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The Journal of Physiology logoLink to The Journal of Physiology
. 2006 Jun 29;575(Pt 2):455–468. doi: 10.1113/jphysiol.2006.114231

Modulation of calcium wave propagation in the dendrites and to the soma of rat hippocampal pyramidal neurons

Shigeo Watanabe 1, Min Hong 1, Nechama Lasser-Ross 1, William N Ross 1
PMCID: PMC1819440  PMID: 16809362

Abstract

Repetitive synaptic stimulation in the stratum radiatum (SR) evokes large amplitude Ca2+ waves in the thick apical dendrites of hippocampal CA1 pyramidal neurons. These waves are initiated by activation of metabotropic glutamate receptors (mGluRs), which mobilize inositol-1,4,5-trisphospate (IP3) and release Ca2+ from intracellular stores. We explored mechanisms that modulate the spatial properties of these waves. Higher stimulus current evoked waves of increasing spatial extent. Most waves did not propagate through the soma; the majority stopped close to the junction of the soma and apical dendrite. Pairing strong stimulation with one electrode and subthreshold stimulation with another (associative activation) extended the waves distally but failed to extend waves into the cell body. Pairing synaptic stimulation with backpropagating action potentials enhanced the likelihood of wave generation but did not extend the waves to the somatic region. Priming the stores with Ca2+ entry through voltage dependent channels modulated wave properties but did not extend them past the dendrites. These results are consistent with propagation failing due to the dilution of synaptically generated IP3 as it diffuses into the large volume of the soma (impedance mismatch). Synaptically activating waves in the presence of low concentrations of carbachol, which probably increased the tonic level of IP3 throughout the cell, enhanced the extent of propagation and generated waves that invaded the soma, as long as low-affinity indicators were used to detect the [Ca2+]i changes. Consistent with this explanation direct injection of IP3 into the soma promoted wave propagation into this region. Ca2+ waves that propagated through the cell body were interesting because they did not fill the volume of the soma, but passed through the centre, often with large amplitude. These waves may be particularly effective in activating gene expression and protein synthesis.

In most neurons of the CNS, synaptic activity evokes postsynaptic [Ca2+]i increases in addition to electrical responses. The three main pathways for evoking [Ca2+]i changes are Ca2+ entry through voltage gated channels, Ca2+ entry through ligand gated channels, and release of Ca2+ from intracellular stores. These three mechanisms generate [Ca2+]i increases with different spatial and temporal characteristics (Nakamura et al. 2002; Augustine et al. 2003). In hippocampal pyramidal neurons, the ligand gated [Ca2+]i increase is primarily due to entry through NMDA receptors on spines on the oblique dendrites; the voltage gated entry is predominantly due to backpropagating action potentials and is found throughout the dendrites; and Ca2+ release from stores, mediated by mGluR mobilization of IP3, is most prominent in the primary apical dendrites (Nakamura et al. 2002). The amplitude and spatial extent of each of these sources of Ca2+ is variable and can be modulated by intrinsic conductances and the pattern of stimulation.

The sites of Ca2+ release from stores may be particularly interesting because the large amplitude (several micromolar) and duration (0.5–1.5 s) of the [Ca2+]i increases from this source suggest that they could be effective in activating a number of downstream signalling mechanisms. Some inhibitory neurons target the apical dendrite close to the cell body and the IPSPs they evoke are known to be modulated by large postsynaptic Ca2+ signals (e.g. Wilson & Nicoll, 2001; Alger, 2002). In addition, several papers (Spacek & Harris, 1997; Berridge, 1998; Oertner & Svoboda, 2002; Nakamura et al. 2002) have suggested that synaptically activated waves could propagate through this region to the soma where large [Ca2+]i changes could affect protein synthesis, gene expression (Ghosh & Greenberg, 1995; Hardingham et al. 2001) and long-term synaptic plasticity (e.g. Yeckel et al. 1999).

Examination of previous results indicates that there is variability in the reported extent of Ca2+ wave propagation in the dendrites and soma of pyramidal neurons. Jaffe & Brown (1994) induced waves in these cells with ionophoretic glutamate pulses. While they did not systematically analyse these waves, examples in their paper show propagation through the soma. Similarly, Power & Sah (2002) evoked Ca2+ waves that entered the soma and passed through the nucleus following acute application of muscarine or carbachol (CCh). They also found that synaptic activation of cholinergic fibres enhanced spike-evoked Ca2+ signals in the soma, although the effect was small. In contrast, in our anecdotal experience (Nakamura et al. 1999, 2002; Zhou & Ross, 2002) most synaptically activated Ca2+ waves appeared to be confined to the dendritic region and rarely entered the soma. Therefore, we decided to examine more systematically the extent of wave propagation and to investigate conditions that modulate the spatial properties of Ca2+ release, in particular those mechanisms that could extend propagation into and through the cell body.

