Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons

Abstract

In rat cerebellar granule neurons, mRNA and protein levels of the NR2A and NR2C subunits of the NMDA receptor increase during the second postnatal week. At this time, mRNA and protein levels of the NR2B subunit begin to fall. To investigate targeting of NMDA receptor subunits, we performed whole-cell recordings from rat cerebellar granule neurons at different times during development and investigated the pharmacological and biophysical properties of mossy fiber-evoked NMDA EPSCs. Isolated NMDA EPSCs from newly formed synapses in the first postnatal week exhibited partial block by the NR2B subunit-specific antagonist (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606). By the end of the second postnatal week, NMDA EPSCs were virtually unaffected by the NR2B antagonist. In parallel, NMDA EPSC decay times decreased over a similar developmental time course. We compared properties of synaptic NMDA receptors with extrasynaptic receptors that are present on the cell body with rapid application of glutamate to excised nucleated patches. Deactivation of patch responses accelerated with development and closely resembled evoked NMDA EPSCs in rats of the same age. However, patch responses were highly sensitive to CP 101,606 through the second postnatal week, and sensitivity was seen in some neurons up to the fourth postnatal week. Spermine potentiated peak NMDA patch responses from postnatal days 10-14 rats but had little effect on evoked NMDA EPSCs. Our data suggest selective targeting of a distinct NMDA receptor subtype to synaptic receptor populations in cerebellar granule neurons. Later in development, similar changes occur in the extrasynaptic receptor population.

Fig. 1.

Comparison of kinetic and pharmacological properties of evoked NMDA EPSCs in developing granule neurons.A, Top, Evoked NMDA EPSCs from granule neurons in rats at P7 and P21. The averages of 5–10 consecutive sweeps are shown with a superimposed double exponential curve. The individual exponential curves are also shown. Bottom, The two responses are shown superimposed after scaling with an indication of the weighted time constant (τ_w) to emphasize differences in deactivation. B, Top, Average of five to seven traces taken from a P7 rat before (control) and in the presence of 5 μm CP 101,606 (CP). Bottom, Average of six to seven traces taken from a P14 rat before (control) and in the presence of 5 μm CP 101,606 (CP). C, Relationship between percentage control response in the presence of CP 101,606 (5 μm) as a function of the weighted time constant of NMDA EPSCs in P6–P8 and P10–P14 rats.

Fig. 2.

Deactivation of patch responses accelerate with development. A, Single response tol-glutamate (1 mm, 4 msec) in nucleated patches from P7 and P24 rats. B, Top, Averagel-glutamate (1 mm, 4 msec) activated currents in two nucleated patches from P7 and P24 rats. The average of seven consecutive l-glutamate applications are shown with a superimposed double exponential curve. The individual exponential curves are also shown. Bottom, The two responses are shown overlapped after scaling with an indication of the weighted time constant (τ_w) to emphasize differences in deactivation. C, Summary of all individual weighted time constants from NMDA responses in nucleated patches at three developmental age groups (P7–P8, P10–P14, and greater than P18).Horizontal bar located among responses indicates arithmetic mean.

Fig. 3.

Effect of NR1/NR2B antagonists on nucleated patch responses. A, Top, Responses to 1 mml-glutamate before (control), during (CP), and after (wash) application of 5 μm CP 101,606 in a P7 rat. In each panel, at least four traces were averaged.Bottom, As above, with responses elicited from nucleated patches in a P14 rat. In both panels, horizontal barsindicate duration of l-glutamate applications (200 msec).B, A comparison of the effect of NR1/NR2B-specific antagonists between patch responses and NMDA EPSCs of the same age. *p < 0.05, significant differences from 100% (independent t test); #p < 0.01, statistical significance between nucleated patches and NMDA EPSCs of the same age group (independent t test).C, The relationship between the percentage control after CP 101,606 application and the weighted time constant of nucleated patch responses (4 msec pulses) from rats in the three age groups.

Fig. 4.

CP 101,606 blockade of extrasynaptic receptors in the intact granule neuron. A, Top, Spontaneous NMDA channel currents recorded after establishing a whole-cell recording from a P14 rat. Middle, Effect of bath perfusion of 5 μm CP 101,606. Bottom, Trace represents effect of 10 μm CPP on spontaneous NMDA single channel events. CPP was bath applied immediately after CP 101,606. B, Evoked NMDA EPSCs in the presence of 10 μm MK-801 recorded in the same cell after washout of CPP. Stimulus frequency was 0.1 Hz. Responses at different stimulus number (Stim) from the beginning of MK-801 perfusion are illustrated. C, Top, Current sweep taken immediately after Stim 30 in B. MK-801 was allowed to wash for ∼3 min. Middle, Bath perfusion of 100 μm NMDA after MK-801 wash. Bottom, Trace acquired 2 min after application of a solution containing both 100 μm NMDA and 5 μm CP 101,606.

Fig. 5.

Spermine potentiates patch responses but not NMDA EPSCs. A, Top, Evoked NMDA EPSCs from a P7 granule neuron. Left trace represents average of 20 consecutive sweeps in control conditions. Right tracerepresents the average of 15 sweeps in the presence of 500 μm spermine (sp 500).Bottom, Left trace represents single-channel currents from the tail of individual evoked NMDA EPSCs in control conditions. Right trace represents NMDA single-channel currents from the same cell in the presence of 500 μm spermine. In both traces, overlapped channel openings were observed. B, Nucleated patch responses from a P14 granule neuron. Left trace is the average of five consecutive 200 msec l-glutamate applications in control conditions. Right trace is the average of five consecutive 200 msec l-glutamate applications in the presence of 500 μm spermine (sp 500).Horizontal bars indicate duration of thel-glutamate applications. C, Summary illustrating peak NMDA EPSCs in rats at P7 (n = 6 cells) and peak patch currents in P14 rats (n = 8 cells) in the absence and the presence of 500 μmspermine. *p < 0.05, statistical significance between control and spermine responses (paired ttest).