Fig. 1. Development of passive membrane properties of NL neurons. (A) RMP. (B) R_in. (C) Cell capacitance. (D) Membrane time constant (tau). Data were pooled from all cells recorded in this study. In this and subsequent figures, means±1 standard error are shown (note that means and standard deviations are reported in the text), and *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively (ANOVA post hoc tests).

Fig. 2. Development of firing properties of NL neurons in response to prolonged (duration of 100 ms) current injections. (A) Superimposed membrane voltage responses of individual neurons. At all ages except for E18, two response patterns were seen. Some cells fired single APs in response to depolarizing current steps (left column), while the others fired multiple APs (right column). At E18, only the former firing pattern was observed. The RMPs of these cells were −56 and −61 mV (E10); −65 and −61 mV (E11); −60 and −66 mV (E12); −77 and −66 mV (E14); −65 and −60 mV (E16); and −62 mV (E18). Current injections were given in a step of 50 or 100 pA. The scale bars apply to all recordings. The triangle indicates where the amplitude of voltage responses was measured to obtain I/V relationships shown in C. (B) Number of APs plotted against the amplitude of injected currents relative to the threshold current (the minimum current required to elicit APs). The number of cells is indicated in parentheses. (C) Averaged steady-state I/V relationships. For clarity, voltage responses to current injections >1.2 nA are not included. The number of cells is indicated in parentheses. Cells that were unable to fire APs were excluded in B but included in C. (D, E) Slopes of the I/V relationships, calculated from voltage responses below (D) as well as above RMP (E), decrease over age. (F) Rectification, the ratio of the slope above RMP over the slope below RMP, remains unchanged in early ages (E10–12), increases at E14/16 significantly, and then decreases at E18.

Fig. 3. Development of excitability properties. (A) NL neurons become less excitable with age, as shown by the progressively increasing threshold current (threshold I). (B) AP threshold (threshold V) is more positive (higher) at E14/16 than at E10/11. (C) The maximal rising rate (max rise rate) of APs shows a dramatic increase at E16/18. (D) The maximal falling rate (max fall rate) of APs shows almost identical developmental changes to the rising rate. (E) AP height shows small but significant increases at E16/18. (F) Consistent with the changes in the maximal rising and falling rates of APs, AP half-width significantly decreases at E16/18. Note that no significant differences were detected in any parameters tested here among E10, E11, and E12.

Fig. 4. The ability of NL neurons to follow high frequency inputs enhances during early development. Current pulse trains (frequency of 50, 100, 200, 300, 400, or 800 Hz) were applied to the recorded cells. Each pulse train was repeated 10 times. Response patterns are similar from trial to trial, and only one trace from each cell at each frequency is presented. The ability of cells to fire full-size spikes in a one-to-one manner seems to improve with age. The RMPs of these cells were −50, −60, −60, −60, −65, and −63 mV at E10, E11, E12, E14, E16, and E18, respectively.

Fig. 5. I_k in NL neurons significantly increase in amplitude and density after E12. (A) Representative I_k recordings at six different ages. Cells were voltage clamped at −60 mV. A series of voltage commands (duration of 100 ms) was applied in the range between −80 mV and 20 mV in a step of 5 mV. The triangle indicates where the averaged current amplitude (over a period of 5 ms) was measured to obtain the I/V relationships shown in B. The RMPs of the these cells were −68, −56, −61, −59, −65, and −56 mV at E10, E11, E12, E14, E16, and E18, respectively. (B) Averaged I/V relationships. The number of cells is indicated in parentheses. A LVA component started appearing at about −55 mV (see the inset), and continued to increase in amplitude with more depolarizing voltage commands. Activation of a HVA component was seen at about −15 mV. (C) The total I_k measured at 20 mV. (D) The LVA I_k measured at −40 mV. (E, F) Both the total and the LVA I_k densities (currents normalized to cell capacitance) show significant increases at E16/18.

Fig. 6. Postsynaptic excitatory responses in NL neurons are developmentally regulated. (A) Representative recordings of postsynaptic excitatory responses at six different ages. At each age, one EPSP (the thick trace) and one AP (the thin trace) from the same cell were superimposed. Stimulation intensity was set at such a level that a mix of EPSPs and spikes was recorded in response to the same repeated stimulus. In early ages (E10–12), APs are followed by a large ADP. The triangle indicates where the amplitude of the ADP was measured obtain data shown in E. The scale bars apply to all recordings in A. Stimulation artifacts are blanked for clarity, and the onset of stimulation is indicated by an upward arrow. Cells were recorded at their RMPs, which were −56, −53, −57, −62, −51, and −64 mV at E10, E11, E12, E14, E16, and E18, respectively. (B) The 10–90% rise time of EPSPs shows a 14-fold reduction from E10/11 to E18. (C) Correspondingly, EPSP half-width (the width measured at half peak amplitude) progressively decreases over age, showing >37-fold reduction from E10/11 to E18. (D) The amplitude of EPSPs increases by twofold from E10/11 to E14/16, then slightly decreases at E18. (E) ADP amplitude, measured at 20 ms after the onset of APs, significantly declines at E16, and diminishes at E18. (F, G) ADP observed in EPSP (the thin line in F) and AP (the thin line in G) at E10 was largely eliminated by AP5 (100 μM) (the thick lines), an antagonist for NMDA receptors. Stimulation artifacts are truncated for clarity.

Summary of numerical data on intrinsic and synaptic properties of NL neurons during early development

Mean±1 standard deviation are shown. Note that ANOVA P values for each parameter are reported here, while significances between two ages detected by using Fisher's post hoc analyses are indicated in Fig. 1, Fig. 2, Fig. 3, Fig. 5 and Fig. 6. Number of cells is indicated in parentheses.