Glioplasticity in irritable bowel syndrome.

BACKGROUND: Growing evidence indicates a wide array of cellular remodeling in the mucosal microenvironment during irritable bowel syndrome (IBS), which possibly contributes to pathophysiology and symptom generation. Here, we investigated whether enteric glial cells (EGC) may be altered, and which factors/mechanisms lead to these changes. METHODS: Colonic mucosal biopsies of IBS patients (13 IBS-Constipation [IBS-C]; 10 IBS-Diarrhea [IBS-D]; 11 IBS-Mixed [IBS-M]) and 24 healthy controls (HC) were analyzed. Expression of S100ß and GFAP was measured. Cultured rat EGC were incubated with supernatants from mucosal biopsies, then proliferation and Ca2+ response to ATP were analyzed using flow cytometry and Ca2+ imaging. Histamine and histamine 1-receptor (H1R) involvement in the effects of supernatant upon EGC was analyzed. KEY RESULTS: Compared to HC, the mucosal area immunoreactive for S100ß was significantly reduced in biopsies of IBS patients, independently of the IBS subtype. IBS-C supernatants reduced EGC proliferation and IBS-D and IBS-M supernatants reduced Ca2+ response to ATP in EGC. EGC expressed H1R and the effects of supernatant upon Ca2+ response to ATP in EGC were blocked by pyrilamine and reproduced by histamine via H1R. IBS supernatants reduced mRNA expression of connexin-43. The S100ß-stained area was negatively correlated with the frequency and intensity of pain and bloating. CONCLUSION AND INFERENCES: Changes in EGC occur in IBS, involving mucosal soluble factors. Histamine, via activation of H1R-dependent pathways, partly mediates altered Ca2+ response to ATP in EGC. These changes may contribute to the pathophysiology and the perception of pain and bloating in patients with IBS.

L. fermentum CECT 5716 prevents stress-induced intestinal barrier dysfunction in newborn rats.

BACKGROUND: Intestinal epithelial barrier (IEB) dysfunction plays a critical role in various intestinal disorders affecting infants and children, including the development of food allergies and colitis. Recent studies highlighted the role of probiotics in regulating IEB functions and behavior in adults, but their effects in the newborn remain largely unknown. We therefore characterized in rat pups, the impact of Lactobacillus fermentum CECT 5716 (L. fermentum) on stress-induced IEB dysfunction, systemic immune response and exploratory behavior. METHODS: Newborn rats received daily by gavage either L. fermentum or water. Intestinal permeability to fluorescein sulfonic acid (FSA) and horseradish peroxidase (HRP) was measured following maternal separation (MS) and water avoidance stress (WAS). Immunohistochemical, transcriptomic, and Western blot analysis of zonula occludens-1 (ZO-1) distribution and expression were performed. Anxiety-like and exploratory behavior was assessed using the elevated plus maze test. Cytokine secretion of activated splenocytes was also evaluated. KEY RESULTS: L. fermentum prevented MS and WAS-induced IEB dysfunction in vivo. L. fermentum reduced permeability to both FSA and HRP in the small intestine but not in the colon. L. fermentum increased expression of ZO-1 and prevented WAS-induced ZO-1 disorganization in ileal epithelial cells. L. fermentum also significantly reduced stress-induced increase in plasma corticosteronemia. In activated splenocytes, L. fermentum enhanced IFNγ secretion while it prevented IL-4 secretion. Finally, L. fermentum increased exploratory behavior. CONCLUSIONS & INFERENCES: These results suggest that L. fermentum could provide a novel tool for the prevention and/or treatment of gastrointestinal disorders associated with altered IEB functions in the newborn.

Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

IgG response to intracerebral xenotransplantation: specificity and role in the rejection of porcine neurons.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

The use of stem cells in regenerative medicine for Parkinson's and Huntington's Diseases.

