Somatic Spikes Regulate Dendritic Signaling in Small Neurons in the Absence of Backpropagating Action Potentials

Somatic action potential trains elevate sodium levels in axons but not in dendrites of stellate cells

Sodium action potentials elevate sodium within neurons (Jaffe et al., 1992; Regehr, 1997; Rose et al., 1999; Rose, 2003; Kole et al., 2008). We therefore measured sodium levels in SCs to provide an indirect measure of the relative sodium channel densities in axons and dendrites. We detected changes in intracellular sodium levels by recording from SCs with electrodes containing the sodium indicator SBFI (Minta and Tsien, 1989). Because individual action potentials only lead to small changes in intracellular sodium, this approach is not suited to measuring the sodium responses to single action potentials. Instead, we imaged dendritic and axonal regions 50 μm from the soma during trains of action potentials (1 s, 100 Hz) (Fig. 1A,B). Action potential trains produced robust decreases in SBFI fluorescence in axons (Fig. 1B, site 1) (−14 ± 2% ΔF/F, n = 8 neurons) corresponding to substantial increases in axonal sodium. TTX eliminated sodium action potentials (Fig. 1A, bottom) and nearly eliminated train-evoked axonal sodium increases (Fig. 1C) (ΔF/F was reduced from −17 ± 4% to −1.5 ± 1.5% in TTX, n = 4 neurons). In contrast to axons, action potential trains produced only small changes in dendritic fluorescence (Fig. 1B, site 2) (−2.3 ± 0.3% ΔF/F, n = 8 neurons). Thus, intense firing robustly elevates sodium levels in axons, but not in dendrites.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Somatic action potential trains elevate sodium levels in axons but not in dendrites. P17–P19 SCs were loaded with the sodium indicator SBFI (2 mm) through patch pipettes, and stimulus-evoked sodium transients were measured. Sodium elevations decrease SBFI fluorescence. Representative sodium transients (B, C, E) are averages of 3–5 trials (see Materials and Methods). In A and B, action potential trains were evoked in the soma (1 s, 100 Hz, A, bottom), whereas in D and E, PFs were activated (20 stimuli, 50 Hz). A, A representative SC is shown (top) with a black dendritic arbor and a blue axonal arbor. Sodium was measured at an axonal site (site 1) and a dendritic site (site 2). The resulting changes in SBFI fluorescence are indicated (B). C, Axonal sodium transients in control conditions and after wash-in of TTX (500 nm) are shown for a representative experiment (A, bottom, and C). D, Schematic (left) shows configuration for PF activation and somatic responses in control conditions (black trace) and after wash-in of NBQX (10 μm, red trace). The dashed circle indicates the area where dendritic sites were selected for imaging (see Materials and Methods). E, A representative experiment is shown in which PF stimulation evoked robust localized dendritic sodium transients that were blocked by NBQX.

We next examined the sensitivity of our dendritic sodium measurements by activating parallel fibers (PFs) and measuring the resulting local sodium increases. In contrast to somatic action potentials, synaptic activation substantially elevated dendritic sodium (Fig. 1D,E). As shown for a representative experiment, PF activation (20 stimuli, 50 Hz) depolarized the cell (Fig. 1D, right) and elevated dendritic sodium near the site of stimulation (Fig. 1E). The AMPA receptor antagonist NBQX prevented both the somatic depolarization and the elevation of dendritic sodium (Fig. 1E) (ΔF/F was reduced from −19 ± 3% to −1.7 ± 1.3%, n = 4 neurons). This indicates that sodium entry through AMPA receptors can lead to dendritic sodium transients that are readily detected. These results demonstrate that sodium levels in dendrites can be robustly elevated by synaptic stimulation, but not by somatic spiking.

