Dual contribution of ASIC1a channels in the spinal processing of pain information by deep projection neurons revealed by computational modeling.

Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but its exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of rat spinal neurons with computational modeling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a homomeric channels. A computational model of WDR neuron supplemented with different ASIC1a channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using the Texas coral snake ASIC-activating toxin (MitTx) and calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin). This study supports a dual contribution to windup of calcium permeable ASIC1a channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases.

Modulation of GABAergic Synaptic Transmission by NMDA Receptors in the Dorsal Horn of the Spinal Cord.

The dorsal horn (DH) of the spinal cord is an important structure involved in the integration of nociceptive messages. Plastic changes in the properties of neuronal networks in the DH underlie the development of analgesia as well as of hyperalgesia and allodynia in acute and chronic pain states. Two key mechanisms are involved in these chronic pain states: increased electrical activities and glutamate release leading to the recruitment of NMDAr and plastic changes in the synaptic inhibition. Although: (1) the balance between excitation and inhibition is known to play a critical role in the spinal network; and (2) plastic changes in spinal excitation and inhibition have been studied separately, the relationship between these two mechanisms has not been investigated in detail. In the present work, we addressed the role of NMDA receptors in the modulation of GABAergic synaptic transmission in the DH network. Using tight-seal whole-cell recordings on adult mice DH neurons, we characterized the effect of NMDAr activation on inhibitory synaptic transmission and more especially on the GABAergic one. Our results show that, in a subset of neurons recorded in lamina II, NMDAr activation facilitates spontaneous and miniature GABAergic synaptic transmission with a target specificity on GABAergic interneurons. In contrast, NMDA reduced the mean amplitude of evoked GABAergic IPSCs. These results show that NMDAr modulate GABAergic transmission by a presynaptic mechanism of action. Using a pharmacological approach, we investigated the composition of NMDAr involved in this modulation of GABAergic synaptic transmission. We found that the NMDA-induced facilitation was mediated by the activation of NMDAr containing GluN2C/D subunits. Altogether, our results bring new insights on nociceptive information processing in the spinal cord network and plastic changes in synaptic inhibition that could underlie the development and maintenance of chronic pain.

Inhibitory interneurons with differential plasticities at their connections tune excitatory-inhibitory balance in the spinal nociceptive system.

ABSTRACT: Networks of the dorsal horn of the spinal cord process nociceptive information from the periphery. In these networks, the excitation-inhibition balance is critical to shape this nociceptive information and to gate it to the brain where it is interpreted as pain. Our aim was to define whether short-term plasticity of inhibitory connections could tune this inhibition-excitation balance by differentially controlling excitatory and inhibitory microcircuits. To this end, we used spinal cord slices from adult mice expressing enhanced green fluorescent protein (eGFP) under the GAD65 promoter and recorded from both eGFP+ (putative inhibitory) and eGFP- (putative excitatory) neurons of lamina II while stimulating single presynaptic GABAergic interneurons at various frequencies. Our results indicate that GABAergic neurons of lamina II simultaneously contact eGFP- and eGFP+ neurons, but these connections display very different frequency-dependent short-term plasticities. Connections onto eGFP- interneurons displayed limited frequency-dependent changes and strong time-dependent summation of inhibitory synaptic currents that was however subjected to a tonic activity-dependent inhibition involving A1 adenosine receptors. By contrast, GABAergic connections onto eGFP+ interneurons expressed pronounced frequency-dependent depression, thus favoring disinhibition at these synapses by a mechanism involving the activation of GABAB autoreceptors at low frequency. Interestingly, the balance favors inhibition at frequencies associated with intense pain, whereas it favors excitation at frequencies associated with low pain. Therefore, these target-specific and frequency-specific plasticities allow to tune the balance between inhibition and disinhibition while processing frequency-coded information from primary afferents. These short-term plasticities and their modulation by A1 and GABAB receptors might represent an interesting target in pain-alleviating strategies.

Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice.

