Christian Lohr

Molecular Identification and Physiological Characterization of a Novel Monosaccharide Transporter from Arabidopsis Involved in Vacuolar Sugar Transport

The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT–green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter–β-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type–specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH4+, fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.

Regulation of Store-Operated Calcium Entry by Calcium-Independent Phospholipase A2 in Rat Cerebellar Astrocytes

We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 μm) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 μm). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 μm) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 μm) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 μm) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA2, lysophosphatidylcholine (250 nm) and lysophosphatidylinositol (250 nm), but not lysophosphatidic acid (250 nm), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling.

Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: A multiphoton study of cerebellar slices

Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real‐time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several‐fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG‐P can diffuse horizontally across the molecular layer, presumably through gap junctions between Bergmann glial cells. Our main conclusion is that in acute cerebellar slices, the glucose transport capacity and glycolytic rate of Bergmann glia are several‐fold higher than those of Purkinje cells. Given that the cerebellum is largely fueled by glucose and Purkinje neurons are estimated to spend more energy than Bergmann glial cells, these results suggest substantial shuttling of an energy‐rich metabolite like lactate between glial cells and neurons.

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca2+ imaging and two-photon Na+ imaging. GABA evoked Ca2+ transients in astrocytes that persisted in the presence of GABAA and GABAB receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca2+ blocked GABA-induced Ca2+ transients, and depletion of Ca2+ stores with cyclopiazonic acid reduced Ca2+ transients by approximately 90%. This indicates that the Ca2+ transients depend on external Ca2+, but are mainly mediated by intracellular Ca2+ release, conforming with Ca2+-induced Ca2+ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca2+ transients, whereas the InsP3 receptor blocker 2-APB inhibited the Ca2+ transients. GABA also induced Na+ increases in astrocytes, potentially reducing Na+/Ca2+ exchange. To test whether reduction of Na+/Ca2+ exchange induces Ca2+ signaling, we inhibited Na+/Ca2+ exchange with KB-R7943, which mimicked GABA-induced Ca2+ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca2+ transients in astrocytes. The significance of GABAergic Ca2+ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na+ rises that reduce Na+/Ca2+ exchange, thereby leading to a Ca2+ increase sufficient to trigger Ca2+-induced Ca2+ release via InsP3 receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.

Cav1.3 Calcium Channels Are Required for Normal Development of the Auditory Brainstem

Within the Cav1 family of voltage-gated calcium channels, Cav1.2 and Cav1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Cav1.3 subunits in the activity-dependent development of the auditory brainstem. We used Cav1.3-deficient (Cav1.3−/−) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25–59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Cav1.3−/− mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the Kv1.2 subtype. Fura2 imaging provided evidence for functional Cav1.3 channels in the LSO of wild-type mice. Our results imply that Cav1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Cav1.3−/− mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Cav1.3 channels in the neurons plus lack of peripheral input.

Axon-glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulb

Olfactory ensheathing cells (OECs) accompany receptor axons in the olfactory nerve and promote axonal growth into the central nervous system. The mechanisms underlying the communication between axons and OECs, however, have not been studied in detail yet. We investigated the effect of activity‐dependent neuronal transmitter release on Ca2+ signaling of OECs in acute mouse olfactory bulb slices using confocal Ca2+ imaging. TTX‐sensitive axonal activity upon electrical nerve stimulation triggers a rise in cytosolic Ca2+ in OECs, which can be mimicked by application of DHPG, an agonist of metabotropic glutamate receptors (mGluRs). Both stimulation‐ and DHPG‐induced Ca2+ transients in OECs were abolished by depletion of intracellular Ca2+ stores with cyclopiazonic acid (CPA). The mGluR1‐specific antagonist CPCCOEt completely inhibited DHPG‐evoked Ca2+ transients, but reduced stimulation‐induced Ca2+ transients only partly, suggesting the involvement of another neurotransmitter. Application of ATP evoked CPA‐sensitive Ca2+ transients in OECs, which were inhibited by the P2Y1‐specific antagonist MRS2179. Co‐application of CPCCOEt and MRS2179 almost completely blocked the stimulation‐induced Ca2+ transients, indicating that they were mediated by mGluR1 and P2Y1 receptors. Our results show that OECs are able to respond to olfactory nerve activity with an increase in cytosolic Ca2+ due to glutamate and ATP release.

