The BDNF excitatory effect on synaptic transmission is dependent on adenosine receptor activation at the rat diaphragm neuromuscular junction

Pousinha P, Ribeiro JA, Sebastião AM. Institute of Pharmacology and Neuroscience, Faculty of Medicine and Institute of Molecular Medicine, University of Lisbon.

Adenosine modulates evoked acetylcholine release from the phrenic nerve of the rat through both inhibitory (A1) and excitatory (A2A) adenosine receptors (Correia-de-Sá et al., 1991). At the developing neuromuscular junction the excitatory action of brain derived neurotrophic factor (BDNF) on synaptic transmission is facilitated by pre-depolarization (Boulanger et al., 1999). In the hippocampus of young rats, adenosine, through A2A receptor activation, potentiates synaptic actions of BDNF (Diógenes et al., 2004). We now investigate the modulatory action of BDNF on neuromuscular transmission and how adenosine or pre-depolarization influences that effect.

The experiments were carried out on isolated preparations of the phrenic nerve-diaphragm of the rat (male Wistar, 3–4 weeks old). Evoked end plate potentials (e.p.ps) were recorded from single fibres of innervated strips of the diaphragm at room temperature (22-25ºC). The strips ( @ 8 mm wide) were mounted in a Perspex chamber (3ml) through which the solution (mM: NaCl 117; KCl 5; NaH2PO4 1.25; NaHCO3 26; MgCl2 1.2; CaCl2 2.5; glucose 10, 95% O2 / 5% CO2, pH 7.4, ) flowed continuously at a rate of 3ml.min-1. The e.p.ps were recorded with intracellular electrodes filled with KCl (3M) and of 10-20M W resistance. The nerve was stimulated supramaximally with rectangular pulses of 20μs duration applied every 2s. Evoked responses of 60 consecutive stimuli were averaged after amplification with LTP software (Anderson et al., 2001). The resting membrane potential of the muscle fibre was continuously monitored. In these experiments tubocurarine was added to the bath in a concentration (1.5 μM) sufficient to cause complete blockade of muscle contractions. BDNF (20-100 ng/ml) alone was virtually devoid of effect on end-plate potential (e.p.p) amplitude (n=4). However, when applied to innervated diaphragm preparations that had been shortly depolarized by a pulse of K+ ( 10 mM) for 2min, 50min before neurotrophin application, BDNF (20 ng/ml) increased the end-plate potential amplitude by 18.0±2.6% (n=3, p < 0.05 compared with BDNF alone). This effect might be related to A2A receptor activation since it was prevented (n=3) by its specific antagonist, ZM 241385 (50 nM) (Poucher et al., 1995). Moreover, activation of adenosine A2A receptors with 10nM CGS 21680 (Jarvis et al., 1989) triggered the excitatory action of BDNF (20ngml-1; 16.0 ± 1.4% increase, n=4, p < 0.05 compared with BDNF alone), an effect prevented by ZM 241385 (50 nM, n=4) or upon inhibition of TrKB receptors (200nM, n=2) with the TrkB receptor antagonist, K252a (Berg et al., 1992).

The results suggest that pre-depolarization induces increase of extracellular adenosine levels and that activation of A2A receptors unmasks the BDNF excitatory effect on transmission at the rat diaphragm neuromuscular junction.

Anderson W.W. et al. (2001) J. Neuroscience Methods 108: 71-83
Berg M.M. et al. (1992) J Biol Chem 267 : 13-16.
Boulanger, L. et al. (1999) Nature Neurosci., 2: 346-351.
Correia-de-Sá, P. et al. (1991) Br J Pharmacol., 103(2): 1614-20.
Diógenes M.J. et al. (2004) J Neurosci, 24: 2905-2913.
Jarvis M.F. et al. (1989) J. Pharmacol. Exp. Ther., 251: 888-893.
Poucher S.M. et al. (1995) J Pharm Pharmacol., 48: 601-6.

Work supported by FCT. BDNF was a gift by Regeneron.