We found that focal stimulation in the SR evoked waves of variable extent that almost never propagated through the cell body; most waves stopped at the soma–dendrite border. Associative stimulation with a secondary electrode near the soma, pairing synaptic stimulation with backpropagating action potentials, or priming of stores with action potential-evoked Ca2+ entry also failed to extend the waves into the soma. However, bath application of low concentrations of carbachol under appropriate conditions or the direct injection of IP3 into the cell body consistently allowed synaptically activated waves to propagate through the soma. These waves, and the few waves that propagated through the soma without modulators, had high amplitude in the centre of the cell, in contrast to spike-evoked [Ca2+]i increases, which were largest just under the membrane. Some of these results have been published previously in abstract form (Hong et al. 2005).

Methods

Whole-cell recording

Experiments were performed on transverse hippocampal slices (300 μm thick) prepared from 2- to 4-week-old Sprague-Dawley rats (Nakamura et al. 1999, 2002). Animals were anaesthetized with isoflurane and decapitated using procedures approved by the Institutional Animal Care and Use Committee of New York Medical College. Slices were cut in an ice-cold solution consisting of (mm): 120 choline-Cl, 3 KCl, 8 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10–20 glucose. They were incubated for at least 1 h in normal artificial cerebrospinal fluid (ACSF) composed of (mm): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 or 20 glucose, bubbled with a mixture of 95% O2–5% CO2, making the final pH 7.4. The same solution was used for recording.

Submerged and superfused slices were placed in a chamber mounted on a stage rigidly bolted to an air table and were viewed with a 40× or 60× water-immersion lens in an Olympus BX50WI microscope mounted on an X–Y translation stage. Somatic whole-cell recordings were made using patch pipettes pulled from 1.5 mm outer diameter thick-walled glass tubing (1511-M, Friedrich and Dimmock, Millville, NJ, USA). Tight seals on CA1 pyramidal cell somata were made with the ‘blow and seal’ technique using video-enhanced DIC optics to visualize the cells (Sakmann & Stuart, 1995). For most experiments the pipette solution contained (mm): 140 potassium gluconate, 4 NaCl, 4 Mg-ATP, 0.3 Na-GTP, and 10 Hepes, pH adjusted to 7.2–7.4 with KOH. This solution was supplemented with 150–200 μm bis-fura-2 (a high-affinity indicator) or 300 μm furaptra (a low-affinity indicator; Molecular Probes, Eugene, OR, USA). Most experiments were repeated using both indicators. Synaptic stimulation was evoked with 100 μs pulses with glass or tungsten electrodes placed on the slice about 10–30 μm to the side of the main apical dendritic shaft and at varying distances from the soma. The glass electrodes were low resistance patch electrodes (less than 5 MΩ) filled with ACSF with a tungsten wire glued to the side. The bipolar tungsten electrodes (used in only a few experiments) had one sharpened electrode (WPI, model TM33B01KT) with a second tungsten wire glued to the side about 1 mm behind the tip of first electrode (Nakamura et al. 1999). Temperature in the chamber was maintained between 31 and 33°C. trans-1-Amino-cyclopentyl-1,3-dicarboxylate (t-ACPD) was obtained from Tocris-Cookson (Ellisville, MO, USA). All other chemicals were obtained from Fisher Scientific (Piscataway, NJ, USA) or Sigma Chemical (St Louis, MO, USA).

Dynamic [Ca2+]i measurements

Time-dependent [Ca2+]i measurements from different regions of the pyramidal neuron were made as previously described (Lasser-Ross et al. 1991; Nakamura et al. 2002). Briefly, a Photometrics (Tucson, AZ, USA) AT300 or Quantix cooled CCD camera, operated in the frame transfer mode, was mounted on the camera port of the microscope. Custom software (original version described in Lasser-Ross et al. 1991) controlled readout parameters and synchronization with electrical recordings. A second custom program was used to analyse and display the data. Pixels were binned in the cameras to allow frame rates of 30–50 Hz. Fluorescence changes of bis-fura-2 and furaptra were measured with single wavelength excitation (382 ± 10 nm) and emission >455 nm. [Ca2+]i changes are expressed as −ΔF/F, where F is the fluorescence intensity when the cell is at rest and ΔF is the change in fluorescence during activity. Corrections were made for indicator bleaching during trials by subtracting the signal measured under the same conditions when the cell was not stimulated.

To examine the spatial distribution of postsynaptic [Ca2+]i changes, we selected pyramidal neurons that were in the plane of the slice and close to the surface. In these neurons, we could examine [Ca2+]i increases over a range of 140–230 μm with the cameras and lenses selected for these experiments. Increases in different parts of the cell are displayed using either selected regions of interest (ROIs) or a pseudo ‘line scan’ display (Nakamura et al. 2000).