Female reproductive success relies on proper integration of circadian- and ovarian- signals to the hypothalamic-pituitary-gonadal axis in order to synchronize the preovulatory LH surge at the end of the ovarian follicular stage with the onset of the main active period. In this study, we used a combination of neuroanatomical and electrophysiological approaches to assess whether the hypothalamic neurons expressing Arg-Phe amide-related peptide (RFRP-3), a gonadotropin inhibitory peptide, exhibit daily and estrous stage dependent variations in female mice. Furthermore, we investigated whether arginine vasopressin (AVP), a circadian peptide produced by the suprachiamatic nucleus regulates RFRP-3 neurons. The number of c-Fos-positive RFRP-3 immunoreactive neurons is significantly reduced at the day-to-night transition with no difference between diestrus and proestrus. Contrastingly, RFRP neuron firing rate is higher in proestrus as compared to diestrus, independently of the time of the day. AVP immunoreactive fibers contact RFRP neurons with the highest density observed during the late afternoon of diestrus and proestrus. Application of AVP increases RFRP neurons firing in the afternoon (ZT6-10) of diestrus, but not at the same time point of proestrus, indicating that AVP signaling on RFRP neurons may depend on circulating ovarian steroids. Together, these studies show that RFRP neurons integrate both daily and estrogenic signals, which downstream may help to properly time the preovulatory LH surge.

Enhanced analgesic cholinergic tone in the spinal cord in a mouse model of neuropathic pain.

Endogenous acetylcholine (ACh) is an important modulator of nociceptive sensory processing in the spinal cord. An increased level of spinal ACh induces analgesia both in humans and rodents while interfering with cholinergic signaling is allodynic, demonstrating that a basal tone of spinal ACh modulates nociceptive responses in naïve animals. The plasticity undergone by this cholinergic system in chronic pain situation is unknown, and the mere presence of this tone in neuropathic animals is controversial. We have addressed these issues in mice through behavioral experiments, histology, electrophysiology and molecular biology, in the cuff model of peripheral neuropathy. Our behavior experiments demonstrate the persistence, and even increased impact of the analgesic cholinergic tone acting through nicotinic receptors in cuff animals. The neuropathy does not affect the number or membrane properties of dorsal horn cholinergic neurons, nor specifically the frequency of their synaptic inputs. The alterations thus appear to be in the neurons receiving the cholinergic signaling, which is confirmed by the fact that subthreshold doses of acetylcholinesterase (AChE) inhibitors in sham animals become anti-allodynic in cuff mice and by the altered expression of the ß2 nicotinic receptor subunit. Our results demonstrate that endogenous cholinergic signaling can be manipulated to relieve mechanical allodynia in animal models of peripheral neuropathy. Until now, AChE inhibitors have mainly been used in the clinics in situations of acute pain (parturition, post-operative). The fact that lower doses (thus with fewer side effects) could be efficient in chronic pain conditions opens new avenues for the treatment of neuropathic pain. SIGNIFICANCE STATEMENT: Chronic pain continues to be the most common cause of disability that impairs the quality of life, accruing enormous and escalating socio-economic costs. A better understanding of the plasticity of spinal neuronal networks, crucially involved in nociceptive processing, could help designing new therapeutic avenues. We here demonstrate that chronic pain modifies the spinal nociceptive network in such a way that it becomes more sensitive to cholinergic modulations. The spinal cholinergic system is responsible for an analgesic tone that can be exacerbated by acetylcholinesterase inhibitors, a property used in the clinic to relief acute pain (child birth, post-op). Our results suggest that lower doses of acetylcholinesterases, with even fewer side effects, could be efficient to relieve chronic pain.

Action of mefloquine/amitriptyline THN101 combination on neuropathic mechanical hypersensitivity in mice.

