The projections from the anterior cingulate cortex to the nucleus accumbens and ventral tegmental area contribute to neuropathic pain-evoked aversion in rats.

Although the anterior cingulate cortex (ACC) plays a vital role in neuropathic pain-related aversion, the underlying mechanisms haven't been fully studied. The mesolimbic dopamine system encodes reward and aversion, and participates in the exacerbation of chronic pain. Therefore, we investigated whether the ACC modulates aversion to neuropathic pain via control of the mesolimbic dopamine system, in a rat model of chronic constriction injury (CCI) to the sciatic nerve. Using anterograde and retrograde tracings, we confirmed that a subgroup of ACC neurons projected to the nucleus accumbens (NAc) and ventral tegmental area (VTA), which are two crucial nodes of the mesolimbic dopamine system. Combining electrophysiology in juvenile rats 7 days post-CCI, we found that the NAc/VTA-projecting neurons were hyperexcitable after CCI. Chemogenetic inhibition of these projections induced conditioned place preference in young adult rats 10-14 days post-CCI, without modulating the evoked pain threshold, whereas activation of these projections in sham rats mimicked aversive behavior. Furthermore, the function of the ACC projections was probably mediated by NAc D2-type medium spiny neurons and VTA GABAergic neurons. Taken together, our findings suggest that projections from the ACC to the NAc and VTA mediate neuropathic pain-related aversive behavior.

Inhibition of Metabotropic Glutamate Receptor Subtype 1 Alters the Excitability of the Commissural Pyramidal Neuron in the Rat Anterior Cingulate Cortex after Chronic Constriction Injury to the Sciatic Nerve.

BACKGROUND: Inhibition of the metabotropic glutamate receptor subtype 1 in the anterior cingulate cortex has an analgesic effect during sustained nociceptive hypersensitivity. However, the specific changes in different subtypes of anterior cingulate cortex layer 5 pyramidal neurons, as well as the distinct effect of metabotropic glutamate receptor subtype 1 inhibition on different neuronal subtypes, have not been well studied. METHODS: Retrograde labeling combined with immunofluorescence, whole cell clamp recording, and behavioral tests combined with RNA interference were performed in a rat model of chronic constriction injury to the sciatic nerve. RESULTS: Commissural layer 5 pyramidal neurons (projecting to the contralateral cortex) existed in the anterior cingulate cortex. The voltage-gated potassium channel subunit 2-mediated current in these neurons were substantially reduced after chronic constriction injury (current densities at +30 mV for the sham, and chronic constriction injury neurons were [mean ± SD] 10.22 ± 3.42 pA/pF vs. 5.58 ± 2.71 pA/pF, respectively; n = 11; P < 0.01), which increased the spike width and fast afterhyperpolarization potential, resulting in hyperexcitability. Inhibition of metabotropic glutamate receptor subtype 1 alleviated the down-regulation of voltage-gated potassium channel subunit 2 currents (current density increased by 8.11 ± 3.22 pA/pF; n = 7; P < 0.01). Furthermore, knockdown of voltage-gated potassium channel subunit 2 current in the commissural neurons attenuated the analgesic effect of metabotropic glutamate receptor subtype 1 inhibition (n = 6 rats; P < 0.05). CONCLUSIONS: The effect of metabotropic glutamate receptor subtype 1 inhibition on commissural anterior cingulate cortex layer 5 pyramidal neurons is likely different with the modification of previously studied hyperpolarization-activated/cyclic nucleotide-gated channel-dependent neurons but relies on the alteration of voltage-gated potassium channel subunit 2 currents. These results will contribute to a better understanding of the therapeutic role of metabotropic glutamate receptor subtype 1 in chronic pain.

Dendritic development of hippocampal CA1 pyramidal cells in a neonatal hypoxia-ischemia injury model.

It is believed that neonatal hypoxia-ischemia (HI) brain injury causes neuron loss and brain functional defects. However, the effect of HI brain injury on dendritic development of the remaining pyramidal cells of the hippocampus and the reaction of contralateral hippocampal neurons require further studies. The Morris water maze and Golgi-Cox staining were used to evaluate the learning and memory and dendritic morphology of pyramidal cells. The results of Golgi-Cox staining showed CA1 pyramidal neurons of HI injury models with fewer bifurcations and shorter dendrite length than the naive control group. The density of dendritic spines of hippocampal CA1 pyramidal neurons was significantly lower in the HI brain injury group than in controls. With respect to hippocampal function, the HI brain injury group presented cognitive deficits in the reference memory task and probe trail. In the HI group, the pyramidal cells of left hippocampus that did not experienced ischemia but did experience hypoxia had more complex dendrites and higher density of spine than the HI injury side and control. The functional implementation of injured hippocampus might depend mainly on the hypertrophy of contralateral hippocampus after HI brain injury. Corticosterone can partially prevent the hippocampal pyramidal cells from HI injury and reduce the difference of the bilateral hippocampus pyramidal cells, but there was no improvement in learning and memory.