Results

Previous experiments from our laboratory (Nakamura et al. 1999, 2002) showed that synaptically activated Ca2+ release was regenerative and was localized in the main apical dendrite near the stimulating electrode. However, the spatial modulation of Ca2+ release was not studied in detail. In the first series of experiments we examined the effects of increasing the stimulus current on wave parameters. Figure 1A shows a typical experiment where Ca2+ waves were evoked in a pyramidal cell filled with bis-fura-2 using repetitive stimuli of increasing intensity. At threshold intensity (70 μA) a small event was evoked in the apical dendrites near the stimulating electrode, localized at a branch point near the stimulating electrode. As the stimulation intensity increased the amplitude of ΔF/F at the ROI positioned at the initiation site (related to the change in [Ca2+]i; Lev-Ram et al. 1992) increased rapidly and then appeared to saturate (Nakamura et al. 1999). In contrast, the spatial extent of the release wave increased more gradually, as illustrated with the ‘line scan’ plots. For these initial experiments we maintained the amplitude of the summating EPSPs below action potential threshold (in some cases by injecting hyperpolarizing current into the somatic electrode) to avoid the complication of the synergistic action of spike evoked calcium entry and synaptic mobilization of IP3 (Nakamura et al. 1999; see Fig. 5). Subthreshold EPSPs by themselves evoke little [Ca2+]i increase in the proximal apical dendrites or soma (e.g. see Fig. 3). This was the typical pattern of response over many experiments, with some variation mostly related to the position of the stimulating electrode and the branching pattern of the dendrites. A plot of the average signal amplitude and wave extent as a function of the stimulation current for six cells is shown below the traces, confirming that the waves spread over a larger area with increasing intensity.

Figure 1. Synaptic stimulation at increasing intensities evokes Ca2+ waves that extend over increasing lengths of the apical dendrite.

Figure 1

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A, cell image showing a pyramidal neuron filled with 150 μm bis-fura-2. The patch electrode is on the soma and the position of the stimulating electrode is shown with a dotted arrow. The set of pixels through the soma and dendrite indicate the position of the ‘line scan’ in the other panels. The box indicates the ROI (region of interest) at a branch point from where the individual optical traces were recorded. The series of panels show the fluorescence changes and electrical responses to a series of tetanic stimuli (100 Hz for 0.5 s) at increasing intensity. The threshold current was 70 μA and increments above that value are indicated below the pseudocolour images. The optical, electrical and ‘line scan’ panels all have the same time scale. At each intensity the wave initiated at a location close to the branch point covered by the ROI, as determined by the location of the earliest fluorescence change. At this location the amplitude jumped to an approximately steady level for intensities above 5 μA above threshold. However, the extent of the wave, indicated by the region of fluorescence change in the ‘line scans’, increased with higher intensities. The average amplitude and spatial extent of the waves as a function of the stimulus intensity are plotted below. Plots are referenced to the threshold current since the threshold was different for different cells. The large error bars reflect the variation from cell to cell; the plots for individual cells showed smooth increments. B, similar display and plots for five neurons filled with 300 μm furaptra. The amplitude and spatial extent of the waves gradually increased with stimulus intensity. The striping in the images is due to the fact that not all pixels were positioned over the centre of the dendritic processes.

Figure 5. Synaptic stimulation and action potentials together do not promote wave propagation into the soma.

Figure 5

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A, synaptic stimulation (100 Hz for 1 s) followed by 10 intrasomatically evoked spikes at 30 ms intervals. The spikes triggered the wave but the wave did not enter the soma. The [Ca2+]i increases in the soma peaked at the time of the last action potential suggesting that they were due to Ca2+ entry through voltage-dependent channels. B, a similar experiment using the indicator furaptra where a Ca2+ wave was first generated with a subthreshold tetanus (100 Hz for 0.5 s) and then generated with the same tetanus but with a burst of intrasomatically evoked action potentials. In both cases the wave had about the same spatial extent in the dendrites. The pseudocolour images were filtered with a five point interpolation in the spatial dimension.

Figure 3. Associative extension of Ca2+ wave propagation.

Figure 3

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A, left, a pyramidal neuron filled with 300 μm furaptra from the patch electrode on the soma. The positions of two glass stimulating electrodes are indicated in the top image, which also show the pixels underlying the line scans. The bottom image is the same cell with ROIs. Right, the electrical and optical responses when electrode 1 was stimulated (100 Hz for 0.5 s at 70 μA), electrode 2 was stimulated at the same rate at 115 μA or when both electrodes were stimulated together at those intensities (70 and 115 μA). When only electrode 1 was stimulated the line scan image shows that wave propagation extended from the soma to about 75 μm from the soma. Stimulation of electrode 2 caused no detectable [Ca2+]i change in the cell. Stimulation with both electrodes (offset by 5 ms to avoid synergistic activation of presynaptic fibres) evoked a wave that extended about an additional 25 μm into the dendrites. Only this protocol evoked a [Ca2+]i increase at the distal ROI (green box and trace). The electrical, optical, and line scan traces all have the same time scale. B, summary of results for five cells. The limits of wave propagation in the distal and proximal directions relative to the initiation site are shown. Associative stimulation distal to the stimulating electrode extended the wave front distally in all cells but had no effect on propagation in the proximal direction. The limit of wave propagation was defined as the point where the amplitude fell to 30% of the peak amplitude. C, failure to associatively extend wave propagation into the soma. At all tested intensities stimulation by electrode 2, placed close to the soma, failed to evoke a [Ca2+]i increase in the cell and failed to extend the wave generated by stimulation of electrode 1. Note the hyperpolarizing electrical response to stimulation by electrode 2, in contrast to the depolarizing response to the electrode placed in the dendritic region. D, summary of results for eight cells. In five cells the wave front extended proximally but in no case did the wave propagate through the soma.

