Dendritic mechanisms underlying the coupling of the dendritic with the axonal action potential initiation zone of adult rat layer 5 pyramidal neurons

Abstract

1. Double, triple and quadruple whole-cell voltage recordings were made simultaneously from different parts of the apical dendritic arbor and the soma of adult layer 5 (L5) pyramidal neurons. We investigated the membrane mechanisms that support the conduction of dendritic action potentials (APs) between the dendritic and axonal AP initiation zones and their influence on the subsequent AP pattern. 2. The duration of the current injection to the distal dendritic initiation zone controlled the degree of coupling with the axonal initiation zone and the AP pattern. 3. Two components of the distally evoked regenerative potential were pharmacologically distinguished: a rapidly rising peak potential that was TTX sensitive and a slowly rising plateau-like potential that was Cd(2+) and Ni(2+) sensitive and present only with longer-duration current injection. 4. The amplitude of the faster forward-propagating Na(+)-dependent component and the amplitude of the back-propagating AP fell into two classes (more distinctly in the forward-propagating case). Current injection into the dendrite altered propagation in both directions. 5. Somatic current injections that elicited single Na(+) APs evoked bursts of Na(+) APs when current was injected simultaneously into the proximal apical dendrite. The mechanism did not depend on dendritic Na(+)-Ca(2+) APs. 6. A three-compartment model of a L5 pyramidal neuron is proposed. It comprises the distal dendritic and axonal AP initiation zones and the proximal apical dendrite. Each compartment contributes to the initiation and to the pattern of AP discharge in a distinct manner. Input to the three main dendritic arbors (tuft dendrites, apical oblique dendrites and basal dendrites) has a dominant influence on only one of these compartments. Thus, the AP pattern of L5 pyramids reflects the laminar distribution of synaptic activity in a cortical column.

Figure 1. The effect of input time course on initiation zone coupling

A, reconstruction of biocytin-filled L5 pyramidal neuron showing the sites of current injection (D_stim, 930 μm from soma) and recording (860 μm from soma and at the soma). B-E, current injections (double exponential functions, f(t) = (1 - e^-t/τ1)e^-t/τ2, where τ₂= 4τ₁) for which the times to peak were set to 50, 10, 5 and 2 ms, respectively (dashed traces). The different duration current injections elicited variable-length sodium-calcium action potentials (Na⁺-Ca²⁺ APs) at the dendritic recording electrode (black traces) and a variable number and pattern of sodium action potentials (Na⁺ APs) at the soma (blue traces).

Figure 2. Na⁺ and Ca²⁺ components of the dendritic potential

A, reconstruction of biocytin-filled L5 pyramidal neuron showing the sites of current injection (D_stim, 750 μm from soma) and recording (620 μm from soma and at the soma). B, short current injections (time to peak, 1 ms; dashed trace) elicited an all-or-none event, which was short and resembled a dendritic Na⁺ AP (black traces) that did not propagate to the soma (blue traces) but appeared as a ‘boosted EPSP’. Here traces just before and after threshold current injection are shown superimposed. C, application of TTX (1 μm) abolished the event and subsequent addition of Ni²⁺ and Cd²⁺ (100 and 50 μm, respectively) had no further effect. D, longer current injection (time to peak, 10 ms; dashed trace) elicited a complex dendritic potential with three Na⁺ APs with the first peak in the dendrite (black traces) preceding the first back-propagating Na⁺ AP (blue traces), but subsequent dendritic peaks following the somatic potentials. E, application of TTX blocked the fast rising component, the back-propagating Na⁺ APs and the dendritic peaks, leaving only a long potential that could be almost completely blocked by subsequent addition of Cd²⁺ and then further blocked by Ni²⁺.

Figure 3. Forward propagation of the dendritic potential

A, schematic representation of triple recordings (left) in which a dendritic potential was generated with the distally located electrode by injection of current (time to peak, 2-5 ms). The middle electrode was used to determine the maximum depolarization of the first peak and the somatic recording to show the time of the first back-propagating Na⁺ AP to check that the potential recorded at the middle electrode was propagating forwards. Dashed line represents 0 mV. B, the absolute membrane potential (V_m) at the peak of 67 dendritically recorded potentials from 40 neurons travelling towards the soma plotted as function of the distance of the middle electrode from the soma. The distal site varied from 560 to 860 μm from the soma (666 ± 67 μm). Dashed lines were fitted by eye through the two putative classes of forward propagation (labelled Amplification and Attenuation).

