Development of Electrophysiological Properties of Nucleus Gigantocellularis Neurons Correlated with Increased CNS Arousal

Abstract

Many types of data have suggested that neurons in the nucleus gigantocellularis (NGC) in the medullary reticular formation are critically important for CNS arousal and behavioral responsiveness. To extend this topic to a developmental framework, whole-cell patch-recorded characteristics of NGC neurons in brainstem slices and measures of arousal-dependent locomotion of postnatal day 3 (P3) to P6 mouse pups were measured and compared. These neuronal characteristics developed in an orderly, statistically significant monotonic manner over the course of P3-P6: (1) proportion of neurons capable of firing action potential (AP) trains, (2) AP amplitude, (3) AP threshold, (4) amplitude of inward and outward currents, (5) amplitude of negative peak currents, and (6) steady state currents (in I-V plot). These measurements reflect the maturation of sodium and certain potassium channels. Similarly, all measures of locomotion, latency to first movement, total locomotion duration, net locomotion distance, and total quiescence time also developed monotonically over P3-P6. Most importantly, electrophysiological and behavioral measures were significantly correlated. Interestingly, the behavioral measures were not correlated with frequency of excitatory postsynaptic currents or the proportion of neurons showing these currents, responses to a battery of neurotransmitter agents, or rapid activating potassium currents (including IA). Considering the results here in the context of a large body of literature on NGC, we hypothesize that the developmental increase in NGC neuronal excitability participates in causing the increased behavioral responsivity during the postnatal period from P3 to P6.

Fig. 1. Capability of neurons to fire AP train increased over P3 to P6 of age

Percentage of recorded neurons capable of firing AP train increased monotonically and significant statistically (Mann-Kendall, or MKT, test for Trend, enclosed insert) from P3 through P6. Neither the fraction of neurons capable of firing no or single AP, nor that of firing both single and AP train combined was changing monotonically (Table 2). Typical examples of neurons firing no AP (insert a), single AP (insert b), or AP train (insert c) are illustrated in the inserts. The examples in the insert also illustrate general differences between single AP and AP train in amplitude (peak below 10 mV for single vs above 20 mV for train) and the threshold (above −45 mV for single vs below −40 mV for the first AP in the train).

Fig. 2. Example of currents generated by I-V step from holding potential (Vh) at −70 mV and −40 mV

Current traces (A and B) and the corresponding I-V plots (C and D) from both protocols (inserts Cc and Dd) are obtained from a single, typical neuron. Traces in A and the corresponding I-V plot (C) were generated with the protocol (see insert Cc) starting from Vh = −70 mV, while those of B and D were from the protocol (insert d) with Vh= −40 mV. In A, the middle traces, from the second trace below through the third trace above the high-lighted one, the current response is initiated by a rapid upswing (i.e., rapid activating current). These upswing are expanded and illustrated in the insert Aa and reflected as the gaps between PPk and SSt curves in I-V plot C. In contrast, these upswings can no longer be seen in B or the insert Bb, and are reflected as the vanishing of the PPk-SSt gaps from −35 mV through 25mV in I-V plot D. All these indicating that rapid activating currents were inactivated at Vh of −40 mV. Note that after the rapid activating currents disappeared in A or inactivated in B, traces, especially upper ones, are still initiated with a gentle “peak”. These peak currents are slower than the rapid activating currents and are referred to as “fast activating currents”. They are reflected as the PPk-SSt gaps at 40 mV through 85 mV in I-V plots C and D. Traces in A and B are plotted in same scales, which are shown in the upper right corner of B.

Fig. 3. Recording of EPSC from NGC neurons

Some neurons like the one shown in A did not show EPSC. Others showed EPSC of various amplitude and frequency. One example with a large EPSC is illustrated in B.

Fig. 4. Responses of NGC neurons to a battery of transmitter agents

Traces are membrane potential recorded from NGC neurons, which frequently responded with depolarization to the agents. The bar beneath the trace indicates the time and the duration (always 2 min) when the agent indicated below the bar was bath applied. The numbers above or below the end of each trace are the fraction and percentage of the neurons responded. In A through E, neurons were tested with one of the agents. In F, the neuron was treated with the vehicle, ACSF, to serve as control

Fig. 5. Induction of AP by current injection (70 pA) of CA1 neurons from hippocampus of P4 mice

All 8 hippocampal neurons recoded were capable of firing AP in response to an injection of the same amount of current, 70 pA, as those NGC neurons shown in Fig. 1. This responsiveness is similar and surpasses that of P6 NGC neurons in that 100% of hippocampal neurons were capable of firing AP, indicating that hippocampal neurons are more advanced in development.

Fig. 6. Mice quiescence time decreases and movement time gets increases over P3 – P6

Durations of quiescence and total movement were observed for a total of 900 second. Results are presented as average and with SEM as error bar. N = 14 for all age groups.

Fig. 7. Correlations between behavior measures and the capability of NGC neurons to fire AP train

For clarity, three behavior measures are divided into panels A and B. In A, the age scale for latency to movement (dashed line) is reversed. Changes for all measures over the age range are monotonical and significant (MKT, of Mann-Kendall test for Trend, all p<0.05, one-tailed). For each measure, the value on P3 is significantly different from that on P6 (ANOVA, all p<0.01, two-tailed). As presented in Table 3, the monotonical change of neurons with AP train is significantly (Spearman rank correlation coefficient test, all p<0.05, one-tailed) correlated with that for all behavior measures. Error bars represent SEM. For all behavior measures, n = 14; for neurons with AP train, n = 12 to 18.