Active dendrites support efficient initiation of dendritic spikes in hippocampal CA3 pyramidal neurons

Abstract

CA3 pyramidal neurons are important for memory formation and pattern completion in the hippocampal network. It is generally thought that proximal synapses from the mossy fibers activate these neurons most efficiently, whereas distal inputs from the perforant path have a weaker modulatory influence. We used confocally targeted patch-clamp recording from dendrites and axons to map the activation of rat CA3 pyramidal neurons at the subcellular level. Our results reveal two distinct dendritic domains. In the proximal domain, action potentials initiated in the axon backpropagate actively with large amplitude and fast time course. In the distal domain, Na(+) channel-mediated dendritic spikes are efficiently initiated by waveforms mimicking synaptic events. CA3 pyramidal neuron dendrites showed a high Na(+)-to-K(+) conductance density ratio, providing ideal conditions for active backpropagation and dendritic spike initiation. Dendritic spikes may enhance the computational power of CA3 pyramidal neurons in the hippocampal network.

Figure 1

Subcellular patch-clamp recording from dendrites and axons of CA3 pyramidal neurons. (a-c) Dendritic recording from CA3 pyramidal neurons. A confocal image of a CA3 pyramidal cell filled with 100 μM Alexa Fluor 488 via the somatic recording pipette during the experiment is shown in a. The dendritic recording site is 218 μm from the soma. An infrared differential interference contrast video image of the soma (top) and the apical dendrite (bottom) of the same CA3 pyramidal neuron with the patch pipettes attached is shown in b. A light micrograph of a CA3 cell filled with biocytin during recording and labeled using 3,3′-diaminobenzidine (DAB) as chromogen is shown in c. The bottom panel shows an expanded view corresponding to the dashed box shown in the top panel. Arrowheads in c indicate thorny excrescences, the postsynaptic spines of hippocampal mossy fiber synapses. (d-f) Axonal recording from CA3 pyramidal neurons. A confocal image of a CA3 pyramidal neuron filled with 100 μM Alexa Fluor 488, showing an axon bleb at the surface of the slice, is presented in d. The axonal recording site is 90 μm from the soma. A corresponding infrared differential interference contrast video image is shown in e. Post hoc biocytin labeling of the same CA3 cell using DAB as chromogen is shown in f. The axon was unequivocally identified by the complete lack of spines. Arrowhead in f indicates axon bleb. Photomicrographs in a, d and f represent collages of images at slightly different focal planes. Images in a and b were obtained from the same cell; images in d-f were obtained from another cell.

Figure 2

Action potentials backpropagate into the dendrites of CA3 pyramidal neurons with large amplitude and fast time course. (a) Train of action potentials (APs) evoked by somatic current injection, simultaneously recorded at soma and dendrites (left) and soma and axon (right). Black traces represent somatic voltage and corresponding current, red traces represent dendritic voltage and current, and blue traces represent axonal voltage and current. The current intensity was 325 pA (left) and 675 pA (right). Bottom traces show first action potential in the 1-s train on an expanded timescale. The dendritic recording site is 144 μm from the soma and the axonal recording site is 54 μm from the soma. (b,c) Summary plot of action potential peak amplitude measured from threshold (b) and duration at half-maximal amplitude (c) in 43 somatodendritic recordings plotted against distance (positive distance, apical dendrite; negative distance, basal dendrite; zero distance, soma). Somatic rheobase current stimuli were used in all cases. Dashed curves represent a fitted Boltzmann function (b) and the results of linear regression (c) for data points from soma and apical dendrites. (d,e) Summary plot of action potential latency measured at half-maximal amplitude (d) and maximal rate of rise ((dV/dt)_max, e) in 43 dendritic and 11 axonal recordings plotted against distance (positive distance, axon; negative distance, dendrites). Somatic rheobase current stimuli were used in all cases. The green curve represents a third-order polynomial function fitted to the data points. The smallest latencies were measured at a distance of 75 μm from the center of the soma, representing the action potential initiation site.

Figure 3

Dendritic action potential backpropagation in CA3 pyramidal cells shows only moderate activity dependence. (a-c) Action potentials evoked by trains of ten brief current pulses applied to the soma at a frequency of 20, 50 and 100 Hz. Note that action potentials were efficiently propagated even during high-frequency trains. Black represents somatic voltage and red represents dendritic voltage. The recording site on the apical dendrite is 144 μm from the soma. (d) Ratio of action potential amplitude for the fifth action potential over that of the first action potential in the train. Dashed lines represent the results of linear regression. (e) Ratio of action potential half-duration for the fifth action potential over that of the first action potential in the train. The ratio was close to 1, indicating that action potentials during repetitive activity were propagated with fast time course.

Figure 4

High Na⁺-to-K⁺ conductance ratio and distinct conductance gradients in CA3 pyramidal neuron dendrites. (a) Na⁺ current (average from 20–33 single traces, respectively) evoked in outside-out patches from soma and apical dendrite (150 μm). Test pulse potential was 0 mV. Na⁺ current was recorded with Cs⁺-internal solution. Left, soma; right, dendrite; top, control; bottom, currents in the presence of 0.5 μM TTX in the bath. We used outside-out patches; leak and capacitive currents were subtracted by a ‘P over −8’ correction procedure. (b) A-type K⁺ current (average from 9–42 single traces) evoked in outside-out patches from soma and apical dendrite (153 μm). K⁺ current was recorded with K⁺-internal solution. A-current was isolated by subtraction of traces with a −40-mV prepulse from those with a −120-mV prepulse. Left, soma; right, dendrite; top, control; bottom, currents in the presence of 5 mM 4-AP in the bath. (c) Delayed rectifier K⁺ current (average from 5–10 single traces) evoked in outside-out patches from soma and apical dendrite (134 μm). Delayed rectifier K⁺ current was isolated by a −40-mV prepulse. Left, soma; right, dendrite; top, control; bottom, currents in the presence of 20 mM TEA in the bath. Pulse protocols in b and c: prepulse to −120 mV or −40 mV, test pulse to 70 mV. (d-f) Plot of Na⁺ current density (d), A-type K⁺ current density (e) and delayed rectifier K⁺ current density (f) against distance from the soma. Dashed lines represent the results of linear regression of data points from apical dendrite. Data from 12, 12 and 12 somatic (black circles) and 41, 38 and 38 dendritic patches (red circles).

