No abstract

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. We show here that Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy.

Neural progenitor cells often produce distinct types of neurons in a specific order, but the determinants that control the sequential generation of distinct neuronal subclasses in the vertebrate CNS remain poorly defined. We examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain. We found that the temporal specification of these neurons varies along the anterior-posterior axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx-and Hox-class homeodomain proteins. A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign new roles for Nkx, Hox, and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates. Neuronal cell diversity is established by mechanisms that operate in space and over time during central nervous system (CNS) development. Insight has been obtained regarding the initial steps of spatial patterning of neurons along the dorsal-ventral (DV) and anterior-posterior (AP) axes of the neural tube Jessell 2000). Local inductive signals determine the spatial pattern of expression of transcription factors along both these axes, so that neural progenitors at different positions acquire distinct molecular identities. In the ventral neural tube, neuronal fate along the DV axis depends on the Shh-mediated patterning of Nkx-, Dbx-, Pax-, and Irx-class homeodomain (HD) proteins . Along the AP axis, the overlapping, or nested, expression pattern of Hox HD proteins provides positional values that influence the fate of neurons . Despite significant advances, however, DV and AP patterning have generally been analyzed independently, leaving open the issue as to what degree these orthogonal patterning mechanisms are integrated (Davenne et al. 1999;Gaufo et al. 2000). Compared to spatial patterning, little is known about the mechanisms that underlie how neural progenitors produce distinct types of neurons in a specific temporal order. Studies of the retina (Livesey and Cepko 2001) and developing neo-cortex (Monuki and Walsh 2001) suggest that the sequential production of different neuronal subtypes reflects temporal changes in neural progenitors, either in response to extrinsic cues or mechanisms intrinsic to neural progenitor cells. Recent data indicate that modulation of Notch signaling by the bHLH protein Mash1 and the HD proteins Dlx1/2 may control the sequential specification of progenitors in subcortical areas of the telencephalon (Yun et al. 2002). Apart from this, few molecular determinants that influence these temporal processes in the vertebrate CNS have been identified to date. ResultsTo address how spatial and temporal aspects of cell patterning are integrated during development, w...

Most neurons in vertebrates make a developmental choice between two principal neurotransmitter phenotypes (glutamatergic versus GABAergic). Here we show that the homeobox gene Lbx1 determines a GABAergic cell fate in the dorsal spinal cord at early embryonic stages. In Lbx1-/- mice, the presumptive GABAergic neurons are transformed into glutamatergic cells. Furthermore, overexpression of Lbx1 in the chick spinal cord is sufficient to induce GABAergic differentiation. Paradoxically, Lbx1 is also expressed in glutamatergic neurons. We previously reported that the homeobox genes Tlx1 and Tlx3 determine glutamatergic cell fate. Here we show that impaired glutamatergic differentiation, observed in Tlx3-/- mice, is restored in Tlx3-/-Lbx1-/- mice. These genetic studies suggest that Lbx1 expression defines a basal GABAergic differentiation state, and Tlx3 acts to antagonize Lbx1 to promote glutamatergic differentiation.

SUMMARY Itch can be suppressed by painful stimuli, but the underlying neural basis is unknown. We generated conditional null mice in which VGLUT2-dependent synaptic glutamate release from mainly Nav1.8-expressing nociceptors was abolished. These mice showed deficits in pain behaviors including mechanical pain, heat pain, capsaicin-evoked pain, inflammatory pain and neuropathic pain. The pain deficits were accompanied by greatly enhanced itching, as suggested by i) sensitization of both histamine-dependent and histamine-independent itch pathways, and ii) development of spontaneous scratching and skin lesions. Strikingly, intradermal capsaicin injection promotes itch responses in these mutant mice, as opposed to pain responses in control littermates. Consequently, co-injection of capsaicin was no longer able to mask itch evoked by pruritogenic compounds. Our studies suggest that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Elimination of VGLUT2 in these nociceptors creates a mouse model of chronic neurogenic itch.

Astrogliosis is a prominent feature of many, if not all, pathologies of the brain and spinal cord, yet a detailed understanding of the underlying molecular pathways involved in the transformation from quiescent to reactive astrocyte remains elusive. We investigated the contribution of voltage-gated sodium channels to astrogliosis in an in vitro model of mechanical injury to astrocytes. Previous studies have shown that a scratch injury to astrocytes invokes dual mechanisms of migration and proliferation in these cells. Our results demonstrate that wound closure after mechanical injury, involving both migration and proliferation, is attenuated by pharmacological treatment with tetrodotoxin (TTX) and KB-R7943, at a dose that blocks reverse mode of the Na+/Ca2+ exchanger (NCX), and by knockdown of Nav1.5 mRNA. We also show that astrocytes display a robust [Ca2+]i transient after mechanical injury and demonstrate that this [Ca2+]i response is also attenuated by TTX, KB-R7943, and Nav1.5 mRNA knockdown. Our results suggest that Nav1.5 and NCX are potential targets for modulation of astrogliosis after injury via their effect on [Ca2+]i.

Diabetic neuropathic pain imposes a huge burden on individuals and society, and represents a major public health problem. Despite aggressive efforts, diabetic neuropathic pain is generally refractory to available clinical treatments. A structure-function link between maladaptive dendritic spine plasticity and pain has been demonstrated previously in CNS and PNS injury models of neuropathic pain. Here, we reasoned that if dendritic spine remodeling contributes to diabetic neuropathic pain, then (1) the presence of malformed spines should coincide with the development of pain, and (2) disrupting maladaptive spine structure should reduce chronic pain. To determine whether dendritic spine remodeling contributes to neuropathic pain in streptozotocin (STZ)-induced diabetic rats, we analyzed dendritic spine morphology and electrophysiological and behavioral signs of neuropathic pain. Our results show changes in dendritic spine shape, distribution, and shape on wide-dynamic-range (WDR) neurons within lamina IV-V of the dorsal horn in diabetes. These diabetesinduced changes were accompanied by WDR neuron hyperexcitability and decreased pain thresholds at 4 weeks. Treatment with NSC23766 (N 6 -[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride), a Rac1-specific inhibitor known to interfere with spine plasticity, decreased the presence of malformed spines in diabetes, attenuated neuronal hyperresponsiveness to peripheral stimuli, reduced spontaneous firing activity from WDR neurons, and improved nociceptive mechanical pain thresholds. At 1 week after STZ injection, animals with hyperglycemia with no evidence of pain had few or no changes in spine morphology. These results demonstrate that diabetes-induced maladaptive dendritic spine remodeling has a mechanistic role in neuropathic pain. Molecular pathways that control spine morphogenesis and plasticity may be promising future targets for treatment.

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) co-receptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knockout to gain insight into the physiological role of RGMb in the DRG, and address if RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the peripheral and central nervous systems 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 wildtype 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 naïve 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 re-growth after nerve injury in vivo and a negative effect of Noggin.