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Astrocytes are glial cells that have an intimate physical and functional association with synapses in the brain. One of their main roles is to recycle the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), as a component of the glutamate/GABA-glutamine cycle. They perform this function by sequestering neurotransmitters and releasing glutamine via the neutral amino acid transporter SNAT3. In this way, astrocytes regulate the availability of neurotransmitters and subsequently influence synaptic function. Since many plasma membrane transporters are regulated by protein kinase C (PKC), the aim of this study was to understand how PKC influences SNAT3 glutamine transport in astrocytes located immediately adjacent to synapses. We studied SNAT3 transport by whole-cell patch-clamping and fluorescence pH imaging of single astrocytes in acutely isolated brainstem slices, adjacent to the calyx of the Held synapse. Activation of SNAT3-mediated glutamine transport in these astrocytes was reduced to 77 ± 6% when PKC was activated with phorbol 12-myristate 13-acetate (PMA). This effect was very rapid (within ~20 min) and eliminated by application of bisindolylmaleimide I (Bis I) or 7-hydroxystaurosporine (UCN-01), suggesting that activation of conventional isoforms of PKC reduces SNAT3 function. In addition, cell surface biotinylation experiments in these brain slices show that the amount of SNAT3 in the plasma membrane is reduced by a comparable amount (to 68 ± 5%) upon activation of PKC. This indicates a role for PKC in dynamically controlling the trafficking of SNAT3 transporters in astrocytes in situ. These data demonstrate that PKC rapidly regulates the astrocytic glutamine release mechanism, which would influence the glutamine availability for adjacent synapses and control levels of neurotransmission. [ABSTRACT FROM AUTHOR]

The mechanisms by which the excitatory neurotransmitter glutamate is recycled at synapses are currently unknown. By examining the functional expression of plasma membrane transporters at presynaptic terminals, we aim to elucidate some of the mechanisms of glutamate recycling. Using whole-cell voltage-clamp recordings from rat calyx of Held presynaptic terminals, our data show, for the first time, that the glutamate precursor glutamine causes the direct activation of an electrogenic, sodium-dependent presynaptic transporter, which supplies glutamine for generation of presynaptic glutamate and helps sustain synaptic transmission. Interestingly, the functional expression of this transporter at the presynaptic plasma membrane is dynamically controlled by electrical activity of the terminal, indicating that uptake of neurotransmitter precursors is controlled by the demand at an individual terminal. Induction of the transporter current is calcium-dependent and inhibited by botulinum neurotoxin C, demonstrating the involvement of SNARE-dependent exocytosis in inserting transporters into the plasma membrane when the terminal is active. Conversely, inactivity of the presynaptic terminal results in removal of transporters via clathrin-mediated endocytosis. To investigate whether the presynaptic glutamine transporter supplies the precursor for generating the synaptically released glutamate, we measured miniature EPSCs to assess vesicular glutamate content. When the presynaptic glutamate pool was turned over by synaptic activity, inhibiting the presynaptic glutamine transporters with MeAIB reduced the miniature EPSC amplitude significantly. This demonstrates that presynaptic glutamine transport is centrally involved in the production of glutamate and assists in maintaining excitatory neurotransmission. [ABSTRACT FROM AUTHOR]

An essential component of the neural network regulating ingestive behavior is the brain 5-hydroxytryptamine2C receptor (5-HT2CR), agonists of which suppress food intake and were recently approved for obesity treatment by the US Food and Drug Administration. 5-HT2CR-regulated appetite is mediated primarily through activation of hypothalamic arcuate nucleus (ARC) pro opiomelanocortin (POMC) neurons, which are also disinhibited through a 5-HT1BR-mediated suppression of local inhibitory inputs. Here we investigated whether 5-HT2CR agonist anorectic potency could be significantly enhanced by coadministration of a 5-HT1BR agonist and whether this was associated with augmented POMC neuron activation on the population and/or single-cell level. The combined administration of subanorectic concentrations of 5-HT2CR and 5-HT1BR agonists produced a 45% reduction in food intake and significantly greater in vivo ARC neuron activation in mice. The chemical phenotype of activated ARC neurons was assessed by monitoring agonist-induced cellular activity via calcium imaging in mouse POMC-EGFP brain slices, which revealed that combined agonists activated significantly more POMC neurons (46%) compared with either drug alone (--25% each). Single-cell electrophysiological analysis demonstrated that 5-HT2CR/5-HT1BR agonist coadministration did not significantly potentiate the firing frequency of individual ARC POMC-EGFP cells compared with agonists alone. These data indicate a functional heterogeneity of ARC POMC neurons by revealing distinct subpopulations of POMC cells activated by 5-HT2CRs and disinhibited by 5-HT1BRs. Therefore, coadministration of a 5-HT1BR agonist potentiates the anorectic efficacy of 5-HT2CR compounds by increasing the number, but not the magnitude, of activated ARC POMC neurons and is of therapeutic relevance to obesity treatment. [ABSTRACT FROM AUTHOR]