Somatic action potentials elevate dendritic calcium in stellate cells

We next measured dendritic calcium levels to determine the extent of action potential invasion into SC dendrites. Whole-cell recordings of SCs were obtained with electrodes containing the green fluorescent calcium indicator fluo-5F (Brenowitz and Regehr, 2007) (50 μm, K_D = 585 nm) and the red calcium-insensitive fluorescent dye Alexa 594 (50 μm). Single action potentials or trains of action potentials (2 s, 100 Hz) were evoked in current clamp with brief current pulses (2 ms, 1–2 nA). Calcium levels were calculated from the green/red fluorescence obtained using line scans across dendritic sites 50 μm from the soma (Fig. 2A,F) (Yasuda et al., 2004).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Action potentials in SCs elevate dendritic calcium. Recordings of P17–P19 SCs were made with pipettes containing a green calcium-sensitive indicator (fluo-5F, 50 μm) and a red calcium-insensitive dye (Alexa 594, 50 μm). Action potentials were evoked in current clamp from an initial potential of −80 mV (black traces) or −60 mV (gray traces), and calcium was measured at a dendritic site 50 μm from the soma. Representative calcium transients evoked by single action potentials (C, H) are averages of 12–16 trials, whereas transients evoked by 2 s 100 Hz trains (D, I) are averages of 3–5 trials. A–E and F–J represent ∼80% and 20% of neurons, respectively. In images of example neurons (A, F), axons were removed for clarity. In a cell representing the majority of neurons (A), a single action potential (B, left) elevated dendritic calcium by 10 nm, regardless of the initial potential (C, left). TTX eliminated the action potential (B, right) and substantially reduced the dendritic calcium elevations (C, right). D, Action potential trains (2 s, 100 Hz) elevated dendritic calcium to 300 nm (left). TTX reduced the calcium elevations evoked by trains (right). E, Recordings as in A–D are summarized (n = 5 neurons). In a cell representing the remaining neurons (F), a single action potential generated an afterdepolarization when evoked from −80 mV but not when evoked from −60 mV (G, left). Single action potentials elevated dendritic calcium to 40 and 10 nm when evoked from initial potentials of −80 mV and −60 mV, respectively (H, left). TTX eliminated the action potential but did not substantially reduce the afterdepolarization (G, right) or the calcium elevation (H, right) for an initial potential of −80 mV. However, calcium elevations were reduced when the initial potential was −60 mV. I, Action potential trains elevated dendritic calcium to 300 nm in control conditions, regardless of the initial potential (left). TTX reduced calcium elevations evoked by trains (right). J, Recordings as in F–I are summarized (n = 5 neurons).

The neurons we recorded from could be divided into two types of cells that differed with respect to the properties of single action potentials and associated calcium increases, but were similar in dendritic calcium signaling during action potential trains. Most of the neurons we recorded from (∼80%) (Fig. 2A–E) had action potentials with a strong afterhyperpolarization (Fig. 2B). In these cells, single action potentials elevated calcium to ∼10 nm, regardless of the initial potential of the cell (Fig. 2C, left). TTX eliminated the sodium action potential (Fig. 2B, right) and greatly reduced the dendritic calcium responses (Fig. 2C, right). Trains of action potentials (2 s, 100 Hz) elevated dendritic calcium to several hundred nanomolar (Fig. 2D, left). TTX greatly reduced the calcium elevations evoked by trains (Fig. 2D, right). Thus, in the majority of cells, TTX eliminated somatic action potentials, which in turn greatly reduced dendritic calcium elevations (Fig. 2E).

The remaining SCs (∼20%) (Fig. 2F) exhibited a slow afterdepolarization when a single action potential was evoked from −80 mV (Fig. 2G, left, black traces) but not −60 mV (Fig. 2G, left, gray traces). Single action potentials evoked larger dendritic calcium increases when evoked from −80 mV compared with −60 mV (Fig. 2H, left). This voltage dependence is consistent with the involvement of T-type calcium channels, which are present in SCs (Molineux et al., 2005). TTX did not reduce the slow afterdepolarization (Fig. 2G, right), only slightly reduced the magnitude of calcium increases evoked by single action potentials when the cell was initially at −80 mV and greatly reduced the magnitude of the calcium signals when the initial potential was −60 mV (Fig. 2H, right). Trains of action potentials (2 s, 100 Hz) produced similar calcium increases in control conditions, regardless of the initial potential (Fig. 2I, left). This is consistent with the observation that the slow afterdepolarization became much less prominent during trains (data not shown). TTX reduced the calcium elevations regardless of the initial potential (Fig. 2I, right). Thus, although for a holding potential of −80 mV the calcium increases evoked by single action potentials were only weakly TTX sensitive, calcium elevations evoked by trains of action potentials were greatly reduced in TTX (Fig. 2J).

