Modulation of L-lactate release by astrocytic GABA_B receptors and cyclic AMP signalling in vitro

Abstract

Astrocytes are crucial for the maintenance of the excitation/inhibition balance of neural circuits and therefore must sense and respond to local neuronal activity. Their regulation of glutamatergic transmission is currently better understood than that of GABAergic. While GABA_B receptor activation is generally associated with G_i/o protein signalling, previous work in astrocytes suggested an additional IP₃-dependent pathway leading to intracellular Ca²⁺ oscillations. In this study, we set out to investigate whether the GABAB receptor agonist baclofen modulates production and release of the gliotransmitter L-lactate (LL) in dissociated primary astrocyte cultures. Specifically, we focused on the cyclic AMP (cAMP) dependent pathway which has been linked glycogenolysis. We used the FRET-based nanosensor Laconic to monitor intracellular LL dynamics and a fluorimetric LL assay (EnzyFluo) to measure LL release. We found that baclofen (10μM-100μM) inhibited production of LL (88+24.9% ; n=15) in unstimulated astrocytes, and that LL production and release under these conditions was partially glycogen-dependent. However, presumably since basal constitutive LL release was low, an inhibitory effect of baclofen on release was not detectable. Activation of adenylyl cyclase by NKH 477 (6-[3-(dimethylamino)propionyl]-forskolin; 0.3μM-30μM) resulted in LL mobilisation in a concentration dependent manner. Interestingly, intracellular LL increase peaked at 3μM NKH, but LL release from the cells peaked at 10μM NKH, suggesting potentially differential effects of cAMP signalling on LL production and release mechanisms. The cAMP driven LL production was inhibited by activation of GABA_B receptors with baclofen (100μM), or by blocking glycogenolysis with DAB (1,4-Dideoxy-1,4-imino-D-arabinitol; 500μM), or PKA activity with H89 (10μM). Whilst the effect of baclofen on the NKH-stimulated rise of cAMP signalling was abolished by inhibition of G_i/o signalling with Pertussis toxin (PTX), PTX application did not abolish the effect of baclofen on the NKH-stimulated rise of intracellular LL. Our preliminary observations also suggest that inhibition of G_i/o signalling with PTX may increase LL release. Further work is needed to evaluate the effects of any cross-talk between cAMP and Ca²⁺ signalling pathways on LL release.

Date of Award	2 Dec 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Anja G Teschemacher (Supervisor) & Sergey Kasparov (Supervisor)