Lithium reverses mechanical allodynia through a mu opioid-dependent mechanism.

Background Lithium is widely used to treat bipolar disorders and displays mood stabilizing properties. In addition, lithium relieves painful cluster headaches and has a strong analgesic effect in neuropathic pain rat models. Objectives To investigate the analgesic effect of lithium on the cuff model of neuropathic pain. Methods We used behavioral and pharmacological approaches to study the analgesic effect of a single injection of lithium in wild-type and mu opioid receptor (MOR) null cuffed neuropathic mice. Mass spectrometry and enzyme-linked immunosorbent assay allowed to measure the levels of endogenous MOR agonist beta-endorphin as well as monoamines in brain and plasma samples 4 h after lithium administration. Results A single injection of lithium chloride (100 mg/kg, ip) alleviated mechanical allodynia for 24 h, and this effect was absent in MOR null neuropathic mice. Biochemical analyses highlight a significant increase in beta-endorphin levels by 30% in the brain of lithium-treated mice compared to controls. No variation of beta-endorphin was detected in the blood. Conclusions Together, our results provide evidence that lithium induces a long-lasting analgesia in neuropathic mice presumably through elevated brain levels of beta-endorphin and the activation of MORs.

Favouring inhibitory synaptic drive mediated by GABA(A) receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Corticosterone analgesia is mediated by the spinal production of neuroactive metabolites that enhance GABAergic inhibitory transmission on dorsal horn rat neurons.

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic-pituitary-adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3α5α-reduced steroids, which modulate GABAA receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3α5α-reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non-genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C-fiber-mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

An analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray in rats.

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

Calcium imaging and BAPTA loading of amygdala astrocytes in mouse brain slices.

Astrocytes are glial cells that exhibit calcium signaling-mediated activity. Here, we present a protocol to monitor and manipulate astrocyte calcium activity from mouse amygdala slices. In the first part of this protocol, we describe the procedure of astrocyte calcium imaging. In the second part, we detail how to disrupt astrocyte calcium activity by patch-clamp-mediated loading of BAPTA. These two approaches are presented separately but they can also be used simultaneously to monitor the effects of disruption on an astrocyte network. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021).

The Role of Oxytocin in Abnormal Brain Development: Effect on Glial Cells and Neuroinflammation.

The neonatal period is critical for brain development and determinant for long-term brain trajectory. Yet, this time concurs with a sensitivity and risk for numerous brain injuries following perinatal complications such as preterm birth. Brain injury in premature infants leads to a complex amalgam of primary destructive diseases and secondary maturational and trophic disturbances and, as a consequence, to long-term neurocognitive and behavioral problems. Neuroinflammation is an important common factor in these complications, which contributes to the adverse effects on brain development. Mediating this inflammatory response forms a key therapeutic target in protecting the vulnerable developing brain when complications arise. The neuropeptide oxytocin (OT) plays an important role in the perinatal period, and its importance for lactation and social bonding in early life are well-recognized. Yet, novel functions of OT for the developing brain are increasingly emerging. In particular, OT seems able to modulate glial activity in neuroinflammatory states, but the exact mechanisms underlying this connection are largely unknown. The current review provides an overview of the oxytocinergic system and its early life development across rodent and human. Moreover, we cover the most up-to-date understanding of the role of OT in neonatal brain development and the potential neuroprotective effects it holds when adverse neural events arise in association with neuroinflammation. A detailed assessment of the underlying mechanisms between OT treatment and astrocyte and microglia reactivity is given, as well as a focus on the amygdala, a brain region of crucial importance for socio-emotional behavior, particularly in infants born preterm.

Anti-Hyperalgesic Properties of Menthol and Pulegone.

