Extracellular Calcium Modulates Persistent Sodium Current-Dependent Burst-Firing in Hippocampal Pyramidal Neurons

Effects of lowering [Ca²⁺]_o on the firing patterns of CA1 PCs

In standard saline, the proportion of intrinsically bursting CA1 PCs was small and the bursting threshold in these bursters was high, as described previously (Jensen et al., 1994). Of 136 neurons examined, 112 (82%) were nonbursters and 24 (17%) were bursters (of which 20 were HTBs and 4 grade I LTBs). Lowering [Ca²⁺]_o markedly increased the incidence of, and decreased the threshold for, intrinsic bursting in most PCs. In the representative experiment illustrated in Figure 2, changing from standard to 1.2 mm Ca²⁺ saline converted the nonbursting PC (Fig. 2A) into an HTB (Fig.2B). Further reduction of [Ca²⁺]_o to 0.5 mm converted the PC into a grade I LTB (Fig.2C). Finally, in nominally Ca²⁺-free saline the PC changed into a grade II LTB (Fig. 2D).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Effect of lowering [Ca²⁺]_o on the intrinsic firing pattern of a CA1 PC. Recordings were made in a slice perfused with salines containing different concentrations of Ca²⁺.A, In standard saline, the PC was a nonburster, as determined from its responses to 200 msec (a) and 3 msec (b) depolarizing current pulses.B, After a 30 min wash in 1.2 mmCa²⁺-saline, the PC became an HTB, firing a burst in response to a strong 200 msec depolarization (a).C, After a 30 min wash in 0.5 mmCa²⁺-saline, the PC became a grade I LTB, firing a burst also in response to a threshold 200 msec depolarization (a). D, Finally, after a 30 min wash in Ca²⁺-free saline, the PC became a grade II LTB, firing a burst in response to any threshold depolarization (a, b). E, The transition from regular firing to bursting was associated with a progressive decrease in the fast AHP and an increase in size of the spike ADP, as shown in the overlay of the expanded traces from b inA–D.

Similar concentration-dependent effects were seen in 15 of 20 PCs exposed to graded reductions in [Ca²⁺]_o, although the final grade of bursting attained in Ca²⁺-free saline varied among the neurons. As summarized in Figure 3, lowering [Ca²⁺]_o from 2 to 1.2 mm converted 8 of 17 nonbursters to HTBs. Further reduction to 0.5 mm converted many of the HTBs to LTBs. Subsequent exposure to Ca²-free saline further augmented the bursting propensity of these LTBs. Spontaneous intrinsic bursters (grade III LTBs) were encountered only in nominally Ca²⁺-free saline in 5 of the 20 PCs (25%) in this sample.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

The firing patterns of CA1 PCs in different Ca²⁺_o concentrations. The firing patterns of 20 CA1 PCs was monitored while [Ca²⁺]_o was reduced in a stepwise manner from 2 mm (standard saline) to 0 mm(Ca²⁺-free saline). In control conditions, 17 PCs were nonbursters, 2 were HTBs, and only 1 was a grade I LTB. Thelines in the graph depict the direction of change in firing pattern within each subclass of PCs when [Ca²⁺]_o is lowered to the next concentration. Thus, of the 17 native nonbursters, 8 PCs became HTBs in 1.2 mm Ca²⁺ and LTBs in 0.5 mm Ca²⁺, another 4 PCs became HTBs in 0.5 mm Ca²⁺, and the remaining 5 PCs did not change at all.

The waveform of intrinsic bursts in low [Ca²⁺]_o was similar to that of native intrinsic bursts (Jensen et al., 1994, 1996). The number of intraburst spikes varied between three and seven across different cells (averaging 3.5 ± 1.1; n = 40) but was rather constant in any given cell. The initial and mean firing rates during a burst were 178.7 ± 28.8 and 134.7 ± 28.2 Hz (n = 40), respectively.