These findings indicate that action potentials elevate dendritic calcium in SCs. This study is focused primarily on dendritic calcium signaling during trains of action potentials, and such trains elevated calcium in a similar TTX-sensitive manner in all SCs studied (Fig. 2E,J).

Action-potential-evoked dendritic calcium elevations do not require dendritic sodium channels

We next determined whether dendritic sodium channels are required for action-potential-evoked calcium elevations. As shown for a representative dendritic site (50 μm from the soma) (Fig. 3A), a single action potential and a train of action potentials elevated dendritic calcium by 20 and 300 nm, respectively (Fig. 3B, top). TTX prevented the generation of action potentials and greatly reduced the amplitude of calcium elevations (Fig. 3B, middle). In the presence of TTX, voltage clamping the soma with the action potential waveform recorded in control conditions (Stuart and Sakmann, 1994) elevated dendritic calcium to levels that were comparable to those measured in control conditions in current clamp (Fig. 3B, bottom). The similarity of the calcium signals evoked by action potentials in current clamp and by voltage-clamped action potentials in TTX indicates that dendritic sodium channels are not required for somatic action potentials to invade SC dendrites. Instead, passive spread of membrane depolarization is sufficient to convey somatic action potentials to the dendrites of SCs.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Somatic action potentials elevate dendritic calcium in a manner that does not rely on dendritic sodium channels. A, B, As shown for a representative experiment, action potentials were evoked in the soma of a P17–P19 SC, and the resulting calcium signals were measured at the site indicated in A (blue box). The axon is colored blue for clarity. B, Membrane voltage (left) and dendritic calcium signals following a single action potential (middle) and an action potential train (2 s, 100 Hz, right) are shown. Somatic depolarization evoked action potentials in current clamp (CC, top) but failed to do so in the presence of TTX (middle). In the presence of TTX, the cell was then voltage clamped with the action potential waveform (bottom). C, A second experiment is shown in which calcium was measured in an axon located 75 μm from the soma. Calcium signals evoked by either single action potentials (left) or trains of action potentials (right) are shown. D, E, Calcium elevations are summarized for dendritic sites (left) and axonal sites (right) for single action potentials (D) and 2 s action potential trains (E).

In another experiment, single action potentials and trains of action potentials also elevated axonal calcium levels (Fig. 3C, top). TTX eliminated axonal calcium elevations both in current clamp (Fig. 3C, middle) and when the soma was voltage clamped with the action potential waveform (Fig. 3C, bottom). The inability of voltage-clamped action potentials in TTX to evoke axonal calcium signals suggests that, in contrast to dendrites, the passive spread of depolarization associated with a somatic action potential is not effective at evoking calcium signals in axons.

Voltage-clamp rescue of calcium signals in the presence of TTX in dendrites but not axons was a consistent finding for single action potentials (Fig. 3D) and for trains of action potentials (Fig. 3E). For sites between 75 and 100 μm from the soma, the calcium signals evoked by single action potentials in current clamp, current clamp in TTX, and voltage-clamped action potentials in TTX were 11.3 ± 2.4 nm, 1.6 ± 1.8 nm, and 9.6 ± 2.2 nm, respectively (Fig. 3D, left) (n = 5 neurons). Action potential trains at the same sites elevated dendritic calcium by 272 ± 25 nm, 56 ± 22 nm, and 222 ± 20 nm, respectively (Fig. 3E, left). Dendritic calcium signals were slightly smaller with increasing distance from the soma (Fig. 3D,E, left). At axonal sites <100 μm from the soma, single action potentials and action potential trains evoked calcium signals (51 ± 8 nm and 2.06 ± 0.54 μm, respectively, n = 7 neurons) (Fig. 3D,E, right) that were larger than in dendrites (Fig. 3D,E, left). TTX reliably eliminated the axonal calcium elevations in current clamp and in action potential voltage clamp (Fig. 3D,E, right). Thus, somatic action potentials elevate dendritic calcium in a manner that does not rely on sodium channels in the dendrites. Despite the lack of dendritic sodium spikes, trains of somatic action potentials can elevate dendritic calcium to several hundred nanomolar.

