BDNF decreases [3H] GABA uptake in hippocampal nerve terminals, an effect independent of adenosine A2A receptor activation

Sandra H. Vaz, J. Alexandre Ribeiro and Ana M. Sebastião. Institute of Pharmacology and Neurosciences, Faculty of Medicine and Institute of Molecular Medicine, University of Lisbon

Brain-derived neurotrophic factor (BDNF) inhibits γ-gamma-aminobutyric acid (GABA) release from hippocampal synaptosomes by acting on GABA transporters (Canas et al., 2004). Since the principal function of transporters is re-uptake of neurotransmitters from the synaptic cleft, we studied the effect of acutely applied BDNF upon the uptake of GABA by hippocampal nerve terminals (synaptosomes) and the possible transduction pathway operated by BDNF to elicit this action. The influence of adenosine A2A receptor activation upon BDNF action was also evaluated because A2A receptors are required for the excitatory action of BDNF upon synaptic transmission (Diógenes et al., 2004).

Synaptosomal fraction was prepared from hippocampus of 3-4-weeks-old male Wistar rats. GABA uptake was determined using [3H]GABA as a radiotracer. The synaptosomal suspension (0.5 mg protein ml-1) was preincubated at 37ºC for 20 minutes in a total volume of 300 μl, in the presence or absence of testing drugs, and the transport was initiated by addition of 5 μM [3H]GABA (specific activity 0.133 Ci/mmol). After 40 seconds the transport was terminated by the addition of 5 ml ice-cold Krebs-HEPES solution followed by rapid filtration and washing out of the preparation. GABA uptake was calculated as the difference between the total [3H]GABA taken up by synaptosomes and the non-specific component of [3H]GABA uptake, which was determined by preincubation with SKF89976a (0.2mM), an inhibitor of GABA transport. The effect of BDNF was calculated taking as 100% the uptake of GABA in the absence of BDNF in the same experimental conditions. P value was assessed by one-way ANOVA followed by the Dunnett’s post-test. BDNF (10 - 100 ng ml-1) decreased GABA uptake in a concentration dependent manner (% decrease: 9.49 ± 3.918 %, n=6 for 10 ng/ml, 20.92 ± 2.838 %, n=19, P<0.01 for 30 ng ml-1 and 28.91 ± 3.629 %, n=6, P<0.01 for 100 ng ml-1). This effect involves tyrosine-kinase B (TrkB) receptor phosphorylation, since it was abolished by K252a (200nM), which prevents TrkB-mediated phosphorylation. The inhibition of phospholipase C (PLC) with U73122 (3 μM), also prevented BDNF action, suggesting that the transduction pathway activated by BDNF is via PLC. Activation of adenosine A2A receptors with the selective agonist CGS 21680 (30 nM) did not have an appreciable effect on GABA uptake. In the presence of CGS 21680 (30 nM) the effect of BDNF on GABA transport was similar to the effect of BDNF (30 ng/ml) in the absence of CGS 21680 (30 nM). Blockade of adenosine A2A receptors with the selective antagonists SCH 58261 (50 nM) or ZM 241385 (50 nM) did not influence the effect of BDNF (30 ng ml-1) on GABA transport (% of inhibition by BDNF: 17.56 ± 2.861 % in the presence of SCH 58261 (n=5, p<0.001) and 16.60 ± 6.171 % in the presence of ZM 241385 (n=3, p<0.05)).

These results suggest that BDNF, via TrkB and PLC, modulates GABA uptake in hippocampal synaptosomes and that A2A receptors do not influence this effect.

Canas et al., 2004, Brain Res.,1016: 72-78.
Diógenes et al., 2004, J Neurosci 24:2905-2913.

Supported by FCT (POCTI/NSE/37398/2001). BDNF was a gift by Regeneron.