Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N-type calcium channels

Abstract

Sensory signals of widely differing dynamic range and intensity are transformed into a common firing rate code by thalamocortical neurons. While a great deal is known about the ionic currents, far less is known about the specific channel subtypes regulating thalamic firing rates. We hypothesized that different K(+) and Ca(2+) channel subtypes control different stimulus-response curve properties. To define the channels, we measured firing rate while pharmacologically or genetically modulating specific channel subtypes. Inhibiting Kv3.2 K(+) channels strongly suppressed maximum firing rate by impairing membrane potential repolarization, while playing no role in the firing response to threshold stimuli. By contrast, inhibiting Kv1 channels with alpha-dendrotoxin or maurotoxin strongly increased firing rates to threshold stimuli by reducing the membrane potential where action potentials fire (V(th)). Inhibiting SK Ca(2+)-activated K(+) channels with apamin robustly increased gain (slope of the stimulus-response curve) and maximum firing rate, with minimum effects on threshold responses. Inhibiting N-type Ca(2+) channels with omega-conotoxin GVIA or omega-conotoxin MVIIC partially mimicked apamin, while inhibiting L-type and P/Q-type Ca(2+) channels had small or no effects. EPSC-like current injections closely mimicked the results from tonic currents. Our results show that Kv3.2, Kv1, SK potassium and N-type calcium channels strongly regulate thalamic relay neuron sensory transmission and that each channel subtype controls a different stimulus-response curve property. Differential regulation of threshold, gain and maximum firing rate may help vary the stimulus-response properties across and within thalamic nuclei, normalize responses to diverse sensory inputs, and underlie sensory perception disorders.

Figure 8. Role of Kv1, SK and Kv3.2 in the firing response to excitatory postsynaptic currents (EPSCs)

EPSC stimuli were designed based on the kinetics of EPSC events observed in these neurons during whole-cell voltage clamp (−70 mV, Fig. 8B bottom right). The current pulse used to model an EPSC stimulus is shown in the thicker tracing overlay with peak current reached in 0.5 ms and decaying to 40% of peak value in 1 ms. A slower component decays to baseline over a 5 ms period. A, EPSC-like stimuli at 30 Hz (1000 pA peak current) caused an initial burst of action potentials and then largely failed to evoke firing in controls (Ctrl). Washing in apamin (300 nm) to inhibit SK Ca²⁺-activated K⁺ channels in this same control neuron increased the number of spikes within the burst and improved coupling over the first few EPSC-like stimuli. Washing in α-dendrotoxin (100 nm) to block Kv1 K⁺ channels increased the number of spikes within the burst, but also increased EPSC-spike coupling. Bath application of apamin and α-dendrotoxin increased EPSC-spike coupling at 100 Hz (900 pA peak) and 200 Hz (700 pA peak). B, washing in the non-selective Kv3.2 channel blocker (TEA, 1 mm) depolarized the interstimulus membrane potential and attenuated action potential height with 200 Hz EPSC-like stimuli (900 pA, peak). At 100 Hz (900 pA, peak), action potentials at 100 Hz were widened, but only weakly attenuated, by 1 mm TEA (far right, thick trace versus Ctrl, thin trace, 1 ms and 20 mV scale).

Figure 7. Differential contribution of K⁺ channels to action potential repolarization and threshold and membrane potential minimum while firing at high rates (80 Hz)

A, action potential repolarization is slowed when K_v3.2 is deleted (Kv3.2) and unaffected when SK or Kv1 channels are blocked compared to controls (WT). B, action potential threshold (V_th) is lowered when Kv1 or SK channels are blocked, but unaltered when K_v3.2 is deleted compared to controls (WT). C, minimum membrane potential (Min V_m) is more hyperpolarized when Kv1 and SK channels are blocked, but more depolarized when K_v3.2 is deleted compared to controls (WT). D, overlay of firing rate curves for control (WT), SK channel blocker, Kv1 channel blocker, and Kv3.2 gene deletion demonstrate different roles for these K⁺ channels in regulating firing.

Figure 2. High threshold K⁺ channel KV3.2 is required to sustain high firing rates in adult thalamocortical neurons

A, genetically deleting (Kv3.2 KO) or non-selectively inhibiting (TEA, 1 mm) Kv3.2 suppressed maximum firing of thalamocortical neurons (n= 47, control; n= 16, KV3.2 KO; n= 8, TEA). Statistical analyses by repeated-measures ANOVA: P < 0.001 (Ctrl versus TEA); P < 0.001 (Ctrl versus KV3.2 KO). B, at 500 pA, firing rate was suppressed when KV3.2 was inhibited by gene deletion (KV3.2 KO) or a non-selective blocker (TEA, 1 mm, bath) when compared to control (Ctrl). At 900 pA, tonic firing of action potentials failed during the current pulse in Kv3.2 KO. Insets show traces at a higher temporal resolution. C, during 80 Hz firing, K_v3.2 KO mice demonstrate normal action potential threshold, while half-width is elongated, action potential repolarization rate (AP Repol) is slowed, and fast after hyperpolarization (fAHP) is reduced.