Effects of lowering [Ca²⁺]_o on the spike ADP

Lowering [Ca²⁺]_o did not significantly affect resting membrane potential, input resistance, or time constant, nor did it affect spike threshold or amplitude (Table1). However, it strongly modulated the spike afterpotentials, namely the fast AHP and ADP, in most PCs. As illustrated in Figure 2E, lowering [Ca²⁺]_o reduced the fast AHP and augmented the size of the ADP in a concentration-dependent manner. On average, changing from standard to Ca²⁺-free saline reduced the fast AHP by 5.8 ± 4.0 mV and variably augmented the spike ADP by 63.2 ± 54.8% (n = 40) (Table 1).

During the lowering of [Ca²⁺]_o, the size of the spike ADP and the propensity to burst increased in parallel (Fig. 2). Accordingly, the final size of the spike ADP in Ca²⁺-free saline was significantly larger in bursters (275.9 ± 103.7 mV · msec;n = 33) than in nonbursters (196.4 ± 54.2 mV · msec; n = 7). These observations are consistent with the notion that intrinsic bursts are triggered by suprathreshold spike ADPs (Kandel and Spencer, 1961; Jensen and Yaari, 1996).

Effects of I_NaP blockers on low [Ca²⁺]_o-induced intrinsic bursting

In pharmacologically untreated CA1 PCs, somatic intrinsic bursting is driven by I_NaP (Azouz et al., 1996). We examined whether a similar ionic mechanism underlies intrinsic bursting in low [Ca²⁺]_o by testing its sensitivity to three drugs previously shown to reduceI_NaP in CA1 PCs, namely tetrodotoxin (TTX), the anti-epileptic drug phenytoin, and the protein kinase C (PKC) activator PDB. Representative results are shown in Figure4.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Blockers of I_NaPsuppress intrinsic bursting in low [Ca²⁺]_o. Recordings were made from four grade II LTBs in Ca²⁺-free saline. Burst firing was evoked by 3 msec depolarizing stimuli.A, The responses of the neuron are shown before (a) and 5 and 20 min after (b andc, respectively) adding 0.1 μm TTX to the Ca²⁺-free saline, which caused a gradual suppression of intrinsic bursting. B, The responses of the neuron are shown before (a), 30 min after adding 50 μm phenytoin to the Ca²⁺-free saline (b), and 30 min after wash of phenytoin (c). C, The responses of the neuron are shown before (a) and 30 min after (b) adding 5 μm PDB to the Ca²⁺-free saline (b). Both phenytoin and PDB blocked the burst response completely but affected the first spike very little. D, The responses of the neuron are shown before (a) and 45 min after (b) adding 50 μm PDC to the Ca²⁺-free saline. No effect of this drug was noted.

Effects of I_NaP blockers on low [Ca²⁺]_o-enhanced spike ADPs

In a fraction of the PCs, changing to Ca²⁺-free saline enhanced the spike ADP, but not sufficiently to trigger a burst in response to a brief stimulus (i.e., HTBs and grade I LTBs) (Fig.1B,C). We tested in these neurons the effects of the three I_NaP blockers on the enhanced spike ADPs. As illustrated in Figure5, the spike ADP was consistently suppressed by 0.1–0.2 μm TTX (Fig.5A) (n = 4), 50–100 μm phenytoin (Fig. 5B) (n = 9), and 2–10 μm PDB (Fig.5C) (n = 9), but not by 50 μm PDC (n = 3; data not shown). These results are consistent with the notion that the enhanced spike ADPs in low [Ca²⁺]_o are driven primarily by I_NaP.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Blockers of I_NaPsuppress spike ADPs in low [Ca²⁺]_o.A–C, Recordings of single spikes evoked by 3 msec stimuli were made in three CA1 PCs in slices perfused sequentially with standard saline (2 Ca²⁺ ), Ca²⁺-free saline (0 Ca²⁺), and Ca²⁺-free saline containing a drug known to suppressI_NaP. In the three PCs the native spike was followed by an active ADP. Lowering [Ca²⁺]_o caused spike broadening, blocked the fast AHP, and enhanced the active ADP. The latter potential was completely depressed after 20 min exposure to 0.2 μm TTX (A). Likewise, it was markedly reduced after 30 min exposure to 50 μmphenytoin (B) or 5 μm PDB (C).

