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 Fӧrster 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.

Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in real time, by transfecting them with different GDP‐ and GTP‐locked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobility in the presence of wild‐type and Rab mutants. Dominant‐negative and dominant‐positive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L, Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement, and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N, and Rab5 Q79L. Our data indicate that changes in the GDP‐GTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton.

1 We measured membrane capacitance (C_m) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerises cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2 In control cells, cytosol dialysis with 1 μm Ca²⁺_i increased C_m by 23 ± 4 % (n= 11) relative to the resting C_m 400 s after the start of patch rupture. In CST-treated cells the increase in C_m was 32 ± 5 % (n= 15), not significantly different from controls. The rate of C_m increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of C_m increase was 4.27 ± 0.85 fF s⁻¹ (n= 12; measured 200 s after the start of patch rupture) in controls and 8.0 ± 1.35 fF s⁻¹ (n= 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3 In control cells cytosol dialysis with 0 μm Ca²⁺_i decreased C_m by 9 ± 3 % (n= 7), in CST-treated cells C_m increased by 11 ± 3 % (n= 7) relative to resting C_m 400 s after the start of cytosol dialysis. The rate of change in C_m remained constant (controls: -1 to -2 fF s⁻¹; CST treatment: 1-2 fF s⁻¹). 4 Transient and sustained effects of CST treatment on changes in C_m at high or low [Ca²⁺]_i, respectively, suggest a distinct role of cytoskeleton in Ca²⁺-dependent and Ca²⁺-independent changes in C_m. Transient enhancement of the rate of C_m by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca²⁺-independent changes in C_m suggests cytoskeletal involvement in endocytosis.