Cell transplantation has been proposed as a means of replacing specific cell populations lost through neurodegenerative processes such as that seen in Parkinson's or Huntington's diseases. Improvement of the clinical symptoms has been observed in a number of Parkinson and Huntington's patients transplanted with freshly isolated fetal brain tissue but such restorative approach is greatly hampered by logistic and ethical concerns relative to the use of fetal tissue, in addition to potential side effects that remain to be controlled. In this context, stem cells that are capable of self-renewal and can differentiate into neurons, have received a great deal of interest, as demonstrated by the numerous studies based on the transplantation of neural stem/progenitor cells, embryonic stem cells or mesenchymal stem cells into animal models of Parkinson's or Huntington's diseases. More recently, the induction of pluripotent stem cells from somatic adult cells has raised a new hope for the treatment of neurodegenerative diseases. In the present article, we review the main experimental approaches to assess the efficiency of cell-based therapy for Parkinson's or Huntington's diseases, and discuss the recent advances in using stem cells to replace lost dopaminergic mesencephalic or striatal neurons. Characteristics of the different stem cells are extensively examined with a special attention to their ability of producing neurotrophic or immunosuppressive factors, as these may provide a favourable environment for brain tissue repair and long-term survival of transplanted cells in the central nervous system. Thus, stem cell therapy can be a valuable tool in regenerative medicine.

Assessing the potential clinical utility of transplantations of neural and mesenchymal stem cells for treating neurodegenerative diseases.

Treatments for neurodegenerative diseases have little impact on the long-term patient health. However, cellular transplants of neuroblasts derived from the aborted embryonic brain tissue in animal models of neurodegenerative disorders and in patients have demonstrated survival and functionality in the brain. However, ethical and functional problems due to the use of this fetal tissue stopped most of the clinical trials. Therefore, new cell sources were needed, and scientists focused on neural (NSCs) and mesenchymal stem cells (MSCs). When transplanted in the brain of animals with Parkinson's or Huntington's disease, NSCs and MSCs were able to induce partial functional recovery by promoting neuroprotection and immunomodulation. MSCs are more readily accessible than NSCs due to sources such as the bone marrow. However, MSCs are not capable of differentiating into neurons in vivo where NSCs are. Thus, transplantation of NSCs and MSCs is interesting for brain regenerative medicine. In this chapter, we detail the methods for NSCs and MSCs isolation as well as the transplantation procedures used to treat rodent models of neurodegenerative damage.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH-IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH-IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 microm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP-IR but not nNOS-IR enteric neurons. The KCl-induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 microm), of noradrenaline (1 microm) and of the alpha(2)-adrenoreceptor agonist clonidine (1 microm). Combining pharmacological and calcium imaging studies, we have further shown that L-type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal-regulated kinases were required for the increase in the proportion of TH-IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.

Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.

The neuropeptide Y receptors, Y1 and Y2, are transiently and differentially expressed in the developing cerebellum.

Neuropeptide Y (NPY), a peptide widely expressed in the brain, acts through the protein G-coupled receptors Y1, Y2 and Y5. In the adult rat, this peptide modulates many important functions such as the control of energy balance and anxiety. Its involvement in brain development has been less investigated. In the present study, we have analysed the expression of Y1 and Y2 in the developing rat cerebellum using RNase protection assay. Both receptors were detected in the embryo but at very low levels. Their expression then increased, reaching a peak at postnatal day 10. At later stages, we observed a down-regulation of both Y1 and Y2 mRNA levels. This pattern of expression was delayed in hypothyroid rats, suggesting that the regulation of NPY receptors was strictly related to cerebellar development stages. In situ hybridisation and immunohistochemistry analyses revealed specific localisations of the receptors. Y1 was exclusively expressed by Purkinje cells while Y2 was found mostly in granule cells of the internal granule cell layer. These observations argue in favour of specific roles for Y1 and Y2 in the developing cerebellum. In an initial attempt to characterise these roles, we have determined the number of apoptotic cells in the developing cerebellum of Y2(-/-) mice and analysed the effects of NPY on primary cultures of cerebellar granule neurones. Our data showed that the absence of Y2 did not increase cell death in the internal granule cell layer of the developing cerebellum, and that NPY by itself did not prevent the death of differentiated granule cells cultured in serum-free medium. However, we found that co-treatment of the cells by NPY and neuromediators such as NMDA or GABA strongly promoted the survival of granule neurones. Taken together, these observations suggest an involvement of the NPY receptors in cerebellar ontogenesis that remains to be demonstrated in vivo.

