Oligodendroglial Signal Transduction Systems Are Developmentally Regulated

Abstract: Studies from several laboratories indicate that oligodendroglia exhibit signal transduction systems that can be activated by classical neurotransmitters. Previous studies from this laboratory indicate that oligodendroglia express neuroligand receptors linked to the regulation of Ca²⁺_i. Experiments presented in this article were designed to determine if developmental processes that influence the ability of oligodendroglia to respond to neuroligands with an increase in Ca²⁺_i proceed either in vitro or in vivo. Findings support the view that developmental processes markedly affected the sensitivity of these cells to both purinergic and cholinergic receptor agonists, whereas their responsiveness to either histamine or bradykinin appeared relatively stable over time. Approximately 90 and 75% of oligodendroglia responded to ATP or carbachol, respectively, after 4 days in vitro, whereas <10% of these cells responded to either of these neuroligands after 8 days in vitro. The decrease in the percentage of oligodendroglia responding to ATP, but not carbachol, could be prevented by including dibutyryl cyclic AMP in the culture medium during the final 4 days in vitro. However, once the loss in responsiveness to ATP had occurred, it could not be reversed by exposure to dibutyryl cyclic AMP. Developmental changes in the ATP sensitivity of oligodendroglia occurred in cells expressing galactocerebroside and myelin basic protein. The neuroligand sensitivity of oligodendroglia isolated from either neonatal, 2-, 3-, or 5-week-old spinal cord was examined to determine if developmental changes in oligodendroglial Ca²⁺ regulation occurred in vivo. The results of these experiments indicate that the percentage of oligodendroglia responding to either ATP or carbachol markedly decreased as a function of the age of the animal used to prepare the cultures; this was not the case for the stimulation of Ca²⁺_i by histamine. The decreased sensitivity of oligodendroglia isolated from older animals could not be reversed through the addition of dibutyryl cyclic AMP. Overall, the results of these experiments indicate that developmental processes selectively influence the sensitivity of oligodendroglia to specific neuroligands and suggest that oligodendroglial processes unrelated to myelin formation may be regulated by neuroligands in vivo.     

Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue, we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells (RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom, dose-dependently elicited neurite activation (i.e., Ca2+ mobilization) and, after a lag period, RBL Ca2+ mobilization. Neither bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature.     

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture

Transductionmechanisms between target cells within the intestinal wall andperipheral terminals of extrinsic primary afferent neurons are poorlyunderstood. The purpose of this study was to characterize theinteractions between smooth muscle cells from the rat distal colon andlumbar dorsal root ganglion (DRG) neurons in coculture. DRG neuronsvisually appeared to make contact with several myocytes. We show thatbrief mechanical stimulation of these myocytes resulted inintracellular Ca²⁺ concentration([Ca²⁺]_i)transients that propagated into 57% of the contacting neurites. Directmechanical stimulation of DRG neurites cultured without smooth musclehad no effect. We also show that colonic smooth muscle cells expressmultiple connexin mRNAs and that these connexins formed functional gapjunctions, as evidenced by the intercellular transfer of Luciferyellow. Furthermore, thapsigargin pretreatment and neuronal heparininjection abolished the increase in neurite [Ca²⁺]_i,indicating that the neuronal Ca²⁺signal was triggered by inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellularstores. Our results provide evidence for intercellular chemicalcommunication between DRG neurites and intestinal smooth muscle cellsthat mediates the exchange of second messenger molecules betweendifferent cell types.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.     

Modulation of neuritogenesis by astrocyte muscarinic receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.     

Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

Both neurotrophins (NTs) and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT), calcitonin-gene related peptide (CGRP), neurofilament 200 (NF-200), and microtubule associated protein 2 (MAP-2) in dorsal root ganglion (DRG) sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG) neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF) or neurotrophin-3 (NT-3) on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.     

ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma

Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-gamma. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X(7) purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-gamma increased ATP release and potentiated the P2X(7)-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.     

Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.     

Control of the intracellular Ca(2+)-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: evidence for different kinetics of Ca(2+)-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production

Carbachol, through a muscarinic receptor, thyrotropin-releasing hormone (TRH), prostaglandin F2 alpha (PGF2 alpha), bradykinin, and adenosine triphosphate (ATP) increased the apparent [Ca2+]i (intracellular free Ca2(+)-concentration) of dog thyrocytes in primary culture. The [Ca2+]i measured by the Quin-2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+]i declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2 alpha and in the case of bradykinin, TRH, and ATP, the [Ca2+]i returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+]i followed by a decline of the [Ca2+]i often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF2 alpha, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2 alpha, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2 alpha, bradykinin, and ATP through their action on the Ca2(+)-phosphatidylinositol cascade.     

