Extracellular ATP inhibits excitatory synaptic input on parvalbumin positive interneurons and attenuates gamma oscillations via P2X4 receptors.

BACKGROUND AND PURPOSE: P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular ATP released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable with NMDA receptors. Although P2X4Rs have been reported to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. EXPERIMENTAL APPROACH: We investigated the effects of P2X receptors at network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mouse hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. KEY RESULTS: The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB-12062 and was not observed in P2X4-/- mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin-dependent internalization of AMPA receptors. CONCLUSIONS AND IMPLICATIONS: The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and presumably the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer's disease and schizophrenia.

Microglial P2X4 receptors are essential for spinal neurons hyperexcitability and tactile allodynia in male and female neuropathic mice.

In neuropathic pain, recent evidence has highlighted a sex-dependent role of the P2X4 receptor in spinal microglia in the development of tactile allodynia following nerve injury. Here, using internalization-defective P2X4mCherryIN knockin mice (P2X4KI), we demonstrate that increased cell surface expression of P2X4 induces hypersensitivity to mechanical stimulations and hyperexcitability in spinal cord neurons of both male and female naive mice. During neuropathy, both wild-type (WT) and P2X4KI mice of both sexes develop tactile allodynia accompanied by spinal neuron hyperexcitability. These responses are selectively associated with P2X4, as they are absent in global P2X4KO or myeloid-specific P2X4KO mice. We show that P2X4 is de novo expressed in reactive microglia in neuropathic WT and P2X4KI mice of both sexes and that tactile allodynia is relieved by pharmacological blockade of P2X4 or TrkB. These results show that the upregulation of P2X4 in microglia is crucial for neuropathic pain, regardless of sex.

Microglial P2X4 receptors promote ApoE degradation and contribute to memory deficits in Alzheimer's disease.

Numerous evidences support that microglia contributes to the progression of Alzheimer's disease. P2X4 receptors are ATP-gated channels with high calcium permeability, which are de novo expressed in a subset of reactive microglia associated with various pathological contexts, contributing to microglial functions. P2X4 receptors are mainly localized in lysosomes and trafficking to the plasma membrane is tightly regulated. Here, we investigated the role of P2X4 in the context of Alzheimer's disease (AD). Using proteomics, we identified Apolipoprotein E (ApoE) as a specific P2X4 interacting protein. We found that P2X4 regulates lysosomal cathepsin B (CatB) activity promoting ApoE degradation; P2rX4 deletion results in higher amounts of intracellular and secreted ApoE in both bone-marrow-derived macrophage (BMDM) and microglia from APPswe/PSEN1dE9 brain. In both human AD brain and APP/PS1 mice, P2X4 and ApoE are almost exclusively expressed in plaque-associated microglia. In 12-month-old APP/PS1 mice, genetic deletion of P2rX4 reverses topographical and spatial memory impairment and reduces amount of soluble small aggregates of Aß1-42 peptide, while no obvious alteration of plaque-associated microglia characteristics is observed. Our results support that microglial P2X4 promotes lysosomal ApoE degradation, indirectly altering Aß peptide clearance, which in turn might promotes synaptic dysfunctions and cognitive deficits. Our findings uncover a specific interplay between purinergic signaling, microglial ApoE, soluble Aß (sAß) species and cognitive deficits associated with AD.

Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?

Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.

A P2rx7 Passenger Mutation Affects the Vitality and Function of T cells in Congenic Mice.

Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several transgenic mice carry a P2rx7 passenger mutation that affects the function of T cells. By the example of P2rx4tm1Rass we demonstrate that P2X4ko T cells express higher levels of P2X7 and are more sensitive toward the P2X7 activators ATP and NAD+, rendering these cells more vulnerable toward NAD-induced cell death (NICD) compared with wild type (WT). The enhanced NICD sensitivity confounded functional assays e.g. cytokine production and cell migration. Our results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes.

Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4.

The P2X4 channel is involved in different physiological and pathological conditions and functions in the nervous system. Despite the existence of several mouse models for which the expression of the gene was manipulated, there is still little information on the expression of the protein at the cellular level. In particular, supposedly specific available antibodies have often proved to recognize unrelated proteins in P2X4-deficient mice. Here, we used an in vivo DNA vaccine approach to generate a series of monoclonal antibodies and nanobodies specific for human, mouse, and rat P2X4 channels. We further characterized these antibodies and show that they solely recognize the native form of the proteins both in biochemical and cytometric applications. Some of these antibodies prove to specifically recognize P2X4 channels by immunostaining in brain or sensory ganglia slices, as well as at the cellular and subcellular levels. Due to their clonality, these different antibodies should represent versatile tools for further characterizing the cellular functions of P2X4 in the nervous system as well as at the periphery.