Figure 4. Two components of the forward-propagating potential

A, schematic diagram showing the electrode placement at 630 μm (1), 300 μm (2), 200 μm (3) and the soma (4) on a L5 pyramidal cell. The recording was made in two stages (first electrodes 1, 2 and 4 and then electrodes 1, 3 and 4; i.e. electrode 2 was moved to the position of electrode 3). B, injection of an EPSP-shaped current (bottom trace) was used to elicit a Na⁺-Ca²⁺ AP in the distal dendrite. The initial component failed to propagate forwards as recorded at electrodes 2 and 3 (*; 300 and 200 μm from the soma), but there was a second propagating component, also starting at the most distal electrode, that decreased in amplitude up to 300 μm (**) and later increased to 200 μm(***) before reaching full height at the soma. C, the boxed region from B on an enlarged time scale showing the sequence of events.

Figure 5. Modulating the amplitude of the forward-propagating potential

A, schematic diagram showing the electrode placement on a L5 pyramidal cell: distally, proximally and at the soma (P1, P2 and P3, respectively). B, isolated dendritic potential that was converted to a propagating potential. B1, current injected at the distal dendritic electrode (P1) caused a Na⁺-Ca²⁺ AP in the distal dendrite that failed to propagate fully to the soma. B2, a very small current injection (200 pA pulse for 50 ms; onset 30 ms before EPSP waveform current injection at distal electrode) at the proximal electrode (300 μm from soma) caused the forward-propagating event to propagate well, leading to Na⁺ APs at the soma and a dendritic Na⁺-Ca²⁺ AP complex. C, AP propagating well that was converted to an isolated dendritic potential. C1, an example of a cell with well-propagating forward potentials under control conditions. C2, hyperpolarizing current (-450 pA) injected at the proximal electrode (P2; 300 μm from the soma) caused the forward-propagating potential to fail. Current injection waveforms are shown beneath the recorded potentials.

Figure 6. Timing of the input to the proximal region

A, current injected at the proximal electrode (300 μm) caused the forward-propagating dendritic Na⁺-Ca²⁺ AP evoked by an EPSP-shaped waveform at the distal electrode (650 μm) to fail completely towards the soma. B, in this same cell the forward-propagating Na⁺-Ca²⁺ AP was in an intermediate state of propagation under control conditions and showed two components. The second rising component was interrupted by the presence of a back-propagating Na⁺ AP from the soma. C, depolarizing current at the proximal electrode caused the forward propagation to be completely active and only one component was then apparent. Further current injection led to more Na⁺ APs. D, the onset of an EPSP-shaped waveform injected at the distal electrode was moved relative to the end of the 50 ms step pulse injection at the proximal dendritic electrode. Here, we show data from 20 ms before the end of the proximal injection (•), and 5 ms (○) and 35 ms (▵) after the end of the proximal injection (shown above the graph schematically). The least current required to improve propagation of the dendritic Na⁺-Ca²⁺ AP was needed when it was injected during the proximal current injection (•). E, the sharp cutoff between APs that propagated well and those that propagated badly for a number of cells (n = 7) is shown by aligning the last point still under the half-amplitude point to 0 pA. The actual current injected for this point varied from -500 to +400 pA (mean, 100 ± 290 pA) depending on the initial conditions of coupling for the cell. The shift between good propagation and failure to propagate occurred within ≈50 pA.

Figure 7. Critical region for forward propagation

A, reconstruction of biocytin-filled L5 pyramidal neuron showing the locations of dendritic recording. The experiment consisted of two triple recordings with the proximal electrode (250 μm) exchanged for the less proximal electrode (500 μm) but the distal (700 μm) and somatic electrodes remaining in place. B, an EPSP-shaped current injection at the distal electrode (shown in E) of 1.4 nA at the peak failed to evoke a dendritic Na⁺-Ca²⁺ AP. C, however, at the threshold of 1.6 nA, the dendritic Na⁺-Ca²⁺ AP was evoked and propagated well towards the soma. The rising phase clearly had two components at 500 μm and probably also at 250 μm (points of inflection indicated by arrows). D, with further current injection (2.2 nA) the potential failed at the previous turning point (marked by arrows) and rapidly decreased towards the soma, indicating the presence of a critical region in the apical dendrite that needs to be active for complete forward propagation.