Gq-protein-coupled receptors (GqPCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ release from intracellular stores via the phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) signaling pathway. Because early GqPCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study, we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate GqPCR signaling in neurons. Our results demonstrate that GqPCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca2+. Depolarization has a bidirectional effect on GqPCR signaling, potentiating thapsigargin-sensitive Ca2+responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating, and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP3/GPIP2 (glycerophosphoryl-myo-inositol 4,5-bisphosphate) places the voltage sensor before the level of the Ca2+store, and measurements using the fluorescent bioprobe eGFP-PHPLCPLCδ(enhanced green fluorescent protein-pleckstrin homology domain-PLCδ) directly demonstrate that voltage affects muscarinic signaling at the level of the IP3 production pathway. The sensitivity of GqPCR IP3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial. [ABSTRACT FROM AUTHOR]

The article studies the role performed by glutamate transporters in the homeostasis of glutamate in the brain. Glutamate transporters have a crucial function in controlling the extracellular concentration of glutamate in the brain. They remove glutamate from extracellular space and help terminate glutamatergic synaptic transmission. This prevents the extracellular concentration of glutamate from rising to neurotoxic levels.

Intracellular Ca2+ store release contributes to activity-dependent synaptic plasticity in the central nervous system by modulating the amplitude, propagation, and temporal dynamics of cytoplasmic Ca2+ changes. However, neuronal Ca2+ stores can be relatively insensitive to increases in the store-mobilizing messenger inositol 1,4,5-trisphosphate (IP3). Using a fluorescent biosensor we have visualized M1 muscarinic acetylcholine (mACh) receptor signaling in individual hippocampal neurons and observed increased IP3 production in the absence of concurrent Ca2+ store release. However, coincident glutamate-mediated synaptic activity elicited enhanced and oscillatory IP3 production that was dependent upon ongoing mACh receptor stimulation and S-α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid receptor activation of Ca2+ entry. Moreover, the enhanced levels of IP3 now mobilized Ca2+ from intracellular stores that were refractory to the activation of mACh receptors alone. We conclude that convergent ionotropic and metabotropic receptor inputs can facilitate Ca2+ signaling by enhancing IP3 production as well as augmenting release by Ca2+-induced Ca2+ release. [ABSTRACT FROM AUTHOR]

An essential component of the neural network regulating ingestive behavior is the brain 5-hydroxytryptamine2C receptor (5-HT2CR), agonists of which suppress food intake and were recently approved for obesity treatment by the US Food and Drug Administration. 5-HT2CR-regulated appetite is mediated primarily through activation of hypothalamic arcuate nucleus (ARC) pro-opiomelanocortin (POMC) neurons, which are also disinhibited through a 5-HT1BR-mediated suppression of local inhibitory inputs. Here we investigated whether 5-HT2CR agonist anorectic potency could be significantly enhanced by coadministration of a 5-HT1BR agonist and whether this was associated with augmented POMC neuron activation on the population and/or single-cell level. The combined administration of subanorectic concentrations of 5-HT2CR and 5-HT1BR agonists produced a 45% reduction in food intake and significantly greater in vivo ARC neuron activation in mice. The chemical phenotype of activated ARC neurons was assessed by monitoring agonist-induced cellular activity via calcium imaging in mouse POMC-EGFP brain slices, which revealed that combined agonists activated significantly more POMC neurons (46%) compared with either drug alone (∼25% each). Single-cell electrophysiological analysis demonstrated that 5-HT2CR/5-HT1BR agonist coadministration did not significantly potentiate the firing frequency of individual ARC POMC-EGFP cells compared with agonists alone. These data indicate a functional heterogeneity of ARC POMC neurons by revealing distinct subpopulations of POMC cells activated by 5-HT2CRs and disinhibited by 5-HT1BRs. Therefore, coadministration of a 5-HT1BR agonist potentiates the anorectic efficacy of 5-HT2CR compounds by increasing the number, but not the magnitude, of activated ARC POMC neurons and is of therapeutic relevance to obesity treatment.

Gq-protein-coupled receptors (GqPCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ release from intracellular stores via the phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) signaling pathway. Because early GqPCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study, we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate GqPCR signaling in neurons. Our results demonstrate that GqPCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca2+. Depolarization has a bidirectional effect on GqPCR signaling, potentiating thapsigargin-sensitive Ca2+ responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating, and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP3/GPIP2 (glycerophosphoryl-myo-inositol 4,5-bisphosphate) places the voltage sensor before the level of the Ca2+ store, and measurements using the fluorescent bioprobe eGFP-PH(PLCdelta) (enhanced green fluorescent protein-pleckstrin homology domain-PLCdelta) directly demonstrate that voltage affects muscarinic signaling at the level of the IP3 production pathway. The sensitivity of GqPCR IP3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial.