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Differential Cholinergic Modulation of Ca²⁺ Transients Evoked by Backpropagating Action Potentials in Apical and Basal Dendrites of Cortical Pyramidal Neurons

¹Department of Physiology, College of Medicine, and ²Catholic Neuroscience Center, The Catholic University of Korea, Seoul, South Korea

Submitted 18 January 2008; accepted in final form 14 April 2008

	ABSTRACT

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The effect of the cholinergic agonist carbachol (CCh) on backpropagating action potential (bAP)–evoked Ca²⁺ transients in distal apical and basal dendrites of layer 2/3 pyramidal neurons in the primary visual cortex of rats was studied using whole cell recordings and confocal Ca²⁺ imaging. In the presence of CCh (20 µM), initial bAP-evoked Ca²⁺ transients were followed by large propagating secondary Ca²⁺ transients that were restricted to proximal apical dendrites 40 µm from the soma. In middle apical dendrites (41–100 µm from the soma), Ca²⁺ transients evoked by AP bursts at 20 Hz, but not by single APs, were increased by CCh without secondary transients. CCh failed to increase the bAP-evoked Ca²⁺ transients in distal apical dendrites (101–270 µm from the soma). In contrast, in basal dendrites, CCh increased Ca²⁺ transients evoked by AP bursts, but not by single APs, and these transients were relatively constant over the entire length of the dendrites. CCh further increased the enhanced bAP-evoked Ca²⁺ transients in the presence of 4-aminopyridine (200 µM), an A-type K⁺ channel blocker, in basal and apical dendrites, except in distal apical dendrites. CCh increased large Ca²⁺ transients evoked by high-frequency AP bursts in basal dendrites, but not in distal apical dendrites. CCh-induced increase in Ca²⁺ transients was mediated by InsP₃-dependent Ca²⁺-induced Ca²⁺-release. These results suggest that cholinergic stimulation differentially increases the bAP-evoked increase in [Ca²⁺]_i in apical and basal dendrites, which may modulate synaptic activities in a location-dependent manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Action potentials (APs) initiated in the axon hillock of cortical pyramidal neurons actively propagate into dendrites (Stuart and Sakmann 1994) and evoke Ca²⁺ influx in dendrites through voltage-dependent Ca²⁺ channels (Markram et al. 1995; Spruston et al. 1995). These backpropagating AP (bAP)–evoked dendritic Ca²⁺ transients are thought to be involved in dendritic excitability, synaptic plasticity, restoration of intracellular Ca²⁺ ([Ca²⁺]_i) stores, and intracellular metabolic processes (Egorov et al. 1999; Hausser et al. 2001; Magee and Johnston 1997; Waters et al. 2005; Williams and Stuart 2000; Zhou et al. 2005). The amplitude of bAP in apical dendrites of pyramidal neurons decreases with the distance from the soma to the dendrite (Larkum et al. 2007; Spruston et al. 1995). The resulting dendritic Ca²⁺ transients increase in proximal dendrites and then decrease in distal dendrites (Waters et al. 2003). However, the amplitude of bAP-evoked Ca²⁺ transients in distal basal dendrites is similar to that in proximal basal dendrites of hippocampal (Hoogland and Saggau 2004) and layer 5 pyramidal neurons (Nevian et al. 2007). In basal dendrites of layer 2/3 pyramidal neurons, the amplitude of bAP-evoked Ca²⁺ transients in distal dendrites has been reported to be both smaller (Antic 2003) or larger (Cho et al. 2006) than that in proximal dendrites. Synaptic inputs from different areas of the brain are relatively segregated and terminate in isolated dendritic areas (Feldmeyer et al. 2002). Therefore the dendritic location of individual synapses may be critical for the modulation of synaptic activities by the interaction of presynaptic inputs with bAPs and by the resulting Ca²⁺ influx (Koester and Sakmann 1998; Waters et al. 2003).

Cholinergic stimulation facilitates the backpropagation of AP trains (Tsubokawa and Ross 1997) and increases Ca²⁺ transients in apical dendrites of hippocampal (Beier and Barish 2000; Nakamura et al. 2000; Tsubokawa and Ross 1997) and neocortical pyramidal neurons (Larkum et al. 2003; Yamamoto et al. 2000). Under cholinergic stimulation, initial bAP-evoked Ca²⁺ influx induces large secondary propagating Ca²⁺ waves, which are confined to the thick primary apical dendritic shaft and the soma in hippocampal (Nakamura et al. 2000) and neocortical pyramidal neurons (Larkum et al. 2003; Yamada et al. 2004) and in basolateral amygdala (BLA) neurons (Power and Sah 2007). These large secondary Ca²⁺ waves are released from Ca²⁺ stores via inositol 1,4,5-trisphosphate (InsP₃)–receptor activation (Larkum et al. 2003; Nakamura et al. 1999, 2000; Power and Sah 2007). However, little information is available concerning the effect of cholinergic stimulation on Ca²⁺ transients evoked by bAP in fine dendritic arbors such as distal apical and basal dendrites of cortical pyramidal neurons, where the majority of excitatory synaptic inputs terminate in the primary visual cortex (Larkman 1991). Because the postsynaptic modification of each input is known to depend on its dendritic location (Froemke et al. 2005; Hausser et al. 2001; Letzkus et al. 2006), it is important to more thoroughly understand the dendritic location-dependent cholinergic modulation of bAP-evoked Ca²⁺ transients.

The present study investigated the location-dependent effects of cholinergic stimulation on bAP-evoked Ca²⁺ transients in distal apical and basal dendrites of layer 2/3 pyramidal neurons. Results of the present study were previously reported in part in abstract form (Cho et al. 2005).

	METHODS

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Slice preparation

Coronal slices of primary visual cortex were prepared from Sprague–Dawley rats at postnatal days 21–27. Animal care and surgical procedures were conducted with the approval of the Catholic Ethics Committee of the Catholic University of Korea and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After anesthetization with chloral hydrate (400 mg/kg, administered intraperitoneally), the brains were quickly isolated and then immersed in ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 1 CaCl₂, 2 MgSO₄, and 10 D-glucose and aerated with a mixture of 95% O₂-5% CO₂. Coronal sections (300-µm thick) containing the visual cortex were prepared on a vibroslicer (HM650V, Microm, Walldorf, Germany) and allowed to recover in a submerged slice chamber for 30 min at 37°C. The slices were then maintained at room temperature (22–24°C) in the same ACSF prior to recording. The slices were transferred to the recording chamber and superfused continuously (1–1.5 ml/min) with the same solution, except for the addition of 2 mM CaCl₂ and 1 mM MgSO₄. All experiments were performed at 32–33°C.

Whole cell patch-clamp recording

A standard whole cell patch-clamp technique with a bridge amplifier (BVC-700A, Dagan, Minneapolis, MN) was used to record the membrane potential and to evoke somatic APs. The patch electrodes (4–6 M) pulled from borosilicate glass were filled with a solution containing (in mM) 130 K-gluconate, 10 KCl, 3 Mg-ATP, 10 Na₂-phosphocreatine, 0.3 Na₃-GTP, and 10 Hepes (pH 7.25/KOH), supplemented with 50 µM Oregon Green 488 BAPTA-1 (OGB-1; K_d = 170 nM; Molecular Probes, Eugene, OR), 200 µM Fluo-5F (K_d = 2.3 µM, Molecular Probes), or 200 µM Fluo-4FF (K_d = 9.7 µM, Molecular Probes) as a Ca²⁺ indicator. Pyramidal neurons in layer 2/3 of the primary visual cortex (Paxinos and Watson 1997) were recorded using infrared differential interference contrast video-microscopy with an upright microscope (BX51-WI fitted with a x40/0.80 NA water-immersion objective; Olympus, Tokyo, Japan) and their regular spiking patterns and the morphology of the bifurcated apical dendrites were confirmed. Typical access resistance was 15–20 M. Membrane potentials were not corrected for an approximately 14-mV junction potential. Command generation, data acquisition, and analyses were performed with the pClamp 9.2 Suite software (Axon Instruments, Foster City, CA). Data were filtered at 5 kHz, sampled at 20 kHz, and saved to a computer hard drive (Pentium PC).

Cells with soma 350 µm from the surface of the pia were selected. Cells were used when the resting membrane potential (RMP) was sufficiently negative (less than –68 mV). When carbachol (CCh) was present in the bath solution, some cells were depolarized by 5–7 mV. To eliminate the effect of this membrane depolarization, a hyperpolarizing current was injected via the recording pipette, restoring the RMP. In some experiments, CCh was applied locally near (50 µm) the dendritic tree of interest using a patch pipette filled with ACSF. The location of the pipette relative to the dendritic tree of interest and the flow of CCh-containing pipette solution were monitored by using Alexa Fluor 594 (50 µM, Molecular Probes) in the pipette and a 532-nm laser line.

Ca²⁺ imaging

Fluorescence imaging was performed 20 min after obtaining the cells using laser-scanning confocal microscopy (FV-300, Olympus). Light from an argon ion laser (488 nm) was used for illumination and the emitting fluorescence was filtered with a 510-nm long-pass filter. Dendrites were traced from the soma using a fluorescent Ca²⁺ indicator. The intensity of the laser used for excitation of the indicator was adjusted to minimize phototoxic damage. Dendritic Ca²⁺ transients evoked by bAPs, which were generated by brief (2- or 10-ms) current injections into the soma, were measured. The fluorescence signals were obtained using either the line-scan (every 1.4–1.6 ms) or area-scan mode (every 17–24 ms). For the line-scanned data, every 10 data points were averaged; for the area-scan data, dendritic areas 10 µm in length were averaged. The distance of the measured area from the soma was calculated from the center of the soma. Fluorescence signals were background-corrected and traces were expressed as the relative change in fluorescence [F/F₀ = (F – F₀)/F₀], where F₀ is the prestimulus fluorescence. The peak amplitude of the Ca²⁺ transients was determined at the largest F/F₀ value of the transients.

Statistics

All data are expressed as the means ± SE. A Student's t-test and ANOVA with a post hoc Tukey test were used for statistical comparisons. A value of P < 0.05 was considered to be statistically significant.

Drugs

CCh, low-molecular weight heparin, atropine, 4-aminopyridine (4-AP), and all other chemicals, except for ruthenium red (Tocris, Ellisville, MO) and -dendrotoxin (Alomone Labs, Jerusalem, Israel), were purchased from Sigma (St. Louis, MO).

	RESULTS

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Cholinergic modulation of bAP-evoked Ca²⁺ transients along apical dendrites

Whole cell recordings were made from the soma of layer 2/3 pyramidal neurons 350 µm from the surface of the pia in the visual cortex. The RMP of the pyramidal neurons was –79.1 ± 0.5 mV and the input resistance was 138 ± 5 M (n = 63). Somatic APs were generated using a positive-step current injection (10 ms, 0.5–1.1 nA) and the resulting dendritic Ca²⁺ transients evoked by bAPs were measured along the dendrite (Fig. 1A). The peak amplitude of the Ca²⁺ transients evoked by a single AP and by bursts of three and five APs at 20 Hz increased with the distance from the soma in thick apical dendrites and then declined in distal apical dendrites beyond the point of bifurcation (Fig. 1, B and C). This result was consistent with previous reports from our group (Cho et al. 2006) and from Waters et al. (2003).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Location-dependent modulation of carbachol (CCh) on backpropagating action potential (bAP)–evoked Ca2+ transients in apical dendrites. A: a reconstructed layer 2/3 pyramidal neuron of the primary visual cortex after the experiments. The scale bar represents 100 µm. B: Ca2+ transients were evoked by one, 3, and 5 somatic APs (20 Hz, bottom traces) in apical dendrites (shown in black). OGB-1 (50 µM) was used as a calcium dye. The top 3 rows of traces show fluorescence changes (F/F0) by bAPs measured by line scanning for apical dendrites at the positions indicated in A. In the presence of 20 µM CCh in the bath (shown in red), a large secondary Ca2+ increase was observed after bAP-evoked Ca2+ transients in proximal dendrites (p). In middle apical dendrites (m), CCh treatment increased the bAP-evoked Ca2+ transients. In distal apical dendrites (d), CCh failed to increase Ca2+ transients. The increases in Ca2+ transients were abolished by the addition of 10 µM atropine to the bath (shown in blue). C: spatial profile of the peak amplitudes of Ca2+ transients evoked by one (top), 3 (middle), and 5 APs (bottom) in controls and in the presence of CCh. Data were obtained from 44 cells and the number of cells at each distance from the soma is indicated in parentheses. *P < 0.05. D: summary of the effect of CCh on the peak amplitudes of bAP-evoked Ca2+ transients in proximal (10–40 µm from the soma), middle (41–100 µm from the soma), and distal (101–270 µm from the soma) apical dendrites. Data are the percentage increases relative to the control. *P < 0.05, **P < 0.01, ***P < 0.001.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Inositol 1,4,5-trisphosphate (InsP3)–dependent Ca2+-induced Ca2+ release by CCh in proximal and middle apical dendrites. A: traces for the control experiment were obtained with the intracellular application of heparin (1 mg/ml) or ruthenium red (50 µM) (shown in black). CCh (20 µM, shown in gray) had no effect on bAP-evoked Ca2+ transients in the presence of heparin in proximal (30 µm from the soma) and middle (60 µm from the soma) apical dendrites. However, in the presence of ruthenium red, bAP and CCh induced secondary Ca2+ transients and an increase in initial Ca2+ transients in proximal (25 µm from the soma) and middle apical dendrites (60 µm from the soma), respectively. The effects of heparin and ruthenium red were determined by using OGB-1 (50 µM) and large Ca2+ transients are evoked by high-frequency APs (5 APs at 100 Hz, dashed line). B: summary of the effect of heparin and ruthenium red on the CCh-induced increase in Ca2+ transients evoked by 5 APs in proximal (32 ± 2 µm from the soma, n = 6, and 22 ± 2 µm from the soma, n = 7, respectively) and middle apical dendrites (70 ± 5 µm from the soma, n = 5, and 63 ± 7 µm from the soma, n = 4, respectively). Data are average increases relative to the control in response to CCh treatment in the presence of heparin or ruthenium red. CCh data were taken from Fig. 1D for comparison. **P < 0.01 vs. CCh.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Spatial profile of cholinergic increase in Ca2+ transients in the presence of 4-aminopyridine (4-AP). A: spatial profile of cholinergic increases in Ca2+ transients evoked by bAP. Cells showing dendritic structure 150 µm using an area scan were included in the analysis. Either OGB-1 (50 µM, n = 5) or Fluo-5F (200 µM, n = 5) was used as a Ca2+ dye. Gray bars and lines afterward represent the main apical trunk before the main bifurcation and the other parts of the whole dendritic trees, respectively. Symbols denote the most distal location where the analyzed area showed an apparent Ca2+ increase. The panels on the right show a representative cell (asterisk in the left panel) and Ca2+ transients measured at the indicated dendritic area (81 µm from the soma) with area-scanned fluorescent images. CCh (20 µM, gray traces) increased only Ca2+ transients evoked by bursts of 5 APs at this location (black traces). B: spatial profile of cholinergic increase in 4-AP–enhanced Ca2+ transients. Fluo-5F (200 µM) was used as a Ca2+ dye. The panel on the right shows representative Ca2+ transients analyzed at 87 µm from the soma, in a cell indicated with an asterisk in the left panel. 4-AP (1 mM, black traces) enhanced the Ca2+ transients evoked by bAPs (dotted traces). CCh further increased Ca2+ transients evoked by single AP and AP bursts at this location (gray traces). The scale bars in the area-scanned images represent 20 µm.