Effects of I_NaP blockers on low [Ca²⁺]_o-enhanced subthreshold ADPs

When CA1 PCs are depolarized with a brief current pulse to near threshold potential, the membrane potential decays back to resting potential more slowly than expected from the membrane time constant. These subthreshold ADPs are readily blocked by TTX, but not by Ca²⁺ channel blockers (Azouz et al., 1996), suggesting that they are generated byI_NaP, the activation threshold of which is slightly more negative than spike threshold (French et al., 1990). We tested the effects of lowering [Ca²⁺]_o on these potentials. In all cases (n = 22), as illustrated in Figure 6, deleting Ca²⁺_o markedly enhanced the subthreshold ADPs. Thus, the waveform of these potentials increased significantly from 134.9 ± 50.3 to 333.5 ± 71.2 mV · msec after changing from standard to Ca²⁺-free saline. Consistent with the notion that they are generated byI_NaP, these potentials were readily blocked by 0.1–0.2 μm TTX (Fig.6A) (n = 4), 50–100 μm phenytoin (Fig. 6B) (n = 9), and 2–10 μm PDB (Fig.6C) (n = 9).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

Blockers of I_NaPsuppress subthreshold ADPs in low [Ca²⁺]_o. A–C, Recordings of subthreshold ADPs evoked by 3 msec threshold-straddling stimuli were made in three CA1 PCs in slices perfused sequentially with standard saline (2 Ca²⁺ ), Ca²⁺-free saline (0 Ca²⁺), and Ca²⁺-free saline containing a drug known to suppressI_NaP. In the three PCs, when the stimulus failed to trigger a spike, it evoked a slow depolarizing potential that declined more slowly to resting potential than expected from passive membrane charging. Lowering [Ca²⁺]_o induced a subthreshold ADP in these PCs. The latter potential was suppressed by 0.2 μm TTX (20 min; A), 50 μm phenytoin (30 min; B), and 2 μm PDB (40 min; C).

Effects of Ca²⁺ current block with Ni²⁺

The results described above established that low [Ca²⁺]_o-induced intrinsic bursting is most likely driven byI_NaP. However, they did not indicate the mechanism by which lowering [Ca²⁺]_o augments the slow potentials that trigger the burst discharge. One possibility is that lowering [Ca²⁺]_o reduces outward Ca²⁺-activated K⁺ currents, thereby increasing the depolarizing impact of I_NaP (Azouz et al., 1996). Alternatively, lowering [Ca²⁺]_o may upmodulate I_NaP itself. The first hypothesis predicts that blocking Ca²⁺channels pharmacologically also would induce intrinsic bursting. We tested this notion in four nonbursters by adding 1 mm Ni²⁺ (in replacement of 1 mmMg²⁺) to the standard saline that perfused the slices. Consistent results were obtained in all cases and are illustrated in Figure 7. Adding Ni²⁺ suppressed the fast AHP (Fig. 7, compare a in A and B), as expected from block of the fast Ca²⁺-activated K⁺ current (I_c) (Storm, 1987). Likewise, it suppressed the slow AHP that follows a train of several spikes, which is generated by the slow Ca²⁺-activated K⁺ current (I_AHP) (Madison and Nicoll, 1984), as well as the associated spike frequency accommodation (Fig. 7, compareb in A and B). Despite the apparent block of these K⁺ currents, the nonbursting pattern of the PC did not change (Fig. 7, comparec in A and B). Yet, bursting was readily induced in the Ni²⁺-containing saline by deleting Ca²⁺ from the solution (Fig. 7C, c).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 7.

Block of Ca²⁺-activated K⁺ currents with Ni²⁺ does not induce intrinsic bursting. Recordings were obtained from a native nonbursting CA1 PC. Shown are the responses of the neuron to 4 msec (a), 400 msec (b), and 200 msec depolarizing stimuli (c) in three conditions.A, In standard saline, the solitary spike was followed by distinct fast AHP and ADP (a), and repetitive firing was followed by a medium AHP and slow AHPs (b). B, After the change to standard saline containing 1 mm Ni²⁺, the fast and slow AHPs were suppressed, whereas the medium AHP was preserved (a, b). Despite the reduction in spike frequency accommodation (b), the nonbursting firing pattern of the PCs was unaltered (c). C, Changing to Ca²⁺-free saline containing 1 mm Ni²⁺(b) converted the PC to a grade I LTB (c).

