Multiple Conductances Cooperatively Regulate Spontaneous Bursting in Mouse Olfactory Bulb External Tufted Cells

Intracellular calcium buffering stabilizes spontaneous bursting activity

Spontaneous burst firing of rat olfactory bulb ET cells runs down rapidly in whole-cell recording mode (Hayar et al., 2004a). A similar phenomenon was observed in mouse ET cells (Fig. 1F,H). Spontaneous bursting gradually decreased and then disappeared within 15 min after whole-cell break-in. The averaged decline in burst frequency from five cells over 20 min of whole-cell recording was fitted by a single exponential decay function with a time constant (τ) of 3.1 min. After 20 min of recording, the MMP was hyperpolarized (Fig. 1F) by ∼10 mV (from −55.3 ± 0.5 to −65.4 ± 0.7 mV; n = 5; p < 0.01), the input resistance reduced by 35 ± 7% (Fig. 1I) from 241.4 ± 22.7 to 159.4 ± 29.2 MΩ (n = 5; p < 0.01) and bursting could not be restored by steady current injection. Because this rapid rundown precluded investigation of bursting mechanisms, it was necessary to obviate the problem. Intracellular Ca²⁺ buffer plays an important role in maintaining resting membrane potential and input resistance (Krnjevic et al., 1975; Milesi et al., 1999). Therefore, we increased the concentration of EGTA in the internal solution from 0.2 to 5.5 mm (see Materials and Methods) and added 0.5 mm Ca²⁺ to yield an estimated free Ca²⁺ concentration of 20 nm. Bursting activity recorded with this internal solution did not significantly change after 20 min of whole-cell recording (Fig. 1G,H). The average burst frequency from five cells decreased only from 2.6 ± 0.5 to 2.4 ± 0.5 Hz after 20 min whole-cell recording with a time constant (τ) of 729.8 min when fitted by a single exponential decay function. In addition, there was minimal change of MMP from −56.2 ± 1.7 to 57.5 ± 2.6 mV (n = 5; p > 0.05) and input resistance from 258.2 ± 23.3 to 251.6 ± 22.3 MΩ (n = 5; p > 0.05) (Fig. 1G,I). In many cases, stable bursting was maintained for >60 min. These results showed that optimal intracellular Ca²⁺ buffer prevented rundown of spontaneous bursting and suggested a Ca²⁺ dependence of bursting activity in ET cells. This internal solution was used for the remaining experiments.

Persistent Na⁺ channels are active at MMP and AMP and essential for intrinsic bursting

The TTX-sensitive persistent sodium current (I_NaP) exists in a variety of CNS neurons (Crill, 1996). I_NaP is present in rat ET cells and is necessary for spontaneous burst initiation (Hayar et al., 2004a). To investigate I_NaP in mouse ET cells, we used a ramp protocol to depolarize the membrane from −75 to −40 mV at a velocity (30 mV/s) sufficiently slow to prevent activation of fast or transient sodium channels (I_NaT) (Fig. 2A). This protocol reliably disclosed a TTX-sensitive inward current (Fig. 2A). The average TTX-sensitive inward current from four cells was 13.6 ± 1.5 pA at −55.3 mV (MMP) and 42.1 ± 2.5 pA at −51.3 mV (AMP), had an activation voltage of about −60 mV, and reached its maximal magnitude (96.4 ± 7.2 pA; n = 4) at −45 mV (Fig. 2B). This shows that mouse ET cells have an active I_NaP even at their MMP. This conductance increases monotonically with membrane depolarization.

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Figure 2.

Persistent Na⁺ current is active at minimal and average membrane potentials and is required for spontaneous bursting. A, TTX-sensitive persistent Na⁺ current, I_NaP, revealed by voltage ramp protocol from −75 to −40 mV at 30 mV/s. B, Averaged I–V curve from four ET cells showing that I_NaP is active at MMP. C, Bath application of TTX (1 μm) terminates the spontaneous bursting, which cannot be restored by either positive or negative steady current injection. Burst frequency declines and voltage humps (arrowheads) appear between bursts before termination (expanded traces). D, Bath application of a lower concentration of TTX (10 nm; expanded traces a and g) reversibly terminates the spontaneous bursting, which cannot be restored by positive steady current injection. Inset, I–V curves revealed by the same voltage ramp protocol as in A at different time points 1, 2, and 3. Note that 10 nm TTX significantly blocks I_NaP (inset, trace 2 vs trace 1) but does not block the action potentials evoked by a hyperpolarization voltage step (expanded trace e). Spontaneous voltage humps only appear during the early phase of TTX wash-in (expanded trace b) and late phase of washout (expanded trace f) when significant I_NaP has been recovered (trace 3 in the inset). Only a single hump can be evoked by each depolarizing current step (expanded trace d) in the presence of TTX.

To investigate the role of I_NaP in bursting, we treated ET cells with 1 μm TTX after a stable bursting recording was established. As shown in Figure 2C, in addition to hyperpolarizing the membrane from −57.4 ± 1.1 to −60.3 ± 0.9 mV (n = 5; p < 0.005), spontaneous bursting slowed and small depolarizing “humps” (arrowheads in the expanded trace) appeared between bursts. Both bursts and humps were rapidly (2.1 ± 0.2 min; n = 5) terminated by TTX. Low-voltage-activated (LVA) Ca²⁺ spikes could be evoked in the presence of TTX (see Figs. 6⇓–8), but neither action potentials nor spontaneous depolarizing events were restored by depolarizing the membrane potential with steady current injection (Fig. 2C), indicating that I_NaP is required to trigger the depolarizing envelope in mouse ET cells. The presence of the small spontaneous voltage humps during early phase of 1 μm TTX application suggested that they might persist at lower concentration TTX because it takes time for the toxin to reach maximal concentration at the recorded cells. At 10 nm TTX (Fig. 2D), the majority of I_NaP (82% measured at −45 mV) (Fig. 2D, inset) was blocked but rebound action potentials persisted (Fig. 2De). Spontaneous bursting was terminated (Fig. 2D) and could not be restored by depolarizing current injection (Fig. 2Dd). Spontaneous voltage humps were again present only during the early phase of 10 nm TTX wash-in (Fig. 2Db) and during the late phase of washout (Fig. 2Df) when I_NaP had partially recovered (Fig. 2D, inset, I–V curve 3). This suggests that I_NaP is necessary to generate the humps. Although the humps were abolished (Fig. 2Dc) when majority of I_NaP was blocked by TTX, single humps could be evoked by depolarizing current steps (Fig. 2Dd). This indicates that the role of I_NaP is to depolarize the membrane to the activation voltage of Ca²⁺ currents (see below and Fig. 6), and suggests that the spontaneous humps are depolarizing envelopes that inactivate before reaching the threshold voltage required to launch action potentials.