Microglia in Alzheimer Disease: Well-Known Targets and New Opportunities.

Microglia are the resident macrophages of the central nervous system. They play key roles in brain development, and physiology during life and aging. Equipped with a variety of molecular sensors and through the various functions they can fulfill, they are critically involved in maintaining the brain's homeostasis. In Alzheimer disease (AD), microglia reaction was initially thought to be incidental and triggered by amyloid deposits and dystrophic neurites. However, recent genome-wide association studies have established that the majority of AD risk loci are found in or near genes that are highly and sometimes uniquely expressed in microglia. This leads to the concept of microglia being critically involved in the early steps of the disease and identified them as important potential therapeutic targets. Whether microglia reaction is beneficial, detrimental or both to AD progression is still unclear and the subject of intense debate. In this review, we are presenting a state-of-knowledge report intended to highlight the variety of microglial functions and pathways shown to be critically involved in AD progression. We first address both the acquisition of new functions and the alteration of their homeostatic roles by reactive microglia. Second, we propose a summary of new important parameters currently emerging in the field that need to be considered to identify relevant microglial targets. Finally, we discuss the many obstacles in designing efficient therapeutic strategies for AD and present innovative technologies that may foster our understanding of microglia roles in the pathology. Ultimately, this work aims to fly over various microglial functions to make a general and reliable report of the current knowledge regarding microglia's involvement in AD and of the new research opportunities in the field.

Blocking α2δ-1 Subunit Reduces Bladder Hypersensitivity and Inflammation in a Cystitis Mouse Model by Decreasing NF-kB Pathway Activation.

Bladder pain is frequently associated with bladder inflammation, as in conditions like interstitial cystitis (IC), for which current analgesic therapies have limited efficacy. The antinociceptive effect of alpha-2-delta (α2δ) ligands on inflammation-associated visceral pain like that experienced in cystitis has been poorly investigated. To investigate the effect of pregabalin (PGB), an α2δ ligand, we evaluated its impact on mechanical hyperalgesia in a mouse model of cystitis induced by cyclophosphamide (CYP). We further studied its effect on inflammation and NF-kB pathway activation. Acute cystitis was induced by intraperitoneal injection of 150 mg kg-1 of CYP in C57Bl/6J male mice. PGB was subcutaneously injected (30 mg kg-1) 3 h after CYP injection. The effect of PGB on CYP-induced mechanical referred hyperalgesia (abdominal Von Frey test), inflammation (organ weight, cytokine production, α2δ subunit level, NF-kB pathway activation) were assessed 1 h after its injection. In parallel, its effect on cytokine production, α2δ subunit level and NF-kB pathway activation was assessed in vitro on peritoneal exudate cells (PECs) stimulated with LPS. PGB treatment decreased mechanical referred hyperalgesia. Interestingly, it had an anti-inflammatory effect in the cystitis model by reducing pro-inflammatory cytokine production. PGB also inhibited NF-kB pathway activation in the cystitis model and in macrophages stimulated with LPS, in which it blocked the increase in intracellular calcium. This study shows the efficacy of PGB in hypersensitivity and inflammation associated with cystitis. It is therefore of great interest in assessing the benefit of α2δ ligands in patients suffering from cystitis.

Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte-derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα-driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

Inflammatory-induced spinal dorsal horn neurons hyperexcitability is mediated by P2X4 receptors.

INTRODUCTION: Purinergic ionotropic P2X receptors (P2RX) are involved in normal and pathological pain transmission. Among them, P2X4 are expressed in dorsal root ganglion and in the spinal cord. Their activation during nerve injury or chronic peripheral inflammation modifies pain sensitivity that leads to the phenomenon of allodynia and hyperalgesia. OBJECTIVES: We study here, in vivo, the role of P2X4 on the excitability of dorsal horn neurons (DHNs) in naive or pathological context. METHODS: We recorded DHNs in vivo in anesthetized wild-type or P2RX4-/- mice. We measured nociceptive integration and short-term sensitization by DHNs both in naive and inflamed mice. RESULTS: Our results indicate that P2X4 alter neuronal excitability only in the pathological context of peripheral inflammation. Consequently, excitability of DHNs from inflamed P2RX4-/- mice remains similar to naive animals. CONCLUSION: These results confirm the prominent role of P2X4 in inflammatory pain context and demonstrate that P2X4 are also involved in the hyperexcitability of DHNs.