Since bis-fura-2 is a strong Ca2+ buffer we were concerned that it might affect regenerative release and propagation of the Ca2+ wave (Nakamura et al. 2002). Therefore, we repeated the experiments using the low-affinity Ca2+ indicator furaptra (Fig. 1B). Using this indicator the spatial extent increased gradually, similar to the results with bis-fura-2, showing that the extent of propagation was not significantly affected by the affinity of the indicator. In experiments with either indicator the waves appeared to be confined to the proximal apical dendritic region of the pyramidal neurons (Nakamura et al. 1999).

A second reason for repeating the experiments with furaptra was that the signal amplitude saturated in experiments using the high-affinity indicator bis-fura-2, Using furaptra the amplitude, as expected, did not saturate and grew gradually after an initial jump as the stimulus intensity increased. This pattern is consistent with regenerative release although it is not strictly ‘all-or-none’. The peak change of 15% corresponds to a [Ca2+]i increase of about 7 μm (Nakamura et al. 1999) and is much higher than can be detected accurately with bis-fura-2 (Larkum et al. 2003).

As discussed previously (Nakamura et al. 2002), a reasonable hypothesis for the extent of wave propagation is that increasing stimulus intensity activates more synapses, which mobilize increasing amounts of IP3 distributed over a region largely determined by the synaptic sites contacted by the activated presynaptic fibres. The regenerative propagating wave fails when it reaches a region with low IP3 concentration ([IP3]i).

Since the region of Ca2+ wave activation was generally near the location of the stimulation electrode (Nakamura et al. 1999) and this region expanded with increasing stimulation intensity, we thought that stimulating near the soma with sufficiently high intensity would evoke waves that entered the soma. However, that was generally not the case. Figure 2A shows a typical response that generated large amplitude [Ca2+]i increases in the dendrite close to the cell body, but almost no increase in the soma. Figure 2B summarizes the results of many experiments where stimulation was near the soma (exact positions shown in the figure). Almost every wave stopped near the soma–dendrite border when the high-affinity indicator bis-fura-2 was included in the patch pipette. Using the low-affinity indicator furaptra to reduce Ca2+ buffering of regenerative propagation slightly extended the zone of propagation but did not change the general conclusion (Fig. 2C). However, a few waves propagated through the soma to the basal dendrites. These will be discussed below.

Figure 2. Most synaptically activated Ca2+ waves stop near the soma–dendrite boundary.

Figure 2

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A, [Ca2+]i changes recorded at three locations near the soma in a pyramidal neuron in response to tetanic stimulation at 100 Hz for 0.5 s. At the two dendritic locations (red and green boxes) the Ca2+ release signal was large, with almost 40% change in bis-fura-2 fluorescence. However, at the somatic location (blue box) the change was much smaller. The wave clearly failed at a location between the red and blue boxes, where the dendrite joins the soma. B, distribution of stop locations of synaptically activated propagating Ca2+ waves when bis-fura-2 (150 μm) was used as the calcium indicator. Stop location was defined as the point where the amplitude of the wave fell to 30% of the peak amplitude. For each cell the stop location and the position of the stimulating electrode are shown. Histogram binning of these locations is shown below against a canonical pyramidal neuron with cell body length of 25 μm. Only a few waves propagated into the centre of the soma. C, the same analysis for experiments where furaptra (300 μm) was used as the indicator. A similar distribution of stop locations was found.

We tested four physiologically relevant protocols that might allow wave propagation into the soma. One possibility is that associative stimulation of additional synapses could modulate the spatial extent of wave generation. The underlying idea is that these additional inputs mobilize IP3 that, by itself, rises to a concentration below threshold level for initiating a Ca2+ release wave. However, if this bolus of IP3 is in the path of a propagating wave it would combine with the regeneratively released Ca2+ to extend the wave into this region since activation of the IP3 receptor requires both Ca2+ and IP3 (Iino, 1990; Bezprozvanny et al. 1991; Finch et al. 1991). Figure 3A shows a successful example of associative extension in the distal direction. Focal tetanic stimulation from electrode 1 evoked a wave in the proximal apical dendrites. Focal stimulation from electrode 2 (at the specified intensity) failed to generate Ca2+ release at any location in the dendrites. However, when both electrodes were tetanically activated together the wave extended further into the apical dendrites. We recorded Ca2+ waves from five neurons that were extended in this manner (summarized in Fig. 3B). In one cell the wave was extended towards the soma when electrode 2 was placed more proximal to electrode 1. In general, this was not a robust phenomenon. The intensity of stimulation with the second electrode had to be set just below threshold for evoking a wave with that electrode in order to get associative extension. However, the extension was reliable enough that we could repeat the sequence of three conditions at least once in each successful cell. In five other tested cells we failed to increase the region of propagation with this protocol. Varying the duration of the tetanus or offsetting the timing of the tetani from the two electrodes did not improve the likelihood of associative propagation.