I_h current contributes to setting membrane potential and is required for spontaneous bursting

The hyperpolarization-activated cation current (I_h) has been identified in a variety of mammalian neurons (Pape, 1996; Robinson and Siegelbaum, 2003). One function of I_h is to generate pacemaker activity. Of the four hyperpolarization-activated, cation-nonselective (HCN1–4) genes that encode I_h channels (Robinson and Siegelbaum, 2003) HCN2 and HCN4 mRNA are highly expressed in the olfactory bulb of rat and mouse (Monteggia et al., 2000; Santoro et al., 2000), and HCN1, HCN2, and HCN3 immunoreactivity is present in glomeruli (Holderith et al., 2003; Notomi and Shigemoto, 2004). As shown in Figure 3A, hyperpolarizing current steps in current clamp generated depolarizing sags, indicating activation of I_h channels in ET cells. In cells voltage clamped at −55 mV, hyperpolarizing steps revealed slowly developing inward currents that were blocked by bath-applied 3 mm Cs⁺ (Fig. 3B,C), a nonselective blocker of I_h channels. The amplitude of these Cs⁺-sensitive inward currents steadily increased with membrane hyperpolarization from 88 ± 37.3 pA (n = 4) at −70 mV to 434.5 ± 54.2 pA (n = 4) at −110 mV (Fig. 3C). The selective I_h channel blocker ZD7288 (100 μm) also blocked this current (Fig. 3D,E), confirming that it is mediated by I_h channels (Luthi et al., 1998).

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Figure 3.

ET cells have a prominent I_h current that is active at MMP and AMP. A, Current-clamp recording showing robust depolarizing sag (arrow) indicating the presence of I_h. B, Voltage-clamp recording in the same cell in the presence of TTX (1 μm) showing hyperpolarization-activated inward current, I_h (holding potential, −55 mV). C, Average I–V curves of I_h measured as in B with and without CsCl (3 mm in the bath; n = 4 cells). D, Voltage-clamp recording with membrane potential hyperpolarized by voltage steps (5 mV/step) from −40 to −120 mV in the presence of TTX (1 μm). E, Same cell in the presence of I_h channel blocker, ZD7288 (100 μm). F, D minus E shows the ZD7288-sensitive I_h current. G, I–V curve of steady-state ZD7288-sensitive I_h current fitted with Boltzmann equation (n = 5 cells). H, Magnification of G in the voltage range from −60 to −40 mV, showing that there is ∼25 pA of I_h active in the voltage range between MMP and AMP. I, ZD7288 (100 μm) increased input resistance in all cells tested (n = 5) consistent with the reduction of a steady-state conductance (measured at −55 mV). Bar graphs show the averaged value from five cells. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

To determine whether I_h contributes to setting membrane potential, ET cells were held at −40 mV and 500 ms hyperpolarizing voltage steps were used to elicit ZD7288-sensitive inward currents (Fig. 3 D–G). When plotted against their test voltages (Fig. 3F,G), considerable ZD-sensitive currents, 23.5 pA at −51.3 mV to 28.2 pA at −55.3 mV (n = 5), were revealed (Fig. 3H), suggesting that I_h is active between MMP and AMP (i.e., at “resting” membrane potentials). Consistent with this, blocking I_h channels with ZD7288 significantly hyperpolarized the MMP from 56.2 ± 1.4 to 62.5 ± 0.8 mV (n = 5; p < 0.01) (Fig. 4A,E,I) and increased input resistance (measured at −55 mV) from 245.8 ± 11.4 to 271.2 ± 12.3 MΩ (p < 0.01; n = 5) (Fig. 3I). Thus, I_h contributes ∼6 mV to the MMP of ET cells.

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Figure 4.

I_h contributes to spontaneous bursting. A, Spontaneous bursting slows (3 and 4) and is eventually terminated by the I_h channel blocker, ZD7288 (100 μm), (top panel). Inset 1 and 2, ZD7288 abolishes the depolarizing sag (arrowheads) and the MMP was hyperpolarized indicating that I_h is active at MMP (see also Fig. 3G,H). 5, Bursting rescued in the presence of ZD7288 when the MMP restored to original level by depolarizing current injection. B–E, Graphs showing the progressive effects of ZD7288 on burst frequency (B), envelope duration (C), spikes per burst (D), and MMP (E) of cell in A. F–I, Average changes for five cells. Error bars indicate SEM. **p < 0.01; ***p < 0.001.

What is the role of I_h current in spontaneous bursting? In all five ET cells tested, bursting was terminated by 100 μm ZD7288 (Fig. 4 A–D,F–H). Before the termination of bursting, the frequency decreased [Fig. 4A (insets 3 and 4), B,F] from 2.5 ± 0.4 Hz in control to 0.9 ± 0.2 Hz at 2 min after ZD7288 treatment (n = 5; p < 0.01), and the duration of the burst envelope increased (Fig. 4C,G) from 120 ± 3.7 ms in control to 204 ± 23.6 ms at 2 min after ZD7288 treatment (n = 5; p < 0.01). After its termination, bursting activity was restored (Fig. 4 A–D,F–H) by current injection sufficient to depolarize the membrane potential its original level (Fig. 4A,E,I). However, the current-rescued bursting exhibited a lower frequency (Fig. 4B,F) at 0.4 ± 0.1 Hz (n = 5) and a significant increase in both envelope duration (499 ± 73.2 ms; n = 5; p < 0.001) and spikes per burst (6.8 ± 1.1; n = 5; p < 0.001), compared with control, suggesting that I_h also contributes to bursting frequency and burst duration. Together, these results indicate that, in addition to promoting burst generation by repolarizing the membrane potential from its hyperpolarizing level after burst termination, I_h also helps depolarize the membrane potential to a range that facilitates the activation of other conductances, such as I_NaP, that are required for burst generation.