Sensory neuronal P2RX4 receptors controls BDNF signaling in inflammatory pain.

Chronic inflammatory and neuropathic pains are major public health concerns. Potential therapeutic targets include the ATP-gated purinergic receptors (P2RX) that contribute to these pathological types of pain in several different cell types. The purinergic receptors P2RX2 and P2RX3 are expressed by a specific subset of dorsal root ganglion neurons and directly shape pain processing by primary afferents. In contrast the P2RX4 and P2RX7 are mostly expressed in myeloid cells, where activation of these receptors triggers the release of various pro-inflammatory molecules. Here, we demonstrate that P2RX4 also controls calcium influx in mouse dorsal root ganglion neurons. P2RX4 is up-regulated in pain-processing neurons during long lasting peripheral inflammation and it co-localizes with Brain-Derived Neurotrophic Factor (BDNF). In the dorsal horn of the spinal cord, BDNF-dependent signaling pathways, phosphorylation of Erk1/2 and of the GluN1 subunit as well as the down regulation of the co-transporter KCC2, which are triggered by peripheral inflammation are impaired in P2RX4-deficient mice. Our results suggest that P2RX4, expressed by sensory neurons, controls neuronal BDNF release that contributes to hyper-excitability during chronic inflammatory pain and establish P2RX4 in sensory neurons as a new potential therapeutic target to treat hyperexcitability during chronic inflammatory pain.

Alleviating pain hypersensitivity through activation of type 4 metabotropic glutamate receptor.

Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain.

Development of NMDAR antagonists with reduced neurotoxic side effects: a study on GK11.

The NMDAR glutamate receptor subtype mediates various vital physiological neuronal functions. However, its excessive activation contributes to neuronal damage in a large variety of acute and chronic neurological disorders. NMDAR antagonists thus represent promising therapeutic tools that can counteract NMDARs' overactivation. Channel blockers are of special interest since they are use-dependent, thus being more potent at continuously activated NMDARs, as may be the case in pathological conditions. Nevertheless, it has been established that NMDAR antagonists, such as MK801, also have unacceptable neurotoxic effects. Presently only Memantine is considered a safe NMDAR antagonist and is used clinically. It has recently been speculated that antagonists that preferentially target extrasynaptic NMDARs would be less toxic. We previously demonstrated that the phencyclidine derivative GK11 preferentially inhibits extrasynaptic NMDARs. We thus anticipated that this compound would be safer than other known NMDAR antagonists. In this study we used whole-genome profiling of the rat cingulate cortex, a brain area that is particularly sensitive to NMDAR antagonists, to compare the potential adverse effects of GK11 and MK801. Our results showed that in contrast to GK11, the transcriptional profile of MK801 is characterized by a significant upregulation of inflammatory and stress-response genes, consistent with its high neurotoxicity. In addition, behavioural and immunohistochemical analyses confirmed marked inflammatory reactions (including astrogliosis and microglial activation) in MK801-treated, but not GK11-treated rats. Interestingly, we also showed that GK11 elicited less inflammation and neuronal damage, even when compared to Memantine, which like GK11, preferentially inhibits extrasynaptic NMDAR. As a whole, our study suggests that GK11 may be a more attractive therapeutic alternative in the treatment of CNS disorders characterized by the overactivation of glutamate receptors.

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus.

Within the central nervous system, functions of the ATP-gated receptor-channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)-induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R-deficient mice, behavioral responses during KA-induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage-dependent potassium channels were impaired in P2X4R-deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity-dependent degenerative processes, the CA1 area was partially protected from SE-induced neuronal death in P2X4R-deficient mice compared with wild-type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation.

P2X2 and P2X5 subunits define a new heteromeric receptor with P2X7-like properties.

Ligand-gated ion channels are prototypic oligomeric membrane proteins whose stoichiometry determines their functional properties and subcellular localization. Deciphering the quaternary structure of such protein complexes is an arduous task and usually requires the combination of multiple approaches. ATP-gated P2X receptors are formed by the association of three subunits, but the quaternary arrangement of the seven P2X subunits at the plasma membrane remains poorly characterized. By combining bioluminescence resonance energy transfer, bifunctional fluorescence complementation and protein biochemistry, we developed an experimental approach that allows precise determination of rat P2X receptor quaternary assembly. We found that P2X5 subunits associate with P2X1, P2X2, and P2X4 subunits. We demonstrate that P2X5 and P2X2 subunits interact to form as yet uncharacterized heteromeric receptors with alternate stoichiometries, both present at the plasma membrane. P2X2/5 receptors display functional properties such as pore dilatation, membrane blebbing, and phosphatidylserine exposure that were previously thought to be characteristic hallmarks of the P2X7 receptor. In mouse, P2X2 and P2X5 subunits colocalize and physically interact in specific neuronal populations suggesting that other P2X receptors might contribute to cellular responses typically attributed to P2X7 receptor.