We tried to use the same protocol to extend the waves into the soma without success. We tested eight neurons (four using bis-fura-2 and four using furaptra), with many trials on each cell. The second electrode was positioned next to the soma or next to the main dendrite near the soma dendrite border. Figure 3C shows an example of this effort and the results from all eight cells are shown in Fig. 3D. The synaptic response from stimulating electrode close to the soma was hyperpolarizing in some cases (n = 3), reflecting the predominance of inhibitory contacts on this region of the pyramidal neuron (Megias et al. 2001). Therefore, it appears that neither directly activating synapses near the soma nor associatively activating synapses in this region is an effective mechanism for generating waves that propagate through the cell body region.

A second possible mechanism to modulate wave propagation is to vary the extent that stores are filled. Previous experiments in pyramidal neurons (Jaffe & Brown, 1994; Pozzo-Miller et al. 1996; Stutzmann et al. 2003; Power & Sah, 2005) and Purkinje cells (Finch & Augustine, 1998) indicated that Ca2+ release from stores was enhanced by priming that filled the stores before stimulation. If regenerative release is enhanced it might also extend the region of active wave propagation. Figure 4A shows typical results of this kind of experiment. The cell was tetanically stimulated at the same intensity 8 times every 2 min. Action potentials were evoked intrasomatically at 4 Hz in the interval preceding some trials (‘Primed’). Other trials were not preceded by priming. In this cell priming slightly extended the range of wave propagation and made it more likely to observe waves. The extent was reduced or the wave failed altogether when there was no priming. A cumulative analysis from nine cells is presented in Fig. 4B. In general, priming increased the spatial extent of the waves when the previous trial evoked a wave and stimulation evoked a wave of about the same size or smaller when priming was not used. None of these waves extended significantly into the soma. A more extensive analysis of the effects of priming will be presented elsewhere.

Figure 4. Priming of stores extends the range of wave propagation but not into the soma.

Figure 4

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A, the image shows a bis-fura-2 filled pyramidal neuron with marked pixels for the line scans. Each panel shows the spatial and temporal extent of a Ca2+ wave stimulated with identical protocols (100 Hz for 0.5 s, indicated by a bar under the first image) every 2 min. Before certain trials (indicated by ‘Primed’) intrasomatically evoked spikes were evoked at 4 Hz during the interval between trials; for the other trials there was no stimulation. The time of each trial is indicated below each image. The pseudocolour images are scaled to the peak amplitude observed during the 18 trials (33%). In general, the spatial extent, indicated by the vertical limits of the coloured regions, was larger following priming. However, none of the waves extended into the soma (the boundary between the soma and dendrites is indicated by dotted lines under the images). B, distribution of the effects of priming or no priming on wave extent in 26 trials in 9 cells. Trials were divided into two categories: those following priming (left panel) and those without priming (right panel). For example, we included the change between 9 and 11 min in the experiment in A in the left panel in B and the change between 13 and 15 min in the right panel. We did not include all-or-none changes like the event between 15 and 17 min or the one between 17 and 19 min. Wave extent was compared to the previous trial. In general, priming increased the spatial extent of the waves.

A third possible mechanism is that voltage gated calcium entry into the soma could promote wave propagation into this region. Previously (Nakamura et al. 1999), we showed that pairing backpropagating action potentials with synaptic stimulation synergistically enhanced wave generation. The most likely explanation for this synergism is that spike-evoked Ca2+ entry and synaptically mobilized IP3 act together to open IP3 receptors. From this perspective the higher [Ca2+]i level in the soma due to spikes could overcome the lower IP3 concentration in that region to promote further Ca2+ release and wave propagation. However, three kinds of experiments failed to demonstrate propagation into the cell body when spikes were generated or even show clear evidence that the spikes extended the range of wave propagation. In the first experiment (Fig. 5A) we repeated the synergistic pairing protocol with action potentials following synaptic stimulation (n = 6 using bis-fura-2 as the indicator as shown in the figure and n = 5 using furaptra). In the second experiment (Fig. 5B; n = 5 with furaptra and n = 10 with bis-fura-2) we stimulated a burst of action potentials by intrasomatic depolarization during the synaptic stimulation. In the third experiment (not shown; n = 6) we increased the stimulus intensity and removed all holding current to allow the generation of a strong spike burst during tetanic stimulation. Although all three protocols produced significant increases in [Ca2+]i in the soma they probably can be attributed to the action potentials alone since the somatic increases started with the first spike and peaked at the time of the last spike. In contrast, as shown in the figures, Ca2+ release usually starts with a delay and outlasts the stimulus (Nakamura et al. 1999).

One possibility for failure to propagate to the soma is that the patch electrode on the cell body washed out components that were essential for allowing Ca2+ release from stores in that region. However, two different control experiments argue against that conclusion. First, we found that patching the cell on the dendrites about 50 μm from the soma and stimulating the cell with an extracellular electrode near the soma evoked waves proximal to the patch electrode that failed to enter the cell body (n = 2; data not shown). When the stimulating electrode was distal to the recording electrode the waves passed through the location of the dendritic patch without any effect on the amplitude or propagation velocity of the wave (n = 3). Second, we found that we could evoke waves that propagated into the soma by intrasomatically generating action potentials in ACSF containing 30 μm t-ACPD (Nakamura et al. 1999, 2000).