ET cells have a prominent low-voltage-activated Ca²⁺ current that is mediated by T- and/or L-type Ca²⁺ channels

Because the rundown experiments indicated a Ca²⁺ dependence of bursting in ET cells, we next investigated the role of voltage-activated Ca²⁺ channels in spontaneous bursting. To isolate Ca²⁺ currents in ET cells, we used Cs⁺-methanesulfonate-based intracellular solution containing TEA (20 mm) to block K⁺ currents, and added TTX (1 μm) and ZD7288 (100 μm) to the aCSF to eliminate Na⁺ and I_h conductances, respectively.

With a holding potential at −70 mV, depolarizing voltage steps (5 mV/step) (Fig. 5B) elicited inward currents (Fig. 5A) that were abolished by removal of extracellular Ca²⁺ (Fig. 5B) with addition of equivalent Mg²⁺ to maintain osmolarity/charge. This Ca²⁺-dependent current had an activation voltage at approximately −50 mV (n = 4) and reached its mean maximum amplitude of 637.0 ± 51.4 pA (n = 4) at −30 mV before it decreased with membrane depolarization to 562.8 ± 58.1 pA at −10 mV (Fig. 5D, inset). The time-to-peak of this current was voltage dependent (Fig. 5C, inset), decreasing from 26.5 ± 3.1 ms at −45 mV to 10.9 ± 0.8 ms at −35 mV (n = 4). To better estimate the activation voltage of this current, a conductance–voltage (G–V) curve (Fig. 5D) was constructed by dividing the current by its driving voltage and fitted with the Boltzmann equation: Single-channel conductance depends on channel types and even varies for the same type of Ca²⁺ channel from different tissues. For example, with Ca²⁺ as the charge carrier reported single-channel conductance of T-type Ca²⁺ channels and L-type Ca²⁺ channels range ∼4–9 pS (Huguenard, 1996; Perez-Reyes, 2003) and ∼4–7 pS (Catterall et al., 2005), respectively. For the present purpose, a single-channel conductance of 6 pS was used. The resulting Boltzmann equation gave a Ca²⁺ current an estimated activation threshold voltage of −61 mV. The activation voltage of this LVA Ca²⁺ current is consistent with that of T-type calcium channels, although its inactivation kinetics (Fig. 5C), with a time constant of 61.9 ± 3.2 ms (n = 4) for test voltage at −30 mV, is slower than classical T current.

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Figure 5.

ET cells have a prominent LVA Ca²⁺ current mediated by L- and/or T-type Ca²⁺ channels. A, Inward currents generated by depolarizing voltage steps with membrane potential held at −70 mV in aCSF containing TTX (1 μm) and ZD7288 (100 μm). B, Same cell with aCSF containing 0 mm Ca²⁺/2.6 mm Mg²⁺ and 0.3 mm EGTA. C, A minus B shows Ca²⁺ current. D, G–V curve of the peak Ca²⁺ current from five cells fitted with Boltzmann equation. Inset, The corresponding I–V curve. E, Steady-state inactivation of Ca²⁺ current. A −30 mV test pulse was preceded by incremental (5 mV/step) hyperpolarizing pulses (10 s) from −100 mV. F, Activation (V_1/2 = −37.1 mV) and inactivation (V_1/2 = −59.2 mV) I–V curves of five cells expressed as a function of membrane voltage and fitted with Boltzmann equations shows very little Ca²⁺ current at MMP. G, I–V curves of peak Ca²⁺ currents with holding potential at −55 mV showing that NNC 55-0396 (50 μm), a selective T-type Ca²⁺ channel blocker, significantly reduces I_Ca2+ (n = 4 cells). H, I–V curves of the peak Ca²⁺ current showing that nimodipine (20 μm), an L-type Ca²⁺ channel blocker, significantly reduces I_Ca2+. After nimodipine washout with addition of Bay K8644 (5 μm), an L-type Ca²⁺ channel activator, I_Ca2+ is restored (n = 5 cells). I, I–V curves of the peak Ca²⁺ current showing that Bay K8644 (5 μm) increases I_Ca2+, and its activation voltage shifts from −50 to −55 mV. After Bay K8644 washout, addition of nimodipine (20 μm) reduces I_Ca2+ (n = 5 cells). Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001 compared with control.

LVA current is generally attributed to the activation of T-type Ca²⁺ channels (Huguenard, 1996). Immunohistochemical and in situ hybridization studies indicate that neurons in the olfactory bulb glomerular layer express several T-channel subtypes (Talley et al., 1999; Yunker et al., 2003). Thus, I_LVA in ET cells may be mediated, at least in part, by T-channels. To investigate this possibility, we first examined the effect of Ni²⁺, which blocks T-type calcium channels rapidly and reversibly. I_LVA evoked by a 500 ms voltage step from −70 to −30 mV was reversibly blocked by Ni²⁺ in a dose-dependent manner (Fig. 6C) with 49.7 ± 3.2% (n = 5) blocked at concentrations of 100 μm.

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Figure 6.