Effects of buffering intracellular Ca²⁺with BAPTA

As an additional test for the role of Ca²⁺-activated currents in shaping the firing mode of CA1 PCs, we injected seven neurons with the Ca²⁺ chelator BAPTA by applying negative current pulses (up to 0.5 nA; 10–30 min). The effects of BAPTA were similar in all cases. As illustrated in Figure8, BAPTA injection expectedly suppressed the fast AHP (Fig. 8, compare a in A andB), the slow AHP, and the associated spike frequency accommodation (Fig. 8, compare b in A andB). Despite these effects, BAPTA injection did not induce intrinsic bursting (Fig. 8, compare c in A andB). However, bursting was readily induced in the BAPTA-injected neuron by removing Ca²⁺_o (Fig.8C, c).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 8.

Buffering of intracellular Ca²⁺concentration with BAPTA does not induce intrinsic bursting. Recordings were obtained from a native nonbursting CA1 PC. Shown are the responses of the neuron to 4 msec (a), 400 msec (b), and 200 msec depolarizing stimuli (c) in three conditions.A, In standard saline, the solitary spike was followed by distinct fast AHP and ADP (a), and repetitive firing was followed by a medium and slow AHPs (b). B, Injecting BAPTA into the neuron suppressed the fast and slow AHPs and reduced spike frequency accommodation (a, b), but the nonbursting firing pattern of the PC was unaltered (c).C, Changing to Ca²⁺-free saline converted the PC to a grade I LTB (c).

Effect of low [Ca²⁺]_o on slow spikes

Perfusing CA1 PCs with standard saline containing 10 mm TEA, which blocks various voltage-gated K⁺ currents in these neurons (e.g., delayed rectifier and M-current) (Storm, 1990), induces compound slow (duration 100–400 msec) and fast spike responses to brief depolarizing stimuli. Deleting Ca²⁺_o converts the slow spikes to “plateau” potentials lasting 1–2 sec (Garcı́a-Muñoz et al., 1993; Jensen et al., 1996). These potentials are blocked by TTX at low concentrations that do not affect the fast spikes, indicating that they are generated byI_NaP. Accordingly, they are also blocked by phenytoin (H. Su and Y. Yaari, unpublished observations) and PKC activation (Alroy et al., 1999).

We tested whether blocking Ca²⁺-activated K⁺ currents with Ni²⁺ also induces plateau potentials in TEA-containing saline. A representative experiment is illustrated in Figure 9A. In standard saline containing 10 mm TEA, a brief depolarizing pulse evoked a complex of several fast spikes riding on a slow spike, followed by a slow AHP (Fig. 9A, a). Adding 1 mm Ni²⁺ abolished the slow AHP and slightly enhanced the slow spike, but did not induce a plateau potential (Fig. 9A, b). However, deleting Ca²⁺ from the Ni²⁺-containing saline induced a prolonged (∼1.8 sec) plateau potential (Fig. 9A, c). This slow potential was blocked entirely by 0.2 μmTTX before the primary fast spike was affected (Fig. 9A,d). Similar results were obtained in all experiments (n = 4).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 9.

Lowering of [Ca²⁺]_o, but not block of Ca²⁺-activated K⁺ currents, induces TTX-sensitive plateau potentials. A, In this experiment the slice was perfused with standard saline containing 10 mm TEA to block several voltage-gated K⁺currents. A constant negative current was injected into the cell to maintain the native resting membrane potential (−63 mV). Suprathreshold responses were evoked by 5 msec positive current pulses. Salines were exchanged every 30 min. In standard saline containing TEA, the PC generated a compound response, consisting of fast spikes riding on a slow spike, and followed by a slow AHP (a). Adding 1 mm Ni²⁺ to the saline suppressed the slow AHP completely and slightly prolonged the slow spike (b). Deleting Ca²⁺ from the saline induced a prolonged plateau potential lasting ∼1.8 sec (c), which was suppressed by 0.2 μmTTX within 10 min of exposure (d).B, In another slice perfused with salines containing 10 mm TEA, [Ca²⁺]_o was reduced in a stepwise manner every 30 min. The responses of the PC to 5 msec pulse stimulation are shown in standard saline (a), 1.2 mm Ca²⁺saline (b), 0.5 mmCa²⁺ saline (c), and Ca²⁺-free saline (d). The control response comprised fast and slow spikes (a). The duration of the plateau potential increased as [Ca²⁺]_o decreased (b–d).