ABSTRACT: Tricyclic antidepressants that inhibit serotonin and noradrenaline reuptake, such as amitriptyline, are among the first-line treatments for neuropathic pain, which is caused by a lesion or disease affecting the somatosensory nervous system. These treatments are, however, partially efficient to alleviate neuropathic pain symptoms, and better treatments are still highly required. Interactions between neurons and glial cells participate in neuropathic pain processes, and importantly, connexins-transmembrane proteins involved in cell-cell communication-contribute to these interactions. In a neuropathic pain model in rats, mefloquine, a connexin inhibitor, has been shown to potentiate the antihyperalgesic effect of amitriptyline, a widely used antidepressant. In this study, we further investigated this improvement of amitriptyline action by mefloquine, using the cuff model of neuropathic pain in mice. We first observed that oral mefloquine co-treatment prolonged the effect of amitriptyline on mechanical hypersensitivity by 12 hours after administration. In addition, we showed that this potentiation was not due to pharmacokinetic interactions between the 2 drugs. Besides, lesional and pharmacological approaches showed that the prolonged effect was induced through noradrenergic descending pathways and the recruitment of α2 adrenoceptors. Another connexin blocker, carbenoxolone, also improved amitriptyline action. Additional in vitro studies suggested that mefloquine may also directly act on serotonin transporters and on adenosine A1 and A2A receptors, but drugs acting on these other targets failed to amplify amitriptyline action. Together, our data indicate that pharmacological blockade of connexins potentiates the therapeutic effect of amitriptyline in neuropathic pain.

[Nociception pain and autism]. / Nociception, douleur et autisme.

Autistic subjects frequently display sensory anomalies. Those regarding nociception and its potential outcome, pain, are of crucial interest. Indeed, because of numerous comorbidities, autistic subjects are more often exposed to painful situation. Despite being often considered as less sensitive, experimental studies evaluating this point are failing to reach consensus. Using animal model can help reduce variability and bring, regarding autism, an overview of potential alterations of the nociceptive system at the cellular and molecular level.

TITLE: Nociception, douleur et autisme. ABSTRACT: Les sujets autistes présentent fréquemment des anomalies sensorielles. Celles concernant la nociception ainsi que sa potentielle résultante, la douleur, sont d'un intérêt capital. En effet, du fait de nombreuses comorbidités, les sujets autistes sont plus souvent exposés à des situations douloureuses que la population générale. Alors qu'ils sont souvent considérés comme moins sensibles, les études expérimentales sur ce point sont loin de faire consensus. Utiliser des modèles animaux pourrait permettre de s'affranchir de certaines sources de variabilité et d'apporter, dans le cadre de l'autisme, une vue d'ensemble des altérations potentielles du système nociceptif aux niveaux cellulaire et moléculaire.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

A Nonpeptide Oxytocin Receptor Agonist for a Durable Relief of Inflammatory Pain.

Oxytocin possesses several physiological and social functions, among which an important analgesic effect. For this purpose, oxytocin binds mainly to its unique receptor, both in the central nervous system and in the peripheral nociceptive terminal axon in the skin. However, despite its interesting analgesic properties and its current use in clinics to facilitate labor, oxytocin is not used in pain treatment. Indeed, it is rapidly metabolized, with a half-life in the blood circulation estimated at five minutes and in cerebrospinal fluid around twenty minutes in humans and rats. Moreover, oxytocin itself suffers from several additional drawbacks: a lack of specificity, an extremely poor oral absorption and distribution, and finally, a lack of patentability. Recently, a first non-peptide full agonist of oxytocin receptor (LIT-001) of low molecular weight has been synthesized with reported beneficial effect for social interactions after peripheral administration. In the present study, we report that a single intraperitoneal administration of LIT-001 in a rat model induces a long-lasting reduction in inflammatory pain-induced hyperalgesia symptoms, paving the way to an original drug development strategy for pain treatment.

Cav3.2 T-type calcium channels shape electrical firing in mouse Lamina II neurons.