Effect of Ni²⁺ on spontaneous bursting. A, Bath application of 100 μm NiCl₂ reversibly terminates the spontaneous bursting, which can be restored by 10 pA depolarizing steady current injection to bring the membrane potential to its original level (expanded trace c). Spontaneous voltage humps (expanded trace b) persist. B, Bath application of 1 mm NiCl₂ reversibly terminates spontaneous bursting, which cannot be restored by depolarizing steady current injection. Note that there are no spontaneous or evoked voltage humps in the presence of 1 mm NiCl₂ (expanded traces b and c). C, Dose–response curve from five cells showing the inhibitory effect of NiCl₂ on Ca²⁺ currents evoked by voltage step to −30 mV with holding potential at −70 mV. D, I–V curve from four cells showing that I_h is blocked by 2 mm CsCl but not affected by 1 mm NiCl₂ at all tested voltages when membrane was held at −55 mV. Error bars indicate SEM. E, Action potentials are evoked by a depolarizing current step in the presence of 1 mm NiCl₂.

Next, we investigated the role of this Ni²⁺-sensitive LVA Ca²⁺ current in spontaneous bursting. In 100 μm NiCl₂, which blocked ∼50% of I_Ca2+, spontaneous bursts were terminated but small depolarizing humps persisted (Fig. 6Ab). Bursting could be restored by depolarizing current injection (Fig. 6Ac). In contrast, 1 mm NiCl₂, which blocked >90% of I_Ca2+ with no effect on I_h and action potential generation (Fig. 6D,E), reversibly eliminated both spontaneous bursts and depolarizing humps (Fig. 6 Ba–d). Neither bursting nor the humps were restored by depolarizing current. These results suggest that Ca²⁺ current underlies both the depolarizing envelopes, which generate bursts of action potentials, and the smaller depolarizing humps, which may represent depolarizing envelopes that fail to reach the threshold for action potentials.

Considering the nonselective effect of Ni²⁺ (Tsien et al., 1991; Zamponi et al., 1996), we next investigated the effect of (1S,2S)-2-(2-(N-[(3-benzimidazol- 2-yl) propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride [NNC 55-0396 (NNC)], a selective T-type calcium channel blocker that does not affect L-type Ca²⁺ channels at concentration up to 100 μm (Huang et al., 2004; Li et al., 2005). Similar to its hydrolysable analog, mibefradil (Bezprozvanny and Tsien, 1995; McDonough and Bean, 1998), the potency of NNC 55-0396 on T-type Ca²⁺ currents (I_T) has a strong voltage dependence (Huang et al., 2004; Li et al., 2005) and increases with membrane depolarization. This was confirmed in the present study. The effect of NNC on Ca²⁺ current elicited by incremental depolarizing voltage steps (5 mV/step) was therefore examined with holding potential at −55 mV, which is the MMP for most ET cells. NNC (50 μm) significantly reduced the LVA calcium current in all ET cells (n = 4) at all tested voltages (Fig. 5G). At −30 mV NNC (50 μm; 10 min application) inhibited the LVA Ca²⁺current by 77.7 ± 2.5% (n = 4), indicating that most of the LVA current in ET cells was mediated by T-type Ca²⁺ channels (Fig. 5G).

Window T-type Ca²⁺current-mediated bistability underlies the expression of the slow oscillatory or rhythmic activity as well as high-frequency bursting firing in some neurons (Crunelli et al., 2005). However, the mean half-activation and half-steady-state inactivation voltages of the LVA Ca²⁺ were −37.1 and −59.2 mV (n = 5), respectively. This gave an estimated window current in ET cells at MMP (approximately −55 mV) of 3.8 ± 2.9 pA (Fig. 5F), indicating little estimated contribution (∼0.9 mV) to the MMP. Thus, the LVA Ca²⁺ window current plays no obvious role in ET cell bursting.

The glomerular layer also shows immunoreactivity to L-type Ca²⁺ channels (Grunnet and Kaufmann, 2004), some of which also have low activation voltages (Lipscombe et al., 2004). Murphy et al. (2005) reported that L-type Ca²⁺ channels mediate LVA I_Ca in PG cells. Thus, we investigated the effect of nimodipine, an L-type Ca²⁺ channel blocker on ET cells. At 20 μm, nimodipine reduced I_Ca2+ at all voltages tested (Fig. 5H). Nimodipine reduced peak Ca²⁺ current from 45.1 ± 2.5 to 19.3 ± 6.9 pA (n = 5; p < 0.05) at −50 mV and from 409.6 ± 7.3 to 113.1 ± 42.3 pA (n = 5; p < 0.01) at −30 mV, respectively. This reduction was reversed on nimodipine washout and addition of 1,4-dihydro-2,6-dimethyl-5-nitro-4[trifluoromethyl-phenyl]-3-pyridinecarboxylic acid methylester [Bay K8644 (BayK)], an L-type Ca²⁺ channel activator (Fig. 5H). Ten minutes after BayK (5 μm) application, I_Ca2+ increased from 19.3 ± 6.9 to 72.6 ± 13.3 pA (n = 5; p < 0.01) at −50 mV and from 113.1 ± 42.3 to 472.2 ± 122.4 pA (n = 5; p < 0.05) at −30 mV. In separate experiments, slices were treated first with BayK followed by washout of BayK plus addition of nimodipine (Fig. 5I). BayK (5 μm; 5 min) increased peak Ca²⁺ current from 35.6 ± 12.4 to 62.2 ± 19.9 pA (n = 5; p < 0.05) at −50 mV and from 425.1 ± 70.2 to 821.4 ± 96.2 pA (n = 5; p < 0.01) at −30 mV, respectively. When BayK was replaced with nimodipine (20 μm), I_Ca2+ was reduced from 62.2 ± 19.9 to 9.3 ± 2.4 pA (n = 5; p < 0.05) at −50 mV and from 821.4 ± 96.2 to 125.5 ± 61.6 pA at −30 mV (n = 5; p < 0.01).

R-type calcium currents contribute to bursting in hippocampal neurons (Metz et al., 2005) and are also sensitive to low concentrations of Ni²⁺. However, LVA Ca²⁺ currents in ET cells were insensitive to SNX-482 (400 nm), a selective R-type calcium channel blocker (Bourinet et al., 2001) (data not shown) (mean peak I_Ca in five cells changing from 585.5 ± 49.0 pA in control to 559.7 ± 42.0 pA after SNX-482 measured at −30 mV with holding potential at −70 mV), indicating no involvement of SNX-482-sensitive R-type channels.