Context: Menthol, the main monoterpene found in Mentha piperita L. (M. piperita) is known to modulate nociceptive threshold and is present in different curative preparations that reduce sensory hypersensitivities in pain conditions. While for pulegone, a menthol-like monoterpene, only a limited number of studies focus on its putative analgesic effects, pulegone is the most abundant monoterpene present in Calamintha nepeta (L.) Savi (C. nepeta), a plant of the Lamiaceae family used in traditional medicine to alleviate rheumatic disorders, which counts amongst chronic inflammatory diseases. Objectives: Here, we analyzed the monoterpenes composition of C. nepeta and M. piperita. We then compared the putative anti-hyperalgesic effects of the main monoterpenes found, menthol and pulegone, in acute inflammatory pain conditions. Methods: C. nepeta and M. piperita extracts were obtained through pressurized liquid extraction and analyzed by gas chromatography-mass spectrometry. The in vitro anti-inflammatory activity of menthol or pulegone was evaluated by measuring the secretion of the tumour necrosis factor alpha (TNF α) from LPS-stimulated THP-1 cells. The in vivo anti-hyperalgesic effects of menthol and pulegone were tested on a rat inflammatory pain model. Results: Pulegone and menthol are the most abundant monoterpene found in C. nepeta (49.41%) and M. piperita (42.85%) extracts, respectively. In vitro, both pulegone and menthol act as strong anti-inflammatory molecules, with EC50 values of 1.2 ± 0.2 and 1.5 ± 0.1 mM, respectively, and exert cytotoxicity with EC50 values of 6.6 ± 0.3 and 3.5 ± 0.2 mM, respectively. In vivo, 100 mg/kg pulegone exerts a transient anti-hyperalgesic effect on both mechanical (pulegone: 274.25 ± 68.89 g, n = 8; vehicle: 160.88 ± 35.17 g, n = 8, p < 0.0001), thermal heat (pulegone: 4.09 ± 0.62 s, n = 8; vehicle: 2.25 ± 0.34 s, n = 8, p < 0.0001), and cold (pulegone: 2.25 ± 1.28 score, n = 8; vehicle: 4.75 ± 1.04 score, n = 8, p = 0.0003). In a similar way, 100 mg/kg menthol exerts a transient anti-hyperalgesic effect on both mechanical (mechanical: menthol: 281.63 ± 45.52 g, n = 8; vehicle: 166.25 ± 35.4 g, n = 8, p < 0.0001) and thermal heat (menthol: 3.65 ± 0.88 s, n = 8; vehicle: 2.19 ± 0.26 s, n = 8, <0.0001). Conclusion: Here, we show that both pulegone and menthol are anti-inflammatory and anti-hyperalgesic monoterpenes. These results might open the path towards new compound mixes to alleviate the pain sensation.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

A Nonpeptide Oxytocin Receptor Agonist for a Durable Relief of Inflammatory Pain.

Oxytocin possesses several physiological and social functions, among which an important analgesic effect. For this purpose, oxytocin binds mainly to its unique receptor, both in the central nervous system and in the peripheral nociceptive terminal axon in the skin. However, despite its interesting analgesic properties and its current use in clinics to facilitate labor, oxytocin is not used in pain treatment. Indeed, it is rapidly metabolized, with a half-life in the blood circulation estimated at five minutes and in cerebrospinal fluid around twenty minutes in humans and rats. Moreover, oxytocin itself suffers from several additional drawbacks: a lack of specificity, an extremely poor oral absorption and distribution, and finally, a lack of patentability. Recently, a first non-peptide full agonist of oxytocin receptor (LIT-001) of low molecular weight has been synthesized with reported beneficial effect for social interactions after peripheral administration. In the present study, we report that a single intraperitoneal administration of LIT-001 in a rat model induces a long-lasting reduction in inflammatory pain-induced hyperalgesia symptoms, paving the way to an original drug development strategy for pain treatment.

Unveiling the Impact of Morphine on Tamoxifen Metabolism in Mice in vivo.