The T-type calcium channel, Cav3.2, is necessary for acute pain perception, as well as mechanical and cold allodynia in mice. Being found throughout sensory pathways, from excitatory primary afferent neurons up to pain matrix structures, it is a promising target for analgesics. In our study, Cav3.2 was detected in ~60% of the lamina II (LII) neurons of the spinal cord, a site for integration of sensory processing. It was co-expressed with Tlx3 and Pax2, markers of excitatory and inhibitory interneurons, as well as nNOS, calretinin, calbindin, PKCγ and not parvalbumin. Non-selective T-type channel blockers slowed the inhibitory but not the excitatory transmission in LII neurons. Furthermore, T-type channel blockers modified the intrinsic properties of LII neurons, abolishing low-threshold activated currents, rebound depolarizations, and blunting excitability. The recording of Cav3.2-positive LII neurons, after intraspinal injection of AAV-DJ-Cav3.2-mcherry, showed that their intrinsic properties resembled those of the global population. However, Cav3.2 ablation in the dorsal horn of Cav3.2GFP-Flox KI mice after intraspinal injection of AAV-DJ-Cav3.2-Cre-IRES-mcherry, had drastic effects. Indeed, it (1) blunted the likelihood of transient firing patterns; (2) blunted the likelihood and the amplitude of rebound depolarizations, (3) eliminated action potential pairing, and (4) remodeled the kinetics of the action potentials. In contrast, the properties of Cav3.2-positive neurons were only marginally modified in Cav3.1 knockout mice. Overall, in addition to their previously established roles in the superficial spinal cord and in primary afferent neurons, Cav3.2 channel appear to be necessary for specific, significant and multiple controls of LII neuron excitability.

WNK1 kinase and its partners Akt, SGK1 and NBC-family Na+/HCO3- cotransporters are potential therapeutic targets for glioblastoma stem-like cells linked to Bisacodyl signaling.

Glioblastoma is a highly heterogeneous brain tumor. The presence of cancer cells with stem-like and tumor initiation/propagation properties contributes to poor prognosis. Glioblastoma cancer stem-like cells (GSC) reside in hypoxic and acidic niches favoring cell quiescence and drug resistance. A high throughput screening recently identified the laxative Bisacodyl as a cytotoxic compound targeting quiescent GSC placed in acidic microenvironments. Bisacodyl activity requires its hydrolysis into DDPM, its pharmacologically active derivative. Bisacodyl was further shown to induce tumor shrinking and increase survival in in vivo glioblastoma models. Here we explored the cellular mechanism underlying Bisacodyl cytotoxic effects using quiescent GSC in an acidic microenvironment and GSC-derived 3D macro-spheres. These spheres mimic many aspects of glioblastoma tumors in vivo, including hypoxic/acidic areas containing quiescent cells. Phosphokinase protein arrays combined with pharmacological and genetic modulation of signaling pathways point to the WNK1 serine/threonine protein kinase as a mediator of Bisacodyl cytotoxic effect in both cell models. WNK1 partners including the Akt and SGK1 protein kinases and NBC-family Na+/HCO3- cotransporters were shown to participate in the compound's effect on GSC. Overall, our findings uncover novel potential therapeutic targets for combatting glioblastoma which is presently an incurable disease.

NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain after Peripheral Nerve Injury.

Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Bax confirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain.

Neuronal networks and nociceptive processing in the dorsal horn of the spinal cord.

The dorsal horn (DH) of the spinal cord receives a variety of sensory information arising from the inner and outer environment, as well as modulatory inputs from supraspinal centers. This information is integrated by the DH before being forwarded to brain areas where it may lead to pain perception. Spinal integration of this information relies on the interplay between different DH neurons forming complex and plastic neuronal networks. Elements of these networks are therefore potential targets for new analgesics and pain-relieving strategies. The present review aims at providing an overview of the current knowledge on these networks, with a special emphasis on those involving interlaminar communication in both physiological and pathological conditions.

Inhibition of the kinase WNK1/HSN2 ameliorates neuropathic pain by restoring GABA inhibition.

