Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study - PubMed (original) (raw)

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study

Marco Canepari et al. J Physiol. 2007.

Abstract

The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca(2+) imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca(2+)](i) changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca(2+)](i) signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP-AP pairing evoked supra-linear summation of [Ca(2+)](i) transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca(2+) influx through NMDA receptors (0 to -15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.

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Figures

Figure 1

Figure 1. Pharmacological effects and photodynamic damage of the voltage-sensitive dye

A, electrode recordings from the soma. The resting membrane potential and AP shape and size were not altered significantly after the 40 min staining period and additional 90 min of incubation at room temperature (dye equilibration time; grey trace) from the original values at the start of the recording (black trace). B, summary data for spike amplitude, voltage threshold, and width at half-height determined for 4 neurons before (black bars) and after (grey bars) staining and dye equilibration. The differences were not statistically significant. C, the 1st and the 14th 100 ms optical recording trial (TR 1 and TR 14) of the evoked bAP signals from a selected region (white circle) on the dendritic arbor of a stained neuron shown on the left. D, the 1st and the 52nd 100 ms optical recording trial of the evoked bAP signals from the dendritic arbor of another stained neuron. E, relative change in the amplitude of the last single-trial recording of the bAP signal normalized to the size of the first recording shown for two groups of experiments. In one group (black bars) the total number of trials was in the range of 8–16 in different experiments (_n_=38). In the other group the total number of trials was in the range of 30–54 in different experiments (_n_=5). The differences were not statistically significant. F, normalized EPSP amplitude before and after paired stimulation showing the average magnitude and the time course of LTP for control neurons (_n_=4) and the data for two neurons stained with the voltage-sensitive dye. Error bars are

s.d.

values.

Figure 7

Figure 7. Correlation between _V_m signals and [Ca2+]i transients during paired activity

A, scatter-plot of the supra-linear [Ca2+]i signal (○) and its NMDA component (•) versus change in bAP baseline-to-peak amplitudes evoked by paired activity does not show significant correlation (correlation coefficients, _r_=0.0984 and −0.0675; P > 0.5). B, scatter-plot of the change in the NMDA-R-dependent component of the supra-linear [Ca2+]i signal versus change in the peak _V_m depolarization. The signals evoked by paired activity are expressed relative to the signals recorded during EPSP stimulation alone. _r_=0.3319; P > 0.2.

Figure 2

Figure 2. _V_m and [Ca2+]i signals in response to repeated stimulation are stable

A, electrical recordings from the soma during evoked bAP, evoked train of 5 EPSPs, and paired stimulation. B, fluorescent image of a CA1 pyramidal neuron (bis-fura-2 excitation); white circle indicates recording location; somatic patch-electrode and extracellular electrode are shown schematically. C, optical recording of local _V_m and [Ca2+]i signals in response to repeated application of paired stimulation. Four recording trials (grey) and an average result (black) are superimposed for both signals. Note the difference in the time scale.

Figure 3

Figure 3. Comparison of _V_m and [Ca2+]i signals from same dendritic locations

A, a composite image of a neuron (voltage-sensitive dye excitation). B, low resolution images (bis-fura-2 excitation) of the dendritic arbor in recording position. Representative recording locations marked by white ovals; the position of the extracellular electrode shown schematically. C, _V_m signals and [Ca2+]i signals related to bAP (black traces), EPSP train (blue traces, shown only for [Ca2+]i signals), and paired EPSP–AP activity (red traces). _V_m signals (100 ms) during paired activity are superimposed with the recordings of bAP signals alone. Calcium recordings (400 ms) are shown above _V_m traces – signals during three stimulation protocols (bAPs, EPSP train, paired) are superimposed. D, _V_m recordings of the first bAP on the expanded time scale superimposed on the same baseline to show region-specific changes in the peak membrane depolarization during paired activity (upper traces). Lower traces: spike signals aligned to indicate the difference in baseline-to-peak amplitude (EPSP signal subtracted). Traces are shifted horizontally with respect to each other for clarity. E, _V_m signal amplitude comparison: four trials were averaged for both paired and unpaired stimulation pattern. The peak of the relevant spike was used as the reference time point for averaging to remove the effect of trial-to-trial temporal jitter in the timing of AP generation on the shape and size of the averaged signal. In all experiments individual trials were inspected to verify that averaging did not introduce errors.