P2X4 receptors mediate PGE2 release by tissue-resident macrophages and initiate inflammatory pain.

Prostaglandin E2 (PGE2) is a key mediator of inflammation and contributes to pain hypersensitivity by promoting sensory neurons hyperexcitability. PGE2 synthesis results from activation of a multi-step enzymatic cascade that includes cyclooxygenases (COXs), the main targets of non-steroidal anti-inflammatory drugs (NSAIDs). Although NSAIDs are widely prescribed to reduce inflammatory symptoms such as swelling and pain, associated harmful side effects restrict their long-term use. Therefore, finding new drugs that limit PG production represents an important therapeutic issue. In response to peripheral inflammatory challenges, mice lacking the ATP-gated P2X4 channel (P2X4R) do not develop pain hypersensitivity and show a complete absence of inflammatory PGE2 in tissue exudates. In resting conditions, tissue-resident macrophages constitutively express P2X4R. Stimulating P2X4R in macrophages triggers calcium influx and p38 MAPK phosphorylation, resulting in cytosolic PLA2 (cPLA2) activation and COX-dependent release of PGE2. In naive animals, pain hypersensitivity was elicited by transfer into the paw of ATP-primed macrophages from wild type, but not P2X4R-deficient mice. Thus, P2X4Rs are specifically involved in inflammatory-mediated PGE2 production and might therefore represent useful therapeutic targets.

The 19th Ion Channel Meeting. September 2008, France.

The annual meeting of the French Ion Channels Society, held on the Mediterranean coast of France, is aimed at gathering the international scientific community working on various aspects of ion channels. In this report of the 19th edition of the meeting, held in September 2008, we summarize selected symposia on aspects of the ion channel field from fundamental to clinical research. The meeting is an opportunity for leading investigators as well as young researchers to present and discuss their recent advances and future challenges in the ion channel field.

Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain.

ATP is a known mediator of inflammatory and neuropathic pain. However, the mechanisms by which specific purinergic receptors contribute to chronic pain states are still poorly characterized. Here, we demonstrate that in response to peripheral nerve injury, P2X(4) receptors (P2X(4)R) are expressed de novo by activated microglia in the spinal cord. Using in vitro and in vivo models, we provide direct evidence that P2X(4)R stimulation leads to the release of BDNF from activated microglia and, most likely phosphorylation of the NR1 subunit of NMDA receptors in dorsal horn neurons of the spinal cord. Consistent with these findings, P2X4-deficient mice lack mechanical hyperalgesia induced by peripheral nerve injury and display impaired BDNF signaling in the spinal cord. Furthermore, ATP stimulation is unable to stimulate BDNF release from P2X(4)-deficient mice microglia in primary cultures. These results indicate that P2X(4)R contribute to chronic pain through a central inflammatory pathway. P2X(4)R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.

Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling.

Microglia cells are the resident macrophages of the CNS, and their activation plays a critical role in inflammatory reactions associated with many brain disorders, including ischemia, Alzheimer's and Parkinson's diseases, and epilepsy. However, the changes of microglia functional properties in epilepsy have rarely been studied. Here, we used a model of status epilepticus (SE) induced by intraperitoneal kainate injections to characterize the properties of microglial cells in hippocampal slices from CX3CR1(eGFP/+) mice. SE induced within 3 h an increased expression of inflammatory mediators in the hippocampus, followed by a modification of microglia morphology, a microglia proliferation, and a significant neurodegeneration in CA1. Changes in electrophysiological intrinsic membrane properties of hippocampal microglia were detected at 24-48 h after SE with, in particular, the appearance of new voltage-activated potassium currents. Consistent with the observation of an upregulation of purinergic receptor mRNAs in the hippocampus, we also provide pharmacological evidence that microglia membrane currents mediated by the activation of P2 receptors, including P2X(7), P2Y(6), and P2Y(12), were increased 48 h after SE. As a functional consequence of this modification of purinergic signaling, motility of microglia processes toward a source of P2Y(12) receptor agonist was twice as fast in the epileptic hippocampus. This study is the first functional description of microglia activation in an in vivo model of inflammation and provides evidence for the existence of a particular microglial activation state after a status epilepticus.

Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model.

The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.