HSN2is a nervous system predominant exon of the gene encoding the kinase WNK1 and is mutated in an autosomal recessive, inherited form of congenital pain insensitivity. The HSN2-containing splice variant is referred to as WNK1/HSN2. We created a knockout mouse specifically lacking theHsn2exon ofWnk1 Although these mice had normal spinal neuron and peripheral sensory neuron morphology and distribution, the mice were less susceptible to hypersensitivity to cold and mechanical stimuli after peripheral nerve injury. In contrast, thermal and mechanical nociceptive responses were similar to control mice in an inflammation-induced pain model. In the nerve injury model of neuropathic pain, WNK1/HSN2 contributed to a maladaptive decrease in the activity of the K(+)-Cl(-)cotransporter KCC2 by increasing its inhibitory phosphorylation at Thr(906)and Thr(1007), resulting in an associated loss of GABA (γ-aminobutyric acid)-mediated inhibition of spinal pain-transmitting nerves. Electrophysiological analysis showed that WNK1/HSN2 shifted the concentration of Cl(-)such that GABA signaling resulted in a less hyperpolarized state (increased neuronal activity) rather than a more hyperpolarized state (decreased neuronal activity) in mouse spinal nerves. Pharmacologically antagonizing WNK activity reduced cold allodynia and mechanical hyperalgesia, decreased KCC2 Thr(906)and Thr(1007)phosphorylation, and restored GABA-mediated inhibition (hyperpolarization) of injured spinal cord lamina II neurons. These data provide mechanistic insight into, and a compelling therapeutic target for treating, neuropathic pain after nerve injury.

Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Peripheral nerve injury produces a sustained shift in the balance between glutamate release and uptake in the dorsal horn of the spinal cord.

Peripheral nerve injury provokes heightened excitability of primary sensory afferents including nociceptors, and elicits ectopic activity in lesioned and neighboring intact nerve fibers. The major transmitter released by sensory afferents in the superficial dorsal horn of the spinal cord is glutamate. Glutamate is critically involved in nociceptive signaling and the development of neuropathic pain. We recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II of the rat dorsal horn to assess spontaneous synaptic activity after spared nerve injury (SNI), a model of chronic neuropathic pain. Following SNI, the frequency of mEPSCs doubled, indicating heightened glutamate release from primary afferents or spinal interneurons. Consistent with this finding, glutamate concentrations in the cerebrospinal fluid were elevated at 1 and 4 weeks after SNI. Transmitter uptake was insufficient to prevent the rise in extracellular glutamate as the expression of glutamate transporters remained unchanged or decreased. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride, an antagonist of metabotropic glutamate receptor 5 (mGluR5), reduced the frequency of mEPSCs to its preinjury level, suggesting a positive feedback mechanism that involves facilitation of transmitter release by mGluR5 activation in the presence of high extracellular glutamate. Treatment with the ß-lactam antibiotic ceftriaxone increased the expression of glutamate transporter 1 (Glt1) in the dorsal horn after SNI, raised transmitter uptake, and lowered extracellular glutamate. Improving glutamate clearance prevented the facilitation of transmitter release by mGluR5 and attenuated neuropathic pain-like behavior. Balancing glutamate release and uptake after nerve injury should be an important target in the management of chronic neuropathic pain.

The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling.

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

Reduced sodium channel Na(v)1.1 levels in BACE1-null mice.