Figure 4

Figure 4. Supra-linear component of the dendritic calcium signal is spatially and temporally correlated with the increase in the peak membrane depolarization evoked by paired activity

A, calcium signals: the spatial maps of the peak [Ca2+]i transient evoked by the three stimulation protocols shown in the colour-coded display on the left. Corresponding signals from four representative locations (indicated in the top panel) are shown on the right (red traces – somatic _V_m recording). In this and all subsequent figures, the relative colour coding (minimum-to-maximum signal amplitude) was applied separately to each measurement. A mask was applied (with the threshold set to 200% of the average background light intensity) that revealed only detectors that received light from the stained neuron. The bottom panel (Supra-linear Ca2+) shows the relative spatial map and the recordings of the supra-linear component of the [Ca2+]i signal during paired activity. B, comparison of the somatic _V_m recordings (lower 3 traces; AP truncated in a) and calcium signals at the stimulation site (upper 4 traces) in response to bAP (black), a train of 5 EPSPs (blue) and paired activity (red). The trace on top is the isolated supra-linear component of the [Ca2+]i signal marked d. C, summary result (_n_=19): supra-linear component of the [Ca2+]i signal at the stimulation site (grey bar) normalized to the sum of the bAP and EPSP calcium signals (black bar). On average, 43% of the total calcium signal was the result of the non-linear increase in Ca2+ influx during paired activity. D, _V_m signals: the relative spatial maps constructed for the peak of the bAP, EPSP train and paired activity shown in the colour-coded displays on the left; corresponding signals from 4 locations are shown on the right. In the middle panel (EPSP), spike signals were removed artificially to isolate parts of the recording used to generate a spatial map of the peak EPSP signal alone. E, bAP signals superimposed on the same baseline with paired EPSP–bAP signals to reveal region-specific changes in the peak membrane depolarization. F, bAP signals aligned with paired EPSP–bAP signals to reveal region-specific differences in action potential baseline-to-peak amplitudes (EPSP signals subtracted). Traces are shifted horizontally with respect to each other for clarity. G, a fluorescence image of the CA1 pyramidal neuron (bis-fura-2 excitation).

Figure 5

Figure 5. Boosting of bAP amplitude was not directly correlated with the NMDA-dependent component of the supra-linear calcium change evoked by LTP-forming stimuli

A, _V_m signals: recordings during the three stimulation protocols (bAP, EPSP train, paired activity). The colour-coded relative spatial maps shown on the left; corresponding trace displays of signals from 5 representative locations shown on the right. To isolate parts of the recording used to generate a spatial map of the peak EPSP signal, spike signals were removed artificially in the middle panel (EPSP). B, _V_m signals related to bAP (black traces) are superimposed with paired EPSP–bAP signals (red traces) to reveal region-specific changes in the peak membrane depolarization. C, bAP signals aligned with paired EPSP–bAP signals to reveal region-specific differences in action potential threshold to the peak amplitudes (EPSP signals subtracted). Traces are shifted horizontally with respect to each other for clarity. D, fluorescent images of the dendritic arbor (bis-fura-2 excitation); five recording regions (1–5) and the stimulating electrode position are indicated. E, frequency distribution histogram of the change in the bAP baseline-to-peak amplitude evoked by LTP-forming stimuli. Green bars, boosting (_n_=17). White bar, no change (_n_=9). Yellow bars, depression (_n_=18). Dendritic stimulus location: blue, apical trunk (_n_=10); red, oblique or apical branch (_n_=8). F and G, [Ca2+]i signals during the three stimulation protocols in control solution and in the presence of 100 μm of AP-5 in the bath. The spatial maps of the peak signal shown on the left; corresponding trace displays are shown on the right with the somatic _V_m traces shown in red. AP-5 completely eliminated supra-linear component of the calcium signal in this experiment. H, somatically recorded EPSP train under control conditions and in the presence of 100 μm AP-5.

