We have made patch-clamp recordings from slices of fetal and postnatal rat neocortex in order to study the initial expression and activation of NMDA channels. Recordings from both whole cells and outside-out patches indicated that functional NMDA channels are expressed on neurons within the cortical plate, but not on younger cells within the ventricular zone. The NMDA channels on cortical plate neurons had a unitary conductance of approximately 40 pS, had a mean open time of approximately 6 msec, required glycine to open, and were blocked in a voltage-dependent manner by magnesium. These precocious channels were present before the appearance of functional synaptic activity, yet like NMDA channels in the mature neocortex, they were spontaneously activated by an agonist within brain slices. These results demonstrate that NMDA channels are initially expressed on neocortical neurons some time between the last mitotic division within the ventricular zone and completion of migration into the cortical plate. These early NMDA channels have properties characteristic of NMDA channels on more mature neurons and are similarly activated by an endogenous agonist in situ. Their early appearance and activation indicate that NMDA channels may play a role during early stages of cortical development.

1. The properties of receptors for amino acid neurotransmitters expressed by developing cortical neurons were studied with the use of whole-cell recording in the intact cerebral cortex of embryonic turtles in vitro. The inhibitory agonist gamma-aminobutyric acid (GABA) and the excitatory agonist glutamate were focally applied to single cells under voltage clamp, and the ionic dependence, voltage dependence, and pharmacological sensitivity of the responses were characterized. The responses mediated by a glutamate receptor subtype, the N-methyl-D-aspartate (NMDA) receptor, produced by glutamate and by evoked release of an endogenous excitatory agonist, were compared further. Fluctuation analysis was used to characterize the properties of the NMDA channels and the mechanism of action of receptor antagonists. 2. When postmitotic neurons first appeared at stage 15, all neurons tested responded to GABA with a current that reversed at the equilibrium potential for chloride ions and that was sensitive to the GABAA receptor antagonist bicuculline methiodide (BMI). As development proceeded, an increasing proportion of neurons also responded with a BMI-insensitive current that reversed near the equilibrium potential for potassium ions. This current was blocked by the GABAB receptor antagonist 3-amino-2-propyl phosponic acid (phaclofen). The GABAB agonist baclofen, however, failed to produce a detectable postsynaptic current. 3. Neurons at stage 15 showed a biphasic response to glutamate that reversed at the equilibrium potential for cations. All neurons tested showed a slow, sustained response associated with an increase in current variance compared with background, and, as development proceeded, an increasing proportion also exhibited a fast, transient response. Both fast and slow responses varied linearly with voltage in the absence of Mg2+ ions, but the addition of Mg2+ ions to the bathing medium attenuated the slow response at hyperpolarized potentials. As a result, the current-voltage relation of the slow response in the presence of Mg2+ ions exhibited a region of negative slope conductance, like that of currents mediated by NMDA receptors. 4. The fast and slow responses to glutamate differed in their pharmacological sensitivity. The fast responses were sensitive to the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the slow responses were sensitive to the NMDA receptor antagonist D(-)-2-amino-5-phosphonovalerate (D-APV). 5. When cells were held at -70 mV, glutamate evoked a fluctuating current consisting of channel currents with a mean open time, tau, of 4.42 +/- 0.47 (SE) ms in early postmitotic neurons at stage 15 and 4.99 +/- 0.38 ms at stages 17-20.(ABSTRACT TRUNCATED AT 400 WORDS)

We used loose-patch and whole-cell recording techniques to study the development of spontaneous action potential activity and spontaneous excitatory and inhibitory synaptic currents in embryonic neurons in the cerebral hemispheres of turtles. Sporadic action potential activity appeared early in development at stage 17, soon after morphologically identifiable pyramidal and nonpyramidal neurons were first observed in the cortex. As the cortical plate matured in midembryonic stages, action potential activity became more regular and fell into one of two distinct patterns, tonic and intermittent high-frequency firing. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) appeared at developmental stages 18 and 20, respectively, after action potential activity was established. EPSCs and IPSCs exhibited characteristic ionic dependence and pharmacology throughout development. EPSCs reversed in direction at the equilibrium potential for cations and were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA type of glutamate receptor. IPSCs reversed at the equilibrium potential for chloride and were sensitive to bicuculline methiodide, a GABAA receptor antagonist. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous EPSCs appeared at stage 18 and increased progressively in frequency, from 0.2 +/- 0.1 Hz at stage 20 to 3.2 +/- 2.0 Hz at stage 26 (hatching), while spontaneous IPSCs appeared at stage 20 and surpassed EPSCs in frequency, increasing to 7.1 +/- 1.6 Hz at stage 26. EPSCs exhibited stable amplitudes during development, with a mean conductance of 126 +/- 20 pS at stage 26, while IPSCs increased in mean amplitude, from 180 +/- 12 pS at stage 18 to 260 +/- 44 pS at stage 26. The rise time to peak conductance of both types of synaptic currents increased with developmental time, for EPSCs increasing from 1.5 +/- 0.5 msec at stage 20 to 2.7 +/- 0.6 msec at stage 26 and for IPSCs increasing from 2.9 +/- 0.2 msec at stage 18 to 6.2 +/- 0.8 msec at stage 26. While the decay time constants increased for EPSCs, from 3.9 +/- 1.2 msec at stage 20 to 8.7 +/- 2.3 msec at stage 26, decay time constants for IPSCs showed a decreasing trend from 24.0 +/- 5.2 msec at stage 18 to 18.4 +/- 5.3 msec at stage 26. The excitatory and inhibitory synaptic currents were sensitive to the sodium channel blocker TTX and were thus dependent, in part, on spontaneous action potential activity.(ABSTRACT TRUNCATED AT 400 WORDS)