SUMMARYNeuropathic pain is a chronic debilitating disease that results from nerve damage, persists long after the injury has subsided, and is characterized by spontaneous pain and mechanical hypersensitivity. Although loss of inhibitory tone in the dorsal horn of the spinal cord is a major contributor to neuropathic pain, the molecular and cellular mechanisms underlying this disinhibition are unclear. Here, we combined pharmacogenetic activation and selective ablation approaches in mice to define the contribution of spinal cord parvalbumin (PV)-expressing inhibitory interneurons in naive and neuropathic pain conditions. Ablating PV neurons in naive mice produce neuropathic pain-like mechanical allodynia via disinhibition of PKCγ excitatory interneurons. Conversely, activating PV neurons in nerve-injured mice alleviates mechanical hypersensitivity. These findings indicate that PV interneurons are modality-specific filters that gate mechanical but not thermal inputs to the dorsal horn and that increasing PV inter-neuron activity can ameliorate the mechanical hypersensitivity that develops following nerve injury.

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Na v 1.

The effects of prostaglandin E 2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP 3 and the EP 4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP 3 and EP 4 . A perinuclear localization of EP 3␣ and EP 4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP 3␣ and EP 4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP 3 receptors.Prostaglandin E 2 (PGE 2 ) 1 is one of the most abundant prostanoids in the brain (1) and plays an important role in many cerebral functions, particularly in the newborn (2). PGE 2 also influences mitogenesis (3), promotes growth and metastasis of tumors (4), and stimulates gene transcription (5). To date, the biological actions of PGE 2 have been attributed to result from its interaction with plasma membrane G protein-coupled receptors termed EP, which include EP 1 , EP 2 , EP 3 , and EP 4 subtypes (6). Recent studies have shown that the nuclear membrane contains high levels of cyclooxygenase-1 and -2 and of PGE 2 (7). Possible intracellular sites of action for prostanoids are also suggested by other data. For example, a transporter that mediates the influx of prostanoid has been identified (8). Cytosolic phospholipase A 2 undergoes a calcium-dependent translocation to the nuclear envelope (9), and cyclooxygenase-2 has been shown to translocate to the nucleus in response to certain growth factors (10). It is thus possible that prostanoids may exert some of their effects via intracellular EP receptors, to have a direct nuclear action as recently proposed by Goetzl et al. (11), and Morita et al. (12).It has generally been assumed that the signal transduction cascades are initiated at the plasma membrane and not the nuclear membranes. However, recent studies have disclosed that the nuclear envelope plays a major role in signal transduction cascades (13,14). In fact, a novel nuclear lipid metabolism that is a part of unique nuclear signaling cascade termed NEST (nuclear envelope signal transduction) has been hypothesized (15). Both heterotrimeric and low molecular weight G proteins (15, 16), phospholipase C (13), phospholipase D (15), and adenylate cyclase (17) have shown to be localized at the nucleus. Th...

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA 1 R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA 1 R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron-and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA 1 Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA 1 R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA 1 R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitricoxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPAgenerating phospholipase A 2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca 2؉ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca 2؉ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.In the mammalian system, LPA 1 signaling cascades regulate important cellular processes, including gene expression, cell proliferation and growth, cell survival and death, and cell motility and secretion (1-3). These plethora of activities are characteristic features of inflammation that occur in various physiological as well as pathological states (e.g. ontogenic change, wound healing, cancer, etc.) (1-3). In humans, physiological responses induced by LPA arise from specific interactions with at least three genetically identified receptors designated LPA 1 , LPA 2 , and LPA 3 (formerly referred to as EDG 2 , EDG 4 , and EDG 7 receptors, respectively), which belong to the heptahelical transmembrane-spanning G protein-coupled receptor (GPCR) superfamily (4). These receptors show a broad, virtually distinct distribution and may couple in a cell-dependent manner to numerous heterotrimeric G proteins. In this context, LPA 1 and LPA 2 receptors have been shown to interact with G i/o , G q/11/14 , and G 12/13 proteins, whereas the LPA 3 receptor combines with G i/o and G q/11/14 proteins (5). Although many responses induced by extracellular LPA can result from its interaction with plasma membrane GPCRs, there is circumstantial evidence for an intracrine mode of action of LPA. For instance, putative biogenesis (e.g. secretory and cytosolic calcium-depen...

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

Prostaglandin E 2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP 1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP 1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP 1 fused to green fluorescent protein. High-resolution immunostaining of EP 1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Systematic examination of photomontages revealed two types of synaptic glomeruli in laminae II-III. Type I glomeruli have a dark small central (C) terminal of indented contour with closely packed spherical vesicles of variable diameter and few mitochondria. Among the peripheral terminals there are dendritic spines and a few presynaptic dendritic spines (V1 terminals) and axon endings rich in discoid vesicles (V2). These glomeruli occur in groups which are particularly evident in parasagittal sections in which successive C terminals are connected by narrower portions or dark unmyelinated profiles. Type II glomeruli have an electron-lucent and large C terminal of regular contour with less packed synaptic vesicles of more uniform diameter, more mitochondria, and sometimes neurofilaments. Presynaptic dendrites are fewer and axon endings more numerous. C terminals in type II glomeruli are fusiform in longitudinal section, rarely occurring in groups. Lamina I is virtually devoid of glomeruli. Within lamina II, glomeruli are rare in the dorsalmost 20-micrometers band and abundant in the immediately ventral 20-micrometers band in which type I glomeruli are prevalent (approximately 79%). In ventral lamina II, type II glomeruli predominate (66%), being practically exclusive in lamina III where most contain neurofilaments. Considering the distribution of terminations of primary afferents, it is suggested that type I C terminals originated from unmyelinated primary afferents, type II C terminals without neurofilaments from direct myelinated fibers, the those with neurofilaments from recurrent large fibers. The distinct numbers of presynaptic dendritic and axonal endings suggest different modulatory mechanisms functioning in the two types and in ventral vs. dorsal areas of this region.

Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA B receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 μm 2 ) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.homodimerization | quantitative immunocytochemistry | fluorescence fluctuations analysis | GABAB | EGF receptor