Persistent pulsatile release of glutamate induced by N‐methyl‐D‐aspartate in neonatal rat hippocampal neurones.

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Abstract
1. Intracellular recordings were made from CA3 hippocampal neurones in vitro, during the first ten days of postnatal life and in adulthood. 2. Repeated (three to six) applications of N‐methyl‐D‐aspartate (NMDA), in the presence of tetrodotoxin (TTX, 1‐3 microM) and K+ channel blockers (tetraethylammonium chloride or bromide (TEA), 10 mM, and Cs+, 2 mM; or 4‐aminopyridine (4‐AP), 30‐50 microM, and Cs+, 2 mM) induced in neonatal but not in adult neurones, periodic inward currents (PICs) which persisted for several hours after the last application of NMDA. 3. PICs which were due to non‐specific cation currents had a frequency of 0.10 +/‐ 0.04 Hz, and an amplitude of 1.1 +/‐ 0.28 nA at holding potentials between ‐40 and ‐50 mV. The amplitude was a linear function of the membrane potential over the range ‐70 to +20 mV. They reversed polarity at 4.1 +/‐ 9.8 mV. 4. K+ channel blockers alone failed to induce PICs. Repeated (three to six) brief applications of high (12 mM) K+ medium also induced PICs. The frequency and amplitude of K(+)‐induced PICs were however considerably reduced by concomitant applications of the NMDA receptor antagonist D,L‐3‐[( +/‐ )‐2‐carboxypiperazin‐4‐yl‐]propyl‐1‐phosphonic acid (CPP, 20 microM). PICs could be induced also by caffeine (1 mM) in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methyl‐xanthine (IBMX, 200 microM), TTX, TEA and Cs+. 5. Intracellular injection of the calcium chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA) did not prevent the induction of PICs by NMDA. However PICs were blocked by removal of the external calcium and by the calcium antagonists cobalt (2 mM) and cadmium (50 microM). 6. In spite of blockade of propagated synaptic activity by TTX, PICs were synchronous in a pair of intracellularly recorded cells. They were also synchronous with extracellular spikes recorded by electrodes located into stratum pyramidal or stratum radiatum. 7. Once established, PICs were unaffected by NMDA receptor antagonists D(‐)2‐amino‐5‐phosphonovaleric acid (AP‐5, 50 microM), CPP (20 microM) and the NMDA channel blocker ketamine (10 microM). They were reversibly blocked by the broad spectrum excitatory amino acid antagonist kynurenic acid (1 mM) and by the selective non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 microM). 8. It is concluded that PICs are generated in neonatal neurones by a synchronous, pulsatile release of glutamate from presynaptic nerve terminals, secondary to oscillations in intracellular calcium.