By comparison, relatively little is known of the role of excitatory PVINs (ePVINs) in sensory processing. Here, we use neuroanatomical and optogenetic approaches to show that ePVINs comprise a larger proportion of the PVIN population than previously reported and that both ePVIN and inhibitory PVIN populations form synaptic connections among (and between) themselves. We find that these cells contribute to neuronal networks that influence activity within several functionally distinct circuits and that aberrant activity of ePVINs under pathological conditions is well placed to contribute to the development of mechanical hypersensitivity.

Postnatal development of inner lamina II interneurons of the rat medullary dorsal horn.

ABSTRACT: Pain processing in young mammals is immature. Despite the central role of the medullary dorsal horn (MDH) in processing orofacial sensory information, the maturation of the neurons within the MDH has been largely overlooked. Combining in vitro electrophysiological recordings and 3D morphological analysis over the first postnatal month in rats, we investigated the age-dependent development of the neurons within the inner lamina II (IIi) of the MDH. We show the lamina IIi neuronal population transition into a more hyperpolarized state, with modification of the action potential waveform, and a shift from single spiking, at early postnatal ages, to tonic firing and initial bursting at later stages. These physiological changes are associated with a strong structural remodelling of the neuronal morphology with most of the modifications occurring after the third postnatal week. Among the lamina IIi neuronal population, the subpopulation of interneurons expressing the γ isoform of the protein kinase C (PKCγ+) are key elements for the circuits underlying facial mechanical allodynia. How do they develop from the rest of the lamina IIi constitute an important question that remained to be addressed. Here, we show that PKCγ+ interneurons display electrophysiological changes over time comparable with the PKCγ- population. However, they show a distinctive increase of the soma volume and primary branches length, as opposed to the PKCγ- population. Together, our data demonstrate a novel pattern of late postnatal maturation of lamina IIi interneurons, with a spotlight on PKCγ+ interneurons, that may be relevant for the development of orofacial sensitivity.

The coming together of allosteric and phosphorylation mechanisms in the molecular integration of A2A heteroreceptor complexes in the dorsal and ventral striatal-pallidal GABA neurons.

The role of adenosine A2A receptor (A2AR) and striatal-enriched protein tyrosine phosphatase (STEP) interactions in the striatal-pallidal GABA neurons was recently discussed in relation to A2AR overexpression and cocaine-induced increases of brain adenosine levels. As to phosphorylation, combined activation of A2AR and metabotropic glutamate receptor 5 (mGluR5) in the striatal-pallidal GABA neurons appears necessary for phosphorylation of the GluA1 unit of the AMPA receptor to take place. Robert Yasuda (J Neurochem 152: 270-272, 2020) focused on finding a general mechanism by which STEP activation is enhanced by increased A2AR transmission in striatal-pallidal GABA neurons expressing A2AR and dopamine D2 receptor. In his Editorial, he summarized in a clear way the significant effects of A2AR activation on STEP in the dorsal striatal-pallidal GABA neurons which involves a rise of intracellular levels of calcium causing STEP activation through its dephosphorylation. However, the presence of the A2AR in an A2AR-fibroblast growth factor receptor 1 (FGFR1) heteroreceptor complex can be required in the dorsal striatal-pallidal GABA neurons for the STEP activation. Furthermore, Won et al. (Proc Natl Acad Sci USA 116: 8028-8037, 2019) found in mass spectrometry experiments that the STEP splice variant STEP61 can bind to mGluR5 and inactivate it. In addition, A2AR overexpression can lead to increased formation of A2AR-mGluR5 heterocomplexes in ventral striatal-pallidal GABA neurons. It involves enhanced facilitatory allosteric interactions leading to increased Gq-mediated mGluR5 signaling activating STEP. The involvement of both A2AR and STEP in the actions of cocaine on synaptic downregulation was also demonstrated. The enhancement of mGluR5 protomer activity by the A2AR protomer in A2AR-mGluR5 heterocomplexes in the nucleus accumbens shell appears to have a novel significant role in STEP mechanisms by both enhancing the activation of STEP and being a target for STEP61.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Differential Activation of Colonic Afferents and Dorsal Horn Neurons Underlie Stress-Induced and Comorbid Visceral Hypersensitivity in Female Rats.

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions. PERSPECTIVE: Chronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity.

Lamina-specific properties of spinal astrocytes.

Astrocytes are indispensable for proper neuronal functioning. Given the diverse needs of neuronal circuits and the variety of tasks astrocytes perform, the perceived homogeneous nature of astrocytes has been questioned. In the spinal dorsal horn, complex neuronal circuitries regulate the integration of sensory information of different modalities. The dorsal horn is organized in a distinct laminar manner based on termination patterns of high- and low-threshold afferent fibers and neuronal properties. Neurons in laminae I (L1) and II (L2) integrate potentially painful, nociceptive information, whereas neurons in lamina III (L3) and deeper laminae integrate innocuous, tactile information from the periphery. Sensory information is also integrated by an uncharacterized network of astrocytes. How these lamina-specific characteristics of neuronal circuits of the dorsal horn are of functional importance for properties of astrocytes is currently unknown. We addressed if astrocytes in L1, L2, and L3 of the upper dorsal horn of mice are differentially equipped for the needs of neuronal circuits that process sensory information of different modalities. We found that astrocytes in L1 and L2 were characterized by a higher density, higher expression of GFAP, Cx43, and GLAST and a faster coupling speed than astrocytes located in L3. L1 astrocytes were more responsive to Kir4.1 blockade and had higher levels of AQP4 compared to L3 astrocytes. In contrast, basic membrane properties, network formation, and somatic intracellular calcium signaling were similar in L1-L3 astrocytes. Our data indicate that the properties of spinal astrocytes are fine-tuned for the integration of nociceptive versus tactile information.

α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice.

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.