In summary, ET cells have an LVA Ca²⁺ current that is mediated by T- and/or L-type Ca²⁺ channels. Its sensitivity to NNC 55-0396 and its relatively low activation voltage suggest that the isolated LVA Ca²⁺ current is mediated by T-type Ca²⁺ channels. However, unlike classical T-type Ca²⁺ currents, the ET cell LVA Ca²⁺ current exhibited slow inactivation kinetics (τ = ∼62 ms for test voltage at −30 mV). This could be attributable to Ca_V3.3 (α1I) T-type channels, which exhibit slow inactivation kinetics (Park et al., 2004) and relatively low sensitivity of Ni²⁺ (Zamponi et al., 1996). Ca_V3.3 mRNA and protein are expressed at high levels in olfactory bulb glomeruli (Talley et al., 1999; Yunker et al., 2003).

The contribution of L-type Ca²⁺ channels to the LVA current in ET cells is supported by its slow inactivation and its sensitivity to the dihydropyridine agonist, Bay K8644 and the antagonist, nimodipine. L-type channels have traditionally been regarded as high-voltage-activated (HVA) Ca²⁺ channels, although, dihydropyridine-sensitive LVA Ca²⁺ currents have been reported in a number of neurons, including rat olfactory bulb PG cells (Avery and Johnston, 1996; Magee et al., 1996; Murphy et al., 2005). Of the four L-type channels (Cav1.1/α_1S, Cav1.2/α_1C, Cav1.3/α_1D, and Cav1.4/α_1F), Cav1.3/α_1D expresses ubiquitously in the mammalian brain including the bulb (Hell et al., 1993; Ludwig et al., 1997) and activates at relatively low membrane potentials (approximately −55 mV) (Koschak et al., 2001; Xu and Lipscombe, 2001; Lipscombe et al., 2004). In addition, channels containing Cav1.3/α_1D have a relatively lower sensitivity to dihydropyridine antagonists, including nimodipine. The ET cell LVA Ca²⁺ current was partially blocked by 20 μm but not 10 μm nimodipine. This may be because most of the current is mediated by T-type channels and/or because it is partly mediated by Cav1.3 L-type channels. Involvement of L-type channels in the ET cell LVA current would also account for its slow kinetics because all L-type channels inactivate slowly (Catterall et al., 2005).

Activation of T-type Ca²⁺ channels is required for low-threshold Ca²⁺ spikes and spontaneous bursting

In agreement with the voltage-clamp experiments, low-threshold Ca²⁺ spikes (LTSs) were evoked by current pulses in current clamp with holding potential at −62 to −65 mV in the presence of TTX (Figs. 7A, 8A, 9A). The activation threshold for these all-or-none LTSs was −46.9 ± 0.7 mV (n = 15). NNC 55-0396 (50 μm) (Fig. 7B) abolished LTSs in all five cells, indicating the requirement of T-type Ca²⁺ current.

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Figure 7.

Blocking T-type Ca²⁺ channel eliminates LTSs and terminates spontaneous bursting. A, All-or-none Ca²⁺ spikes were evoked by intracellular current injection in the presence of TTX (1 μm). B, Blocking T-type Ca²⁺ channels with NNC 55-0396 (50 μm) abolishes LTSs. C, Current-clamp recording (no TTX) showing that NNC 55-0396 (50 μm) slows and then terminates spontaneous bursting. Bursting is not restored by either positive or negative current injection, but action potentials are elicited by positive current injection in the presence of NNC. D–G, Graphs showing the effect of NNC 55-0396 on burst frequency (D), envelope duration (E), spikes per burst (F), and MMP (G) of the cell in C. H–K, Averaged data (n = 5 cells) showing the effect of NNC 55-0396 on the same burst parameters. Error bars indicate SEM. **p < 0.01.

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Figure 8.

Blocking L-type channels eliminates evoked LTSs and reduces number of spikes per spontaneous burst. A, All-or-none calcium spikes evoked by intracellular current injection in the presence of TTX (1 μm). B, Blocking L-type Ca²⁺ channels with nimodipine (20 μm) abolishes LTSs. C, Washout of nimodipine with addition of Bay K8644 (5 μm) partially rescues LTSs. D, Current-clamp recording of spontaneous bursting (no TTX) showing that nimodipine (20 μm) reduces both envelope duration and spikes per burst. Effect on spikes per burst is not attributable to membrane depolarization because it was not reversed by injection of steady hyperpolarization current to restore the MMP to pre-nimodipine levels. Addition of BayK (5 μm) during nimodipine washout rescues bursting (traces below expanded timescale). E–H, Graphs showing effect of nimodipine and washout of nimodipine plus BayK on burst frequency (E), envelope duration (F), spikes per burst (G), and MMP (H) of cell in D. I–L, Mean burst frequency (I), envelope duration (J), spikes per burst (K), and MMP (L) (n = 5 cells) immediately before (con), 9 min after nimodipine (nim), nimodipine with hyperpolarizing current injection (nim+hyper), and 12 min after washout of nimodipine plus BayK (Bay K). Averages were measured for 20 s for each condition for each cell. Error bars indicate SEM. *p < 0.05; **p < 0.01.

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Figure 9.

