Astrocytes contain glycogen, an energy buffer, which can bridge local short-term energy requirements in the brain. Glycogen levels reflect a dynamic equilibrium between glycogen synthesis and glycogenolysis. Many factors that include hormones and neuropeptides, such as insulin and insulin-like growth factor 1 (IGF-1) likely modulate glycogen stores in astrocytes, but detailed mechanisms at the cellular level are sparse. We used a glucose nanosensor based on Förster resonance energy transfer to monitor cytosolic glucose concentration with high temporal resolution and a cytochemical approach to determine glycogen stores in single cells. The results show that after glucose depletion, glycogen stores are replenished. Insulin and IGF-1 boost the process of glycogen formation. Although astrocytes appear to express glucose transporter GLUT4, glucose entry across the astrocyte plasma membrane is not affected by insulin. Stimulation of cells with insulin and IGF-1 decreased cytosolic glucose concentration, likely due to elevated glucose utilization for glycogen synthesis.
COBISS.SI-ID: 31936985
Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
COBISS.SI-ID: 32590041
In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P ( 0.05), the frequency of ATP-evoked transient (P ( 0.001) and full-fusion exocytotic (P ( 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca2+ activity in astrocytes by ~70 % (P ( 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca2+ excitability.
COBISS.SI-ID: 32520921
During the arousal and startle response, locus coeruleus neurons, innervating practically all brain regions, release catecholamine noradrenaline, which reaches neural brain cells, including astrocytes. These glial cells respond to noradrenergic stimulation by simultaneous activation of the [alpha]- and [beta]-adrenergic receptors (ARs) in the plasma membrane with increasing cytosolic levels of Ca2+ and cAMP, respectively. AR-activation controls a myriad of processes in astrocytes including glucose metabolism, gliosignal vesicle homeostasis, gene transcription, cell morphology and antigen-presenting functions, all of which have distinct temporal characteristics. It is known from biochemical studies that Ca2+ and cAMP signals in astrocytes can interact, however it is presently unclear whether the temporal properties of the two second messengers are time associated upon AR-activation. We used confocal microscopy to study AR agonist-induced intracellular changes in Ca2+ and cAMP in single cultured cortical rat astrocytes by real-time monitoring of the Ca2+ indicator Fluo4-AM and the fluorescence resonance energy transfer-based nanosensor A-kinase activity reporter 2 (AKAR2), which reports the activity of cAMP via its downstream effector protein kinase A (PKA). The results revealed that the activation of [alpha]1-ARs by phenylephrine triggers periodic (phasic) Ca2+ oscillations within 10 s, while the activation of [beta]-ARs by isoprenaline leads to a -10-fold slower tonic rise to a plateau in cAMP/PKA activity devoid of oscillations. Thus the concomitant activation of [alpha]- and [beta]-ARs triggers the Ca2+ and cAMP second messenger systems in astrocytes with distinct temporal properties, which appears to be tailored to regulate downstream effectors in different time domains.
COBISS.SI-ID: 32522457
Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.