These results indicated that blocking Ca²⁺-activated K⁺ currents itself is not sufficient to induce plateau potentials in TEA-containing saline. Rather, Ca²⁺_o removal per se is required for this effect. Indeed, we found that even partial Ca²⁺_o replacement is effective in prolonging the slow spike component in TEA-containing saline (n = 4). As illustrated in Figure 9B, reducing [Ca²⁺]_oin a stepwise manner caused a concentration-dependent growth of the plateau potential (Fig. 9B, a–d).

Effect of low [Ca²⁺]_o on inward rectification

CA1 PCs display inward rectification, manifested as an apparent increase in input resistance near action-potential threshold (Hotson et al., 1979). A component of inward rectification persists after suppression of Ca²⁺ currents and is blocked by TTX, indicating that it is caused by activation ofI_NaP (Benardo et al., 1982). If low [Ca²⁺]_oupmodulates I_NaP, then it should augment TTX-sensitive inward rectification. We tested this notion in five slices perfused with saline containing 1 mmNi²⁺ by injecting slow current ramps (duration 0.9 sec) of increasing magnitude into the PCs. In the example illustrated in Figure 10, the current was linearly increased from 0 to 160 pA, causing the neuron to depolarize from −72 mV to spike threshold potential (approximately −60 mV). In standard saline containing Ni²⁺, inward rectification was seen at membrane potentials more positive than −63.8 mV (Fig.10A, a). After changing to Ni²⁺-containing Ca²⁺-free saline, inward rectification occurred already at −67.1 mV (Fig. 10, trace b). Adding 1 μm TTX completely blocked the inward rectification (Fig. 10, trace c). Similar results were obtained in all five experiments. On average, the membrane potential at which the membrane began to rectify shifted from −62.0 ± 2.9 to −67.2 ± 2.0 mV after removal of Ca²⁺_o (a significant difference of −5.2 ± 1.4 mV).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 10.

Lowering [Ca²⁺]_oenhances TTX-sensitive inward rectification. Recordings were obtained from a native nonbursting CA1 PC. The neuron was injected with current ramps from 0 to 160 pA (duration 0.9 sec). Plotted are the subthreshold voltage responses of the neuron to these ramps. Each trace is an average of four consecutive responses. The slice was first perfused with standard saline containing 1 mmNi²⁺ to block voltage-sensitive Ca²⁺ currents. In these conditions (trace a), the membrane potential first depolarized linearly from its resting level (−72 mV) until −63.8 mV, after which it depolarized exponentially until spike threshold was attained (at −60 mV). After 30 min of perfusion with Ni²⁺-containing Ca²⁺-free saline (trace b), the nonlinear response began already at −67.1 mV and was steeper than in control. Addition of 0.1 μm TTX (trace c) completely suppressed the nonlinear subthreshold response of the neuron in 10 min.

Effects of increasing [Ca²⁺]_o on native intrinsic bursting and slow spikes

In three experiments we tested how elevating [Ca²⁺]_o affects native intrinsic bursting. A representative experiment is illustrated in Figure 11A. In standard saline, strong positive current pulses evoked an initial burst response of three spikes (Fig. 11A, a). Raising [Ca²⁺]_o to 4 mm (by substituting 2 mmMg²⁺ with 2 mmCa²⁺) blocked the burst response at all stimulation intensities (Fig. 11A, b). This effect reversed after wash with standard saline (Fig.11A, c). Similar results were obtained in two additional PCs. The suppression of intrinsic bursting by doubling [Ca²⁺]_o was associated with a small (∼3 mV) increase in spike threshold.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 11.

Raising [Ca²⁺]_oblocks native intrinsic bursting and slow spikes. A, In this experiment recordings were obtained from a native HTB. Shown are the responses of the neuron to 200 msec depolarizing stimuli in standard saline (a), 30 min after switching to saline containing 4 mm Ca²⁺(b), and 30 min after changing back to standard saline (c). Raising [Ca²⁺]_o reversibly increased the threshold for evoking spikes and suppressed the initial burst response to the depolarizing pulses. B, In another slice perfused with salines containing 10 mm TEA, [Ca²⁺]_o was increased to 4 mm. The responses of the PC to 5 msec pulse stimulation are shown in standard saline (a), after 20 min in 4 mm Ca²⁺ saline (b), and after 30 min wash back to standard saline (c). The slow spike was reversibly suppressed by doubling [Ca²⁺]_o(a–c).

We also tested the effects of raising [Ca²⁺]_o on the slow spikes in TEA-containing saline (n = 3). As shown in Figure 11B, raising [Ca²⁺]_o to 4 mm selectively and reversibly blocked the slow spike component but spared the fast spike (Fig. 11B,a–c). Consequently, the slow AHP also was reversibly blocked by doubling [Ca²⁺]_o. Similar results were obtained in all three experiments.

Dye coupling and firing patterns of CA1 PCs in low [Ca²⁺]_o

Previous work in CA1 PCs suggested that bursters are more likely to be electrically coupled to each other via gap junctions than nonbursters (Church and Baimbridge, 1991). It was suggested that there may be a causal relationship between electrical coupling and intrinsic bursting. Presumably, the spread of spikes within a small network of electrically coupled neurons can generate a burst response in each neuron, which would appear as an “intrinsic” burst. Because lowering [Ca²⁺]_oreportedly increases gap junctional communication and, by implication, electrotonic coupling between CA1 PCs (Perez Velazquez et al., 1994), we examined whether this effect may contribute to low [Ca²⁺]_o-induced intrinsic bursting. To this end, we injected 46 PCs with the dye tracer biocytin during perfusion with nominally Ca²⁺-free saline. Of these, nine neurons (21.4%) were found to be dye coupled to one, two, or three other PCs (mean number of PCs in a coupled cluster 2.6 ± 0.9). However, we found no significant correlation between the firing pattern of the neuron and the incidence of dye coupling (Table2). Figure12 illustrates two exemplary biocytin-injected neurons, a grade II LTB (Fig. 12A,a and b), which was not coupled to other neurons (Fig. 12A, c), and a regular firing cell (Fig. 12B, a and b), which was coupled to two other PCs (Fig. 12B,c).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 12.

Dissociation between dye coupling and intrinsic bursting in low [Ca²⁺]_o.A, B, Recordings from two native nonbursters 30 min after changing to Ca²⁺-free saline. Both PCs were stained with biocytin to detect dye coupling. A, This neuron became a grade II LTB in low [Ca²⁺]_o, as is evident from its burst responses to 200 msec (a) and 3 msec depolarizing current pulses (b). As shown in the photomicrograph of the neuron (c), it was not dye coupled to other cells. B, This neuron remained nonbursting in low [Ca²⁺]_o (a,b). Biocytin injection into this cell resulted in the staining of three PCs (c), suggesting that the neuron formed gap junctions with one or two other PCs.

It has been argued recently that in some neurons dye coupling may not be a reliable indicator for the presence of electrical coupling via gap junctions (Gibson et al., 1999). Therefore, we also tested the effect of the gap junctional blocker doxyl-stearic acid (Strata et al., 1997;Zahs and Newman, 1997) on low [Ca²⁺]_o-induced intrinsic bursting. This drug was chosen because unlike other commonly used gap junction blockers tested (propionate, octanol, halothane, and carbenoxolone), it exerted no deleterious effects on intrinsic neuronal properties at concentrations that block neuronal gap junctions (Su and Yaari, unpublished observations). Addition of 50 μmdoxyl-stearic acid to the Ca²⁺-free saline did not affect intrinsic bursting during 45–60 min of perfusion in all cases (n = 6; data not shown).