Figure 8. Attenuation of back-propagating action potentials

A, schematic diagram showing the placement of 4 electrodes on a L5 pyramidal neuron simultaneously. The 3 dendritic recordings were made at 460, 620 and 820 μm from the soma. B, traces corresponding to the 4 electrodes arranged vertically and sequentially. Current pulses of 2 ms in duration were injected at the somatic electrode to elicit APs at 20 Hz. The first back-propagating Na⁺ AP showed little reduction over the interval 460-820 μm. The last Na⁺ AP, however, was significantly reduced at 820 μm relative to the initial AP but not at 460 μm with an intermediate amplitude at 620 μm. Dashed lines are superimposed to emphasize the rate of decay of the back-propagating APs. C, percentage amplitude of the last AP (compared to the first AP) in a train shown as a function of distance from the soma. Only cases where the first AP in the train propagated well (> 40 mV) are shown, for clarity. Here, the inactivation of the AP propagation during trains starts to occur around 300 μm and is proportionately more attenuated at distal locations. D, amplitudes of 112 back-propagating APs shown as a function of distance from the soma for both the first AP in a train (•) and the last AP (□). The amplitude of the first back-propagating AP becomes progressively more variable with distance. The last back-propagating AP in a train becomes progressively smaller after around 300 μm, diverging from the amplitude of the first back-propagating AP to form two classes of propagation as in the forward-propagating case. The amplitude of the somatic AP is shown (○) with standard deviation at 0 μm. E, the latency of the time of half-amplitude of the first dendritic back-propagating AP in a train (•) and last AP (□) after the time of half-amplitude at the soma, plotted as a function of distance. The dashed line represents a straight line fit to the first AP in each train. The slope representing the speed of propagation was 0.508 m s⁻¹.

Figure 9. Two classes of back propagation

A, schematic diagram showing the placement of 3 electrodes on a L5 pyramidal neuron simultaneously. B, a train of back-propagating APs was elicited by current injections at the soma at 70 Hz with progressively larger hyperpolarizing current (-600 to -700 pA) injected at the proximal electrode (300 μm from the soma). (Somatic recording, P3, blue; proximal recording (300 μm), P2, black; and distal recording, 630 μm, P1, dashed line.) The hyperpolarization eventually caused the third AP in the train to decrease in amplitude, revealing the presence of a mechanism maintaining the amplitude of the back propagation of APs. C, expanding the time scale for the boxed region in B and overlaying the traces shows the two-component nature of the back-propagating AP at the distal site just before inactivation (1), during (2) and after full inactivation (3), black traces. A small depolarization is visible at the proximal site (black traces) following the peak at the proximal site. D, by subtracting trace 3 from trace 1, the regenerative component of the back-propagating AP is revealed to arise first at the distal location and reflected back towards the soma. Peaks are shown by vertical lines. E, a different cell in which the back-propagating AP clearly had two components under control conditions. Recordings were made at 170 μm (dashed trace), 420 μm (black trace) and 630 μm (blue trace) from the soma. The point of inflection revealing the existence of a further regenerative component on the recording from the 420 μm site is shown by an arrow.

Figure 10. Dependence of back-propagating AP on dendritic V_m

A, reconstruction of a biocytin-filled L5 pyramidal neuron showing the locations of dendritic recordings. The electrode at 930 μm (D_stim) was used for dendritic current injection and the electrode at 860 μm for the dendritic recording. A train of back-propagating APs was generated by injecting 600 pA current for 400 ms at the soma (C, lower traces). B, a series of current injections (100 pA steps) at the dendritic location for 500 ms caused the first back-propagating AP to progressively increase in size. It eventually initiated a burst of APs at the soma followed by a calcium spike in the dendrite. C, the cell fired regularly when no current was injected at the dendritic site. Current injection is represented below the AP traces. D, the amplitude of the back-propagating AP recorded at the dendritic location is plotted as a function of the current injected at the nearby dendritic location.

Figure 11. Depolarization enhances bursting

A, triple dendritic patch recording of a dendritic Na⁺-Ca²⁺ AP initiated by injection of an EPSP-shaped waveform (1.6 nA, shown below E) at a distal dendritic location (P1, 650 μm from soma; dashed line) that led to a Na⁺-Ca²⁺ AP and a single back-propagating AP, recorded at a more proximal location (P2, 310 μm; black line) and the soma (P3; blue line). B, EPSP waveform current injection at a distal dendritic location. C, addition of an extra 5 mm KCl to the extracellular solution (total of 7.5 mm KCl) caused progressive depolarization of the resting membrane potential. At 6 mV more depolarized than the control, the same current injection at the distal dendritic location now caused 2 APs. D, at 12 mV more depolarized than the control, the same current injection caused a burst of 3 APs. E, current injection at all three pipettes causing 10 mV depolarization had a similar effect.

