Derek C. Molliver

IB4-BINDING DRG NEURONS SWITCH FROM NGF TO GDNF DEPENDENCE IN EARLY POSTNATAL LIFE

We have tested the role of glial cell line-derived neurotrophic factor (GDNF) in regulating a group of putatively nociceptive dorsal root ganglion (DRG) neurons that do not express calcitonin gene-related peptide (CGRP) and that downregulate the nerve growth factor (NGF) receptor tyrosine kinase, TrkA, after birth. We show that mRNA and protein for the GDNF receptor tyrosine kinase, Ret, are expressed in the DRG in patterns that differ markedly from those of any of the neurotrophin receptors. Most strikingly, a population of small neurons initiates expression of Ret between embryonic day 15.5 and postnatal day 7.5 and maintains Ret expression into adulthood. These Ret-expressing small neurons are selectively labeled by the lectin IB4 and project to lamina IIi of the dorsal horn. Ret-expressing neurons also express the glycosyl-phosphatidyl inositol-linked (GPI-linked) GDNF binding component GDNFR-alpha and retrogradely transport 125I-GDNF, indicating the presence of a biologically active GDNF receptor complex. In vitro, GDNF supports the survival of small neurons that express Ret and bind IB4 while failing to support the survival of neurons expressing TrkA and CGRP. Together, our findings suggest that IB4-binding neurons switch from dependence on NGF in embryonic life to dependence on GDNF in postnatal life and are likely regulated by GDNF in maturity.

Gene Targeting Reveals a Critical Role for Neurturin in the Development and Maintenance of Enteric, Sensory, and Parasympathetic Neurons

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity

Dissociated primary sensory neurons are commonly used to study growth factor–dependent cell survival, axon outgrowth, differentiation and basic mechanisms of sensory physiology and pain. Spinal or trigeminal sensory neurons can be collected from embryos, neonates or adults, treated with enzymes that degrade the extracellular matrix, triturated and grown in defined media with or without growth factors and additional animal sera. Production of cultures can take as little as 2.5 h. Cells can be used almost immediately or maintained for as long as 1 month. Ease of production and the ability to control growth conditions make sensory neuron culture a powerful model system for studying basic neurobiology of central and peripheral nervous systems.

Glial Cell Line-Derived Neurotrophic Factor Family Members Sensitize Nociceptors In Vitro and Produce Thermal Hyperalgesia In Vivo

Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10–100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund’s adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRα3 (for GDNF family receptor α) was seen in DRG, and GFRα3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.

ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons

BackgroundASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses.ResultsLess than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 μm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus.ConclusionOur data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

Nerve growth factor receptor trkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons

Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is required for the survival of all small and many intermediate‐sized murine dorsal root ganglion (DRG) neurons during development, accounting for 80% of the total DRG population. Surprisingly, NGF/TrkA‐dependent neurons include a large population that does not express TrkA in adult mice (Silos‐Santiago et al., 1995). This finding suggests two hypotheses: Neurons lacking TrkA in the adult may express TrkA during development, or they may be maintained through a paracrine mechanism by TrkA‐expressing neurons. To determine whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood, we examined the distribution of TrkA protein during development. We show here that TrkA expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at postnatal day (P) 21. Because the period of TrkA down‐regulation corresponds with a critical period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell [1993] Trends Neurosci. 16:353‐359), we examined whether NGF modulates the down‐regulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the extent of TrkA down‐regulation. Our results demonstrate a novel form of regulation of neurotrophin receptor expression that occurs late in development. All DRG neurons that require NGF for survival express TrkA during embryogenesis, and many continue to express TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The subsequent down‐regulation of TrkA is likely to be importantly related to functional distinctions among nociceptive neurons in maturity. J. Comp. Neurol. 381:428‐438, 1997.

Artemin Overexpression in Skin Enhances Expression of TRPV1 and TRPA1 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Heat and Cold

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRα3 and the receptor tyrosine kinase Ret. Expression of the GFRα3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRα3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRα3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

TRPV1 and TRPA1 Function and Modulation Are Target Tissue Dependent

The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.

Gi- and Gq-coupled ADP (P2Y) receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior

BackgroundInvestigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception.ResultsWe detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freunds adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia.ConclusionsWe report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

The P2Y agonist UTP activates cutaneous afferent fibers

&NA; The majority of adenosine triphosphate (ATP)‐induced nociceptive transduction and pain has been attributed to ionotropic P2X3 receptors. Metabotropic P2Y receptors, some of which bind pyrimidines as well as purines, have received little attention. Here we have examined the ability of P2Y receptor signaling to evoke action potential firing in functionally identified afferent fibers using the skin nerve preparation from adult mouse. The P2Y2/P2Y4 ligand UTP activated sustained action potential firing in 54% of C fibers in a concentration‐dependent manner. The effect was specific for P2Y2/P2Y4 receptors, as the P2Y6 ligand UDP never activated C fibers. In comparison to C fibers, few thinly myelinated A‐mechanoreceptors (AM) (12%) were activated by UTP. The majority (70–80%) of the UTP‐sensitive C and A&dgr; fibers responded to the algogen capsaicin with a barrage of action potentials, whereas the UTP‐insensitive fibers were largely unresponsive to capsaicin. Furthermore, 86% of the UTP‐sensitive C fibers and 100% of the UTP‐sensitive AM fibers also responded to the P2X agonist &agr;,&bgr;‐methylene ATP, indicating that P2Y and P2X receptors are widely co‐expressed. Surprisingly, a significant proportion (20–40%) of low threshold slowly and rapidly adapting A&bgr; fibers were also activated by UTP and &agr;,&bgr;‐methylene ATP. These data indicate that P2Y receptors on the terminals of capsaicin‐sensitive cutaneous sensory neurons effectively evoke nociceptive transmission, and support the hypothesis that UTP may be an endogenous nociceptive messenger. Furthermore, P2Y signaling may contribute to mechanotransduction in low threshold A&bgr; fibers under normal or pathological conditions.