This last control suggested the fourth possibility, that the range of wave propagation might be extended by increasing the basal level of [IP3]i by applying neuromodulators (Nash et al. 2004). This strategy was successful as shown in Fig. 6A. Synaptically stimulated waves in normal ACSF were confined to the apical dendrites as shown in Fig. 2. However, when 10 μm CCh was added to the ACSF the waves consistently (n = 6/6) spread into and through the cell body. Even 3 μm CCh was effective in 2/3 experiments. This effect did not involve voltage gated calcium entry since the synaptic response was kept below spike threshold. Interestingly, these successful experiments required a low affinity Ca2+ indicator in the pipette (in this case 300 μm furaptra). When the usual 150 μm bis-fura-2 was used the waves spread over a larger region of the dendrites but still failed to fully invade the soma in the presence of 10 μm CCh (Fig. 6B; n = 5/5). This indicates that regenerative propagation in this region is very sensitive to Ca2+ buffering (see also Nakamura et al. 2000). In this experiment bath application of CCh by itself did not evoke Ca2+ release (Nakamura et al. 2000) although acute application at higher concentration evoked waves (Power & Sah, 2002). Similar experiments using 30 μm t-ACPD showed that this modulator also extended waves into the soma if furaptra was used as the Ca2+ indicator (n = 2; data not shown). However, it was hard to synaptically evoke Ca2+ release in the presence of t-ACPD in most cells, even though release was easily evoked by spikes (Nakamura et al. 2000) or could be synaptically evoked after the t-ACPD was washed out. The failure to evoke release in t-ACPD was probably because this bath-applied agonist desensitized the mGluRs preventing their activation by synaptically released glutamate (e.g. Dale et al. 2002). It is also possible that t-ACPD acted through group II presynaptic mGluRs to depress the release of glutamate (e.g. Vignes et al. 1995).

Figure 6. Extension of Ca2+ wave propagation into and through the soma by carbachol.

Figure 6

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A, wave extension monitored with 300 μm furaptra in the pipette. The left panel shows the furaptra-filled pyramidal neuron with the position of the stimulating electrode, ROIs and pixels for ‘line scan’ display. Following tetanic stimulation (100 Hz for 0.5 s) in normal ACSF a Ca2+ wave was generated (middle panel) that spread in a region within the apical dendrites and did not invade the soma. When 10 μm CCh was added to the ACSF (middle panel) the same stimulation evoked a wave that entered and passed through the soma. After washout a restricted wave was again recorded (right panel). B, similar experiment using 150 μm bis-fura-2 as the Ca2+ indicator. In this case the wave in 10 μm CCh spread over a larger region of the dendrites than in normal ACSF but did not pass through the soma.

To test whether wave extension into the soma in the presence of CCh might be due to higher levels of IP3 in this region we directly added this messenger to the cell. Figure 7 shows two cells that were patched with electrodes containing 100 μm IP3. In both cases synaptic stimulation evoked waves that propagated through the soma, supporting this hypothesis. Similar results were obtained in other experiments (1/3 with 50 μm IP3; 5/6 with 100 μm; 3/3 with 500 μm). The experiment in Fig. 7A was typical, i.e. synaptic stimulation evoked a single release event. However, in one cell (Fig. 7B) the release wave was followed by a series of [Ca2+]i oscillations that continued for several minutes and were most prominent in the somatic region. These oscillations resembled those found in other preparations following agonist stimulation that mobilizes the IP3 pathway (e.g. Foskett & Wong, 1992; Johnston et al. 2005; Foreman et al. 2006) but are never seen in pyramidal neurons with normal intracellular composition following synaptic stimulation.

Figure 7. Injection of IP3 into the soma promotes wave propagation into this region.

Figure 7

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A, a cell patched with 100 μm IP3 and 150 μm bis-fura-2 in the pipette. Following tetanic stimulation (100 Hz for 0.5 s) a Ca2+ wave was evoked that spread into the cell body. B, a similar experiment in another cell. In this case the synaptically stimulated wave was followed by a series of [Ca2+]i oscillations that were largest in the somatic region near the base of the apical dendrite. Note the slower time scale of this part of the figure. In both experiments the cell was hyperpolarized to −95 mV with current injection to prevent synaptically evoked action potentials.

In addition to these modulator or IP3-extended Ca2+ waves, a few waves (n = 7 out of more than 200 tested cells) propagated into and through the soma in normal ACSF. One example is shown in Fig. 8A (see the online Supplemental material for a movie of this wave). Interestingly, the [Ca2+]i changes generated by waves that passed through the soma (in normal ACSF and in CCh) were generally larger in the centre of the cell than at the edge (Fig. 8A and B). In contrast, the [Ca2+]i increases evoked by spikes were, on average, larger at the edges of the cell than in the centre since they resulted from Ca2+ entry through voltage-gated channels on the plasma membrane (Hernandez-Cruz et al. 1990). The true difference between the centre/edge ratios for the two protocols is probably greater than shown in Fig. 8B since the centre position includes [Ca2+]i changes from the top and bottom of the cell. Confocal or two-photon scans through the centre of the cell (Hernandez-Cruz et al. 1990; Power & Sah, 2002) would probably show a larger difference.

Figure 8. Wave propagation through the soma evokes [Ca2+]i increases with a different spatial distribution than [Ca2+]i increases evoked by intrasomatically stimulated action potentials.