Mechanism of cholinergic modulation of bAP-evoked Ca²⁺ transients in apical dendrites

The synergistic activation of the mAChR with a bAP-evoked Ca²⁺ influx causes release of Ca²⁺ from InsP₃-sensitive Ca²⁺ stores in proximal apical dendrites and in the soma of neocortical and hippocampal pyramidal neurons (Nakamura et al. 2000; Power and Sah 2002; Yamada et al. 2004). Thus experiments were conducted to determine whether these mechanisms are also responsible for the increase in bAP-evoked Ca²⁺ transients in middle apical dendrites, which are devoid of secondary Ca²⁺ transients. The CCh-induced increase in initial Ca²⁺ transients in middle apical dendrites, as well as secondary Ca²⁺ transients in proximal apical dendrites, was not observed when the solution in the pipette contained heparin (1 mg/ml) (Fig. 3A). The average percentage increases in Ca²⁺ transients evoked by five APs in proximal and middle apical dendrites were –1.1 ± 1.6% (n = 6) and –1.1 ± 3.3% (n = 5) relative to the control, respectively (Fig. 3B). Meanwhile, the application of heparin did not change the amplitude of bAP-evoked Ca²⁺ transients in the absence of CCh (data not shown). Thus the CCh-induced increases in bAP-evoked Ca²⁺ transients were completely blocked by the InsP₃-receptor inhibitor heparin both in middle and in proximal apical dendrites.

To examine the possible involvement of the ryanodine receptor, which is another Ca²⁺-release channel, ruthenium red (50 µM) was added to the solution in the pipette. CCh induced secondary Ca²⁺ transients in proximal apical dendrites and increased initial Ca²⁺ transients in middle apical dendrites in the presence of ruthenium red (Fig. 3A). The average percentage increases in Ca²⁺ transients evoked by five APs in proximal and middle apical dendrites were 32.6 ± 4.1% (n = 7) and 21.9 ± 3.2% (n = 4) relative to the control, respectively. These values are similar to those obtained after CCh treatment in the absence of blockers (Fig. 3B). These results are consistent with previous reports that, in apical dendrites, the CCh-induced enhancement of bAP-evoked Ca²⁺ transients is due to the release of Ca²⁺ from intracellular stores mediated by InsP₃ receptors, but not by ryanodine receptors (Nakamura et al. 1999; Yamamoto et al. 2000). Power and Sah (2007) also reported that the same mechanism is responsible for the increase in initial Ca²⁺ transients evoked by bAPs in BLA neurons. Thus the results of the present study indicate that InsP₃-dependent Ca²⁺-induced Ca²⁺ release (CICR) is responsible for the increases in bAP-evoked Ca²⁺ transients in middle apical dendrites as well as for secondary Ca²⁺ transients in proximal apical dendrites.

Cholinergic modulation of bAP-evoked Ca²⁺ transients in basal dendrites

The amplitude of the Ca²⁺ transients increased with the distance from the soma along basal dendrites when the cells were stimulated with AP bursts [F_(5,57) = 3.29, P < 0.05 for three APs; F_(5,72) = 5.18, P < 0.001 for five APs] (Fig. 4C), consistent with our previous report (Cho et al. 2006). On CCh application, delayed secondary Ca²⁺ transients were observed in only a few cells (8 of 51 cells), which were located within about 20 µm of the soma, in basal dendrites (data not shown). Whereas CCh failed to increase Ca²⁺ transients evoked by single APs, the peak amplitudes of Ca²⁺ transients evoked by AP bursts were enhanced by CCh in most regions of basal dendrites (Fig. 4), similar to the response observed in middle apical dendrites. The effects of CCh in basal dendrites were abolished by addition of atropine to the bath (Fig. 4B) and were reversible on wash-out with normal ACSF (data not shown). Basal dendrites were also classified into two regions for quantitative analysis (Fig. 4D). In proximal basal dendrites (10–60 µm from the soma), CCh increased Ca²⁺ transients evoked by AP bursts (23.1 ± 4.6%, P < 0.001 for three APs and 21.7 ± 2.9%, P < 0.001 for five APs), but not by single APs (3.0 ± 3.5%, P = 0.94). In distal basal dendrites (61–130 µm from the soma), CCh increased only burst-evoked Ca²⁺ transients relative to the control as follows: 11.1 ± 2.5% for three APs (P < 0.05) and 17.7 ± 2.3% for five APs (P < 0.001). These values were similar to those obtained in proximal basal dendrites. These results show that the CCh-induced increases in Ca²⁺ transients evoked by AP bursts occur in all regions of basal dendrites, in contrast to the location-dependent response observed in apical dendrites.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Spatial profile of the effect of CCh on bAP-evoked Ca2+ transients in basal dendrites. A: a reconstructed layer 2/3 pyramidal neuron of the primary visual cortex after the experiments. The scale bar represents 50 µm. B: Ca2+ transients evoked by one, 3, and 5 APs (20 Hz, bottom traces) were recorded in proximal (p; 15 µm from the soma) and distal (d; 80 µm from the soma) basal dendrites, as indicated in A. OGB-1 (50 µM) was used as the calcium dye. In the presence of 20 µM CCh in the bath (shown in red), Ca2+ transients evoked by bursts of 3 and 5 APs (shown in black), but not by a single AP, were enhanced, in both proximal and distal basal dendrites. The increases in Ca2+ transients were abolished by addition of 10 µM atropine to the bath (shown in blue). C: spatial profile of the peak amplitude of Ca2+ transients evoked by one (left), 3 (middle), and 5 APs (right) in the absence (Con) or presence of CCh. Data were obtained from 27 cells and the number of cells at each distance is indicated in parentheses. *P < 0.05. D: summary of the effect of CCh on the peak amplitudes of bAP-evoked Ca2+ transients in proximal (10–60 µm from the soma) and distal (61–130 µm from the soma) basal dendrites. Data are the percentage increases relative to the control. *P < 0.05, **P < 0.01, ***P < 0.001.

