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ABSTRACT: While much is known about the functional properties of cutaneous nociceptors, relatively little is known about the comprehensive functional properties of group III and IV muscle afferents. We have developed a mouse ex vivo forepaw muscle, median and ulnar nerves, DRG, spinal cord recording preparation to examine the functional response properties, neurochemical phenotypes and spinal projections of individual muscle afferents. We found that the majority of group III and IV muscle afferents were chemosensitive (52%) while only 34% responded to mechanical stimulation and fewer (32%) responded to thermal stimuli. The chemosensitive afferents could be grouped into those that responded to a "low" metabolite mixture containing amounts of lactate and ATP at pH 7.0 simulating levels observed in muscle during exercise (metaboreceptors) and a "high" metabolite mixture containing lactic acid concentrations and ATP at pH 6.6, mimicking levels observed during ischemic contractions (metabo-nociceptors). While the majority of the metabo-nociceptive fibers responding to the higher concentration levels were found to contain acid sensing ion channel 3 (ASIC3) and/or transient receptor potential vanilloid type I (TRPV1), metaboreceptors responding to the lower concentration levels lacked these receptors. Anatomically, group III muscle afferents were found to have projections into lamina I, II(o) and deeper laminae in the spinal cord, while all functional types of group IV muscle afferents projected primarily into both lamina I and II. These results provide novel information about the variety of sensory afferents innervating the muscle and provide insight to the types of fibers that may exhibit plasticity after injuries.
Journal of Neurophysiology 02/2013; · 3.32 Impact Factor
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ABSTRACT: Peripheral injury leads to a significant increase in the prevalence of mechanically insensitive, heat-sensitive C-fibers (CH) that contain the heat transducing TRPV1 (transient receptor potential vanilloid type I) channel in mice. We have recently shown that this recruitment of CH fibers is associated with increased expression of the receptor for GDNF (glial cell line-derived neurotrophic factor) family neurotrophic factor artemin (GFRα3), and that in vivo inhibition of GFRα3 prevented the increase in TRPV1 expression normally observed following axotomy. Here we have directly tested the hypothesis that the recruitment of functional CH fibers following nerve regeneration requires enhanced TRPV1 levels. We used in vivo siRNA-mediated knockdown to inhibit the injury-induced expression of TRPV1 coupled with ex vivo recording to examine response characteristics and neurochemical phenotypes of different functionally defined cutaneous sensory neurons after regeneration. We confirmed that inhibition of TRPV1 did not affect the axotomy-induced decrease in polymodal C-fiber (CPM) heat threshold, but transiently prevented the recruitment of CH neurons. Moreover, a recovery of TRPV1 protein was observed following resolution of siRNA-mediated inhibition that was correlated with a concomitant rebound in CH neuron recruitment. Thus dynamic changes in TRPV1 expression, not absolute levels, may underlie the functional alterations observed in CH neurons and may contribute to the development of heat hyperalgesia after nerve injury.
Journal of Neuroscience 12/2012; 32(49):17869-17873. · 7.11 Impact Factor
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Lishi Li,
Michael Rutlin,
Victoria E Abraira,
Colleen Cassidy,
Laura Kus,
Shiaoching Gong, Michael P Jankowski,
Wenqin Luo,
Nathaniel Heintz,
H Richard Koerber,
C Jeffery Woodbury,
David D Ginty
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ABSTRACT: Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aβ-, Aδ-, and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin (guard, awl/auchene, and zigzag hairs) is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aβ-, Aδ-, and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aβ-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.