New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes

Purinergic receptors play a key role in neuron‐glia and glia‐neuron interactions. In the present study, we have recorded cytosolic Ca2+ responses using confocal imaging in astrocytes of acute olfactory bulb slices from mice (postnatal days 3–8). By application of agonists and antagonists, we identified two types of receptors, P2Y1 and A2A, that mediated Ca2+ responses attributable to Ca2+ release from intracellular stores in the astrocytes. Both receptor types were activated by application of ATP and ADP;however, when enzymatic ATP degradation was suppressed by the alkaline phosphatase inhibitor levamisole, ATP only activated MRS2179‐sensitive P2Y1 but not ZM241385‐sensitive A2A receptors. The dose‐response curve for A2A receptors activated by adenosine revealed an EC50 of 0.3 μM, one order of magnitude smaller than the EC50 of 5 μM determined for P2Y1 receptors activated by ADP. Electrical stimulation of the olfactory nerve in the presence of glutamate receptor blockers to suppress excitation of postsynaptic neurons evoked Ca2+ responses in most of the astrocytes, which were inhibited by blocking both P2Y1 and A2A receptors. Our results indicate that olfactory nerve terminals release not only glutamate, but also ATP, which activates P2Y1 receptors and, after degradation of ATP to adenosine, A2A receptors in astrocytes.—Doengi, M., Deitmer, J. W., Lohr, C. New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes. FASEB J. 22, 2368–2378 (2008)

Blockage of voltage-gated calcium signaling impairs migration of glial cells in vivo.

Migration of glial cells is an essential step in the development of the antennal lobe, the primary olfactory center of insects, to establish well‐defined borders between olfactory glomeruli required for odor discrimination. In the present study, we used two‐photon microscopy to visualize calcium signaling in developing antennal lobe glial cells of the sphinx moth Manduca sexta. We found a correlation between the upregulation of functional voltage‐gated calcium channels and the onset of glial cell migration. In addition, glial cells migrating into the center of the antennal lobe express larger voltage‐gated calcium transients than glial cells that remain at the periphery. Migration behavior and calcium signaling of glial cells in vivo were manipulated either by deafferentation, by injection of the calcium channel blockers diltiazem, verapamil, and flunarizine, or by injection of the calcium chelators BAPTA‐AM and Fluo‐4‐AM. In deafferented antennal lobes, glial cells failed to express functional voltage‐gated calcium channels and did not migrate. Calcium channel blockage or reducing glial calcium signals by calcium chelators prevented glial cell migration and resulted in antennal lobes lacking glial borders around glomeruli, indicating that voltage‐gated calcium signaling is required for the migration of antennal lobe glial cells and the development of mature olfactory glomeruli.

Leech giant glial cell: functional role in a simple nervous system

The giant glial cell in the central nervous system of the leech Hirudo medicinalis has been the subject of a series of studies trying to link its physiological properties with its role in neuron–glia interactions. Isolated ventral cord ganglia of this annelid offer several advantages for these studies. First, single giant glial cells can easily be identified and are quite accessible to electrophysiological and microfluorometric studies. Second, only two giant macroglial cells are located in the neuropil of each ganglion, rendering them well suited for studying neuron–glia interactions. Third, many neurons can be identified and are well known with respect to their physiology and their roles in controlling simple behaviors in the leech. This review briefly outlines the major recent findings gained by studying this preparation and its contributions to our knowledge of the functional role of glia in nervous systems. Emphasis is directed to glial responses during neuronal activity and to the analysis of intracellular Ca2+ and H+ transients mediated by neurotransmitter receptors and ion‐driven carriers. Among its numerous properties, the leech giant glial cell prominently expresses a large K+ conductance, voltage‐dependent Ca2+ channels, ionotropic non‐NMDA glutamate receptors, and an electrogenic, reversible Na+‐HCO3− cotransporter. GLIA 28:175–182, 1999.

Ectopic vesicular neurotransmitter release along sensory axons mediates neurovascular coupling via glial calcium signaling

Neurotransmitter release generally is considered to occur at active zones of synapses, and ectopic release of neurotransmitters has been demonstrated in a few instances. However, the mechanism of ectopic neurotransmitter release is poorly understood. We took advantage of the intimate morphological and functional proximity of olfactory receptor axons and specialized glial cells, olfactory ensheathing cells (OECs), to study ectopic neurotransmitter release. Axonal stimulation evoked purinergic and glutamatergic Ca2+ responses in OECs, indicating ATP and glutamate release. In axons expressing synapto-pHluorin, stimulation evoked an increase in synapto-pHluorin fluorescence, indicative of vesicle fusion. Transmitter release was dependent on Ca2+ and could be inhibited by bafilomycin A1 and botulinum toxin A. Ca2+ transients in OECs evoked by ATP, axonal stimulation, and laser photolysis of NP-EGTA resulted in constriction of adjacent blood vessels. Our results indicate that ATP and glutamate are released ectopically by vesicles along axons and mediate neurovascular coupling via glial Ca2+ signaling.