Background: Tamoxifen is used to treat breast cancer and cancer recurrences. After administration, tamoxifen is converted into two more potent antitumor compounds, 4OH-tamoxifen and endoxifen by the CYP3A4/5 and 2D6 enzymes in human. These active compounds are inactivated by the same UDP-glucuronosyltransferase isoforms as those involved in the metabolism of morphine. Importantly, cancer-associated pain can be treated with morphine, and the common metabolic pathway of morphine and tamoxifen suggests potential clinically relevant interactions. Methods: Mouse liver microsomes were used to determine the impact of morphine on 4OH-tamoxifen metabolism in vitro. For in vivo experiments, female mice were first injected with tamoxifen alone and then with tamoxifen and morphine. Blood was collected, and LC-MS/MS was used to quantify tamoxifen, 4OH-tamoxifen, N-desmethyltamoxifen, endoxifen, 4OH-tamoxifen-glucuronide, and endoxifen-glucuronide. Results: In vitro, we found increased K m values for the production of 4OH-tamoxifen-glucuronide in the presence of morphine, suggesting an inhibitory effect on 4OH-tamoxifen glucuronidation. Conversely, in vivo morphine treatment decreased 4OH-tamoxifen levels in the blood while dramatically increasing the formation of inactive metabolites 4OH-tamoxifen-glucuronide and endoxifen-glucuronide. Conclusions: Our findings emphasize the need for caution when extrapolating results from in vitro metabolic assays to in vivo drug metabolism interactions. Importantly, morphine strongly impacts tamoxifen metabolism in mice. It suggests that tamoxifen efficiency could be reduced when both drugs are co-administered in a clinical setting, e.g., to relieve pain in breast cancer patients. Further studies are needed to assess the potential for tamoxifen-morphine metabolic interactions in humans.

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.

Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord.

BACKGROUND: Growing evidence in the literature shows that oxytocin (OT) has a strong spinal anti-nociceptive action. Oxytocinergic axons originating from a subpopulation of paraventricular hypothalamic neurons establish synaptic contacts with lamina II interneurons but little is known about the functional role of OT with respect to neuronal firing and excitability. RESULTS: Using the patch-clamp technique, we have recorded lamina II interneurons in acute transverse lumbar spinal cord slices of rats (15 to 30 days old) and analyzed the OT effects on action potential firing ability. In the current clamp mode, we found that bath application of a selective OT-receptor agonist (TGOT) reduced firing in the majority of lamina II interneurons exhibiting a bursting firing profile, but never in those exhibiting a single spike discharge upon depolarization. Interestingly, OT-induced reduction in spike frequency and increase of firing threshold were often observed, leading to a conversion of the firing profile from repetitive and delayed profiles into phasic ones and sometimes further into single spike profile. The observed effects following OT-receptor activation were completely abolished when the OT-receptor agonist was co-applied with a selective OT-receptor antagonist. In current and voltage clamp modes, we show that these changes in firing are strongly controlled by voltage-gated potassium currents. More precisely, transient IA currents and delayed-rectifier currents were reduced in amplitude and transient IA current was predominantly inactivated after OT bath application. CONCLUSION: This effect of OT on the firing profile of lamina II neurons is in good agreement with the antinociceptive and analgesic properties of OT described in vivo.

A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System.

Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram.

Stable isotope-labelled morphine to study in vivo central and peripheral morphine glucuronidation and brain transport in tolerant mice.

BACKGROUND AND PURPOSE: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels. EXPERIMENTAL APPROACH: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms. KEY RESULTS: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study. CONCLUSIONS AND IMPLICATIONS: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.

Effects of brain-derived neurotrophic factor (BDNF) on activity mediated by NMDA receptors in rat spinal cord cultures.

Brain-derived neurotrophic factor (BDNF) is involved in the differentiation and the survival of neurons. It has also been shown to be associated with the regrowth of neurons of damaged spinal cord and the modulation of ionic currents by acting on sodium channels and NMDA receptors through tyrosine kinase B (TrkB) receptors. We investigated the effects of BDNF on rhythm generation induced by disinhibition in dissociated cultures from embryonic rat spinal cord (E14), with extracellular multisite recordings (MultiElectrode Arrays, MEAs) or intracellular patch-clamp recordings. Exogenous BDNF had only minor effects on the bursting by increasing the activity during the burst. This increase of activity is suggested to be mediated by a potentiation of the postsynaptic NMDA receptors because it has been found that BDNF potentiates the NMDA-evoked depolarization in cultures incubated with BDNF for 10 min. Possible direct effects of BDNF on sodium channels were also investigated by local application of BDNF to the soma of patched neurons but no depolarization was observed. Long-term application of BDNF strongly decreased the activity during the burst and also the number of active electrodes, possibly due to a decrease in network density.

Plasma glucocorticoids differentially modulate phasic and tonic GABA inhibition during early postnatal development in rat spinal lamina II.