Cell 12/2011; 147(7):1615-27. · 32.40 Impact Factor
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ABSTRACT: We have recently found that, following complete Freund's adjuvant (CFA)-induced inflammation, cutaneous polymodal nociceptors (CPM) lacking the transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli. In order to determine possible mechanisms playing a role in this change, we examined gene expression in the L2/L3 sensory ganglia following CFA injection into the hairy hind paw skin and found that G-protein-coupled purinoreceptor P2Y1 expression was increased. This receptor is of particular interest, as most CPMs innervating mouse hairy skin bind isolectin B4, which co-localizes with P2Y1. Additionally, our recent findings have shown that cutaneous CPMs in P2Y1-/- mice displayed significantly reduced thermal sensitivity. Together, these findings suggested a possible role for P2Y1 in inflammation-induced heat sensitization in these fibers. To test this hypothesis, we utilized our in vivo small interfering RNA technique to knock down the inflammation-induced increase in P2Y1 expression and then examined the functional effects using ex vivo recording. We found that the normal reduction of heat thresholds in CPM fibers induced by CFA was completely blocked by inhibition of P2Y1. Surprisingly, inhibition of P2Y1 during inflammation also significantly increased the number of CPM neurons expressing TRPV1 without a change in the total number of TRPV1-positive cells in the L2 and L3 dorsal root ganglia. These results show that the inflammation-induced enhanced expression of P2Y1 is required for normal heat sensitization of cutaneous CPM fibers. They also suggest that P2Y1 plays a role in the maintenance of phenotype in cutaneous afferent fibers containing TRPV1.
Pain 11/2011; 153(2):410-9. · 5.78 Impact Factor
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ABSTRACT: P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold.
To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice.
These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.
Molecular Pain 02/2011; 7:13. · 3.53 Impact Factor
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ABSTRACT: We have shown recently that following saphenous nerve transection and successful regeneration, cutaneous polymodal nociceptors (CPMs) lacking transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli and that mechanically insensitive, heat-sensitive C-fibers (CHs) that contain TRPV1 increase in prevalence. Target-derived neurotrophic factor levels were also enhanced after axotomy and regeneration. In particular, the glial-cell line-derived neurotrophic factor (GDNF) family member artemin was found to be significantly enhanced in the hairy hindpaw skin and its receptor GDNF family receptor α3 (GFRα3) was increased in the L2/L3 dorsal root ganglia (DRGs) following nerve injury. In this study, we assessed the role of enhanced artemin/GFRα3 levels on the changes in mouse cutaneous CH neurons following saphenous nerve regeneration. We used a newly developed siRNA-mediated in vivo knockdown strategy to specifically inhibit the injury-induced expression of GFRα3 and coupled this with an ex vivo recording preparation to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons after injury. We found that inhibition of GFRα3 did not affect the axotomy-induced decrease in CPM threshold, but transiently prevented the recruitment of CH neurons. Western blot and real-time PCR analysis of hairy hindpaw skin and L2/L3 DRGs after saphenous nerve regeneration suggested that inhibition of the potential initial injury-induced increase in enhanced target-derived artemin signaling resulted in dynamic changes in TRPV1 expression after regeneration. These changes in TRPV1 expression may underlie the functional alterations observed in CH neurons after nerve regeneration.
Journal of Neuroscience 12/2010; 30(48):16272-83. · 7.11 Impact Factor
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ABSTRACT: Previous studies have shown that the TRPV1 ion channel plays a critical role in the development of heat hyperalgesia after inflammation, as inflamed TRPV1-/- mice develop mechanical allodynia but fail to develop thermal hyperalgesia. In order to further investigate the role of TRPV1, we have used an ex vivo skin/nerve/DRG preparation to examine the effects of CFA-induced-inflammation on the response properties of TRPV1-positive and TRPV1-negative cutaneous nociceptors.
In wildtype mice we found that polymodal C-fibers (CPMs) lacking TRPV1 were sensitized to heat within a day after CFA injection. This sensitization included both a drop in average heat threshold and an increase in firing rate to a heat ramp applied to the skin. No changes were observed in the mechanical response properties of these cells. Conversely, TRPV1-positive mechanically insensitive, heat sensitive fibers (CHs) were not sensitized following inflammation. However, results suggested that some of these fibers may have gained mechanical sensitivity and that some previous silent fibers gained heat sensitivity. In mice lacking TRPV1, inflammation only decreased heat threshold of CPMs but did not sensitize their responses to the heat ramp. No CH-fibers could be identified in naïve nor inflamed TRPV1-/- mice.
Results obtained here suggest that increased heat sensitivity in TRPV1-negative CPM fibers alone following inflammation is insufficient for the induction of heat hyperalgesia. On the other hand, TRPV1-positive CH fibers appear to play an essential role in this process that may include both afferent and efferent functions.
Molecular Pain 01/2010; 6:58. · 3.53 Impact Factor
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ABSTRACT: The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd-expressing (Mrgprd(+)) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd(+) afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd(-/-) and Mrgprd(+/-) mice. These studies revealed a higher rheobase in neurons from Mrgprd(-/-) mice than from Mrgprd(+/-) mice. Furthermore, the application of the Mrgprd ligand beta-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd(+/-) mice but was without effect in neurons from Mrgprd(-/-) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli.