Distinct roles of the Y1 and Y2 receptors on neuropeptide Y-induced sensitization to sedation.

Intracranial injection of neuropeptide Y (NPY) increases the sensitivity to sodium pentobarbital and ketamin sedation and has similar properties as GABA agonists on sleep. Mice sensitive to sedation have increased levels of NPY in many brain regions and Y1(-/-) mice show a marked resistance to barbiturates. Here we characterized the role of the NPY Y receptors in anesthetic-induced sedation. We show that Y1 and Y2, but not Y5, receptors participate in the modulation of sedation. Administration of a Y1 agonist increased the sodium pentobarbital-induced sedation and Y1(-/-) mice were less sensitive to this anesthetic. However, Y2(-/-) mice display increased sensitivity, showing that Y2 modulates GABAergic induced sedation both pharmacologically and physiologically and has a functionally opposing role to the Y1 receptor. Analysis of Y1(-/-)/Y2(-/-) double mutant mice show that increased sensitivity by Y1 occurs independent of the Y2 receptor, while the decreased sensitivity mediated by Y2 depend on an intact Y1 receptor. In contrast to sodium pentobarbital, both Y1 and Y2 receptors increase the sensitivity in a collaborative fashion to NMDA antagonist-induced sedation. These data demonstrate the physiological and pharmacological impact of the Y1 and Y2 receptors on sedation.

Neuropeptide Y alters sedation through a hypothalamic Y1-mediated mechanism.

Neuropeptide Y (NPY) has been reported to profoundly influence and regulate brain circuits involved in a number of behaviours, like anxiety, alcohol intake, pain and energy homeostasis. Here we show that NPY increases sedation induced by different types of anaesthetics through interactions with the Y1 receptor. Consistently, in Y1-/- (homozygote knockout) mice NPY does not potentiate the pentobarbital-induced sedation. Similar results were obtained for avertin but not for ketalar- (NMDA antagonist) induced sedation. Local microinjection of NPY exhibited the strongest potentiating effect on pentobarbital-induced sedation in the posterior hypothalamic area and Y1 expression was found in the dorsal-premammillary and medial part of medial mammillary nuclei. These results show that Y1 is essential for NPY-induced enhancement of sedation and place this activity of NPY in the posterior hypothalamic area, a region of the brain previously implicated in the regulation of the wake-sleep cycle.

Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFR alpha-1 and GFR alpha-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFR alpha-1 and GFR alpha transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFR alpha-2 mRNA after 24 hr of treatment with 10(-10) M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFR alpha-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFR alpha-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFR alpha-1 and GFR alpha-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma.

Reduced antinociception and plasma extravasation in mice lacking a neuropeptide Y receptor.

Neuropeptide Y (NPY) is believed to exert antinociceptive actions by inhibiting the release of substance P and other 'pain neurotransmitters' in the spinal cord dorsal horn. However, the physiological significance and potential therapeutic value of NPY remain obscure. It is also unclear which receptor subtype(s) are involved. To identify a possible physiological role for the NPY Y1 receptor in pain transmission, we generated NPY Y1 receptor null mutant (Y1-/-) mice by homologous recombination techniques. Here we show that Y1-/- mice develop hyperalgesia to acute thermal, cutaneous and visceral chemical pain, and exhibit mechanical hypersensitivity. Neuropathic pain is increased, and the mice show a complete absence of the pharmacological analgesic effects of NPY. In the periphery, Y1 receptor activation is sufficient and required for substance P release and the subsequent development of neurogenic inflammation and plasma leakage. We conclude that the Y1 receptor is required for central physiological and pharmacological NPY-induced analgesia and that its activation is both sufficient and required for the release of substance P and initiation of neurogenic inflammation.

Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor.

Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

Complementary and overlapping expression of Y1, Y2 and Y5 receptors in the developing and adult mouse nervous system.

Neuropeptide Y, a 36 amino acid peptide, mediates its biological effects by activating the Y1, Y2, Y5 and Y6 receptors, which are also receptors for the structurally related peptide YY. Different classes of receptors have been suggested to be involved in different neuropeptide Y functions. In this report, we have characterized the developmental regulation and compared the cellular localization of these receptors in the developing and in the adult central and peripheral nervous systems of the mouse. RNase protection assays revealed that Y1, Y2 and Y5 messenger RNAs were expressed very early in spinal cord, brain, cerebellum and dorsal root ganglion development and were often down-regulated at times corresponding to their acquirement of the adult function in neurotransmission. In situ hybridization of the adult brain showed that Y1 was widely expressed, Y2 displayed a more restricted pattern, Y5 was expressed at very low levels and only in a few brain nuclei and Y6 was not expressed. Virtually all areas containing neurons positive for Y5 also expressed Y1, whereas many Y1-positive cells clearly did not express Y5. In contrast, Y2 was not expressed by the neurons expressing Y1 or Y5. These findings suggest that neuropeptide Y signaling in the brain could be mediated by simultaneous Y1 and Y5 activation. Similar results were also obtained in peripheral sensory neurons. Furthermore, our results suggest that neuropeptide Y/peptide YY receptors play an important role in nervous system development and that selective receptor combinations are responsible for signaling the different effects of neuropeptide Y in the peripheral and central nervous systems.

Low affinity NGF receptor expression in the central nervous system during experimental allergic encephalomyelitis.

In the central nervous system (CNS), p75, or low-affinity nerve growth factor receptor (LNGFR), is assumed to play a critical role in mediating the effects of neurotrophins on neuronal survival. Recent studies have shown that nerve growth factor (NGF) can act also on immune cells through its binding to p75. Using immunohistochemistry, we have investigated the expression of the p75 receptor in the CNS during chronic relapsing experimental allergic encephalomyelitis (EAE) of the Lewis rat, an animal model of multiple sclerosis (MS). We report here a sequential expression of p75, first in Purkinje cells during the first attack, and secondly on both endothelial and perivascular cells in the latter stages of the disease. Moreover, starting from the second attack, p75 was also expressed on glial ensheathing cells, likely myelinating cells, located primarily in the dorsal roots. These data suggest that during EAE, LNGFR may play an important role in leukocyte-endothelial cell interactions and in the maintenance of Purkinje cells survival.

Expression and regulation of GFRalpha3, a glial cell line-derived neurotrophic factor family receptor.

We report the identification of an additional member of the glial cell line-derived neurotrophic factor (GDNF) family receptor, termed GFRalpha3, that is homologous to the previously identified GDNF and neurturin ligand binding receptors GFRalpha1 and GFRalpha2. GFRalpha3 is 32% and 37% identical to GFRalpha1 and GFRalpha2, respectively. RNase protection assays show that whereas gfralpha1 and gfralpha2 are abundant in both developing and adult brain, gfralpha3 is exclusively expressed during development. All receptors are widely present in both the developing and adult peripheral nervous system and in peripheral organs. For instance, in situ hybridization shows that the developing liver, stomach, intestine, kidney, and sympathetic chain, which all contain ret-expressing cells, transcribe unique complementary and overlapping patterns of most or all of the GDNF family receptors and ligands. In sensory neurons of the trigeminal ganglion gfralpha2 and gfralpha3 are expressed in different subpopulations of neurons, whereas gfralpha1 is coexpressed in some gfralpha2 and gfralpha3-positive neurons. We find that the gfralpha1 population of trigeminal neurons is absent in GDNF null mutant mice, suggesting that GDNF signals in vivo by interacting with GFRalpha1. Thus, our results show that there are at least three members in the GDNF family of ligand binding receptors and that these receptors may be crucial in conferring ligand specificity in vivo. The unique complementary and overlapping expression of gfralpha3 implies distinct functions in the developing and adult mouse from that of GFRalpha1 and GFRalpha2.