AAV5-GFAP-GFP

Feeding behavior is a complex process that depends on the brain's ability to integrate hormonal and nutrient signals, such as glucose. One glucose-sensing mechanism relies on the glucose transporter 2 (GLUT2) in the hypothalamus, particularly in radial glia-like cells, called tanycytes. Here, researchers analyzed whether a GLUT2-dependent glucose-sensing mechanism is required for the normal regulation of feeding behavior in GFAP-positive tanycytes. Genetic inactivation of Glut2 in GFAP-expressing tanycytes was performed using Cre/Lox technology. The efficiency of GFAP-tanycyte targeting in the anterior-posterior and dorso-ventral axes was analyzed by assessing GFP fluorescence. Feeding behavior, hormone levels, neuronal activity using c-Fos, and neuropeptide expression were also analyzed during the transition from fasting to refeeding. Under basal conditions, mice with Glut2 inactivation had normal food intake and meal patterns. Imposing a pre-fasting period resulted in a reduction in total food intake and delayed meal times during the refeeding period. Furthermore, Glut2 inactivation increases the number of c-Fos-positive cells in the ventromedial nucleus in response to fasting and deregulates Pomc expression during the transition from fasting to resumption of feeding.Thus, GLUT2-dependent glucose-sensing mechanisms in GFAP-tanycytes are necessary to control food consumption and promote meal timing after a fasting period.

To delete the Glut2 gene Slc2a2^loxP/loxp from GFAP-positive elongated cells in zones 1 to 6, adeno-associated virus serotype 5 (AAV5)-Gfap-Cre-GFP and control viral vector AAV5-Gfap-GFP were stereotaxically injected in 3V, where GFAP-expressing elongated cells project to the DMN, VMN, and ARC in similar proportions (Figure 1A). Injection of AAV5-Gfap-CRE-GFP induced the expected recombination of the Glut2 allele (Figure 1B). The presence of the non-recombined floxed allele corresponding to the 281 bp product is due to the presence of non-transduced cells in this zone. To confirm that recombination occurred only in elongated cells, the GFP signal and the percentage of GFAP/GFP-positive elongated cells in the hypothalamus of mice transduced for 2 weeks were assessed by confocal microscopy. As expected, GFP was detected in the apical region and processes of elongated cells (Figure 1C, arrows). In addition, GFP fluorescence (green) was observed in GFAP-positive elongated cells (yellow, arrows), which contained button-shaped endfeet and were in close contact with other cells present in the hypothalamic parenchyma (Figure 1D, arrows). To determine the percentage of transduction, the number of GFAP-expressing elongated cells positive for GFP fluorescence was quantified. As shown in Figure 1E, the percentage of GFAP-expressing elongated cells positive for GFP fluorescence was close to 28.5%. In all analyzed sections, only one GFAP-positive astrocyte was detected to be transduced.

Figure 1. Glut2 gene inactivation in GFAP-expressing tanycytes and its in situ evaluation. (Barahona M J, et al., 2022)