Mechanism of cholinergic modulation on bAP-evoked Ca²⁺ transients in basal dendrites

The role of InsP₃ receptors in the CCh-induced increases in bAP-evoked Ca²⁺ transients in basal dendrites was investigated (Fig. 5). When heparin was added to the pipette solution (1 mg/ml), CCh failed to increase bAP-evoked Ca²⁺ transients after five APs in proximal (1.1 ± 2.0% relative to the control, n = 5) and distal (0.5 ± 3.0% relative to the control) basal dendrites, respectively. However, when ruthenium red (50 µM) was added to the solution in the pipette, CCh increased the Ca²⁺ transients evoked by five APs 18.9 ± 7.5 and 12.1 ± 1.9% in proximal and distal basal dendrites, respectively (n = 5). These values were not different from those obtained in the control experiment without heparin or ruthenium red. These results indicate that the CCh-induced increase in bAP-evoked Ca²⁺ transients in basal dendrites is mediated by InsP₃ receptors, but not by ryanodine receptors. Therefore the mechanism of intracellular Ca²⁺ release by cholinergic stimulation does not differ between basal and apical dendrites.

View larger version (25K): [in this window] [in a new window]   FIG. 5. InsP3-dependent Ca2+-induced Ca2+ release by CCh in basal dendrites. A: traces for the control experiment were obtained by the intracellular application of heparin (1 mg/ml) or ruthenium red (50 µM) (shown in black). The CCh-induced (20 µM, shown in gray) increase in bAP-evoked Ca2+ transients was blocked by heparin in proximal (30 µm from the soma) and distal (70 µm from the soma) basal dendrites. However, the CCh-induced increase in bAP-evoked Ca2+ transients was not blocked by ruthenium red in proximal (15 µm from the soma) and distal (62 µm from the soma) basal dendrites. B: summary of the effects of heparin and ruthenium red on the CCh-induced increase in bAP-evoked Ca2+ transients in proximal (21 ± 6 µm from the soma, n = 5, and 28 ± 3 µm from the soma, n = 3, respectively) and distal basal dendrites (72 ± 8 µm from the soma, n = 5, and 74 ± 7 µm from the soma, n = 5, respectively). Data are average increases relative to the control in response to CCh treatment in the presence of heparin or ruthenium red. CCh data were taken from Fig. 4D for comparison. *P < 0.05 vs. CCh.

