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. 2016 Mar 30;10:80.
doi: 10.3389/fncel.2016.00080. eCollection 2016.

Developmental Profile of Ion Channel Specializations in the Avian Nucleus Magnocellularis

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Free PMC article

Developmental Profile of Ion Channel Specializations in the Avian Nucleus Magnocellularis

Hui Hong et al. Front Cell Neurosci. .
Free PMC article

Abstract

Ultrafast and temporally precise action potentials (APs) are biophysical specializations of auditory brainstem neurons; properties necessary for encoding sound localization and communication cues. Fundamental to these specializations are voltage dependent potassium (KV) and sodium (NaV) ion channels. Here, we characterized the functional development of these ion channels and quantified how they shape AP properties in the avian cochlear nucleus magnocellularis (NM). We report that late developing NM neurons (embryonic [E] days 19-21) generate fast APs that reliably phase lock to sinusoidal inputs at 75 Hz. In contrast, early developing neurons (<E12) have slower and less reliable APs that preferentially fire to lower frequencies (5-10 Hz). With development, the membrane time constant of NM neurons became faster, while input resistance and capacitance decreased. Change in input resistance was due to a 2-fold increase in KV current from E10 to E21 and when high-voltage activated potassium (K(+) HVA) channels were blocked, APs for all ages became significantly slower. This was most evident for early developing neurons where the ratio of K(+) HVA current accounted for ~85% of the total KV response. This ratio dropped to ~50% for late developing neurons, suggesting a developmental upregulation of low-voltage activated potassium (K(+) LVA) channels. Indeed, blockade of K(+) LVA eliminated remaining current and increased neural excitability for late developing neurons. We also report developmental changes in the amplitude, kinetics and voltage dependence of NaV currents. For early developing neurons, increase in NaV current amplitude was due to channel density while channel conductance dominated for late developing neurons. From E10 to E21, NaV channel currents became faster but differed in their voltage dependence; early developing neurons (<E16) had similar NaV channel inactivation voltages while late developing NM neurons (>E19) contained NaV channels that inactivate at more negative voltages, suggesting alterations in NaV channel subtypes. Taken together, our results indicate that the refinement of passive and active ion channel properties operate differentially in order to develop fast and reliable APs in the avian NM.

Keywords: action potential; auditory brainstem; development; neural excitability; nucleus magnocellularis; voltage dependent potassium ion channel; voltage dependent sodium ion channel.