Figure 12. Dendritic depolarization controls the coupling of initiation zones

To test the relationship between bursting and dendritic depolarization, triple recordings were made with one distally located electrode, a proximally located electrode and a somatic electrode. A, reconstruction of a biocytin-filled L5 pyramidal neuron showing the locations of dendritic recordings (690 and 310 μm from the soma) and the recording at the soma. The three recordings shown in B-D represent different levels of depolarizing current injection (I₁, I₂ and I₃) at the proximal electrode, arranged vertically to correspond to the location of the electrodes. B, a single back-propagating AP was generated at the soma with an EPSP-shaped waveform (dashed trace at bottom). No current was injected at the proximal electrode (I₁). C, 500 pA current (I₃) injected at the proximal dendritic location starting 30 ms before the somatic current injection (current injection shown as dashed trace below middle recording trace) caused a burst of Na⁺ APs at the soma and a long depolarizing potential in the dendrite. D, the traces from B and C are overlaid with the subthreshold current injection of 400 pA (I₂) showing the progressive activation of current in the dendrite. The shaded areas highlight the difference between the control and the trace just before threshold from which it can be seen that the increase is greatest in the proximal region.

Figure 13. Dendritic Na⁺-Ca²⁺ AP but not bursting is dependent on Ca²⁺

A, schematic diagram showing the same experimental configuration as in Fig. 12. Here the electrodes were placed at 590 μm (P1, dashed line) and 280 μm (P2; black) from the soma and at the soma (P1; blue). B, an initial somatic Na⁺ AP was elicited by current injection (1.1 nA) at the soma (current traces shown below). C, current injection at the proximal location (400 pA) caused the generation of a burst and a Na⁺-Ca²⁺ AP at the dendritic location. D, application of 100 μm Ni²⁺ and 50 μm Cd²⁺ blocked the Na⁺-Ca²⁺ AP and the third Na⁺ AP in the burst but not the initial doublet, showing that the distal dendritic site was not responsible for the initial doublet.

Figure 14. Timing of the combined input to the soma and proximal dendritic region

A, reconstruction of a biocytin-filled L5 neocortical pyramidal neuron overlaid on a photograph of the somatosensory cortex from a slice of rat brain during the experiment shown in B, with the silhouettes of the 4 electrodes indicating their positions. The layers of the cortex are indicated on the left-hand side. B1-4, current was injected at the electrode in layer 4 (electrode on the left, recording not shown) in the form of a 50 ms square pulse (0.4 nA) for each trace (1-4). Current injected at the somatic electrode (blue) with an EPSP waveform, which normally elicited a single Na⁺ AP, elicited a doublet for the times indicated under B4 showing the current pulses (blue shaded time interval). This doublet caused a broadened potential after the second AP at the most proximal electrode (black) and especially at the distal electrode (dashed line).

Figure 15. The propagation of regenerative potentials along the apical dendrite

A, the amplitude of Na⁺ APs propagating from the soma back into the dendrites varies from cell to cell as a function of distance with increasing variability after the proximal apical zone (> 300 μm). The first AP in a train (continuous lines) sometimes maintained approximately the same amplitude from ≈300 μm to well into the tuft. On the other hand, the subsequent APs in a train become progressively inactivated (dashed line) and failed to propagate fully to the tuft. B, dendritic Na⁺-Ca²⁺ APs propagating towards the soma from the distal dendritic initiation zone fall into 2 categories: those that increase in amplitude until they reach the soma and those that fail somewhere in the proximal apical region and cause only a slight depolarization at the soma.

Figure 16. Simplified representation of a L5 neocortical pyramidal neuron and the relationship between input location and AP pattern

A, representation of a L5 neocortical pyramidal neuron with 3 functional compartments compared to the morphology of a reconstructed biocytin-filled L5 pyramidal neuron from the somatosensory cortex. Compartment A corresponds to the distal apical initiation zone and the tuft dendrites. Compartment B corresponds to the axonal initiation zone and the basal dendrites. Compartment C corresponds to the proximal apical dendrite and oblique dendrites. B, input to the distal dendrite tends to cause a burst of Na⁺ APs whereas the same input to the soma tends to cause single APs or regularly spaced spikes. Typical examples are shown here of the response of the same neuron to a 10 ms time to peak EPSP waveform (dashed traces) injected in either compartment A or compartment C at threshold current levels. Input to compartment B tends to improve the coupling between compartments A and C. After priming the proximal dendritic compartment (B) with a small current injection, a short EPSP waveform (time to peak of 3 ms) at the soma leads to a burst of Na⁺ APs (bottom traces).