Journal of Neuroscience 08/2009; 29(26):8612-9. · 7.11 Impact Factor
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ABSTRACT: Damage to peripheral nerves is known to contribute to chronic pain states, including mechanical and thermal hyperalgesia and allodynia. It is unknown whether the establishment of these states is attributable to peripheral changes, central modifications, or both. In this study, we used several different approaches to assess the changes in myelinated (A) and unmyelinated (C) cutaneous nociceptors after transection and regeneration of the saphenous nerve. An ex vivo recording preparation was used to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons. We found that myelinated nociceptors had significantly lower mechanical and thermal thresholds after regeneration, whereas C-polymodal nociceptors (CPMs) had lower heat thresholds. There was a significant increase in the percentage of mechanically insensitive C-fibers that responded to heat (CHs) after regeneration. Immunocytochemical analysis of identified afferents revealed that most CPMs were isolectin B4 (IB4) positive and transient receptor potential vanilloid 1 (TRPV1) negative, whereas CHs were always TRPV1 positive and IB4 negative in naive animals (Lawson et al., 2008). However, after regeneration, some identified CPMs and CHs stained positively for both markers, which was apparently attributable to an increase in the total number of IB4-positive neurons. Real-time PCR analysis of L2/L3 DRGs and hairy hindpaw skin at various times after saphenous nerve axotomy suggested multiple changes in neurotrophic factor signaling that correlated with either denervation or reinnervation of the cutaneous target. These changes may underlie the functional alterations observed after nerve regeneration and may explain how nerve damage leads to chronic pain conditions.
Journal of Neuroscience 03/2009; 29(6):1636-47. · 7.11 Impact Factor
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ABSTRACT: The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.
Brain research 01/2009; 1256:43-54. · 2.46 Impact Factor
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ABSTRACT: Studies of human brain indicate that both the ventromedial prefrontal cortex (PFC) and the dorsal raphe nucleus (DRN) may be dysfunctional in major depressive illness, making it important to understand the functional interactions between these brain regions. Anatomical studies have shown that the PFC projects to the DRN, although the synaptic targets of this excitatory pathway have not yet been identified. Electrophysiological investigations in the rat DRN report that most serotonin neurons are inhibited by electrical stimulation of the PFC, suggesting that this pathway is more likely to synapse onto neighboring gamma-aminobutyric acid (GABA) neurons than onto serotonin cells. We tested this hypothesis by electron microscopic examination of DRN sections dually labeled for biotin dextran amine anterogradely transported from the PFC and immunogold-silver labeling for tryptophan hydroxylase (TrH) or for GABA. In the DRN, the majority of PFC axons either synapsed onto unlabeled dendrites or failed to form detectable synapses in single sections. Other PFC axons synapsed onto either TrH- or GABA-immunolabeled processes. Considerably more tissue sampling was necessary to detect PFC synapses onto TrH- than onto GABA-labeled dendrites, suggesting that the latter connections are more common. In other cases, PFC terminals and TrH- or GABA-immunoreactive dendrites either were closely apposed, without forming detectable synapses, or were separated by glial processes. These results provide potential anatomical substrates whereby the PFC can both directly and indirectly regulate the activity of serotonin neurons in the DRN and possibly contribute to the pathophysiology of depression.