The substantial calcium increases that can be evoked by somatic depolarization in the absence of sodium channels suggest that there is significant passive spread of potential within SC dendrites from P17–P19 rats. We therefore analyzed dendritic morphology, which is a major determinant of the passive spread of potential. In P17–P19 SCs, primary dendrites were short (Fig. 4A), with a mean length of 60 ± 2 μm (n = 247 dendrites) (Fig. 4C,D). There were 4.7 ± 0.1 primary dendrites per cell (n = 54 neurons) (Fig. 4E,F). The dendrites were not extensively branched, with only 1.7 ± 0.1 secondary branches per primary dendrite (Fig. 4G,H). The short dendrites and simple branching of P17–P19 SCs makes them well suited to support passive spread of somatic action potentials into the dendritic arbor.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Morphology of stellate cell dendrites. SCs from P17–P19 or P30–P33 rats were loaded with Alexa 594, Z stacks were obtained, and dendrite morphology was analyzed using NeuronJ (Meijering et al., 2004). A, B, Shown are images of P18 (A) and P30 (B) SCs. Axons were removed for clarity. C, D, Histograms (C) and cumulative histograms (D) of SC dendrite lengths are shown for P17–P19 rats (C, left and D, black trace, n = 247 dendrites) and P30–P33 rats (C, right and D, blue trace, n = 193 dendrites). Histograms of the number of primary dendrites per SC (E) and the number of secondary branches per primary dendrite (G) are shown for P17–P19 rats (left, n = 54 neurons) and P30–P33 rats (right, n = 50 neurons). F, H, Experiments as in E and G are summarized.

Dendritic signaling in stellate cells of P30–P33 rats

It is possible that developmental changes in dendritic morphology or sodium channel density alter dendritic signaling in the SCs of older animals. We therefore extended our studies of dendritic signaling to SCs from older rats (P30–P33). The dendrites were more variable in length (Fig. 4B,C) and the average length of primary dendrites was longer than in young animals (90 ± 3 μm, n = 193 dendrites) (Fig. 4C,D). SCs from P30–P33 rats had 3.8 ± 0.2 primary dendrites (n = 50 neurons) (Fig. 4E,F) and maintained a simple branching pattern, with 2.4 ± 0.2 secondary branches per primary dendrite (Fig. 4G,H). We also observed numerous short protrusions from the dendrites (Fig. 4B), but as these did not appear to be full-fledged dendrites, they were excluded from analysis (see Materials and Methods). Thus, in P30–P33 SCs some of the dendrites have short, simple morphologies that favor the spread of passive potentials, as in P17–P19 SCs, but the rest of the dendrites are longer and more attenuation is possible with distance from the soma.

We characterized action potential-evoked dendritic calcium increases in P30–P33 SCs (Fig. 5). Action potentials were evoked in the soma and calcium levels were measured at various dendritic locations, as in Figure 3. As shown for a representative experiment (Fig. 5A–D), single action potentials and 2 s 100 Hz action potential trains in control conditions elevated dendritic calcium near the soma comparably to younger SCs (Fig. 5C,D, site 1, black traces). However, calcium signals strongly attenuated with distance from the soma (Fig. 5C,D, sites 2 and 3). On average, dendritic calcium elevations at 75–100 μm from the soma were ∼25% of those observed in dendrites near the soma (Fig. 5E,F, black bars). TTX eliminated the action potential (Fig. 5B, blue trace) and substantially reduced dendritic calcium elevations (Fig. 5C–F, blue). The large TTX-insensitive calcium signals observed in some P17–P19 SCs (Fig. 2F–J) were not observed in any P30–P33 SCs. Thus, action potentials elevate dendritic calcium in older SCs, but on average somatic action potentials are ineffective at elevating calcium in distal compartments.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Action-potential-evoked calcium signals attenuate with distance from the soma in the dendrites of P30–P33 stellate cells. A–D, As shown for a representative SC obtained from a P30 rat, action potentials were evoked in the soma, and calcium levels were imaged at locations indicated by the numbered boxes (A, axon removed for clarity). Somatic membrane voltage in response to a single action potential (B) and dendritic calcium signals evoked by either single action potentials (C) or 2 s 100 Hz action potential trains (D) are shown for dendritic sites indicated in A. Blue traces indicate responses after application of TTX. Calcium elevations evoked by single action potentials (E) or action potential trains (F) are summarized. G, Data from the two most proximal regions in E and F are replotted for short dendrites (40–50 μm long, black bars) and long dendrites (>100 μm long, gray bars). Numbers above the bars indicate the number of dendrites analyzed.