Activation of L-type Ca²⁺ channels prolongs evoked LTSs and spontaneous bursts. A, All-or-none calcium spikes evoked by intracellular current injection in the presence of TTX. B, Bay K8644 5 (μm) increases LTS duration and reduces LTS activation threshold from 44.5 ± 1.7 to −48.5 ± 1.5 mV (n = 5; p < 0.01). C, BayK washout with addition of nimodipine (20 μm) abolishes LTSs. D, In spontaneous bursting (no TTX), BayK (5 μm) increases both burst duration and spikes per burst without changing burst frequency. Effects are not attributable to BayK-induced membrane hyperpolarization because they persist during depolarizing current injection that restores MMP to pre-BayK baseline. Washout of BayK plus nimodipine (20 μm) restores bursting to control values (expanded traces below). E–H, Graphs showing the progressive effects of BayK, BayK plus 30 pA depolarizing current, and BayK washout plus nimodipine on the burst frequency (E), envelope duration (F), spikes per burst (G), and MMP (H) of the cell in D. I–L, Mean burst frequency (I), envelope duration (J), spikes per burst (K), and MMP (L) from five cells in control (con) and 4 min after BayK, BayK plus depolarization (BayK+dep), and 10 min after washout of BayK plus nimodipine (nim), respectively. Error bars indicate SEM. *p < 0.05; **p < 0.01.

LTSs have been implicated in pacemaker function because an LTS typically triggers a burst of action potentials (Huguenard, 1996; Perez-Reyes, 2003). To investigate the role of the T-type calcium channel-mediated LTS in spontaneous bursting, we tested the effect of NNC 55-0396 (50 μm; 10 min) on ET cells recorded in current clamp. NNC irreversibly abolished spontaneous bursting with no effect on MMP (Fig. 7C,G,K). Bursting could not be restored either by depolarizing or hyperpolarizing current injection (Fig. 7C). NNC did not alter the ability of the cells to fire action potentials (Fig. 7C, blowup). Before termination of bursting, burst frequency (Fig. 7C,D,H), envelope duration (Fig. 7E,I), and spikes per burst (Fig. 7F,J) all decreased with time in NNC. Measured 6 min after NNC 55-0396 treatment, burst frequency decreased from 2.7 ± 0.6 to 0.6 ± 0.2 Hz (n = 5; p < 0.01), envelope duration from 111.1 ± 9.2 to 64.3 ± 6.0 ms (n = 5; p < 0.01), and spikes per burst from 3.4 ± 0.3 to 1.8 ± 0.1 (n = 5; p < 0.01). These results support an essential role of T-type calcium channels in spontaneous ET cell burst generation.

L-type Ca²⁺ channels are required for LTSs and modulate spontaneous bursting

Because the voltage-clamp experiments also provided evidence for the involvement of L-type Ca²⁺ channels in LVA I_Ca, we next investigated their role in LTSs. After exposure to nimodipine (20 μm; 10 min), LTSs were abolished in all five ET cells tested (Fig. 8B). This was partially reversed after replacement of nimodipine with Bay K8644 (5 μm; 15 min) (Fig. 8C). In separate experiments, LTSs were significantly prolonged in duration by BayK (5 μm) (Fig. 9A,B); LTS duration measured at −40 mV increased from 112.4 ± 6.7 to 205.9 ± 8.7 ms (n = 5; p < 0.01). The prolongation of LTSs was reversed by washout of BayK with addition of nimodipine (20 μm) (Fig. 9C). These results support the involvement of L-type Ca²⁺ channels in LTSs.

We next investigated the role of L-type channels in spontaneous bursting. Nimodipine (20 μm) decreased both envelope duration [Fig. 8D (blowups), F,J] and spikes per burst [Fig. 8D (blowups),G,K]. The mean envelope duration (Fig. 8J) and spikes per burst (Fig. 8K) in five cells decreased from 105.1 ± 13.6 to 76.0 ± 9.4 ms (n = 5; p < 0.05) and 2.4 ± 0.1 to 1.0 ± 0.01 (n = 5; p < 0.01), respectively. Although nimodipine did not eliminate bursting, the attenuated depolarizing envelopes generated only single spikes (Fig. 8D,G,K). If we count envelopes with only single spikes as “bursts,” burst frequency was not significantly affected by nimodipine (Fig. 8E,I). Nimodipine also depolarized the membrane potential (Fig. 8D,H,L) from −57.1 ± 1.2 to −54.0 ± 1.2 mV (n = 5; p < 0.01). Restoring the MMP to its original level by hyperpolarizing current injection increased envelope duration, but did not increase spikes per burst, indicating that those changes were not entirely attributable to nimodipine-induced membrane depolarization.

All changes in bursting were reversed after replacing nimodipine with Bay K8644 (5 μm; 12 min) (Fig. 8D,F–H,J–L). Specifically, envelope duration (Fig. 8J), spikes per burst (Fig. 8K), and MMP (Fig. 8L) changed from 76.0 ± 9.4 to 126.6 ± 7.7 ms (n = 5; p < 0.05), 1.0 ± 0.01 to 2.5 ± 0.1 (n = 5; p < 0.01), and −54.0 ± 1.2 to 56.9 ± 1.4 mV (n = 5; p < 0.01), respectively. In separate experiments, BayK was applied first, and then nimodipine was added during washout. In this condition (Fig. 9D), BayK (5 μm) increased envelope duration (Fig. 9F,J) and spikes per burst (Fig. 9G,K) over control values from 89.4 ± 10.7 to 160.8 ± 19.5 ms (n = 5; p < 0.05) and 2.8 ± 0.2 to 6.6 ± 0.5 (n = 5; p < 0.01), respectively. The MMP was hyperpolarized from −55.3 ± 1.4 to −59.3 ± 3.7 mV (Fig. 9H,L) with little effect on burst frequency (Fig. 9E,I). Washout of BayK plus addition of 20 μm nimodipine (Fig. 9D,F–H,J–L) reversed all effects of BayK except burst frequency, which was reduced from 3.2 ± 0.1 to 1.3 ± 0.2 Hz (n = 5; p < 0.01). However, neither envelope duration nor spikes per burst was reversed by restoring MMP to its original level with depolarizing current (Fig. 9D,F,G,J,K), suggesting that the effect of BayK on bursting was not attributable to a change of membrane potential. Together, these results support the idea that L-type Ca²⁺ channels prolong the duration of the depolarizing envelope and increase the number of spikes per burst.