Bradykinin and prostaglandin E₁ regulate calcitonin gene-related peptide expression in cultured rat sensory neurons

Primary cultures of adult rat dorsal root ganglia (DRG) sensory neurons were used to determine whether bradykinin and prostaglandins E? (PGE?), E? (PGE?) or I? (PGI?) stimulate long-term calcitonin gene-related peptide (CGRP) mRNA accumulation and peptide release. Treatment (24 h) of neurons with either bradykinin or PGE?, significantly increased CGRP mRNA content and iCGRP release. However, PGE? or PGI? was without effect. Exposure of the cultured neurons to increasing concentrations of bradykinin or PGE? demonstrated that the stimulation of CGRP expression was concentration-dependent, while time-course studies showed that maximal levels of CGRP mRNA accumulation and peptide release were maintained for at least 48 h. Treatment of the neuronal cultures with a bradykinin B? receptor antagonist significantly inhibited the bradykinin-induced increase in CGRP expression and release. In addition, preincubation of neuronal cultures with the cyclooxygenase inhibitor indomethacin did not alter the PGE?-mediated stimulation of CGRP but blocked completely the bradykinin-induced increase in CGRP production. Therefore, these data indicate that bradykinin and PGE? can regulate the synthesis and release of CGRP in DRG neurons and that the stimulatory effects of bradykinin on CGRP are mediated by a cyclooxygenase product(s). Thus, these findings suggest a direct relationship between chronic alterations in bradykinin/prostaglandin production that may arise from pathophysiological causes and long-term changes in CGRP expression.     

Neurochemical Characteristics of a Novel Dorsal Root Ganglion X Neuroblastoma Hybrid Cell Line, F-11

We investigated the properties of the novel dorsal root ganglion (DRG) hybrid cell line F-11 to see how closely these cells resembled normal DRG cells. Under normal growth conditions, F-11 cells appeared to contain several short neurite-like processes. However, these cells could also be grown under conditions in which they showed a much more extensive neuronal morphology, exhibiting many long neurites. Several differentiated features of DRG cells were present on F-11 cells. These included the presence of delta-opioid receptors, receptors for prostaglandins and bradykinin, and dihydropyridine-sensitive calcium channels. F-11 cells also synthesized and released a substance P-like compound, as determined by immunoreactivity. Both the number of bradykinin receptors and the voltage-sensitive calcium influx increased on cell differentiation. Opioid agonists (delta-specificity) were found to decrease cyclic AMP levels in F-11 cells in a naloxone- and pertussis toxin-reversible fashion. Bradykinin stimulated the synthesis of inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate. Ca2+ channel agonists stimulated voltage-sensitive Ca2+ influx in a dose-dependent, stereospecific manner, whereas Ca2+ channel antagonists inhibited Ca2+ influx. F-11 cells should, therefore, prove useful as models for authentic DRG neurons.     

Bronchial responsiveness to nonantigenic bronchoconstrictors in awake sheep

The experiments were designed to further characterize pulmonary responsiveness to nonantigenic aerosol bronchoconstrictors in unanesthetized sheep. The distribution of aerosol histamine responsiveness was described among 55 sheep. Within day reproducibility of aerosol histamine (n = 18) and carbachol (n = 8) responsiveness was studied and aerosol histamine and carbachol responsiveness were compared (n = 9). The effects of cyclooxygenase inhibition with meclofenamate (n = 7) and ibuprofen (n = 8) on pulmonary responsiveness to aerosol histamine was studied as was the effect of ibuprofen (n = 6) on pulmonary responsiveness to aerosol carbachol. A log normal unimodal distribution of pulmonary responsiveness to aerosol histamine was described. Within day pulmonary responsiveness to aerosol histamine was highly reproducible while pulmonary responsiveness to aerosol carbachol decreased slightly, but not significantly, on the second challenge. Pulmonary responsiveness to aerosol histamine correlated with pulmonary responsiveness to aerosol carbachol (r = 0.85, P less than 0.05). Meclofenamate did not significantly attenuate pulmonary responsiveness to aerosol histamine. Ibuprofen attenuated pulmonary responsiveness to aerosol histamine (P less than 0.05) but not to aerosol carbachol. These experiments supply basic information related to pulmonary responsiveness to nonantigenic bronchoconstrictors in awake sheep.     

Direct neurite-osteoblastic cell communication, as demonstrated by use of an in vitro co-culture system

Using an in vitro co-culture approach comprising cultured murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells, we found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca2+ mobilization and, after a lag period, osteoblastic Ca2+ mobilization. SV did not have any direct effect on the osteoblastic cells in the absence of neurites. The addition of an alpha1-adrenergic receptor (AR) antagonist, prazosin, dose-dependently prevented the osteoblastic activation that resulted as a consequence of neural activation by SV. These results demonstrate that osteoblastic activation occurred as a direct response to neuronal activation, which activation was mediated by alpha1-ARs in the osteoblastic cells.     

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.     

Stimulation of Cyclic GMP Production via a Nitrosyl Factor in Sensory Neuronal Cultures by Algesic or Inflammatory Agents

Abstract: Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E₂, substance P, and calcitonin gene-related peptide did not affect basal, capsaicin-stimulated, or bradykinin-stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by l -arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin-sensitive (small diameter) afferent neurons, attenuated capsaicin- and bradykinin-stimulated cyclic GMP production but did not affect basal or sodium nitroprusside-stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin-sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non-neuronal cells.     

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.