To gain insight into whether dendritic length influences calcium signaling, we divided the data from Figure 5, E and F, into groups based on the length of the primary dendrite (Fig. 5G). In dendrites longer than 100 μm, calcium elevations between 25 and 50 μm from the soma were only 20% of those observed at sites <25 μm from the soma (Fig. 5G, gray bars). In contrast, dendrites between 40 and 50 μm long exhibited little attenuation (Fig. 5G, black bars). These findings suggest that calcium signaling is similar in short dendrites of P30–P33 SCs and in dendrites of P17–P19 SCs.

Finally, we determined whether dendritic sodium channels contribute to action-potential-evoked calcium elevations in P30–P33 SCs. We performed voltage-clamp rescue experiments in the presence of TTX at sites between 70 and 100 μm from the soma (Fig. 6). In the presence of TTX, voltage-clamped action potentials elevated dendritic calcium to levels similar to control conditions in current clamp, as shown in a representative experiment (Fig. 6A–D). This finding was consistent among six neurons tested for both single action potentials and 2 s 100 Hz action potential trains (Fig. 6E). Thus, dendritic sodium channels do not contribute to action-potential-evoked dendritic calcium increases in P30–P33 SCs.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Somatic action potentials elevate dendritic calcium in a manner that does not rely on dendritic sodium channels in P30–P33 SCs. A–D, As shown for a representative experiment, action potentials were evoked in the soma and the resulting calcium signals were measured at the site indicated in A (blue box). The axon has been removed for clarity. B, Membrane voltage (left) and calcium elevations following a single action potential (middle) and an action potential train (2 s, 100 Hz, right) are shown. Calcium elevations were substantially smaller compared with SCs obtained from P17–P19 rats. Somatic depolarization evoked action potentials in current clamp (B) but failed to do so in the presence of TTX (C). In the presence of TTX, the cell was then voltage clamped with the action potential waveform (D). E, Calcium elevations are summarized for dendritic sites 70–100 μm from the soma (n = 6 neurons) for single action potentials (left) and action potential trains (right).

Action potential trains and retrograde endocannabinoid signaling

Previous studies have shown that SCs can regulate the strength of their synaptic inputs by releasing eCBs from their dendrites in a calcium-dependent manner (Beierlein and Regehr, 2006; Soler-Llavina and Sabatini, 2006). We tested the role of somatic action potentials in regulating the release of eCBs from SC dendrites. We focused on the dendrites of SCs from P17–P19 rats in these studies (Figs. 7⇓–9) because they had consistently short dendrites and large evoked calcium increases.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Brief, high-frequency action potential trains evoke retrograde eCB signaling in P17–P19 SCs, which is facilitated by mGluR1/5 activation. A, In a representative experiment, PFs were stimulated (0.5 Hz) in voltage clamp. The recording was switched to current clamp, and somatic action potentials were evoked (2 s, 200 Hz). The SC was returned to voltage clamp, and PF stimulation (0.5 Hz) was resumed. Such trials were repeated every minute for 4 min and then the EPSCs were normalized and averaged. B, The time courses of normalized PF EPSCs are shown for control conditions (black circles), in the presence of the mGluR1/5 receptor agonist DHPG (5 μm, red circles) and in the presence of DHPG and the CB1 receptor antagonist AM 251 (AM, 2 μm, blue circles). C, Normalized EPSC amplitudes are summarized for 2 s action potential trains of different frequencies. Numbers above the bars indicate the number of neurons recorded in each condition. D, Representative calcium transients (left) and synaptic responses (right) are shown for action potential trains of 200 Hz (top) and 50 Hz (middle) in control conditions and 50 Hz after wash-in of DHPG (bottom). Calcium transients are averages of four trials, each. E, Relative synaptic strength is plotted against peak calcium during the action potential trains and fit to the Hill equation (EPSC = 1 − 0.95[xⁿ/(K_0.5 + xⁿ)], where 0.95 is the maximal suppression attained with CB1R activation, K_0.5 is the half-maximal calcium, and n is the Hill coefficient). Dotted lines indicate the peak calcium required for half-maximal synaptic suppression (control: K_0.5 = 2.2 ± 0.3 μm, n = 1.9 ± 0.4; DHPG: K_0.5 = 0.8 ± 0.1 μm, n = 1.6 ± 0.4). Symbol shapes indicate the frequency of the action potential trains (diamonds: 25 Hz, squares: 50 Hz, triangles: 100 Hz, circles: 200 Hz).

