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. 2007 Feb 21;27(8):2112-23.
doi: 10.1523/JNEUROSCI.5266-06.2007.

A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents

MacKenzie A Howard  1 , R Michael Burger, Edwin W Rubel
Affiliations

A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents

MacKenzie A Howard et al. J Neurosci. .

Abstract

Mature nucleus magnocellularis (NM) neurons, the avian homolog of bushy cells of the mammalian anteroventral cochlear nucleus, maintain high [Cl-]i and depolarize in response to GABA. Depolarizing GABAergic postsynaptic potentials (GPSPs) activate both the synaptic conductance and large outward currents, which, when coupled together, inhibit spikes via shunting and spike threshold accommodation. We studied the maturation of the synaptic and voltage-dependent components of inhibition in embryonic NM neurons using whole-cell and gramicidin-perforated patch-clamp techniques to measure Cl- reversal potential, GABAergic synaptic responses, and voltage-dependent outward currents. We found that GABA enhanced excitability in immature NM neurons, undergoing a switch to inhibitory between embryonic day 14 (E14) and E18. Low-voltage-activated Kv1-type (dendrotoxin-I sensitive) K+ currents increased in amplitude between E14 and E18, whereas Cl- reversal potential and synaptic conductances remained relatively stable during this period. GABA was rendered inhibitory because of this increase in low-voltage activated outward currents. GPSPs summed with other inputs to increase spike probability at E14. GPSPs shunted spikes at E18, but blocking Kv1 channels transformed this inhibition to excitation, similar to E14 neurons. Subthreshold depolarizing current steps, designed to activate outward currents similar to depolarizing GPSPs, enhanced excitability at E14 but inhibited spiking in E18 neurons. Blocking Kv1 channels reversed this effect, rendering current steps excitatory. We present the novel finding that the developmental transition of GABAergic processing from increasing neuronal excitability to inhibiting spiking can depend on changes in the expression of voltage-gated channels rather than on a change in Cl- reversal potential.