Taken together, these data do not support a role for electrical coupling in the generation of intrinsic bursting in low [Ca²⁺]_o.

Ionic mechanism of intrinsic bursts in low [Ca²⁺]_o

Several slow inward currents have been implicated in the generation of somatic bursts in cortical neurons, including voltage-sensitive Ca²⁺ (Wong and Prince, 1981; Foehring and Waters, 1991; Deisz, 1996) and Na⁺ currents (Franceschetti et al., 1995;Azouz et al., 1996; Mattia et al., 1997), and a Ca²⁺-activated cationic current (Kang et al., 1998). In CA1 PCs in vitro, native somatic bursting, as well as that induced by elevating [K⁺]_o (Jensen et al., 1994), are insensitive to block of Ca²⁺ currents. Rather, they are suppressed by blockers of I_NaP, implicating the latter current in their generation (Azouz et al., 1996;Alroy et al., 1999). Somatic bursting can be induced in these neurons also by exposure to millimolar concentrations of 4-aminopyridine (4-AP), which blocks several K⁺conductances (Magee and Carruth, 1999). Unlike native bursting, 4-AP-induced bursts are suppressed by Ca²⁺channel blockers, consistent with the view that they are generated by dendritic Ca²⁺ spikes (Magee and Carruth, 1999).

Two lines of evidence suggest that somatic bursting in low [Ca²⁺]_o is driven by I_NaP. First, in most experiments bursting was readily induced in nominally Ca²⁺-free saline containing 2 mm Mn²⁺. Blocking Ca²⁺ channels with Mn²⁺ in addition to deleting Ca²⁺ ensured that neither Ca²⁺ currents nor Na⁺ currents through Ca²⁺ channels (Lux et al., 1990) were involved in bursting. Thus, the only slow inward current available in this saline was I_NaP. Second, intrinsic bursting induced by low [Ca²⁺]_o was readily suppressed by TTX, phenytoin, and PDB. Although these drugs interact with voltage-gated Na⁺ channels in different ways (Catterall, 1999), they share a common depressant effect on I_NaP.

We have examined three hypotheses that explain whyI_NaP becomes more effective in producing intrinsic bursting as [Ca²⁺]_o is lowered: (1) suppression of opposing Ca²⁺-activated K⁺ currents, (2) upmodulation ofI_NaP itself, and (3) increase in electrotonic coupling through gap junctions. These hypotheses will be assessed in the following sections.

Suppression of Ca²⁺-activated K⁺ currents

In CA1 PCs, I_C activates during the action potential and contributes largely to the late phase of spike repolarization (Storm, 1987). Thus, the density ofI_C influences the waveform of the spike ADP and may determine whether it will progress to a burst. We compared the effects of three experimental manipulations that causeI_C suppression, namely, lowering [Ca²⁺ ]_o, blocking Ca²⁺ currents with extracellular Ni²⁺, and preventing intracellular Ca²⁺ accumulation with BAPTA. As expected, all of these manipulations reduced theI_C-dependent fast AHP. However, only low [Ca²⁺ ]_oinduced intrinsic bursting, indicating that reduction inI_C (or any other Ca²⁺-activated current) by itself is not sufficient to invoke this firing pattern in CA1 PCs. Rather, lowering [Ca²⁺ ]_o exerts additional effects that are crucial for the induction of bursting.

Upmodulation of I_NaP

An attractive possibility is that low [Ca²⁺ ]_oupmodulates I_NaP. We tested this possibility by monitoring the effects of Ca²⁺_o removal on TTX-sensitive plateau potentials and inward rectification that are generated by I_NaP. Both the plateau potentials and inward rectification were enhanced in this condition, supporting the view that lowering [Ca²⁺]_o upmodulatesI_NaP.