A large-conductance Ca²⁺-dependent K⁺ (BK) current regulates burst duration

The results to this point show that two inward conductances, I_NaP and I_h, contribute to the initiation of bursting by depolarizing the membrane potential to the threshold for LVA Ca²⁺ spikes, which comprise the depolarizing envelope on which a burst of action potentials is generated. What terminates the burst? Burst termination requires a mechanism that hyperpolarizes the membrane potential sufficiently to turn off the inward conductances that support the depolarizing envelope. Conceivably, passive inactivation of inward conductances (I_NaP, I_T/L, or both), engagement of outward conductances that deactivate the inward currents, or a combination of both factors could provide this mechanism. Lacking selective pharmacological tools to manipulate the inactivation of I_NaP or I_T/L, we investigated the possibility that outward conductance regulate the burst duration.

I_T/L, which is active throughout the depolarizing envelope, elevates cytosolic calcium that can trigger or modulate intracellular targets, including activation of Ca²⁺-dependent ion channels. Ca²⁺-activated potassium channels (K_Ca2+) are broadly divided into three subtypes based on their biophysical and pharmacological profiles: small- (SK), intermediate- (IK), and large-conductance (BK) channels. Although activation of any of these three can cause membrane hyperpolarization, the following considerations make BK an attractive candidate for terminating bursting in ET cells: (1) there is strong BK immunoreactivity in the glomerular layer (GL) of mouse olfactory bulb (Sausbier et al., 2006); (2) activation of BK channels is both voltage and Ca²⁺ dependent, so that deactivation of this outward conductance can be very rapid and complete on membrane hyperpolarization; and (3) BK channels have large conductance so that their activation can produce strong membrane hyperpolarization.

We first asked whether ET cells have functional BK channels. To record maximal BK currents, a 300 ms hyperpolarizing (−100 mV) prepulse (to de-inactivate both BK channels and voltage-dependent calcium channels) was applied before incremental depolarizing voltage steps (500 ms; 5 mV/step) from −70 to −15 mV with holding potential at −70 mV (Fig. 10B, inset). With TTX and ZD7288 in the bath to block Na⁺ and I_h currents, this protocol revealed an outward current (Fig. 10A) that activated at −42.0 ± 1.2 mV (n = 5) and increased with membrane depolarization from 44.3 ± 9.5 pA at −35 mV to 573.9 ± 13.6 pA (n = 5) at −15 mV.

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Figure 10.

Large-conductance Ca²⁺-dependent potassium (BK) current regulates evoked burst termination. A, B, Voltage-clamp recording using the protocol shown in B (inset) in aCSF containing TTX (1 μm) with (B) or without (A) IBTX (200 nm), a selective BK channel blocker, to identify BK currents. C, Averaged I–V curve of IBTX-sensitive currents from five cells. Inset, Subtracted traces (A minus B) showing the IBTX-sensitive currents. D, Current-clamp recordings comparing burst duration before (black) and after (red) IBTX (200 nm), showing that burst duration is significantly lengthened by blocking BK channels. Inset, Pooled data from 40 bursts in four cells showing evoked burst duration before and after IBTX. **p < 0.01. E, Pooled data from four cells showing that IBTX (200 nm) does not affect LTSs recorded in current clamp in the presence of TTX (1 μm) and ZD7288 (100 μm). Inset, LTSs evoked by current steps before (control) and after (IBTX) 15 min of IBTX treatment. F, Current-clamp recordings comparing burst duration before (black) and after (green) of ω-conotoxin MVIIC (CTxMVIIC) (5 μm; 10 min), a nonselective blocker of HVA Ca²⁺ channels, showing that burst duration is significantly lengthened by blocking HVA Ca²⁺ channels. Inset, Pooled data from five cells showing evoked burst duration before (control) and after ω-conotoxin MVIIC. **p < 0.01. G, Representative traces of the first three action potentials from evoked bursts before (black) and after IBTX (red; 200 nm; 15 min) or conotoxin MVIIC (green; 5 μm; 10 min) treatment, showing that the falling rather than the rising phases of action potentials are affected by blocking BK or HVA Ca²⁺ channels. H, I, Pooled data showing that IBTX (n = 4 cells) or conotoxin MVIIC (n = 5 cells) significantly reduces the decay slopes of the first three action potentials in each evoked burst. Error bars indicate SEM. *p < 0.05; **p < 0.01.

The BK channel selective blocker, IBTX (200 nm; 15 min), significantly reduced (Fig. 10B) this voltage-dependent outward current from 44.3 ± 9.5 to 19.8 ± 6.8 pA (n = 5; p < 0.05) at −35 mV, and from 573.9 ± 13.6 to 197.4 ± 78.3 pA (n = 5; p < 0.01) at −15 mV. The IBTX-sensitive outward current (Fig. 10C, inset) activated at about −39.0 ± 1.0 mV and increased in amplitude with membrane depolarization (Fig. 10C) (n = 5) from 24.5 ± 4.8 pA at −35 mV to 375.9 ± 118.4 pA at −15 mV, indicating that ET cells have functional BK channels. The residual IBTX-resistant outward currents may reflect incomplete block of BK by IBTX or the involvement of other potassium channels.

Next, we investigated the role of this current in bursting. In current clamp and with the membrane potential held around −70 mV by steady current injection to prevent spontaneous bursting, 400 ms depolarizing current pulses were applied to evoke a single burst per pulse (Fig. 10D); 10 evoked bursts were collected at 0.2 Hz from each ET cell and compared with 10 bursts evoked in the presence of IBTX (200 nm; 15 min treatment). As shown in Figure 10D, IBTX treatment significantly increased the duration of evoked bursts by 45.5% from 112.8 ± 4.7 to 163.5 ± 6.2 ms (n = 4 cells; p < 0.01) without affecting input resistance measured at −55 mV (from 227.5 ± 10.3 MΩ in control to 240.6 ± 12.8 MΩ at 15 min after IBTX; n = 4 cells).