The increases in Ca²⁺ transients induced by InsP₃-dependent CICR were related to the amplitude of the bAP-evoked Ca²⁺ influx (Figs. 1C and 2). Therefore the relatively small increase in the [Ca²⁺]_i peak evoked by bAPs may be insufficient to induce Ca²⁺ release from stores during CCh application in distal apical dendrites (Nakamura et al. 2000; Power and Sah 2007). To test this possibility, 4-AP was added to the bath solution to enhance bAP-evoked Ca²⁺ transients in distal apical dendrites. Fluo-5F (200 µM) was used to prevent saturation of the fluorescence signal. Because apparent increases in Ca²⁺ transients were observed at 100 µM 4-AP in distal apical dendrites (206 ± 14 µm from the soma, n = 7) and the EC₅₀ of bAP-evoked Ca²⁺ transients was about 200 µM (see Supplemental Fig. S2), 200 µM 4-AP was used to evoke large Ca²⁺ transients with reduced background excitation of the slice. With the application of 4-AP, Ca²⁺ transients evoked by five APs were enhanced by 90–450% (256.4 ± 67.4%, n = 7, P < 0.01; mean distance: 198 ± 13 µm from the soma) relative to the control (Fig. 6A). However, CCh (20 µM) did not result in an additional increase in bAP-evoked Ca²⁺ transients (–16.3 ± 5.6% from 4-AP treatment, P = 0.51). In distal basal dendrites, bAP-evoked Ca²⁺ transients were enhanced by 56.4 ± 5.2% relative to the control in the presence of 4-AP (five APs; n = 6, P < 0.001) (Fig. 6B). In contrast to distal apical dendrites, coapplication of 4-AP and CCh increased Ca²⁺ transients by 81.9 ± 10.8% relative to the control, which was significantly greater than the effect of treatment with 4-AP alone (P < 0.05). Similar increases were also observed in proximal basal dendrites (data not shown). In a previous study, bAP-evoked Ca²⁺ transients in basal dendrites of layer 5 pyramidal neurons were not enhanced by treatment with 100 µM 4-AP (Kampa and Stuart 2006). Because -dendrotoxin (250 nM), a D-type K⁺ channel blocker, did not change Ca²⁺ transients in distal apical dendrites (221 ± 13 µm from the soma, n = 8) in this experiment (see Supplemental Fig. S3), 4-AP (200 µM) enhanced dendritic Ca²⁺ transients via inhibition of A-type K⁺ channels in distal apical dendrites. The EC₅₀ of 4-AP on the Ca²⁺ transients in distal apical dendrites (200 µM) in the present experiment was much smaller than the IC₅₀ of 4-AP on the A-type K⁺ channels (1.4–4.2 mM), which were measured by direct electrophysiological recording (Hoffman et al. 1997; Korngreen and Sakmann 2000). The accumulating effect of 4-AP on the backpropagation of somatic APs en route to the distal dendritic location might explain these discrepancies. These results are consistent with a report that A-type K⁺ channels are involved in the backpropagation of somatic APs in basal dendrites of layer 5 pyramidal neurons (Nevian et al. 2007). In summary, CCh further increased 4-AP–enhanced Ca²⁺ transients in whole dendritic trees, except for distal apical dendrites of layer 2/3 pyramidal neurons in the visual cortex.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effect of CCh on enhanced bAP-evoked Ca2+ transients by 4-AP in distal apical and basal dendrites. A: effect of CCh on enhanced Ca2+ transients in distal apical dendrites. Fluo-5F (200 µM) was used to prevent saturation of the fluorescence signal. The left panel shows representative traces of Ca2+ transients in an apical dendrite (210 µm from the soma) evoked by 5 APs at 20 Hz in response to the following treatments: normal artificial cerebrospinal fluid (ACSF; Control, shown in black); 4-AP (200 µM, shown in green); 4-AP and CCh (20 µM) (shown in red); and after wash-out of both drugs (shown in blue). Saturated Ca2+ transients were evoked by high-frequency APs (5 APs at 100 Hz, dashed trace). The right panel shows a summary of the effect of 4-AP and CCh in distal apical dendrites (198 ± 13 µm from the soma, n = 7). B: effect of CCh on enhanced Ca2+ transients in distal basal dendrites. The left panel shows representative traces of Ca2+ transients in a basal dendrite (74 µm from the soma) evoked by 5 APs at 20 Hz in response to the following treatments: normal ACSF (Control, shown in black); 4-AP (shown in green); 4-AP and CCh (shown in red); and after wash-out of both drugs (shown in blue). The right panel shows a summary of the effect of 4-AP and CCh in distal basal dendrites (77 ± 4 µm from the soma, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control; #P < 0.05 vs. 4-AP.

The effects of CCh on Ca²⁺ transients evoked by high-frequency AP bursts on fine distal apical and basal dendrites were investigated in the next experiment. In distal apical dendrites, increasing Ca²⁺ transient amplitudes were evoked by increasing the frequency of the three APs 100 Hz (Fig. 7A ). However, a burst of three APs at 133 Hz evoked smaller-amplitude Ca²⁺ transients than three APs at 100 Hz, suggesting that bursts at frequencies >100 Hz do not efficiently propagate to the fine distal apical dendrites. The normalized ratio of peak Ca²⁺ transients evoked by bursts of three APs at 100 Hz relative to those evoked by single APs showed a supralinear increase in Ca²⁺ transients in distal apical dendrites (5.3 ± 1.0, 186 ± 7 µm from the soma, n = 9) (Fig. 7C). CCh failed to increase the supralinear Ca²⁺ transients evoked by bursts of three APs at high frequencies (P = 0.45) (Fig. 7A). In distal basal dendrites, the increase in Ca²⁺ transients was linear (133 Hz and 110 µm from the soma; data not shown), but was enhanced by CCh application (Fig. 7B). Supralinear Ca²⁺ transients in fine distal apical dendrites in layer 2/3 pyramidal neurons were evoked by additional dendritic electrogenesis above the critical frequency (Larkum et al. 2007). In this experiment, however, the increase in Ca²⁺ transients in basal dendrites was linear, which is different from the supralinear increase in layer 5 pyramidal neurons (Kampa and Stuart 2006). In summary, the differential effect of CCh on bAP-evoked dendritic Ca²⁺ transients in basal and apical dendrites was not dependent on AP frequency.