Figure 8

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A, left panels show image of bis-fura-2 filled pyramidal neuron with positions of stimulating electrode, ROIs and line scan pixels indicated. In response to tetanic synaptic stimulation (100 Hz for 0.5 s) a Ca2+ wave was initiated in the dendrites that propagated through the soma. The optical traces show that the [Ca2+]i increase was largest in the centre with little increase on the sides. In response to spikes evoked intrasomatically, [Ca2+]i increases were generated essentially at the same time at all locations, corresponding to Ca2+ entry evoked by the backpropagating action potentials. The optical traces show that the [Ca2+]i increases were similar at all three locations. B, comparison of the relative ΔF/F values at different locations in the soma following either the synaptic generation of a Ca2+ wave (n = 7) or intrasomatic stimulation of spikes (n = 5). Values are normalized between the highest and lowest values in each cell to emphasize the difference between the two kinds of events.

Discussion

Failure to propagate Ca2+ waves into the soma

There are two likely hypotheses to explain why waves stop near the soma–dendritic boundary. One idea is that the properties of the endoplasmic reticulum (ER), IP3 receptors and their density are approximately uniform in all cell compartments and that the major differences among compartments are their geometry (i.e. the much larger volume of the soma compared to the dendrites) and the distribution of mGluRs on the surface of the cell. Although the distribution of mGluRs has never been accurately determined, the density of spines is low on the soma and main apical shaft (Bannister & Larkman, 1995; Megias et al. 2001). Most mGluRs are located perisynaptically near the base of the spines (Lujan et al. 1996). If this distribution also reflects the likelihood of activating mGluRs then synaptically mobilized IP3 will not be produced significantly in the soma and apical dendrite. Rather it will be produced in the oblique dendrites where it can then diffuse rapidly (Allbritton et al. 1992) to the main shaft. When IP3 then diffuses further into the soma it will be diluted by the large volume of the cell body and may then be below the threshold concentration needed to support regenerative Ca2+ release. This mechanism resembles the ‘impedance mismatch’ that has often been used to explain the failure of action potentials to pass through branch points (e.g. Parnas & Segev, 1979) or to activate the soma (e.g. Mainen et al. 1995). This model also explains why the addition of CCh or t-ACPD enabled the waves to consistently overcome the failure to invade the soma. These bath-applied metabotropic agonists probably produced a low level of IP3 in the pyramidal neuron that reached diffusional equilibrium, limited only by the desensitization of receptors and the rate of IP3 breakdown. The resulting steady state level of [IP3]i in the soma would be sufficient to support wave propagation into this region. Consistent with this hypothesis direct injection of IP3 into the soma promoted wave propagation into this region (Fig. 7). The failure of spikes to promote wave propagation to the soma or even to extend propagation in the dendrites (Fig. 5) suggests that once waves are initiated exogenous Ca2+ plays no further role in wave propagation; this Ca2+ is supplied abundantly by the release process. The critical factor is the supply of IP3, which apparently does not normally rise to threshold levels in the soma.

The second possibility is that the properties and density of IP3 receptor isoforms and associated signalling molecules is different in the soma and in the dendrites. For example, Jacob et al. (2005) found many differences among relevant molecules in cultured hippocampal neurons. The PMCA1 and SERCA pumps were distributed relatively uniformly while PIPKIγ, IP3R1, RyR1 and chromogranin B (CGB) were more concentrated in the somatic region, and these differences affected the pattern of Ca2+ signals. However, it is not clear how any of these patterns found in cultured hippocampal neurons could explain the difficulty in propagating synaptically activated Ca2+ waves into the soma in intact neurons. Furthermore, it is not clear that the same distributions are found in the intact pyramidal neurons we examined in acute slices. Interestingly, the same group (Jacob et al. 2005) found that the distribution of the low threshold IP3R1 was uniform in PC12 neurons while the high threshold IP3R3 was targeted to the soma, a pattern that could help explain our results if it is also found in intact hippocampal neurons.

Associative propagation

It was difficult to associatively extend the range of wave propagation in the distal direction, but the reasons for this difficulty are not clear. Previous experiments suggest that it should be easier to open IP3Rs if both [Ca2+]i and [IP3]i are raised and that a lower level of [IP3]i should be needed if [Ca2+]i is high (Moraru et al. 1999; Mak et al. 2001). If this conclusion is valid for the dendritic environment, then at the wave front, following massive Ca2+ release, a lower [IP3]i should be needed to extend propagation than to initiate regenerative Ca2+ release. The modest success we achieved with this experiment (Fig. 3A) supports this model. However, a simple reading of the single channel data (Moraru et al. 1999) suggests that a significantly lower level of mGluR activation and IP3 mobilization by synaptic activity should have been effective instead of the just subthreshold levels we needed. Most likely there are too many steps and unknown signalling mechanisms in this process to fit neatly into this simple model. In contrast, the failure to associatively extend propagation into the soma is more likely due to the paucity of synaptic contacts on the cell body and the consequent low level of [IP3]i mobilized in that region by the second electrode. This is the same reason why direct synaptic stimulation in this region, even at high intensity, failed to make most waves enter the soma.