Figure 6

Figure 6. NMDA-dependent Ca2+ influx constituted, on average, 68% of the supra-linear component of the recorded signal evoked by LTP-forming stimuli

A, the relative spatial map of the supra-linear signal during paired activity in control solution. B, recordings of the supra-linear calcium signal from 4 selected locations in control solution and with 100 μm AP-5 present in the bath; the somatic _V_m recordings shown in red. AP-5 did not influence supra-linear calcium signals in this experiment indicating that they were mediated by VSCCs. C, superimposed _V_m signals from same 4 locations during bAP (black traces) and during paired EPSP–bAP signals (red traces) indicate region-specific changes in the peak membrane depolarization during paired activity. Traces on the right (shifted horizontally with respect to each other for clarity) are spike signals aligned to reveal region-specific differences in bAP baseline-to-peak amplitudes. D, traces on the left, peak membrane depolarization during pairing of bAPs with large (red trace) and small (blue trace) EPSP depolarization at location 3. Traces are slightly shifted horizontally with respect to each other for clarity. Traces on the right, bAP signals aligned to indicate the difference in baseline-to-peak amplitude; reduced depolarization (blue trace) clearly increased the boosting effect. Black trace, bAP evoked alone. E, dendritic arbor of another neuron in which both sources of calcium (NMDA-R dependent and VSCC dependent) contributed significantly to the composite signal. F, recordings of supra-linear [Ca2+]i signals from 3 selected locations under control conditions, in the presence of 100 μm AP-5 in the bath, and after wash-out of the drug. G, summary plot of the normalized decrease in signal amplitude in the presence of AP-5 (_n_=13) and the effect of the wash-out of the drug (_n_=5).

Figure 8

Figure 8. Spatial correlation between the amplitude of AP-5-insensitive component of the supra-linear [Ca2+]i signal and the peak membrane depolarization during paired activity

A, [Ca2+]i signals and _V_m signals corresponding to bAPs (black traces) and paired EPSP–bAP signals (red traces) from two locations on the dendritic tree of Cell 1 shown in B. Signals are superimposed to reveal the region-specific increase in peak depolarization during paired activity. B, upper panel: fluorescence image of the dendritic arbor of Cell 1. The stimulating electrode is indicated schematically. Middle panel: colour-coded spatial map of the change in [Ca2+]i signal amplitude during paired activity. Bottom panel: spatial map of the change in peak membrane depolarization (_V_m) during paired activity. C, spatial correlation between _V_m and [Ca2+]i signal in two additional neurons. D, scatter-plot of the AP-5-insensitive [Ca2+]i signal measured at the site of EPSP origin versus the change in peak membrane depolarization at the same location induced by paired activity. _r_=0.9 (P < 0.001).

Figure 9

Figure 9. Non-linear summation of _V_m signals during paired activity brings membrane potential to the optimal range for NMDA-R-mediated Ca2+ flux

A, a composite fluorescent image of a neuron. Position of two extracellular stimulating electrodes indicated schematically. B, superimposed _V_m signals related to bAP evoked alone (black trace) and during paired activity (red traces) recorded from location 1 marked by upper red oval. The calibration of optical signals is approximate (see text). Baseline-to-peak bAP amplitude was reduced to 37% (left inset) by paired activity. The resultant peak membrane depolarization closely matched the range that corresponds to the maximum Ca2+ flux through activated NMDA-R (grey area). Voltage dependence of the Ca2+ flux through NMDA receptor channels (dashed trace; scale in arbitrary units) adapted from Garaschuk et al. (1996) is shown on the right. C, baseline-to-peak bAP amplitude, recorded from location 2 marked by lower red oval, was boosted to 125% (left inset) by paired activity. The resultant peak membrane depolarization was brought closer to the optimal range by bAP boosting at location 2.

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