Nociceptive processing is tuned by GABAA receptor-mediated inhibition in the spinal cord dorsal horn that undergoes postnatal maturation in rodents. These GABAergic inhibitory postsynaptic currents (IPSCs) are modulated by 3α5α-reduced steroids during early postnatal development in spinal cord lamina II. Thus an enhanced phasic inhibition is present in neonates and decreases over time. GABA can also activate extrasynaptic receptors, giving rise to tonic inhibition. In this study, we characterized the contribution of plasma corticosterone (CORT) to postnatal maturation of spinal phasic and, for the first time, tonic GABAergic inhibitions. We used Fisher and Lewis rat strains displaying respectively high and low hypothalamic-pituitary-adrenal axis reactivity, compared to control Sprague-Dawley rats. Measured plasma CORT levels were significantly higher in Fisher rats, which also displayed significantly higher mechanical nociceptive thresholds, supporting the hypothesis of an antinociceptive action of CORT. Recorded GABAA IPSCs shortened during maturation in all strains while remaining larger in Fisher rats. Blocking the 5α-reduction of steroids in Fisher rats produced a further decrease of IPSC deactivation time constant. In contrast, GABAA tonic inhibition progressively increased during maturation, without any difference among strains. In conclusion, we show that both phasic and tonic GABAergic inhibitions undergo postnatal maturation in lamina II. Moreover spinal production of 3α5α-reduced steroids that presumably derive from plasma CORT is correlated to spinal GABAA phasic (but not tonic) inhibition and to mechanical nociceptive thresholds.

Etifoxine analgesia in experimental monoarthritis: a combined action that protects spinal inhibition and limits central inflammatory processes.

Inflammatory and degenerative diseases of the joint are major causes of chronic pain. Long-lasting pain symptoms are thought to result from a central sensitization of nociceptive circuits. These processes include activation of microglia and spinal disinhibition. Using a monoarthritic rat model of pain, we tried to potentiate neural inhibition by using etifoxine (EFX), a nonbenzodiazepine anxiolytic that acts as an allosteric-positive modulator of gamma-aminobutyric acid type A (GABAA) receptor function. Interestingly, EFX also can bind to the mitochondrial translocator protein (TSPO) complex and stimulate the synthesis of 3α-reduced neurosteroids, the most potent positive allosteric modulator of GABAA receptor function. Here we show that a curative and a preventive treatment with 50mg/kg of EFX efficiently reduced neuropathic pain symptoms. In the spinal cord, EFX analgesia was accompanied by a reduction in microglial activation and in the levels of proinflammatory mediators. Using electrophysiological tools, we found that EFX treatment not only amplified spinal GABAergic inhibition, but also prevented prostaglandin E2-induced glycinergic disinhibition and restored a "normal" spinal pain processing. Because EFX is already distributed in several countries under the trade name of Stresam for its anxiolytic actions in humans, new clinical trials are now required to further extend its therapeutic indications as pain killer.

Conductance properties of the acetylcholine receptor current of Guinea pig outer hair cells.

The nicotinic acetylcholine receptor (AChR) current of outer hair cells (OHCs) was investigated in isolated and voltage-clamped cells under conditions where co-activating Ca(2+)-activated K(+) currents had been abolished using internal BAPTA, external calcium removal and/or depolarisation to positive voltages. The AChR current activated rapidly and thereafter declined in the continued presence of ACh. Reversal potential measurements indicated that it was a non-specific cation current with a substantial Ca(2+) permeability. It had a characteristic bidirectional rectification with an especially prominent outward component in solutions containing 1 mM Ca(2+). The I-V relation was fitted with a single-energy barrier model. The fit suggests a blocking site within the channel, situated about one third of the way through the membrane from the outside and probably normally occupied by Ca(2+) or Mg(2+). The AChR current was sensitive to the external Ca(2+) since it was reduced, to differing extents, in nominally Ca(2+)-free saline or in high Ca(2+) saline (10 mM). In the presence of a nominally Mg(2+)-free solution containing 0.4 mM Ca(2+), the currents were larger, indicating a potentiated response. This type of behaviour is also shown by recombinant α9α10 AChRs, suggesting a close similarity. The AChR current at both positive and negative voltages was reduced in external solutions where most of the Na(+) had been replaced by NMG(+). The conductance properties of the OHC AChR are compared with α9α10 receptors and nicotinic receptors in other hair cells and discussed in terms of the accepted functional role of providing calcium influx leading to efferent synaptic inhibition of hair cells.