Need for caution when interpreting Xpert® MTB/RIF results for rifampin resistance among children.

BACKGROUND: Recommended by the World Health Organization as an initial diagnostic test for TB in children, Xpert® MTB/RIF is widely implemented in many countries, including Kenya.METHODS: Three hundred HIV-positive and negative children (<5 years) were enrolled in Kisumu County, Kenya, from October 2013 to August 2015. Multiple specimen types were collected from each child and tested using Xpert, liquid culture, and phenotypic drug susceptibility testing (DST). Samples positive for rifampin (RIF) resistance on Xpert were tested using line-probe assay and sequencing.RESULTS: Of 32 children with bacteriologically confirmed TB, 27 had positive Xpert results. Of these, 3/27 (11%, 95% CI 4-28) had RIF resistance detected on Xpert, but not by phenotypic DST, line-probe assay, or sequencing. For these three children, five Xpert tests showed RIF resistance; all five tests had semi-quantitative "very low" results and delay or absence of probe D signal, whereas no Xpert results with higher semi-quantitative results showed RIF resistance. All three children responded well to standard TB treatment.CONCLUSIONS: False RIF resistance may be detected in pediatric specimens. Further study is needed to determine if false RIF resistance is associated with low bacterial load.

Tuberculosis screening outcomes for newly diagnosed persons living with HIV, Nyanza Province, Kenya, 2009.

SETTING: Fifteen human immunodeficiency virus (HIV) clinics in Nyanza Region, Western Kenya. OBJECTIVE: To describe routine tuberculosis (TB) screening and diagnostic practices among newly enrolled people living with HIV (PLHIV) prior to the implementation of World Health Organization recommended TB intensified case finding. DESIGN: Retrospective chart abstraction of PLHIV aged ⩾7 years who were newly enrolled in HIV care in July and August 2009, and who had not received antiretroviral treatment in the preceding 2 years or been diagnosed with TB in the previous year. Factors associated with evidence of TB diagnostic evaluation among symptomatic PLHIV were assessed. RESULTS: Of 1020 patients included in the analysis, 995 (98%) were screened for TB at enrolment and 613 (62%) reported TB symptoms. Ninety-six (16%) patients with symptoms had evidence of referral for TB diagnostic evaluation, including patients at large clinics, those with advanced HIV disease and those reporting multiple TB symptoms. Among the 43 (45%) with documented evaluation results, 26 (60%) were diagnosed with TB. CONCLUSION: Although most PLHIV were screened for TB, very few underwent an evaluation, and the proportion diagnosed with TB was very low. Efforts to improve TB screening should focus on standardizing the intensified case finding algorithm and linkage to, and adequate infrastructure for, TB diagnostic evaluation.

The utility of stool cultures for diagnosing tuberculosis in people living with the human immunodeficiency virus.

BACKGROUND: Delayed diagnosis of tuberculosis (TB) increases mortality. OBJECTIVE: To evaluate whether stool culture improves the diagnosis of TB in people living with the human immunodeficiency virus (PLHIV). DESIGN: We analysed cross-sectional data of TB diagnosis in PLHIV in Cambodia, Thailand and Viet Nam. Logistic regression was used to assess the association between positive stool culture and TB, and to calculate the incremental yield of stool culture. RESULTS: A total of 1693 PLHIV were enrolled with a stool culture result. Of 228 PLHIV with culture-confirmed TB from any site, 101 (44%) had a positive stool culture; of these, 91 (90%) had pulmonary TB (PTB). After adjusting for confounding factors, a positive stool culture was associated with smear-negative (odds ratio [OR] 26, 95% confidence interval [CI] 12-58), moderately smear-positive (OR 60, 95%CI 23-159) and highly smear-positive (OR 179, 95%CI 59-546) PTB compared with no PTB. No statistically significant association existed with extra-pulmonary TB compared with no extra-pulmonary TB (OR 2, 95%CI 1-5). The incremental yield of one stool culture above two sputum cultures (5%, 95%CI 3-8) was comparable to an additional sputum culture (7%, 95%CI 4-11). CONCLUSION: Nearly half of the PLHIV with TB had a positive stool culture that was strongly associated with PTB. Stool cultures may be used to diagnose TB in PLHIV.

Linear dipole behavior in single CdSe-oligo(phenylene vinylene) nanostructures.