Figures

Figure 1
Figure 1
Developing nucleus magnocellularis (NM) neurons showed distinct firing patterns in response to sustained current injections. (A) Representative voltage traces recorded from NM neurons at E20 (left), E15 (middle) and E11 (right) in response to a sequence of sustained current injections shown below the traces (current step = 20 pA, current duration = 100 ms). Symbols (square, circle and diamond) at the end of voltage trace represent the time window of voltage measured and plotted as a function of injected current shown in (C). Schematic representation of stimuli used to evoke responses are shown below the representative traces. (B) Population data showing threshold current for AP generation as a function of age. (C) Population data showing the voltage-current relationship for each age group. (D) Population data showing the resting membrane potential (RMP) as a function of age. Open circles represent an individual neuron and solid bars represent the average for each age group. Error bars = standard error. *p < 0.05, Bonferroni adjusted t-test.
Figure 2
Figure 2
Development of action potential kinetics in NM. (A) Representative voltage traces (30 superimposed) in response to sustained threshold current injections. Threshold current is defined as the minimum amount of current required for NM neurons to generate an AP ~50% of the time across 30 repetitive stimulations. The injected +300 pA current shown below the traces was determined as the threshold current for this E20 NM neuron. Arrow depicts ~50% AP failures. (B) Representative APs for each age group evoked by sustained current injections at strength 25% above threshold current. AP kinetics were calculated and plotted as a function of age and are shown in (C–F). (C–F) Population data showing developmental changes in AP latency (C), AP half width (D), AP rise rate (E), and AP fall rate (in absolute value, F) as a function of age. Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 3
Figure 3
Development of AP reliability in NM. (A) Partial representative AP traces (30 superimposed, interpulse stimulus interval = 2 s) showing the time of peak AP occurrence from NM neurons at E21 (left), E15 (middle) and E11 (right). Corresponding time points of AP peak are plotted as individual dots in the same time scale for all three age groups shown in (B). (B) Representative raster plot showing peak AP occurrence. (C) Population data showing changes in the range of peak AP occurrence as a function of age. Inset showing the enlarged scale of the range for E14–16 (left) and E19–21 (right). Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 4
Figure 4
Developing NM neurons show distinct firing patterns in response to sinusoidal current injections at varying frequencies. (A–C) Representative voltage traces recorded from NM neurons at E11 (A), E15 (B) and E20 (C) in response to 10, 75, 100 and 200 Hz sinusoidal current injections. The strength of sinusoidal current is 150% above threshold current. (D–F) Firing probability per sinusoidal cycle, calculated as the number of APs divided by the total number of sinusoidal cycles, is plotted as a function of stimulus frequency for NM neurons at E10–12 (D), E14–16 (E) and E19–21 (F). Error bars = standard error.
Figure 5
Figure 5
Development of passive membrane properties in NM. (A) Representative voltage traces recorded from an E20 NM neuron (upper, 30 superimposed) in response to a small hyperpolarizing current (lower, −10 pA). A single exponential was fit to a 10 ms time window following the current injection (superimposed red line), in order to calculate passive membrane properties shown in (B–D). (B–D) Population data showing developmental changes in membrane voltage time constant (TM, B), membrane input resistance (RM, C) and membrane capacitance (CM, D) as a function of age. Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 6
Figure 6
Developing NM neurons showed a significant increase in total steady-state KV currents. (A) Representative KV current traces (IK) recorded from NM neurons at E11 (left), E15 (middle) and E21 (right) in response to membrane voltages clamped from −100 to +20 mV (voltage step = 5 mV, voltage duration = 100 ms). Symbols (diamond, circle and square) at the end of current traces represent time window of measured steady-state KV currents (SS IK). (B–D) Population data showing the relationship of total steady-state KV currents (SS IK, B), conductance (SS GK, C) and density (SS ρK, D) to the varying membrane voltages for each age group. Arrows and arrowhead in (C) indicate the apparent activation of distinct KV channel subtypes at different ages. Note that data points at −100 mV are not shown for simplicity.
Figure 7
Figure 7
Reduction of K+HVA current with Flx application was developmentally regulated. (A) Representative KV current traces recorded from NM neurons at E11 (left), E15 (middle) and E20 (right) before and during fluoxetine (Flx, 100 μM) application, in response to membrane voltages clamped from −100 to +20 mV. Flx is a potent KV3.1-containing K+HVA channel blocker. Scale bar values are 2000 pA/20 ms. Data obtained during drug application are shown in red. Symbols (diamond, circle and square) at the end of current traces represent time window of measured steady-state KV currents. (B–D) Population data showing current-voltage relationship before and during Flx application for E10–12 (B), E14–16 (C) and E19–21 (D). Note that data points at −100 mV are not shown for simplicity. (E–G) Population data showing reduction in steady-state KV currents at membrane voltage of +20 (E), −10 (F) and −50 (G) mV measured as percent change and plotted as a function of age. Note that in (G) several negative data points are not shown in the figure (2 points for E10–12, 1 for E14–16 and 1 for E19–21). Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 8
Figure 8
K+LVA channels mediate the majority of Flx-insensitive KV current and regulate neural excitability. (A) Steady-state KV current traces recorded from an E19 NM neuron before (control) and during subsequent bath application of Flx and DTx (0.1 μM). DTx is a potent KV1.1, KV1.2-containing K+LVA channel blocker. Data obtained during drug application are shown in red. Symbols (square and circles) at the end of current traces represent time window of measured steady-state KV currents. (B) Current-voltage relationship before and during subsequent drug application for the E19 NM neuron shown in (A). Note that data points at −100 mV are not shown for simplicity. (C) Representative voltage traces recorded from an E15 NM neuron before and during DTx application, in response to a sequence of sustained current injections (current step = 20 pA, current duration = 100 ms). (D) Population data showing the number of APs recorded from NM neurons at E14–16 and counted under different conditions: control (Cont) or during DTx application (DTx), in response to threshold (Thres) or suprathreshold injected current (SThres). Inset is the population data showing a significant reduction in threshold current before and during DTx application. Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 9
Figure 9
Blockade of K+HVA channels altered AP kinetics of developing NM neurons. (A) Normalized representative APs recorded from NM neurons at E20 (left), E15 (middle) and E11 (right) before (control) and during bath application of low-concentration TEA (1 mM). Data obtained during drug application are shown in red. Sustained current injections are 25% above threshold current. (B–D) Population data showing percent changes in AP half width (B), fall rate (C) and threshold current (D) during TEA application, as a function of age. Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 10
Figure 10
Development of NaV current properties in NM. (A) Representative NaV current traces (INa) recorded from NM neurons at E11, E15 and E21 in response to membrane voltages clamped at 25% above channel activation voltage (−35, 47 and −54 mV for E11, E15 and E21 NM neuron in this figure, respectively). These currents were used to measure the sodium current properties for each age group shown in (B–F). (B–F) Population data showing developmental changes in NaV current amplitude (in absolute value, B), rise rate (in absolute value, C), fall rate (D), half width (E) and reliability range (F) as a function of age. Open circles represent an individual neuron and solid bars represent the average for each age group. *p < 0.05, Bonferroni adjusted t-test.
Figure 11
Figure 11
Voltage dependent NaV current activation and inactivation in developing NM neurons. (A) Representative NaV current traces recorded from an E20 NM neuron in response to membrane voltages clamped from −55 to −5 mV (voltage step = 5 mV, voltage duration = 100 ms). (B–D) Population data showing the relationship of peak NaV current (peak INa, B), conductance (GNa, C) and density (ρNa, D) to the varying membrane voltages for each age group. (E) Representative NaV current traces recorded from an E20 NM neuron in response to depolarization to −30 mV following pre-pulse holding voltages clamped from −90 to −30 mV (voltage step = 10 mV). (F) Population data showing voltage dependence of NaV channel inactivation for each age group. hNa was calculated as the NaV current recorded for each trial normalized to the maximum current across all trials and plotted as a function of the pre-pulse holding voltage. Error bars = standard error.

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