The Journal of Comparative Neurology 02/2004; 468(4):518-29. · 3.81 Impact Factor
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ABSTRACT: NGF is the most commonly studied growth factor in relation to nociceptor sensitization and serves to promote the survival of DRG neurons during development that express its receptor, trkA (Averill et al. 1995; Huang et al. 2001; Patapoutian and Reichardt 2001). These neurons are generally part of the small and medium diameter DRG population, but some larger cells also express trkA (Wright and Snider 1995; Patapoutian and Reichardt 2001). In addition to its role in development and neuronal survival, it promotes sprouting and regulates innervation density of NGF-responsive neurons in peripheral targets in early post-natal and adult life. For example, it has been shown that ligation of a peripheral nerve induces NGF expression in its target area and these elevated levels are associated with sprouting of adjacent, non-injured afferents into the denervated region (Pertens et al. 1999). Other studies analyzing constitutive overexpression of NGF in the skin (NGF-OEs) report enhanced innervation of the epidermis by both sensory and sympathetic neurons (Albers et al. 1994; Davis et al. 1994, 1996; Goodness et al. 1997). Although NGF appears to be necessary and beneficial for development and maintenance of the peripheral sensory neuron system (Diamond et al. 1992), it has also been shown to participate in the development of thermal and mechanical hyperalgesia (i.e., increased pain in response to normally painful stimuli; Malin et al. 2006; Pertens et al. 1999; Andreev et al. 1995; Lewin et al. 1993) and pain in disorders such as bone cancer and interstitial cystitis (Lowe et al. 1997; Sevcik et al. 2005). Rats chronically treated with NGF are hypersensitive to both mechanical and radiant heat stimulation (Lewin et al. 1993; Andreev et al. 1995; Pertens et al. 1999) in a dose-dependent fashion, and injection of NGF directly into the paw of mice induces a decrease in the paw withdrawal latency to radiant heat (Malin et al. 2006). This NGF sensitization is partially dependent on sympathetic neurons, as sympathectomy partly reduces the effect of NGF in causing hyperalgesia (Andreev et al. 1995). NGF also acts indirectly by activating mast cells and neutrophils, which in turn release additional inflammatory mediators causing hypersensitivity (Lewin et al. 1994; Andreev et al. 1995; Amann et al. 1996; Woolf et al. 1996; Bennett et al. 1998; Bennett 2001). Regardless, it is clear that NGF levels in the target tissue participate in sensitization of nociceptors. For example, NGF-OEs display increases in afferent responses to thermal and mechanical stimulation in a skin-nerve preparation. Stucky and Lewin (1999) found that large diameter Aβ non-nociceptive afferents (typically trkA negative) were unaffected by NGF overexpression, but thermal responsiveness was significantly increased in nociceptive afferents as a result of enhanced cutaneous NGF levels. NGF-sensitive, trkA positive neurons co-label with a variety of other molecules thought to be involved in pain processing. trkA overlaps with neurons containing peptides CGRP and SP (Averill et al. 1995; Molliver and Snider 1997), known mediators of pain behaviors (Koltzenburg et al. 1999; Reeh and Kress 2001; Li et al. 2008) shown to induce hyperalgesia (Oku et al. 1987; Nakamura-Craig and Gill 1991; McMahon, 1996; Sann and Pierau 1998). This population also co-labels with TRPV1, crucial for the development of heat hyperalgesia (Caterina et al. 2000). NGF-induced hyperalgesia may also be mediated by sodium channel, Nav1.8. In mice lacking this channel, NGF does not induce heat hyperalgesia (Kerr et al. 2001), although Nav1.8 knockout mice display indistinguishable thermal thresholds under normal conditions compared to wildtypes (WTs). Since many NGF-responsive neurons contain TRPV1, this channel is suspected of a role in NGF-mediated hypersensitivity (Caterina et al. 1997; Tominaga et al. 1998; Michael and Priestley 1999). Cultured DRG neurons treated with NGF display enhanced inward current in response to application of the TRPV1 agonist capsaicin (Shu and Mendell 1999; Caterina et al. 2000; Zhu et al. 2004). NGF can increase TRPV1 expression (Donnerer et al. 2005; Xue et al. 2007) and promote TRPV1 insertion into the plasma membrane (Zhang et al. 2005). Furthermore, anti-NGF antibodies injected into the hindpaw after peripheral inflammation decrease levels of TRPV1 in DRGs and reduce inflammation-induced hyperalgesia (Ji et al. 2002; Cheng and Ji 2008). Given a clear role for NGF in sensory neuron sensitization and hyperalgesia, anti-NGF treatments may constitute an effective means of treating pain in humans (Anand et al. 1997; Lowe et al. 1997; Saldanha et al. 1999; Sena et al. 2006; Jimenez-Andrade et al. 2007). These hypotheses, however, have not been extensively studied (Abdiche et al. 2008) or verified. Perhaps NGF may only affect a small proportion of nociceptors in the DRG, and other molecules and neurotrophic factors most certainly are involved in hyperalgesia and overall sensory neuron sensitization.
ISBN: 9781439812099