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion.

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X(1 -7)). P2X(3) homomultimer and P2X(2/3) heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X(5) receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X(5) receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X(5) receptors in cat. In mouse DRG, the P2X(5) receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X(5) receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X(5) receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X(5)-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X(5)-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X(5) receptors were coexpressed with NF-200 or with NOS. Only a few P2X(5)-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X(5) receptors.

Effect of hypobaric hypoxia on the P2X receptors of pyramidal cells in the immature rat hippocampus CA1 sub-field.

PRIMARY OBJECTIVE: This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells. RESEARCH DESIGN: The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model. METHODS AND PROCEDURE: P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7-day-old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3-day and 7-day. MAIN OUTCOMES AND RESULTS: In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up-regulated in response to 7 days HH injury. CONCLUSIONS: These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long-term function decrease should result in the expression increase of P2X receptors.

Role of P2Y1 receptor in astroglia-to-neuron signaling at dorsal spinal cord.

Several studies have shown that astrocytes release neurotransmitters into the extracellular space that may then activate receptors on nearby neurons. In the present study, the actions of adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS)-activated astrocyte conditioned medium (ADPbetaS-ACM) on cultured dorsal spinal cord neurons were evaluated by using confocal laser scanning microscopy and whole-cell patch-clamp recording. ADPbetaS caused astrocytic glutamate efflux (43 microM), which in turn induced inward currents in dorsal horn neurons with short time in culture. The inward currents were abolished by 2-amino-5-phosphonlanoicacid (AP-5; NMDAR antagonist) plus 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; non-NMDAR antagonist) but were unaffected by MRS2179 (selective P2Y(1) receptor antagonist). Furthermore, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) was used to block glutamate release from astrocytes. As a result, ADPbetaS-ACM-induced inward currents in neurons were significantly blocked. On the other hand, both NMDAR and non-NMDAR were involved in ADPbetaS-ACM (concentration was diluted to one-tenth)-evoked small [Ca(2+)](i) transients in neurons. Under this condition, the values of glutamate concentrations in the medium are close to values for extracellular glutamate concentrations under physiological conditions. For this reason, it is possible that astrocyte-derived glutamate is important for distant neuron under physiological conditions at dorsal spinal cord. These observations indicate that astrocytic P2Y(1) receptor activation triggered glutamate efflux, which acts on distant neurons to elevate calcium levels or acts on nearby neurons to evoke inward current. Finally, our results support the conclusion that the astrocytic P2Y(1) receptor plays an important role in bidirectional communication between astrocytes and neurons.

[Progress in the research of D-serine on neuron-glia communication].

D-serine is an important gliotransmitter in CNS. As an endogenous ligand for glycine-bind site in NR1 subunit of NMDA glutamate receptors, D-serine is more potent than glycine at activating the site. It is synthesized from L-serine via racemization of serine racemase, which is regulated by many factors. D-serine participates in many physiological and pathological progresses, including synaptic plasticity, sensory information transmission, neural development and neurotoxicity, and is supposed as potential therapeutic target for the treatment of nervous system disease like Alzheimer disease. Here, we provide an overview of recent findings on the mechanisms of its synthesis, degradation, release and physiological and pathological functions in CNS.

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes.

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y(1) receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca(2+)](i) increase and glutamate release are mediated by P2Y(1) receptor. Ca(2+) release from IP(3)-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y(1) receptor-evoked glutamate release is regulated by volume-sensitive Cl(-) channels and anion co-transporter, which open up the possibility that P2Y(1) receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)-benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y(1) receptor-evoked glutamate may be mediated via volume-sensitive Cl(-) channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y(1) receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells.

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.

Rapid inhibition of ATP-induced currents by corticosterone in rat dorsal root ganglion neurons.

The effects of corticosterone (CORT), a natural glucocorticoid hormone, on ATP-induced currents in rat dorsal root ganglion (DRG) neurons and the underlying signaling mechanism were studied by using patch-clamp techniques. Three types of currents (fast, slow and mixed) were evoked by ATP in cultured DRG neurons. Pretreatment with CORT (0.01-10 mumol/l) for 30 s could inhibit the fast current and the fast component of the mixed current. In contrast, CORT had no significant effect on the slow current evoked by ATP. The inhibitory effects were concentration dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 micromol/l), but not by GDP-beta-S (0.2 mmol/l), a blocker of G protein activation. Membrane-impermeable bovine serum albumin-conjugated corticosterone failed to mimic the effects of CORT. The inhibitory effects of CORT on ATP-induced currents diminished after adding protein kinase A inhibitor H89 (10 micromol/l), but were not influenced by protein kinase C inhibitor chelerythrine chloride (10 micromol/l). These results suggest that glucocorticoid hormones might participate in the control of pain by modulating P2X(3) receptor-mediated events in sensory neurons, and the effect is mediated by glucocorticoid receptors and the downstream activation of protein kinase A.