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Figures

Figure 1.
Figure 1.
GABAAR currents can be induced in NM neurons as early as E10. In all experiments, GABAAR currents were induced with picospritz application of the GABAAR agonist muscimol in the presence of DNQX and AP5. A, Averaged responses to muscimol from a representative E10 neuron held in whole-cell voltage clamp at −60 mV. Dark bars above the traces illustrate the time course of the muscimol puff. These currents were blocked completely with bath application of Bic. B, GABAAR currents recorded using gramicidin-perforated patch methods from an E14 (top) and E18 neuron. Puffs of muscimol were delivered after holding the neuron for 10 s at voltage steps ranging from −84 to −14 mV in 10 mV steps. For clarity, only responses to steps ranging from −64 to −24 mV are illustrated (numbers to the right label the holding potential for each trace). The asterisk indicates the approximate peak of the E18 traces, at which point measurements of reversal potential were made. Dashed lines indicate the baseline. Trace baseline is adjusted to the average current during the 5 ms period just before muscimol application. The calibration at the bottom applies to both the E14 and E18 traces. Evoked currents were slow to decay but return to baseline after several seconds (data not shown). GABAAR current reversal potential was −41.8 ± 1.4 mV (SEM) for E14 (n = 6) and −37.9 ± 2.2 mV for E18 (n = 4).
Figure 2.
Figure 2.
Properties of mGPSCs change during development in NM neurons. All mGPSPs were recorded at a holding potential of −70 mV during bath application of DNQX and AP5. A, Traces recorded from E10, E12, E14, and E18 show that mGPSC activity first recorded at E12. The E12 trace should not be considered representative (see Results). B, Expanded view of overlaid, averaged mGPSCs, normalized in amplitude. These traces illustrate the difference in rise and decay times for mGPSCs at these ages.
Figure 3.
Figure 3.
GPSC decay kinetics become faster across development. All GPSCs were recorded at a holding potential of −60 mV during bath application of DNQX and AP5. A, Averaged GPSC traces from representative E10, E12, E14, and E18 neurons. GPSCs were first recorded at E14. Preliminary (Pre) GPSCs were completely blocked with bath application of Bic (+Bic), but recovered to normal levels after washout (Rec). Stimulus artifacts have been removed for clarity. B, E18 GPSCs exhibit faster decay kinetics than E14 when current amplitude-normalized GPSCs are overlaid. The overlaid traces illustrate the faster decay kinetics in more mature neurons.
Figure 4.
Figure 4.
Input resistance is reduced and single AP firing is more prevalent across development. These experiments were performed using current clamp in the presence of bath-applied DNQX, AP5, and Bic. A, Depolarizing currents steps ranging from 0 to 750 pA in 50 pA steps. The asterisk marks the time at which steady-state membrane potential was measured. B, Example responses from representative E10, E12, E14, and E18 neurons. The E10 and E12 neurons each exhibit multiple spikes when stimulated with the largest current steps; E14 and E18 neurons do not. In older neurons the spikes occur and decay much faster. C, Voltage–current relations consist of steady-state membrane potential (averaged across the last 5 ms of the currents step) plotted as a function of injected current. The flattened V–I function of E18 neurons (black squares) is indicative of lower input resistance and larger K+ conductances compared with other ages. The change in membrane potential is significantly less in E18 neurons at all current amplitudes (p < 0.05). Error bars for this and all other figures represent SEM.
Figure 5.
Figure 5.
LVA K+ currents increase with age in NM neurons. These experiments were conducted with DNQX, AP5, Bic, TTX, QX-314, and low Ca2+ present in the bath. A, Protocol in which neurons were held at −70 mV and stepped from −75 to −45 mV in 5 mV steps. B, Representative K+ currents recorded from E14 and E18 neurons. The amplitude of these currents is much greater in mature E18 neurons. The asterisk marks the point at which the peak current was measured. C, Current–voltage curves consist of peak current plotted against test potential. E18 (n = 7; filled squares) neurons showed significantly greater current amplitudes than did E14 neurons (n = 7; open symbols) in response to all depolarizing current steps (p < 0.05). DTX-I (circles) greatly reduces these currents at both ages. These functions were leak subtracted using response to the hyperpolarizing step from −70 to −75 mV as a baseline. A large portion of the LVA K+ current was blocked in both E14 (63% ± 18; n = 6) and E18 neurons (69% ± 6; n = 7), indicating a major role for Kv1-type channels in the LVA K+ currents activated by depolarizing GPSPs.
Figure 6.
Figure 6.
GABAergic inputs enhance excitability at E14 but are inhibitory at E18 in NM neurons. A, Neurons held in whole-cell current clamp were stimulated with depolarizing current pulses of variable amplitude. GABAergic synaptic stimulation was performed with a bipolar electrode in the presence of bath-applied DNQX and AP5. B, Under control conditions, GPSPs summated with current pulses in E14 neurons to induce spiking, but shunted current pulses at E18 to prevent spiking (left column; stimulus artifacts reduced for clarity). Addition of DTX-I to the bath (right column) induced summation between GPSP and current pulses in neurons of both ages. C, Rheobase during a GPSP was subtracted from the control condition for E14 (n = 6) and E18 (n = 7) neurons. All E14 neurons (n = 6) exhibit negative changes in rheobase, indicating enhancement of excitability. This effect was significantly enhanced by addition of DTX-I (*p < 0.05). At E18, positive values for change in rheobase indicate shunting and inhibition during GPSPs. DTX-I application reversed this effect, making GPSPs excitatory (***p < 0.001). Upward and downward arrows indicating inhibition and enhancement of excitation are for clarity.
Figure 7.
Figure 7.
GPSP trains enhance excitability at E14 and inhibitory at E18 in NM neurons. A, Neurons held in whole-cell current clamp were stimulated with trains of depolarizing current pulses at 50 Hz. Simultaneously, trains of GPSPs were stimulated extracellularly at frequencies ranging from 10 to 100 Hz (20 Hz shown in B) in the presence of DQX and AP5. B, Summation between the GPSP-induced steady-state depolarization and current pulses causes an E14 neuron (top trace) to fire in response to current pulses that stimulate only subthreshold responses (marked with asterisks) under control conditions. The opposite is true in an E18 neuron (middle trace), in which firing is blocked during most of the GPSP train except when the membrane approaches rest (marked by dashed line). Excitation is enhanced in E18 neurons exposed to DTX (bottom trace), similar to E14. C, Decreases in rheobase during GPSP trains indicate enhanced excitability in E14 (n = 9) neurons and E18 +DTX (n = 3), whereas increases in rheobase at E18 (n = 7) indicate spike inhibition. This pattern is observed at all GPSP frequencies. DTX significantly shifted inhibition to excitation in E18 neurons (***p < 0.001), but enhanced excitability significantly less than GPSPs at E14 (*p < 0.05).
Figure 8.
Figure 8.
Subthreshold depolarizing current injections enhance excitability at E14 and inhibitory at E18 in NM neurons. A, Neurons held in whole-cell current clamp were stimulated with 50 Hz trains of depolarizing current pulses. After the second control pulse a depolarizing current step, ranging from 0.1 to 0.5 nA was activated. B, A representative E14 neuron (top trace) that displayed subthreshold responses to control current pulses (marked with asterisks) fired spikes in response to the same current pulses after the onset of the current step. The middle trace illustrates a representative E18 neuron in which spikes were fired during control conditions but were shunted to subthreshold (asterisks) during the current step. An E18 neuron exposed to DTX (bottom trace) fired repetitively to current pulses during the current step. E14 and E18 examples that exhibited equivalent depolarization in response to the current step were selected for this comparison. I indicates the current step size for each trace. C, Depolarizing current steps of all amplitudes decreased rheobase (were excitatory) in E14 neurons (n = 9). The enhancement of excitability increased significantly as current step size increased. Spikes were inhibited (rheobase increased) during all but the largest depolarizing current steps in E18 neurons (n = 7). Exposure to DTX converted inhibition to excitation in E18 neurons (n = 3), significantly decreasing rheobase (***p < 0.001), while enhancing excitability less than in E14 neurons (***p < 0.001).
Figure 9.
Figure 9.
GPSPs induce the greatest inhibition in E18 neurons. Inhibition/enhancement of excitability by GPSP trains and current step that induce equivalent depolarizations (∼10 mV) are compared. At E14, enhancement of excitation is significantly greater during current steps than GPSP trains because of the lack of the shunting synaptic conductance. At E18, inhibition is strongest during GPSP trains because of shunting by both synaptic and voltage-activated conductances. *p < 0.05; ***p < 0.001.
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