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Prolonged somatic action potential trains promote retrograde eCB signaling. PFs were stimulated in voltage clamp and action potential trains were evoked in current clamp as in Figure 7. A, Representative calcium transients (left) and synaptic responses (right) are shown for an experiment in which somatic action potentials were evoked in SCs at 100 Hz for 15 s in control conditions (top) and in the presence of DHPG (bottom). B, Normalized EPSC amplitudes are summarized for 15 s action potential trains of different frequencies for control conditions (black), DHPG (red), and DHPG in the presence of AM 251 (AM, blue). Numbers above the bars indicate the number of neurons recorded in each condition. C, Relative synaptic strength is plotted against peak calcium during the action potential trains and fit to the Hill equation as in Figure 7E. Dotted lines indicate the peak calcium required for half-maximal synaptic suppression (control: K_0.5 = 1.3 ± 0.2 μm, n = 1.9 ± 0.4; DHPG: K_0.5 = 0.4 ± 0.1 μm, n = 1.4 ± 0.2). Symbol shapes indicate the frequency of the action potential trains (flat diamonds: 10 Hz, diamonds: 25 Hz, squares: 50 Hz, triangles: 100 Hz).

• Download figure
• Open in new tab
• Download powerpoint

Figure 9.

SCs associate postsynaptic and presynaptic activity. A, In a representative experiment, membrane voltage (left), dendritic calcium transients (center), and relative synaptic strength before and after conditioning stimulation (right) are shown. Conditioning stimulation consisted of action potential trains (1 s, 100 Hz, top), PF bursts (5 stimuli, 50 Hz, middle), or PF bursts delivered in the middle of the action potential trains (bottom), with 7 trials averaged for each condition. The calculated sum of the calcium signal during the burst and train is superimposed on the measured calcium signal for combined stimulation (gray trace). Calcium elevation (B) and relative synaptic strength (C) are summarized (n = 6 neurons). Responses evoked by the PF burst alone and the action potential train paired with PF burst were significantly different (asterisk indicates p < 0.01, 1-way ANOVA).

We initially tested whether high-frequency spike trains elevate calcium sufficiently to evoke eCB release and suppress synaptic transmission. As shown in a representative experiment (Fig. 7A), PF EPSCs were first measured in voltage clamp. The recording was then switched to current clamp and action potentials were evoked in the neuron (2 s, 200 Hz). Following the train, the recording was returned to voltage clamp and PF EPSCs were measured again. For 2-s-long action potential trains in control conditions, 200 Hz trains transiently reduced EPSC amplitude to 71 ± 6% of the initial value (Fig. 7B, black circles) (n = 9 neurons), but lower frequency trains did not result in suppression (Fig. 7C, black bars). This suggests that for 2 s trains, SCs must fire at frequencies greater than the 10–60 Hz firing frequencies observed in vivo (Ekerot and Jörntell, 2003).