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes.

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.

Aspirin increases ferroportin 1 expression by inhibiting hepcidin via the JAK/STAT3 pathway in interleukin 6-treated PC-12 cells.

To understand the potential mechanisms involved in the beneficial effects of aspirin (ASA) in mood disorders, Alzheimer's (AD) and Parkinson's disease (PD), we investigated the effects of ASA on the expression of iron transport proteins transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1), and iron storage protein ferritin light chain (Ft-L) in interleukin-6 (IL-6)-treated PC-12 cells. We demonstrated that IL-6 alone could induce a severe decline in Fpn1 expression and cell viability, and an increase in Ft-L protein, while ASA could markedly diminish the effects of IL-6 on these parameters. We also found that IL-6 significantly increased hepcidin expression and janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation, while ASA also observably suppressed these IL-6-induced effects. The data imply that ASA increases Fpn1 expression by inhibiting hepcidin expression via the IL-6/JAK/STAT3 pathway and show that the reduced content of Ft-L is due to the increased Fpn1 and subsequent iron release in the cells. The reduction of iron in neuronal cells by the increased expression of Fpn1 might be partly associated with the beneficial effects of ASA on mood disorders, AD and PD.

[Real-time measurement of noradrenaline release in central nervous system].

In order to investigate the central nervous mechanism and the diseases involved in catecholamine transmitter secretion, the dynamics of catecholamine release is studied in single cell, brain slice or in vivo. Noradrenaline is an important neurotransmitter and modulator in the central nervous system (CNS) and the peripheral nervous system (PNS). In the present paper, we first compared three real-time methods used to measure noradrenaline secretion in single cells (membrane capacitance, amperometry and confocal fluorescence microscopy imaging). Compared to the electrophysiological method and fluorescence microscopy, the basic usage of the carbon fiber electrode (CFE) in neuroscience research was presented as an example. Then, we presented a primary description of ion channels, including voltage-gated Na(+), K(+) and Ca(2+) channels in locus coeruleus (LC) neurons in rat brain slices. Finally, we presented example recordings of combined patch-clamp and amperometry measurements in LC neurons, indicating Ca(2+)-dependent quantal noradrenaline release following Ca(2+) influx through Ca(2+) channels.

Parameter Optimization Analysis of Prolonged Analgesia Effect of tDCS on Neuropathic Pain Rats.

Background: Transcranial direct current stimulation (tDCS) is widely used to treat human nerve disorders and neuropathic pain by modulating the excitability of cortex. The effectiveness of tDCS is influenced by its stimulation parameters, but there have been no systematic studies to help guide the selection of different parameters. Objective: This study aims to assess the effects of tDCS of primary motor cortex (M1) on chronic neuropathic pain in rats and to test for the optimal parameter combinations for analgesia. Methods: Using the chronic neuropathic pain models of chronic constriction injury (CCI), we measured pain thresholds before and after anodal-tDCS (A-tDCS) using different parameter conditions, including stimulation intensity, stimulation time, intervention time and electrode located (ipsilateral or contralateral M1 of the ligated paw on male/female CCI models). Results: Following the application of A-tDCS over M1, we observed that the antinociceptive effects were depended on different parameters. First, we found that repetitive A-tDCS had a longer analgesic effect than single stimulus, and both ipsilateral-tDCS (ip-tDCS) and contralateral-tDCS (con-tDCS) produce a long-lasting analgesic effect on neuropathic pain. Second, the antinociceptive effects were intensity-dependent and time-dependent, high intensities worked better than low intensities and long stimulus durations worked better than short stimulus durations. Third, timing of the intervention after injury affected the stimulation outcome, early use of tDCS was an effective method to prevent the development of pain, and more frequent intervention induced more analgesia in CCI rats, finally, similar antinociceptive effects of con- and ip-tDCS were observed in both sexes of CCI rats. Conclusion: Optimized protocols of tDCS for treating antinociceptive effects were developed. These findings should be taken into consideration when using tDCS to produce analgesic effects in clinical applications.

Activation of mGluR1 contributes to neuronal hyperexcitability in the rat anterior cingulate cortex via inhibition of HCN channels.