We report on linearly polarized absorption and emission from individual (4.3 nm) CdSe quantum dots whose surfaces are coordinated with monodisperse oligo-phenylene vinylene ligands. Shown previously to suppress quantum dot blinking, we demonstrate here that the electronic interaction of photoexcited ligands with the quantum dot core is manifested as a strong polarization anisotropy in absorption (M = 0.5), as well as distinct linear dipole emission patterns from the quantum dot core. Further, there is a correlation between the quantum dot emission moment and polarization orientation corresponding to the absorption maxima that is manifested as fluctuations in emission moment orientation in the X-Y plane. The observed polarization effects can be switched off by tuning the excitation away from the ligand absorption band. We propose a mechanism based on exciton dissociation from the photoexcited ligand, followed by the pinning of electrons at the quantum dot surface. The resulting Stark interaction is sufficiently strong to break the 2D degeneracy of the emission moment within the dot, and may therefore account for the linear dipole emission character.

Monitoring the performance of mycobacteriology laboratories: a proposal for standardized indicators.

SETTING: Thailand Tuberculosis (TB) Active Surveillance Network: Bangkok, Chiang Rai, Phuket, Tak and Ubon-Ratchathani, Thailand. BACKGROUND: Mycobacteriology laboratories in resource-limited, high TB burden settings are expanding to perform conventional solid media culture and broth-based mycobacteriology culture. Indicators that measure how well a laboratory performs sputum microscopy have been developed and broadly implemented. Routine monitoring of sputum culture performance, however, is not as common. DESIGN: We implemented indicators for monitoring the quality of laboratory services in five province-level mycobacteriology culture facilities in Thailand. These indicators were derived from literature review, consultation with subject matter experts and our program experience. CONCLUSIONS: We believe that an international consensus document providing monitoring guidelines for mycobacteriology laboratories is urgently needed.

Fluorescence lifetimes and correlated photon statistics from single CdSe/oligo(phenylene vinylene) composite nanostructures.

We present measurements of fluorescence intensity trajectories and associated excited-state decay times from individual CdSe/oligo(phenylene vinylene) (CdSe-OPV) quantum dot nanostructures using time-tagged, time-resolved (TTTR) photon counting techniques. We find that fluorescence decay times for the quantum dot emitter in these composite systems are at least an order of magnitude shorter than ZnS-capped CdSe quantum dot systems. We show that both the blinking suppression and associated lifetime/count rate behavior can be described by a modified version of the diffusive reaction coordinate model which couples slow fluctuations in quantum dot electron (1Se, 1Pe) energies to Auger-assisted hole trapping processes, hence modifying both blinking statistics and excited-state decay rates.

Blinking suppression and intensity recurrences in single CdSe-oligo(phenylene vinylene) nanostructures: experiment and kinetic model.

We report time-resolved single molecule fluorescence imaging of individual CdSe quantum dots that are functionalized with oligomeric conjugated organic ligands. The fluorescence intensity trajectories from these composite nanostructures display both a strong degree of blinking suppression and intensity fluctuations with characteristic recurrence times on the order of 10-60 s. In addition, fluorescence decay rate measurements of individual hybrid nanostructures indicate significantly modified non-radiative quantum dot decay rates relative to conventional ZnS-capped CdSe quantum dots. We show that a modified diffusive reaction coordinate model with slow fluctuations in quantum dot electron energies (1S(e), 1P(e)) can reproduce the experimentally observed behaviour.

Retroviral inhibition of cAMP-dependent protein kinase inhibits myelination but not Schwann cell mitosis stimulated by interaction with neurons.

Schwann cells are the myelinating glia of the peripheral nervous system. Neuron-Schwann cell contact profoundly affects several aspects of Schwann cell phenotype, including stimulation of mitosis and myelin formation. Many reports suggest that neuronal contact exerts this influence on Schwann cells by elevating Schwann cell cAMP and activating cAMP-dependent protein kinase A (PKA). To elucidate the importance of Schwann cell PKA in neuronal stimulation of Schwann cell mitosis and myelination, the gene encoding the PKA inhibitory protein RIalphaAB or PKIEGFP was delivered to Schwann cells using retroviral vectors. PKA inhibitory retroviral vectors effectively blocked forskolin-stimulated Schwann cell mitosis and morphological change, demonstrating the ability of the vectors to inhibit PKA in infected Schwann cells. Treatment of dorsal root ganglia neuron-Schwann cell cocultures with H-89 (10 microm) or KT5720 (1-10 microm), chemical inhibitors selective for PKA, significantly inhibited neuronal stimulation of Schwann cell mitosis. In contrast, retrovirus-mediated inhibition of Schwann cell PKA had no effect on the ability of neurons to stimulate Schwann cell mitosis. However, markedly fewer myelin segments were formed by Schwann cells expressing PKA inhibitory proteins compared with controls. These results suggest that activation of Schwann cell PKA is required for myelin formation but not for Schwann cell mitosis stimulated by interaction with neurons.