Figure 1. Age regulates peak action potential firing rate in lateral dorsal nucleus thalamocortical neurons

A, adult neurons (3–7-month-old mice, n= 47) displayed a relatively linear increase of firing rate in response to increasing current injections. By contrast, immature neurons of day 15 old mice (d15, n= 6) displayed impaired firing responses reaching a plateau at ∼50 Hz. Representative voltage tracings (inset, top) demonstrate firing in response to a 300 pA current injection. Single action potentials at threshold current injections in 15 day and 3–7-month-old mice are also compared (inset, bottom). B, threshold current to initiate tonic firing (TTF), maximum firing rate (Max), and maximum firing gain were all significantly reduced in thalamic slices from d15 compared to adult mice. Statistical significance in B by t test (*P < 0.05). Calibration top: 20 mV, 200 ms, bottom: 20 mV, 1 ms.

Figure 3. Kv1 K⁺ channel-blocking toxins lower the threshold current required to initiate action potential firing in adult thalamocortical neurons

A, α-dendrotoxin (α-DTX, 100 nm, n= 7) significantly decreased the current that initiated firing in thalamocortical neurons. Inset demonstrates voltage response to a 200 pA current injection before and after application of 100 nmα-DTX. B, α-DTX lowered the threshold current for firing (ΔI_th) and significantly reduced the membrane potential where action potentials fire (ΔV_th). Inset, trace demonstrating α-DTX effect on action potential threshold aligned at V_m= 0 mV (horizontal line). C, maurotoxin (MTX, 10 nm, n= 5), a Kv1.2 channel specific toxin, produced a similar reduction in the current initiating firing. Inset demonstrates voltage response to a 200 pA current injection before and after application of 10 nm MTX.

Figure 4. SK and BK Ca²⁺-activated K⁺ channels inhibit firing in adult thalamocortical neurons

A, apamin (300 nm), an SK1–3 subunit channel blocker, enhanced thalamocortical firing with 100 μm EGTA internal solution. B, apamin (300 nm) enhanced, while 1-EBIO (200 μm), an SK channel activator, suppressed firing with 1 mm EGTA internal solution. C, apamin decreased action potential firing threshold (ΔV_th) at low firing rates (in 100 μm EGTA only), but strongly increased gain (ΔGain) under both conditions. Threshold current to initiate firing (ΔI_th) was not significantly altered by apamin. D, apamin inhibited the mAHP without significantly altering fAHP or sAHP. Inset, traces demonstrating fAHP, mAHP and sAHP before (thin trace) and after apamin (thick trace). E and F, paxilline (2 μm), a BK channel blocker, enhanced firing of thalamocortical neurons with 100 μm EGTA internal solution, but failed to alter firing with 1 mm EGTA internal solution. G, N-type calcium channel blocker ω-CTX GVIA (200 nm, preincubation) enhanced firing responses with 100 μm EGTA internal solution. Apamin is shown in the same plot to demonstrate the similarity between the two antagonists. H, concurrently blocking both SK and N-type Ca²⁺ channels enhances firing responses more than either blocker alone. Insets in each plot show voltage traces under control and drug conditions in response to a 200 pA current injection. All statistical analysis by repeated-measures ANOVA: P < 0.001 for all curves (A, B, E, G and H) but paxilline in 1 mm EGTA (F, P > 0.05). Statistical analysis: *P < 0.05 by t test for all bar graphs (C and D). n= 5–12 each condition.

Figure 5. N-type calcium channels inhibit firing in adult thalamocortical neurons

A, ω-conotoxin GVIA (200 nm, preincubation > 1 h), an N-type Ca²⁺ channel blocker, enhanced firing. B, ω-conotoxin MVIIC (1 μm, wash in), a P/Q and N-type Ca²⁺ channel blocker, enhanced firing. C, ω-agatoxin IVA (100 nm, preincubation > 1 h), a P/Q-type Ca²⁺ channel blocker, did not alter firing. D, nifedipine (10 μm), an L-type Ca²⁺ channel blocker, had minimal effects on firing. E, genetic deletion of the Cav3.1 T-type Ca²⁺ channel (α1G KO), abolished burst firing (1 s, −200 pA current injection, inset), but did not alter tonic firing. F, cadmium (200 μm), a broad-spectrum Ca²⁺ channel blocker, greatly enhanced firing. Statistical analysis by repeated-measures ANOVA: P < 0.001 for cadmium, ω-CTX, and MVIIC; P > 0.05 for Aga IVA and α1G KO; P= 0.03 for nifedipine; *P < 0.05 by t test. n= 5–12 each condition. Insets in each plot show voltage traces under control and drug conditions in response to a 200 pA current injection.

Figure 6. Comparison of the relative role of various low-threshold and inwardly rectifying K⁺ channels on firing in adult thalamocortical neurons

A, α-DTX markedly increased firing responses to threshold current injections. B, apamin had minimal effects on firing at threshold, but markedly increased firing responses to current injections once firing at 10 Hz and beyond. C, 10 μm barium, which inhibits inward-rectifier K⁺ channels, caused a small increase of firing rate. D, KCNQ (M-type) K⁺ channel blocker XE 991 (10 μm) modestly increased the firing rate. E, K-ATP inward-rectifier K⁺ channel (Kir6) activator, diazoxide (Diaz, 100 μm), inhibited firing rate near threshold, while the Kir6 blocker, glibenclamide (Glib, 30 μm), weakly increased firing rates. Statistical analysis by repeated-measures ANOVA: P < 0.001 for each of Ctrl versus XE 991 (n= 7) and 10 μm Ba²⁺ (n= 6); and P= 0.03 for Ctrl versus Glib (n= 9); and P= 0.006 for Ctrl versus Diaz (n= 7). For control data, s.e.m. bars are within the symbols.