The Alzheimer BACE1 enzyme cleaves numerous substrates, with largely unknown physiological consequences. We have previously identified the contribution of elevated BACE1 activity to voltage-gated sodium channel Na(v)1.1 density and neuronal function. Here, we analyzed physiological changes in sodium channel metabolism in BACE1-null mice. Mechanistically, we first confirmed that endogenous BACE1 requires its substrate, the ß-subunit Na(v)ß(2), to regulate levels of the pore-forming α-subunit Na(v)1.1 in cultured primary neurons. Next, we analyzed sodium channel α-subunit levels in brains of BACE1-null mice at 1 and 3 months of age. At both ages, we found that Na(v)1.1 protein levels were significantly decreased in BACE1-null versus wild-type mouse brains, remaining unchanged in BACE1-heterozygous mouse brains. Interestingly, levels of Na(v)1.2 and Na(v)1.6 α-subunits also decreased in 1-month-old BACE1-null mice. In the hippocampus of BACE1-null mice, we found a robust 57% decrease of Na(v)1.1 levels. Next, we performed surface biotinylation studies in acutely dissociated hippocampal slices from BACE1-null mice. Hippocampal surface Na(v)1.1 levels were significantly decreased, but Na(v)1.2 surface levels were increased in BACE1-null mice perhaps as a compensatory mechanism for reduced surface Na(v)1.1. We also found that Na(v)ß(2) processing and Na(v)1.1 mRNA levels were significantly decreased in brains of BACE1-null mice. This suggests a mechanism consistent with BACE1 activity regulating mRNA levels of the α-subunit Na(v)1.1 via cleavage of cell-surface Na(v)ß(2). Together, our data show that endogenous BACE1 activity regulates total and surface levels of voltage-gated sodium channels in mouse brains. Both decreased Na(v)1.1 and elevated surface Na(v)1.2 may result in a seizure phenotype. Our data caution that therapeutic BACE1 activity inhibition in Alzheimer disease patients may affect Na(v)1 metabolism and alter neuronal membrane excitability in Alzheimer disease patients.

Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice.

Itch is the least well understood of all the somatic senses, and the neural circuits that underlie this sensation are poorly defined. Here we show that the atonal-related transcription factor Bhlhb5 is transiently expressed in the dorsal horn of the developing spinal cord and appears to play a role in the formation and regulation of pruritic (itch) circuits. Mice lacking Bhlhb5 develop self-inflicted skin lesions and show significantly enhanced scratching responses to pruritic agents. Through genetic fate-mapping and conditional ablation, we provide evidence that the pruritic phenotype in Bhlhb5 mutants is due to selective loss of a subset of inhibitory interneurons in the dorsal horn. Our findings suggest that Bhlhb5 is required for the survival of a specific population of inhibitory interneurons that regulate pruritus, and provide evidence that the loss of inhibitory synaptic input results in abnormal itch.

Regional differences in the decay kinetics of GABA(A) receptor-mediated miniature IPSCs in the dorsal horn of the rat spinal cord are determined by mitochondrial transport of cholesterol.

We examined the possibility of a differential spatial control in the endogenous production of 3alpha5alpha-reduced steroids and its consequences on GABA(A) receptor-mediated miniature IPSCs (mIPSCs) in laminas II and III-IV of the rat spinal cord dorsal horn (DH). Early in postnatal development [younger than postnatal day 8 (P8)], mIPSCs displayed slow decay kinetics in laminas II and III-IV resulting from a continuous local production of 3alpha5alpha-reduced steroids. This was mediated by the tonic activity of the translocator protein of 18 kDa (TSPO), which controls neurosteroid synthesis by regulating the transport of cholesterol across the mitochondrial membrane system. TSPO activity disappeared in laminas III-IV after P8 and was functionally downregulated in lamina II after P15, resulting in a marked reduction of mIPSC duration in these laminas. TSPO-mediated synthesis of 3alpha5alpha-reduced steroids was spatially restricted, because, at P9-P15, when their production was maximal in lamina II, no sign of spillover to laminas III-IV was apparent. Interestingly, after P8, the enzymes necessary for the synthesis of 3alpha5alpha-reduced steroids remained functional in laminas III-IV and could produce such steroids from various precursors or after a single subcutaneous injection of progesterone. Moreover, induction of an acute peripheral inflammation by intraplantar injection of carrageenan, restored a maximal TSPO-mediated neurosteroidogenesis in laminas III-IV. Our results indicate that the decay kinetics of GABA(A) receptor-mediated mIPSCs in the DH of the spinal cord are primarily controlled by 3alpha5alpha-reduced steroids, which can be produced from circulating steroid precursors and/or in a spatially restricted manner by the modulation of the activity of TSPO.