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.

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ.

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.

A dicistronic retroviral vector and culture model for analysis of neuron-Schwann cell interactions.

A dicistronic retroviral gene delivery system and tissue culture model has been developed for studies of neuron-Schwann cell interactions at the single cell level. The dicistronic retroviral vector contains a multiple cloning site followed by the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES) and a green fluorescent protein gene. This design allows for 5'-cap dependent translation of any gene of interest and 5'-cap independent translation of green fluorescent protein (GFP) from a single dicistronic RNA. The culture model consists of dorsal root ganglia (DRG) explants grown in defined medium. Under these conditions the Schwann cell population is selectively expanded and infected by the retroviral vector, allowing for rapid transfer of genes of interest selectively to a large percentage of Schwann cells in coculture with neurons. Infected cells are subsequently identified in living cultures by their expression of GFP. Infected (GFP expressing) Schwann cells in contact with neurites continued to exhibit: (1) increased mitotic activity, (2) increased sensitivity to elevate intracellular calcium in response to extracellular application of ATP, and (3) myelination. This viral construct has the added advantage that it allows identification of cells expressing transgenes among a heterogeneous population by fluorescence microscopy, FACS, or flow cytometry.

Responses of Bergmann glia and granule neurons in situ to N-methyl-D-aspartate, norepinephrine, and high potassium.

To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N-methyl-D-aspartate (NMDA) with a rise in [Ca2+]i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and alpha-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K+, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca2+, indicating a direct activation of alpha1-adrenergic receptors that mediated release of Ca2+ from intracellular stores. The KCl-induced response in both neurons and glia was dependent on external Ca2+ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.

Astrocytic neurotransmitter receptors in situ and in vivo.

In the brain, astrocytes are associated intimately with neurons and surround synapses. Due to their close proximity to synaptic clefts, astrocytes are in a prime location for receiving synaptic information from released neurotransmitters. Cultured astrocytes express a wide range of neurotransmitter receptors, but do astrocytes in vivo also express neurotransmitter receptors and, if so, are the receptors activated by synaptically released neurotransmitters? In recent years, considerable efforts has gone into addressing these issues. The experimental results of this effort have been compiled and are presented in this review. Although there are many different receptors which have not been identified on astrocytes in situ, it is clear that astrocytes in situ express a number of different receptors. There is evidence of glutamatergic, GABAergic, adrenergic, purinergic, serotonergic, muscarinic, and peptidergic receptors on protoplasmic, fibrous, or specialized (Bergmann glia, pituicytes, Müller glia) astrocytes in situ and in vivo. These receptors are functionally coupled to changes in membrane potential or to intracellular signaling pathways such as activation of phospholipase C or adenylate cyclase. The expression of neurotransmitter receptors by astrocytes in situ exhibits regional and intraregional heterogeneity and changes during development and in response to injury. There is also evidence that receptors on astrocytes in situ can be activated by neurotransmitter(s) released from synaptic terminals. Given the evidence of extra-synaptic signaling and the expression of neurotransmitter receptors by astrocytes in situ, direct communication between neurons and astrocytes via neurotransmitters could be a widespread form of communication in the brain which may affect many different aspects of brain function, such as glutamate uptake and the modulation of extracellular space.

Oligodendroglial signal transduction systems are regulated by neuronal contact.

Previous reports indicate that oligodendrocytes express signaling systems activated by classical neurotransmitters. Several signaling systems linked to mobilization of intracellular calcium have been demonstrated, and some of these are developmentally lost in vitro and in vivo. The experiments described here use oligodendrocyte-neuron cocultures to examine the effects of neuronal contact on the expression of these signaling pathways. Neonatal rat cerebral oligodendrocytes in contact with dorsal root ganglia (DRG) neurites responded to bath application of histamine, ATP, carbachol, glutamate, or bradykinin with increases in intracellular Ca2+ concentration. Similar results were obtained in coculture with superior cervical ganglia neurons. Preventing neuronal contact by transection of DRG neurites significantly reduced the percentage of oligodendrocytes responsive to each ligand, with the exception of bradykinin responsiveness, which was unaffected. Oligodendroglia isolated from adult rat spinal cord were also examined for responsiveness to these neuroligands. Few isolated adult oligodendroglia were responsive to these ligands, and coculture with DRG neurons failed to restore responsiveness. Neuroligand responsiveness was not induced in oligodendrocytes maintained 8 days in purified culture before establishment of cocultures. A significant reduction in the number of neuroligand-responsive oligodendroglia was noted for histamine, carbachol, glutamate, and ATP after including tetrodotoxin for the final 6 days of coculture. These results suggest that both neuronal contact and neuronal activity contribute to the maintenance of functional neurotransmitter-activated signaling pathways coupled to mobilization of intracellular calcium in oligodendrocytes.