The activation of mGluR1/5 can reduce the calcium levels necessary to evoke eCB release by activating postsynaptic the β isoform of phospholipase C (PLCβ) (Varma et al., 2001; Galante and Diana, 2004; Brenowitz and Regehr, 2005; Hashimotodani et al., 2005; Maejima et al., 2005). We tested whether SC spiking would more effectively promote eCB release when paired with activation of mGluR1/5 receptors. Although strong activation of mGluR1/5 can drive eCB release on its own (Maejima et al., 2001), low-level activation can reduce the calcium dependence of eCB release without affecting synaptic transmission (Ohno-Shosaku et al., 2002). We therefore bath applied the mGluR1/5 agonist DHPG at a concentration (5 μm) that alone did not substantially affect synaptic strength (94 ± 10% of control, n = 29). In the presence of DHPG, 200 Hz trains reduced normalized EPSC amplitudes to 46 ± 6% of baseline amplitude (Fig. 7B, red circles) (n = 7 neurons). The strong suppression in the presence of DHPG was prevented by the CB1 receptor antagonist AM 251 (2 μm) (Fig. 7B,C, blue). In the presence of DHPG, 100 Hz and 50 Hz trains also suppressed synaptic strength, but 25 Hz trains did not (Fig. 7C) (p < 0.05, unpaired t tests between control and DHPG for 50, 100, and 200 Hz). These findings indicate that action potential trains in SCs can retrogradely suppress their synaptic inputs by promoting eCB release from their dendrites. mGluR1/5 activation increases the magnitude of suppression and lowers the firing frequencies required for suppression.

To gain insight into how mGluR1/5 activation enhances eCB release, we determined the calcium dependence of spiking-evoked suppression (Fig. 7D,E) (Wang and Zucker, 2001; Brenowitz and Regehr, 2003; Brenowitz et al., 2006). Experiments were performed as in Figure 7A, except that dendritic calcium was also measured during the trains. In control conditions, a 2 s 200 Hz action potential train elevated dendritic calcium to 1.5 μm and reduced synaptic strength by 30% (Fig. 7D, top), and a 50 Hz train elevated calcium to 0.5 μm and did not reduce synaptic strength (Fig. 7D, middle). After wash-in of DHPG, the 50 Hz train reduced synaptic strength by 50% even though it did not alter the calcium signal (Fig. 7D, bottom).

We performed a series of such experiments to determine the calcium dependence of spike-evoked suppression (Fig. 7E). We plotted normalized EPSC amplitudes against peak calcium elevation for 25, 50, 100, and 200 Hz trains and fit the data with the Hill equation. In control conditions, half-maximal suppression occurred at 2.15 ± 0.26 μm calcium, and in DHPG, half-maximal suppression occurred at 0.80 ± 0.11 μm calcium. Thus, mGluR1/5 activation facilitates spiking-evoked eCB signaling by increasing the calcium sensitivity of eCB release.

These results suggest that SCs require lower levels of calcium to evoke eCB release compared with other cell types, where 4–7 μm calcium is required for half-maximal synaptic suppression (Wang and Zucker, 2001, Brenowitz and Regehr, 2003). Importantly, in such studies dendritic calcium was elevated by depolarizing the neuron in voltage clamp with a Cs-based internal solution and is widely termed depolarization-induced suppression of inhibitory (DSI) and excitatory (DSE) synapses (Lenz and Alger, 1999; Kreitzer and Regehr, 2001; Wilson and Nicoll, 2001). Photolysis of caged-calcium has also been used to determine the calcium dependence of eCB release and requires 4 μm calcium to evoke half-maximal synaptic suppression (Wang and Zucker, 2001). The similar calcium sensitivities reported for studies in which depolarization or photolysis of caged-calcium were used to evoke eCB release suggest that bulk calcium rather than local calcium triggers release.

To gain insight into the apparent difference between the calcium dependence of eCB signaling in SCs and other neurons, we determined the calcium dependence of DSE at PF to SC synapses. Here, we performed experiments similar to those of Figure 7, but used a Cs-based internal solution and elevated dendritic calcium by depolarizing the cell from a holding potential of −70 mV to potentials between −50 and 0 mV for 2 s (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Half-maximal DSE occurred at 4.5 ± 0.4 μm. Thus, when eCB signaling at PF to SC synapses is evoked by depolarization in a Cs-based internal solution, similar levels of calcium are required for eCB release compared with other neurons. One possible explanation for the differences in the calcium dependence of eCB signaling for K-based and Cs-based internal solutions is that high local calcium signals (which are not readily detected with our imaging methodology) could be more prominent when eCB release is driven by action potentials using a K-based internal solution, which would cause us to underestimate the calcium levels that trigger eCB release (as opposed to a smaller steady calcium influx in experiments in which a Cs-based internal solution was used).

