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. 2010 Apr 21;103(6):3448–3464. doi: 10.1152/jn.01069.2009

Fig. 1.

Fig. 1.

The experimental preparation, protocol and [Mg]o–block analysis of N-methyl-d-aspartate receptor (NMDAR) currents recorded from patch-clamped suprachiasmatic nucleus (SCN) neurons within the hypothalamic brain slice. A: this ×10 photograph was modified to show the 3rd ventricle (3V) and the optic chiasm (OC) in relation to the recording pipette (R) used during whole cell recordings of neurons within the SCN. B: voltage-clamped SCN neurons were given a voltage command protocol (mV, light gray trace), which served to inactivate voltage-gated conductances (ramp from −70 to 50 mV over 5 s) and to obtain an I-V record (ramp from 50 to −90 mV over 1 s). The black trace is an example of the current response (pA) to this voltage ramp protocol from an SCN neuron exposed to 100 μM NMDA and 100 μM d-Serine in nominally [Mg]o-free (“0” [Mg]o) physiological saline where voltage-gated sodium and L-type calcium channels (1 μM tetrodotoxin and10 μM nifedipine, respectively), GABAA (100 μM picrotoxin), and non-NMDAR ionotropic glutamate receptors (10 μM NBQX) were blocked. C: the NMDAR I-V curves are shown for baseline (without agonists), 0, 1, 10, 100, and 1,000 [Mg]o and d-APV (1,000 μM [Mg]o +100 μM d-APV) conditions recorded from a neuron within the SCN brain slice using 100 μM NMDA and 100 μM d-serine. The reversal potential was measured to be 12.5 ± 1.6 mV (n = 19). D: these currents are replotted for each condition with the d-APV curve deducted and normalized to the largest outward current of the set at 50 mV. The linear regression of the outward current, referred to as the “theoretical 0” condition, is also plotted (black line). E: the normalized current, with respect to the theoretical 0 line, is plotted as a function of [Mg]o for each voltage from −75 to −20 mV (in 5 mV increments). On this plot, the theoretical 0 points are set to 1 nM [Mg]o, and 0 [Mg]o condition is omitted. F: the natural log of the IC50 of [Mg]o (in μM) for each curve in C is plotted with respect to voltage (mV). The curve is fit with a linear regression to obtain the y intercept [IC50(0)] and slope (δzF/RT). The scale is set to C and D to 20 mV (horizontal bar) and 100 pA (vertical bar).