Neuronal hyperexcitability in the anterior cingulate cortex (ACC) is considered as one of the most important pathological changes responsible for the chronification of neuropathic pain. However, the underlying mechanisms remain elusive. In the present study, we investigated the possible mechanisms using a rat model of chronic constriction injury (CCI) to the sciatic nerve. We found a substantial decrease in hyperpolarization-activated/cyclic nucleotide-gated (HCN) currents in layer 5 pyramidal neurons (L5 PNs) in ACC slices, which dramatically increased the excitability of these neurons. This effect could be mimicked in sham slices by activating group 1 metabotropic glutamate receptors, and be blocked in CCI slices by inhibiting metabotropic glutamate receptor subtype 1 (mGluR1). Next, the inhibition of HCN currents was reversed by a protein kinase C (PKC) inhibitor, followed by a reduced neuronal hyperexcitability. Furthermore, HCN channel subtype 1 (HCN1) level was significantly reduced after CCI, whereas mGluR1 level increased. These changes were mainly observed in L5 of the ACC, where HCN1 and mGluR1 were highly colocalized. For behavioral tests, intra-ACC microinjection of mGluR1-shRNA suppressed the CCI-induced behavioral hypersensitivity, particularly thermal hyperalgesia, but not aversive behavior, and this effect was attenuated by the pre-blockade of HCN channels. Taken together, the neuronal hyperexcitability of ACC L5 PNs likely results from an upregulation of mGluR1 and a downstream pathway involving PKC activation and a downregulation of HCN1 in the early phase of neuropathic pain. These alterations may at least in part contribute to the development of behavioral hypersensitivity in CCI rats.

Differential expression of P2X receptors on neurons from different parasympathetic ganglia.

Whole-cell patch clamp recording and immunohistochemistry were used to investigate the expression of P2X receptors on rat parasympathetic ganglion neurons of the otic, sphenopalatine, submandibular, intracardiac and paratracheal ganglia. Neurons from all five ganglia responded to ATP with a rapidly activating, sustained inward current. Neurons of intracardiac and paratracheal ganglia were insensitive to alphabeta-meATP, while all neurons in the otic and some neurons of sphenopalatine and submandibular ganglia responded. Lowering pH potentiated ATP responses in neurons from all five ganglia. Co-application of Zn(2+) potentiated ATP responses in intracardiac, paratracheal and submandibular ganglion neurons. Immunohistochemistry revealed strong and specific staining for the P2X(2) subunit in all five ganglia and strong P2X(3) staining in otic, sphenopalatine and submandibular ganglia. In conclusion, there is heterogeneity in P2X receptor expression in different parasympathetic ganglia of the rat, but the predominant receptor subtypes involved appear to be homomeric P2X(2) and heteromeric P2X(2/3).

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.

Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies.

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.6 nM. Lowering extracellular pH and co-application of Zn(2+) potentiated responses to ATP and alpha beta-meATP. In P2X(3)(-/-) mouse otic ganglion, all neurones tested responded to 100 microM ATP with a sustained current, but none responded to alpha beta-meATP. In P2X(2)(-/-) mice, no sustained currents were observed, but 36% of neurones responded to both ATP and alpha beta-meATP with transient currents. In P2X(2)/P2X(3)(Dbl-/-) mice, no responses to ATP or alpha beta-meATP were detected, suggesting that other P2X subunits were not involved. In rat otic ganglia, 96% of neurones responded to both ATP and alpha beta-meATP with sustained currents, suggesting a greater proportion of neurones expressing P2X(2/3) receptors. The maximum response to alpha beta-meATP was 40-60% of that evoked by ATP in the same cell. Immunohistochemistry revealed staining for P2X(2) and P2X(3) subunits in WT mouse otic ganglion neurones, which was absent in knockout animals. In conclusion, we have shown for the first time that at least two distinct P2X receptors are present in mouse and rat otic neurones, probably homomeric P2X(2) and heteromeric P2X(2/3) receptors.

P2X2 and P2X3 receptor expression in the gallbladder of the guinea pig.

We investigated for the first time, the distribution pattern of P2X2 and P2X3 receptors in the gallbladder of the guinea pig using immunohistochemistry. P2X2 and P2X3 receptor-immunoreactive nerve fibers were observed within the ganglia, in the interganglionic connectives, in the muscularis and in the paravascular plexus. Immunoreactivity for P2X2 and P2X3 was also observed in most neurons in the ganglionated plexus. Double-labeling studies revealed that 58.1% of all P2X2-positive neurons and 54.3% of all P2X3-positive neurons were found to display nitric oxide synthase. Over 90% of the neurons that were immunoreactive for P2X2 and P2X3 receptor were also immunoreactive for calretinin. We also found that 30.5% of P2X2- and 32.6% of P2X3-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide. No P2X2- or P2X3- immunoreactive neurons stained for calcitonin gene-related peptide; a few calcitonin gene-related peptide-immunoreactive nerve fibers also showed immunoreactivity to P2X2 or P2X3 receptors. These results further demonstrate the neurotransmitter diversity of the nerves of the gallbladder and provide an incentive for studies of the actions of these compounds in the gallbladder wall.