Hippocampal astrocytes in situ respond to glutamate released from synaptic terminals.

A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca2+]i) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker omega-conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K+ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca2+]i.

Control of gap-junctional communication in astrocytic networks.

Astrocytes, which constitute the most abundant cell type in mammalian brain, are extensively coupled to one another through gap junctions composed mainly of connexin43. In regions exhibiting high levels of connexin43 expression, tens of astrocytes are labeled following single-cell intracellular injection. Importantly, both the expression and the permeability of gap junctions are tightly regulated. Such long- and short-term regulations indicate that astrocytic networks might be subject to remodeling and to some plasticity. Since evidence for neuro-glial interaction exists, the degree of coupling between astrocytes could participate to set the tone of neuronal activity and to determine the sphere of influenced neurons. Research in this area is still at its early stages and significant progress requires a transition from the understanding of basic properties to the study of function.

Adenosine receptors modulate [Ca2+]i in hippocampal astrocytes in situ.

Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca2+]i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca2+]i that were blocked by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca2+]i, whereas the P2 agonist adenosine 5'-O-(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P1 purinergic receptors coupled to increases in [Ca2+]i, whereas a small minority appear to contain P2 purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca2+]i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.

Receptor-mediated calcium signals in astroglia: multiple receptors, common stores and all-or-nothing responses.

Calcium signals following activation of P2Y purinergic, alpha 1 adrenergic, and muscarinic cholinergic receptors were examined in individual astroglial cells. ATP, phenylephrine and carbachol, each increased intracellular calcium levels ([Ca2+]i) to similar amplitudes in the presence or absence of extracellular Ca2+. The dose-response relationship showed that less than an order of magnitude increase in ligand concentration led to maximal increase in [Ca2+]i from basal levels. Simultaneous application of multiple ligands did not produce additive effects on [Ca2+]i. These data suggested that different ligands released Ca2+ from common stores and that each of the ligands could cause maximal release. Application of a second ligand immediately after the first ligand produced an additional Ca2+ rise, suggesting that the Ca2+ stores were rapidly refilled and that receptor desensitization rather than Ca2+ depletion accounted for the rapid decline of the Ca2+ peak. Caged IP3 produced Ca2+ signals similar to those produced by ligands. For a given cell, both caged IP3 and ligands sometimes produced only one level of partial Ca2+ increases, suggesting the presence of a pool of high IP3-sensitive stores. Together, our results indicate that neuroligands tend to generate an all-or-nothing Ca2+ release from IP3 sensitive stores. The interactions between different receptor systems most likely occur at the level of IP3 accumulation.

GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i.

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca2+]i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium-sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca2+]i within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca2+]i. The increases in [Ca2+]i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca2+]i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca2+]i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca2+]i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [Ca2+]i and are able to respond to neuronally released neurotransmitters.

Schwann cell ATP-mediated calcium increases in vitro and in situ are dependent on contact with neurons.

Schwann cells freshly isolated from the sciatic nerves of neonatal rats respond to exogenously applied ATP with a rapid increase in cytosolic calcium. This increase in [Ca2+]i is mediated by a P2Y-purinergic pathway (Lyons et al.: J. Neurochem. 63:552-560, 1994) and was measured using the calcium indicator dye, fura-2/AM, and a video-enhanced calcium imaging system. The ability to respond to ATP with increases in intracellular calcium is lost over a period of several days in culture; this loss can be prevented or reversed by application of cAMP analogs in a defined medium. We now demonstrate that the direct contact of Schwann cells with neurons also induces and stabilizes this ATP responsiveness. The induction of ATP responsiveness was observed among all Schwann cells contacting neurites, including those forming myelin, and regardless of whether the source of neurons was dorsal root ganglion neurons or superior cervical ganglion neurons. Approximately 85% of Schwann cells responded to ATP over the time studied (72 d in coculture). Addition of axolemma to Schwann cell cultures did not induce ATP responsiveness. We also examined the ATP responsiveness of Schwann cells in situ (excised nerves) using laser-scanning confocal microscopy and the calcium indicator dye, fluo-3/AM. Schwann cells in intact sciatic nerve segments isolated from neonatal and 16-day-old rats exhibited ATP-mediated [Ca2+]i increases. We conclude that neuronal contact is necessary for the expression of the ATP-mediated calcium responses in Schwann cells and that these responses are independent of myelin formation or maintenance.