View larger version (35K): [in this window] [in a new window]   FIG. 7. CCh failed to increase high-frequency bAP-evoked supralinear Ca2+ transients in distal apical dendrites. A: effect of CCh on supralinear Ca2+ transients in distal apical dendrites. Fluo-4FF (200 µM) was used as a fluorescent dye. The inset shows the dendritic location where Ca2+ transients were measured (175 µm from the soma). The scale bar is 100 µm. The left and middle panels show representative traces of Ca2+ transients evoked by 3 APs at 20 and 100 Hz (shown in black), respectively, and the effect of CCh (shown in gray). The right panel shows the summary of the effect of CCh on Ca2+ transients evoked by 3 APs at 20 to 133 Hz in distal apical dendrites. B: effect of CCh on large Ca2+ transients in distal basal dendrites. The inset shows the dendritic location where Ca2+ transients were measured (105 µm from the soma). The scale bar is 50 µm. The left and middle panels show representative traces of Ca2+ transients evoked by 3 APs at 20 and 133 Hz (shown in black), respectively, and the effect of CCh (shown in gray). The right panel shows a summary of the effect of CCh on Ca2+ transients evoked by 3 APs at 20 to 133 Hz in distal basal dendrites. *P < 0.05 vs. the corresponding control. C: normalized ratio of Ca2+ transients evoked by 3 APs relative to a single AP at different frequencies. Three APs at 100 Hz evoked supralinear Ca2+ transients in distal apical dendrites.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Location dependence of InsP₃-dependent CICR

The influx of extracellular Ca²⁺ induces InsP₃-dependent CICR under the stimulation of generating subthreshold InsP₃, at which InsP₃ alone induces no Ca²⁺ release from the InsP₃-sensitive store (Larkum et al. 2003; Nakamura et al. 2000; Power and Sah 2002; Yamamoto et al. 2000). Because this Ca²⁺ release is from InsP₃-sensitive stores, it appears to depend primarily on the density of the InsP₃ receptor (InsP₃R) and on the capacity of the store. Thus there is a correlation between the distribution of InsP₃R1 (Hertle and Yeckel 2007; Sharp et al. 1993) and the spatial profile of the propagating Ca²⁺ wave evoked by a combined increase in InsP₃ and intracellular Ca²⁺ (Hagenston et al. 2008; Nakamura et al. 2002) in the soma and proximal apical dendrites. Moreover, the propagating Ca²⁺ wave starts at a branching point (Nakamura et al. 2002) where InsP₃R1 immunoreactivity is highly clustered (Hertle and Yeckel 2007). The spatial profile of the CCh-induced InsP₃-dependent CICR along the apical dendritic tree to the distal part (270 µm from the soma) in the present study is consistent with the distribution of InsP₃R1 in the hippocampal pyramidal neurons (Hertle and Yeckel 2007), supporting the idea that the density of InsP₃R is crucial for InsP₃-dependent CICR in pyramidal neurons. Because Ca²⁺ acts as a coagonist on InsP₃R1 (Bezprozvanny et al. 1991), the magnitude of the Ca²⁺ influx is also responsible for the extent of InsP₃-dependent CICR, as shown in the present (Fig. 2) and in previous (Larkum et al. 2003) studies. However, a certain level of InsP₃R and Ca²⁺ release pools seems to be a prerequisite for the Ca²⁺ release from InsP₃-sensitive stores because a high increase in [Ca²⁺]_i did not induce Ca²⁺ release in the fine distal apical dendrites in the presence of CCh and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 µM, n = 4; data not shown), a metabotropic glutamate receptor agonist. In addition, the organization of InsP₃ signaling microdomains (Delmas et al. 2002; Jacob et al. 2005) and different affinities of InsP₃R subtypes for InsP₃ (Hagar et al. 1998; Wojcikiewicz and Luo 1998) might also be responsible for the differential effects of CCh along the apical dendritic trees.

In the present study, we observed CCh-induced InsP₃-dependent CICR in young animals (3 wk old). It is known that InsP₃R1 immunoreactivity becomes more evenly distributed along the apical dendritic tree during maturation in hippocampal pyramidal neurons, although the general patterns are similar in both young and adult rats (Hertle and Yeckel 2007). Thus detailed studies on the distribution of InsP₃R in cortical pyramidal neurons and spatial profiles of InsP₃-dependent CICR with age are warranted.

Location-dependent cholinergic regulation of bAP-evoked dendritic Ca²⁺ transients

CCh failed to enhance Ca²⁺ transients evoked by single APs or by AP bursts in distal apical dendrites. The cholinergic effect on bAP-evoked Ca²⁺ transients may require a minimum threshold [Ca²⁺]_i with respect to the duration or amplitude of the Ca²⁺ influx evoked by bAP to activate InsP₃-dependent CICR, as suggested by Nakamura et al. (2000). The correlation between the magnitude of the cholinergic effect and the amplitude of bAP-evoked Ca²⁺ transients in proximal and middle dendrites (Figs. 1 and 2) supports this hypothesis. However, experimental evidence suggests that this scenario is unlikely in distal apical dendrites, where CCh treatment did not enhance the robust increase in [Ca²⁺]_i that resulted from blocking A-type K⁺ channels (Fig. 6). Furthermore, CCh did not modulate the supralinear Ca²⁺ transients that were evoked by high-frequency APs (Fig. 7). Absence of CCh-induced increases in bAP-evoked Ca²⁺ transients has also been reported in distal dendrites of BLA neurons, where uncaging of InsP₃ did not increase bAP-evoked Ca²⁺ transients (Power and Sah 2007). Our results suggest that mAChRs and/or InsP₃-dependent CICR might be insufficient to evoke InsP₃-dependent CICR in distal apical dendrites. It appears that relatively little mAChR immunoreactivity is present in the distal apical dendrites of cortical layer 2/3 pyramidal neurons (Mrzljak et al. 1993; van der Zee and Luiten 1999). Because t-ACPD as well as CCh induced no increase in Ca²⁺ transients in the present study, the downstream pathway from InsP₃ in these locations might be different from that in the soma, proximal apical, and basal dendrites (Hertle and Yeckel 2007; Power and Sah 2007). However, it is of interest whether other experimental conditions, such as synaptic stimulations, could induce Ca²⁺ release from the stores in distal dendrites and spines.