In a previous study in supraoptic hypothalamic neurons, lowering [Ca²⁺ ]_o enhancedI_NaP and the associated spontaneous intrinsic bursting (Li and Hatton, 1996). However, replacement of Ca²⁺ with other divalent cations prevented these effects, suggesting that they are caused by a net decrease in extracellular divalent ion concentration (which would shiftI_NaP activation to more positive potentials by unscreening the external negative membrane surface potential) rather than by a specific decrease in [Ca²⁺ ]_o. In our experiments, the concentrations of divalent cations were maintained constant at 4 mm in all salines, and the induction of intrinsic bursting occurred regardless of the species of divalent cations used to replace Ca²⁺_o. These data suggest that in CA1 PCs, Ca²⁺_o decreasesI_NaP by a mechanism that involves its selective binding to membrane receptors. The receptors that bind Ca²⁺_o may be the Na⁺ channels themselves (Armstrong and Cota, 1991) or G-protein-coupled Ca²⁺-sensing receptors (Yamaguchi et al., 2000), such as metabotropic glutamate receptors (Kubo et al., 1998), that may modulate I_NaP via second messenger cascades. The latter mechanism is less likely, because 100 μm neomycin (n = 3) and 10 nm gadolinium (n = 3), which activate Ca²⁺-sensing receptors (Xiong and MacDonald, 1999), failed to reverse the induction of intrinsic bursting by low [Ca²⁺ ]_o(Su and Yaari, unpublished observations).

Increase in electrical coupling through gap junctions

We also examined whether augmentation of electrotonic coupling via gap junctions contributes to the appearance of intrinsic bursting in low [Ca²⁺]_o. Both experimental (Getting and Willows, 1974) and theoretical (Sherman and Rinzel, 1992) data suggest that electrotonic coupling can cause neurons to burst. Indeed, it was shown that raising extracellular pH enhances both dye coupling (and by implication, electrotonic coupling) and intrinsic bursting in CA1 PCs and that only PCs coupled to other neurons display bursting behavior (Church and Baimbridge, 1991). Although we found significant dye coupling between CA1 PCs bathed in low [Ca²⁺]_o, we could not substantiate a role for electrotonic coupling in the bursting behavior. First, the incidence of dye coupling among bursters and nonbursters was the same. Second, the gap junction blocker doxyl-stearic acid did not affect intrinsic bursting. It should be noted that both the overall incidence of dye coupling (21.4%) and the size of a coupled aggregate (two to four cells) in Ca²⁺-free saline were much lower than in high pH saline (88% and two to nine cells, respectively) (Church and Baimbridge, 1991). Thus, if electrical coupling via gap junctions and intrinsic bursting are causally associated, this mechanism may be more important for intrinsic bursting in high extracellular pH than in low [Ca²⁺]_o.

Functional implications

Despite the strict homeostatic control of [Ca²⁺]_o in the brain, neuronal activity can cause substantial decreases in [Ca²⁺]_o (Heinemann et al., 1977). In hippocampal slices perfused with 2 mmCa²⁺ saline, repetitive orthodromic activation of CA1 PCs at θ frequencies (5–10 Hz) decreases [Ca²⁺]_o down to 1.4 mm (Benninger et al., 1980). This decrease, in combination with an associated increase in [K⁺]_o (up to 12 mm), would convert many nonbursting PCs into intrinsic bursters. This may explain why θ frequency orthodromic activation of CA1 PCs also leads to the development of postsynaptic bursting (Thomas et al., 1998).

The induction of intrinsic bursting by activity-dependent decreases in [Ca²⁺]_o also may be germane to the genesis of epileptic seizures. In vivorecordings of [Ca²⁺]_o in experimental models of epilepsy have shown dramatic decreases in [Ca²⁺]_o (down to 0.2 mm) during seizure activity (Pumain et al., 1985). These decreases would be expected to enhance intrinsic bursting, which in turn would contribute to the explosive development and spread of seizure activity (Jensen and Yaari, 1997). Consistent with this view is the finding that lowering [Ca²⁺]_o to 0.2 mm or less induces spontaneous population bursts in rat hippocampal slices despite the block of chemical synaptic transmission (Jefferys and Haas, 1982; Taylor and Dudek, 1982; Yaari et al., 1983).