Prolonged action potential trains and mGluR1/5 activation promote spiking-evoked endocannabinoid signaling

Prolonged calcium elevations can be more effective than brief calcium increases at evoking eCB release (Brenowitz et al., 2006). We therefore examined the calcium dependence and mGluR1/5 dependence of eCB signaling evoked by 15 s action potential trains.

A 15 s 100 Hz train elevated calcium to 0.5 μm and reduced synaptic strength by 20% (Fig. 8A, top). After wash-in of DHPG, the 100 Hz train reduced synaptic strength by 60% without affecting the dendritic calcium signal (Fig. 8A, bottom). Although 2 s 100 Hz trains did not reduce synaptic strength, 15 s 100 Hz trains reduced synaptic strength by 30 ± 5% on average (Fig. 8B, black bars) (n = 17 neurons). DHPG facilitated synaptic suppression evoked by 15 s trains, permitting frequencies as low as 25 Hz to evoke synaptic suppression (Fig. 8B, red bars) (p < 0.01, unpaired t tests between control and DHPG for 25, 50, and 100 Hz trains). At the highest frequency tested (100 Hz), AM 251 blocked all synaptic suppression (Fig. 8B, blue bar).

We determined the calcium dependence of eCB signaling for prolonged trains by plotting normalized EPSC amplitude against peak calcium elevation during 15 s action potential trains (Fig. 8C). In control conditions for 15 s trains half-maximal suppression occurred at 1.29 ± 0.23 μm calcium, which is approximately a factor of 2 lower than for 2 s trains. In DHPG half-maximal suppression occurred at 0.36 ± 0.03 μm calcium for 15 s trains, which is also about a factor of 2 lower than for 2 s trains.

Thus, 15 s action potential trains are more effective at evoking eCB signaling, compared with 2 s trains. Pairing mGluR1/5 activation with prolonged SC firing is particularly effective at evoking eCB release. Here, physiologically relevant firing frequencies in SCs (Ekerot and Jörntell, 2003) (25–60 Hz) are sufficient to regulate the strength of their PF synaptic inputs.

Stellate cells associate presynaptic and postsynaptic activity

It has been previously shown that presynaptic activity can evoke retrograde eCB signaling in SCs (Beierlein and Regehr, 2006; Soler-Llavina and Sabatini, 2006). Our present results indicate that somatic spiking in SCs can also evoke retrograde eCB signaling. We therefore determined whether presynaptic activity and postsynaptic activity interact to promote eCB signaling at PF to SC synapses.

We measured dendritic calcium and synaptic strength for experiments using three conditioning stimuli: an action potential train (1 s, 100 Hz), a PF burst (5 stimuli, 50 Hz), and coincident stimulation where the PF burst was delivered in the middle of the action potential train. Although more prolonged PF activation can produce large retrograde inhibition (Brown et al., 2003), brief PF activation produced little or no synaptic suppression. In a representative experiment, the spike train elevated dendritic calcium to 0.5 μm and did not change synaptic strength (Fig. 9A, top). The PF burst locally elevated dendritic calcium to 2 μm and reduced EPSC amplitude by 20% (Fig. 9A, middle). Combining the train and burst elevated calcium to 2.5 μm and reduced EPSC amplitude by 40% (Fig. 9A, bottom). For combined stimulation, the calcium increase was well approximated by the linear summation of the calcium elevations for the train alone and the burst alone (Fig. 9A, bottom, gray trace). The linear summation of calcium elevation evoked by presynaptic and postsynaptic activity was consistent among six neurons tested (Fig. 9B). The train and burst generated significantly larger calcium increases compared with the burst alone (p < 0.01, one-way ANOVA).

Action potential trains also consistently promoted synaptic suppression when paired with PF bursts (Fig. 9C). The trains did not substantially reduce synaptic strength on their own (0.89 ± 0.05 normalized EPSC), but pairing them with the PF burst reduced the EPSC amplitude to a greater extent than the PF burst alone (reduced to 0.50 ± 0.08 fraction of baseline, compared with 0.74 ± 0.06, p < 0.01, one-way ANOVA). This indicates that postsynaptic spiking activity facilitates synaptically evoked retrograde eCB signaling. Here, passive spread of somatic action potentials influences dendritic calcium signaling to promote eCB release from SC dendrites during brief bursts of synaptic activity.