Cholinergic regulation of bAP-evoked Ca²⁺ transients in basal dendrites

Whereas only slight amplitude modulation of bAP has been detected in basal dendrites of layer 2/3 pyramidal neurons by the use of voltage-sensitive dyes (Antic 2003), direct patch-clamp recordings demonstrated much greater attenuation of bAP in basal dendrites than that in apical dendrites of layer 5 pyramidal neurons (Nevian et al. 2007). Layer 2/3 and layer 5 pyramidal neurons exhibited differences in dendritic propagation of somatic APs (Larkum et al. 2007). To date, direct electrophysiological recordings of the basal dendrite of layer 2/3 pyramidal neurons have not been reported. However, the greater amplitude of bAP-evoked Ca²⁺ transients in distal basal dendrites observed in this experiment appears to result from the slight amplitude modulation of bAPs (Antic 2003), the increased duration of bAPs, and/or the surface-to-volume ratio (Nevian et al. 2007). In the present study, CCh uniformly increased Ca²⁺ transients evoked by AP bursts along basal dendrites. However, secondary slow Ca²⁺ transients were not observed in basal dendrites, except in the immediate vicinity of the soma (10 µm of initial dendritic branches) in a few cells. As was observed in apical dendrites, cholinergic stimulation increased bAP-evoked Ca²⁺ transients via mAChRs and InsP₃-dependent CICR. Interestingly, this cholinergic effect was observed when the Ca²⁺ transients were evoked by AP bursts, but not by single APs, which is similar to the pattern observed in middle apical dendrites. Although information on frequency-dependent modulation of the bAP amplitude in basal dendrites is equivocal (Antic 2003; Kampa and Stuart 2006), the results of the present study support that cholinergic stimulation reduces the frequency-dependent modulation of AP amplitude in basal dendrites as it does in apical dendrites (Tsubokawa and Ross 1997).

Because the depth from the cut surface of the slices to the measured areas did not differ between basal and distal apical dendrites under the experimental conditions used in the present study, differences in CCh diffusion between the two sites are unlikely. A similar result was observed with local application of CCh onto the dendritic tree (see Supplemental Fig. S1). Again, these findings suggest that the distribution of InsP₃-dependent CICR and/or mAChRs differs between basal and distal apical dendrites.

Physiological implications of the location-dependent effect of cholinergic stimulation

Propagating Ca²⁺ waves in the soma and proximal dendrites control intrinsic excitability and the firing patterns in pyramidal neurons of the prefrontal cortex (Hagenston et al. 2008). Ca²⁺ release evoked by muscarinic activation could control the neuronal excitability (Gulledge et al. 2007; Yamada et al. 2004). Dendritic Ca²⁺ transients evoked by bAPs are heterogeneous in cortical pyramidal neurons due to the decreasing bAP amplitude, the complex geometry of the dendritic tree (Vetter et al. 2001), and the differential distribution of ion channels (Frick et al. 2003; Hoffman et al. 1997; Schiller et al. 1995; Smith et al. 2003) and of intracellular Ca²⁺ stores (Blaustein and Golovina 2001; Johenning et al. 2002; Pozzo-Miller et al. 2000). These mechanisms allow pyramidal neurons to enhance their integration of synaptic activities via the use of multiple compartments (Antic 2003; Berger et al. 2003; Larkum et al. 2001; Poirazi et al. 2003). The complex spatial profile of the cholinergic effect on bAP-evoked dendritic Ca²⁺ transients suggests an additional mechanism for the modulation of synaptic activity, which might be dependent on dendritic location (Froemke et al. 2005).

Because the presynaptic inputs to layer 2/3 pyramidal neurons in the primary sensory cortex appear to be segregated, terminating in localized dendritic areas of different cortical layers (Binzegger et al. 2004; Feldmeyer et al. 2002; Lubke et al. 2003), modulation of synaptic activity by cholinergic stimulation might differ between distal apical and basal dendrites, where the most excitatory synaptic inputs terminate (Larkman 1991). Furthermore, differences between distal apical and basal dendrites in bAP-evoked Ca²⁺ transient profiles and their cholinergic modulation may be involved in experience-dependent changes in altering the dynamics of cortical networks to allow learning of new information (Hasselmo and Bower 1992; Kimura et al. 1999).

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was supported by the Neurobiology Research Program made available by Korea Ministry of Science and Technology Grant M10412000082-04N1200-08210.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. Jens Eilers for critical comments and advice.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The online version of this article contains supplemental data.

Address for reprint requests and other correspondence: D.-J. Rhie, The Catholic University of Korea, College of Medicine, Department of Physiology, 505 Banpo-dong, Seocho-gu, Seoul 137-701, South Korea (E-mail: djrhie{at}catholic.ac.kr)

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