Figure 4.

Mecp2 ^−/y adrenal chromaffin cells exhibit an increase in the catecholamine RRP and evoked release. The RRP was quantified in wild-type and Mecp2 ^−/y chromaffin cells as described previously (Gillis et al., 1996). Ai, Cells were voltage clamped at −80 mV and stimulated with a pair of 100 ms depolarizations, first to 0 mV and then to +5 mV, to balance Ca²⁺ influx. The top trace shows evoked current influx (I_Mon.), and the bottom trace shows cell capacitance (Cm). In this example, the first pulse resulted in a greater capacitance increase than the second pulse (Cm₁ and Cm₂, respectively), reflecting a consumption of release-ready granules by the first pulse, leaving few for the second pulse to access. Formally, the RRP can thus be quantified as follows: RRP = S/(1 − R ²), where S is the sum of Cm₁ and Cm₂ and R is the ratio of Cm₂ to Cm₁. Aii, This dual-pulse protocol was repeated on MeCP2^+/y and MeCP2^−/y cells. The quantified RRP sizes from Mecp2 ^+/y and Mecp2 ^−/y records (n = 36 and 65, respectively) are summarized and show that the RRP was significantly larger in the Mecp2 ^−/y (*p < 0.02, paired Student’s t test). Bi, To correlate the increased RRP to evoked catecholamine release, the amperometric (I Amp.) current was measured from chromaffin cells in control Ringer’s solution and during nicotine stimulation. A representative record from Mecp2 ^+/+ chromaffin tissue is plotted. Bii, Amperometric currents (ΣAmp.) recorded under this protocol from Mecp2 ^+/y and Mecp2 ^−/y cells were integrated to allow relative comparison of catecholamine release (n = 8 and 10 for Mecp2 ^+/y and Mecp2 ^−/y cells, respectively). These data show significant increases in both spontaneous and evoked catecholamine release from Mecp2 ^−/y cells compared with Mecp2 ^+/y (*p < 0.02, paired Student’s t test).