Nevertheless, if this model of associative wave extension has some validity then it may supply an alternative explanation for the associativity that is one of the hallmarks of LTP. In the usual model of LTP induction associativity is explained by having the membrane depolarization achieved by a strong synaptic input supply the potential needed to open the Ca2+-permeable NMDA receptors activated by a weak synaptic input. In the alternative model associativity is achieved by a mechanism independent of a change in membrane potential. Instead, the rise in [Ca2+]i at the wave front lowers the threshold level of [IP3]i needed for Ca2+ release at the site of the weaker input. These models are not mutually exclusive.

Conditions for successful Ca2+ wave propagation into the soma

In a few experiments in normal ACSF we observed Ca2+ wave propagation into the soma. In addition, in the presence of low concentrations of CCh or t-ACPD synaptically activated Ca2+ waves always propagated further than in normal ACSF and usually propagated through the soma. In most of the experiments in this paper these waves were evoked with a tetanus that stimulated EPSPs that were subthreshold for generating action potentials. Since subthreshold EPSPs cause little [Ca2+]i increase from voltage gated Ca2+ entry the extended propagation cannot be due to the synergism of Ca2+ entry with mGluR mobilized IP3 (Nakamura et al. 1999). Furthermore, in a direct test of the effect of synergistic wave activation (Fig. 5) we found that backpropagating action potentials had no detectable effect on the extent of Ca2+ wave propagation. The most likely explanation for wave extension is that (as mentioned above) the bath applied agonists mobilized a level of [IP3]i in the soma that was higher than could be achieved by synaptic activation alone. Since the threshold level of CCh (3 μm) was similar to that required to evoke release by backpropagating action potentials (Nakamura et al. 2000), it is likely that a similar mechanism promotes Ca2+ release in the soma. In this case the Ca2+ is supplied by the propagating wave front instead of entry through voltage gated Ca2+ channels.

We also note that consistent propagation through the soma in the presence of CCh was only observed when the low-affinity indicator furaptra was included in the patch pipette. With low concentrations of bis-fura-2 (150 μm) we could evoke Ca2+ release in normal ACSF and measure an increase in spatial extent of the wave in CCh (Fig. 6B), but these waves did not extend through the soma. Previously (Nakamura et al. 2000), we found that higher concentrations of bis-fura-2 blocked Ca2+ release completely, which we explained by the interference of the indicator with the regenerative Ca2+ release mechanism. These new experiments suggest that regenerative propagation into the soma is particularly sensitive to Ca2+ buffering. In contrast, the experiments examining the modulation of wave propagation within the dendrites produced results that were relatively insensitive to the choice of Ca2+ indicator. Since the spatial extent of the waves was increased in CCh without increasing the stimulus intensity the spatial increase cannot be due to activating more synapses and is most likely a result of an increase in [IP3]i. Following bath application of CCh the simplest assumption is that the concentration of IP3 is the same in the soma and proximal dendrites, which should allow propagation into the soma if all other parameters are the same, as shown in the IP3 injection experiments. The greater sensitivity to Ca2+ buffering in the soma could be explained either by a different level of endogenous buffer in this compartment or if the regenerative release mechanism is different, e.g. if there is a mix of IP3Rs in the soma with lower Ca2+ affinity than the receptors in the apical dendrite (Koulen et al. 2005), or if the receptors are organized in a different pattern (Delmas & Brown, 2002; Shuai & Jung, 2003) that affects their sensitivity to IP3 and/or Ca2+.

We do not have a clear explanation for the few examples of wave propagation into the soma in normal ACSF. Most likely in these cells there was an increase in one of the critical parameters (e.g. [IP3]i or the density of IP3Rs) that allowed the propagating wave to become suprathreshold in the soma. Another possibility is that some cholinergic fibres were activated along with the Schaffer collaterals and they generated IP3 in the soma. Whether this was just normal variation among pyramidal neurons or a separate category of cells is unknown.

Functional significance

Since Ca2+ waves propagate into the soma only in certain conditions these results suggest that the effects of Ca2+ release in pyramidal neurons should be divided into two classes – those effects that are activated by large amplitude [Ca2+]i increases in the proximal apical dendrites and those that are activated by [Ca2+]i increases in the soma. One example in the first group would be endocannabinoid mediated suppression of synaptic inhibition (e.g. Wilson & Nicoll, 2001) since there is a large concentration of inhibitory inputs targeted to this region (Megias et al. 2001). A classic example in the second group is Ca2+-activated gene transcription (Dolmetsch et al. 1998; Hardington et al. 2001). Our results suggest that in most cases, where Ca2+ waves do not reach the soma and nucleus, the consequences are confined to the first group. However, when cholinergic inputs are also activated (Cole & Nicoll, 1984) waves may reach the nucleus and activate gene expression and other signalling mechanisms. Indeed there is evidence that cholinergic modulation enhances protein synthesis induced by synaptic activation (Feig & Lipton, 1993) and enhances late-phase transcription-dependent LTP activated by tetanic stimulation (Dringenberg et al. 2004).

Acknowledgments

This work was supported in part by NIH grant NS-016295

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