What activates I_BK? Because I_L/T plays an important role in spontaneous bursting (Figs. 5⇑⇑⇑–9), I_L/T might conceivably provide both depolarization and intracellular Ca²⁺ [Ca²⁺]_i elevation for BK channel activation. Although our voltage-clamp data already indicated that very little I_BK is activate at membrane potentials more negative than −30 mV (Fig. 10C) and because the depolarizing envelopes never exceed −40 mV, BK channel activation might conceivably be shifted to a more negative voltage range by [Ca²⁺]_i elevation through I_T/L. If this were the case, BK channels should play a role in terminating LTSs. Alternatively, I_T/L may be insufficient to increase [Ca²⁺]_i for BK channel activation within the voltage range of the depolarizing envelope. In this scenario, transient sodium channel (I_NaT)-mediated action potentials rather than the I_L/T-mediated LTSs might be required to activate BK channels because, in addition to strong depolarization, action potentials can also increase [Ca²⁺]_i by activating HVA Ca²⁺ channels (I_HVA).

To test the first hypothesis, we examined the effect of IBTX on evoked LTSs in the presence of TTX (1 μm) and ZD7288 (100 μm) to block action potentials and I_h and found that blocking BK channels with IBTX (200 nm; 15 min) did not change LTS duration (control, 90.8 ± 7.3 ms; IBTX, 91.4 ± 8.5 ms; n = 5) (Fig. 10E). Therefore, the depolarizing voltage and Ca²⁺ associated with I_T/L are insufficient to activate I_BK. Thus, the depolarizing envelope, by itself, is unlikely to engage this conductance.

We next explored the contribution of I_HVA to BK channel activation and the duration of evoked bursts by blocking I_HVA with ω-conotoxin MVIIC, which at high concentration blocks both P/Q- and N-type Ca²⁺ channels (McDonough et al., 1996). As shown in Figure 10F, ω-conotoxin MVIIC (5 μm; 10 min) increased evoked burst duration by 31.7% from 93.7 ± 6.4 to 123.4 ± 2.5 ms (n = 5; p < 0.01). There was no effect on input resistance measured at −55 mV (control, 231.1 ± 19.4 MΩ; conotoxin MVIIC, 236.3 ± 11.2 MΩ; n = 5). The smaller increase in the depolarizing envelope duration by conotoxin MVIIC (31.7%) compared with IBTX (45.5%) may reflect an incomplete block of HVA Ca²⁺ current by conotoxin MVIIC or the possibility that a strong membrane depolarization during action potentials and LVA Ca²⁺ current combine to activate BK channels. This finding indicates that I_HVA is required for BK channel activation and supports the hypothesis that BK channels are activated by both [Ca²⁺]_i increase and strong depolarization during action potentials.

In addition to increasing depolarizing envelope duration, treatment with IBTX or conotoxin MVIIC also reduced the decay slope of action potentials within bursts. As shown in Figure 10, G–I, IBTX (200 nm; 15 min) decreased the decay slope by 29.7% from 74.8 ± 8.2 to 52.6 ± 7.7 mV/ms (n = 5; p < 0.05) for the first action potential, by 37.3% from 53.6 ± 4.3 to 33.6 ± 6.0 mV/ms (n = 5; p < 0.01) for the second one, by 38.3% from 47.8 ± 5.4 to 29.5 ± 3.6 mV/ms for the third one within each burst, respectively. Conotoxin MVIIC (5 μm; 10 min) reduced the decay slope by 18.8% from 72.3 ± 2.7 to 58.7 ± 1.8 mV/ms for the first action potential, by 34.2% from 51.0 ± 1.7 to 33.6 ± 4.2 mV/ms for the second one, and by 40.3% from 43.2 ± 3.0 to 25.8 ± 2.5 mV/ms for the third one within each burst, respectively. These results indicate a contribution of BK and HVA Ca²⁺ currents to the repolarization of action potentials.

Finally, we investigated the role of I_BK in spontaneous bursting. As shown in Figure 11A, IBTX increased both envelope duration (Fig. 11C,G) and spikes per burst (Fig. 11D,H) without significantly affecting either burst frequency (Fig. 11B,F) or MMP (Fig. 11E,I). The average envelope duration (Fig. 11G) from five cells increased by 57.5% from 106.8 ± 16.5 to 167.1 ± 31.1 ms (n = 5; p < 0.05) and spikes per burst (Fig. 11H) increased from 3.0 ± 0.2 to 4.8 ± 0.2 (n = 5; p < 0.01). Together, these results support the hypothesis that BK current regulates burst duration by contributing to burst termination.

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Figure 11.

BK channels regulate spontaneous burst termination. A, Current-clamp recording showing that IBTX (200 nm) increases both envelope duration and spikes per burst (traces below expanded timescale) of spontaneous bursting. B–E, Graphs showing the effect of IBTX on burst frequency (B), depolarizing envelope duration (C), spikes per burst (D), and MMP (E) of the cell in A. F–I, Mean values of burst frequency, duration of depolarizing envelope, spikes per burst, and MMP from five cells before (control) and 10 min after IBTX; four cells before (control) and 10 min after apamin (300 nm), a specific SK channel blocker; and five cells before (control) and 10 min after bicuculline (30 μm), a blocker of apamin-insensitive SK channels, respectively. Error bars indicate SEM. *p < 0.05; **p < 0.01.

SK channel immunostaining has also been detected in the mouse glomerular layer (Sailer et al., 2004). This type of K_Ca channel plays a role in dendrodendritic inhibition in the olfactory bulb by regulating dendritic excitability of mitral cells (Maher and Westbrook, 2005). Therefore, we tested the role of SK in ET cell bursting with both apamin (300 nm), a selective blocker of SK channels, and bicuculline (30 μm), a GABA_A receptor blocker that also blocks both apamin-sensitive and apamin-insensitive SK channels (Khawaled et al., 1999). Because bicuculline was applied at least 10 min after gabazine (10 μm), a specific GABA_A receptor blocker, any effect on bursting should attribute to SK channels. As shown in Figure 11, F–I, none of the bursting parameters measured was significantly affected, indicating that SK channels do not play an obvious role in the regulation of spontaneous bursting of ET cells.