Figure 5

Efficient initiation of dendritic Na⁺ spikes in CA3 pyramidal neurons. (a) Examples of dendritic spikes in CA3 pyramidal neurons. Top traces, 5-ms current pulses (intensity: 600 pA, 960 pA and 1,180 pA). Upper middle traces, subthreshold response. Lower middle traces, isolated dendritic spike. Bottom traces, dendritic spike followed by an axosomatic spike. Black traces represent somatic signals and red traces represent dendritic signals. (b) Dendritic spikes evoked by EPSC-like current waveforms in CA3 pyramidal neurons. Top, EPSC-like currents used as stimuli (rise time constant = 0.25 ms, decay time constant = 5 ms). The peak current was increased from 300 pA to 2,700 pA in 600-pA steps. Bottom, corresponding EPSP-like voltage waveforms. Black traces represent somatic signals and red traces represent dendritic signals. (c) All-or-none characteristics of dendritic spikes. Plot of peak amplitude of the dendritic signal against intensity of the current pulse. Amplitude was measured after subtraction of scaled subthreshold responses. The stepwise increase at ~700 pA corresponds to the initiation of the dendritic spike, whereas the second increase at ~1.2 nA reflects initiation and backpropagation of the axosomatic action potential (bAP). Data shown in a and c were taken from the same cell; data shown in b were taken from a different cell. The recording sites on apical dendrite are 133 μm (a,c) and 142 μm (b) from the soma. (d) Histogram of number of recordings with (gray bars) or without dendritic spikes (open bars) at different distances from the soma. Note that dendritic action potential initiation robustly occurred at distances >100 μm. Stimulation intensity = 100 pA to 3 nA. The blue curve shows the corresponding probability of dendritic spike initiation (right axis), as obtained by fitting with a Boltzmann function. (e,f) Plot of initiation threshold for axosomatic spikes (e) and dendritic spikes (f) as a function of distance. Data in e and f are from the same set of recordings. In distal recordings, current stimuli easily evoked dendritic spikes, but occasionally failed to trigger axosomatic spikes even at high intensity (no corresponding data points in e). Open circles indicate threshold values for 5-ms current pulses. Filled circles indicate threshold values for EPSC-like current waveforms.

Figure 6

Dendritic spikes are mediated by voltage-gated Na⁺ channels. (a) Schematic illustration of the current-clamp recording configuration (CC), combined with bath and local application of TTX. Encircled characters indicate correspondence between scheme and subsequent figure panels. (b) Bath application of 0.5 μM TTX blocked both dendritic and axosomatic action potentials evoked by a 5-ms dendritic current pulse (950 pA). The recording site on the apical dendrite is 164 μm from the soma. (c,d) Local application of TTX to the dendrite near the recording pipette tip selectively blocked the dendritic spike, but left the axosomatic action potential unaffected. Responses to current pulses with three different intensities (400 pA, 700 pA and 1100 pA) are shown overlayed. Black traces represent somatic voltage and red traces represent dendritic voltage. Note that the dendritic action potential was blocked, whereas the axosomatic action potential was largely unaffected. This confirms the local nature of the application. The recording site on the apical dendrite is 209 μm from the soma. (e,f) Bath application of 200 μM Cd²⁺ did not affect dendritic spikes. Responses to current pulses with three different intensities (300 pA, 550 pA and 800 pA) are shown overlayed. Black traces represent somatic voltage and red traces represent dendritic voltage. The recording site on the apical dendrite is 263 μm from the soma. Note that Cd²⁺ only had negligible effects on dendritic spikes, suggesting that the contribution of Ca²⁺ channels is minimal. Scale bars also apply to c and e.

Figure 7

Dendritic spikes increase the efficacy of axosomatic action potential initiation. (a) Schematic illustration of the recording configuration. Encircled characters indicate correspondence between scheme and subsequent figure panels. (b) Dendritic spikes evoked by EPSC-like current waveforms in CA3 pyramidal neurons. Top, EPSC-like currents were used as stimuli. The peak currents were 150, 600, 1,200, 1,800 and 2,400 pA. Center, corresponding EPSP-like waveforms. Bottom, expanded view of the somatic EPSP-like waveforms. Black traces represent somatic signals and red traces represent dendritic signals. The recording site on the apical dendrite is 142 μm from the soma. (c) Local application of 1 μM TTX to the dendrite prolonged the rising phase of the somatic EPSP-like voltage waveform and increased the initiation threshold of axosomatic spikes. EPSC-like current waveform; peak current was 150 pA and 300–2,100 pA in 300-pA increments. The recording site on the apical dendrite is 294 μm from the soma. Data in b and c were from different cells. Arrows indicate acceleration of rising phase produced by dendritic spikes. (d) Dendrosomatic propagation of dendritic spikes. Amplitude of dendritic spikes (DS) at the soma, normalized to that at the dendrite, was plotted against distance. The amplitude of the dendritic spike was measured after subtraction of scaled subthreshold responses. The attenuation of the backpropagated action potential (bAP) is replotted for comparison. (e) Summary graph of the average 20–80% rise time of EPSP-like voltage waveforms recorded at the soma without (−) or with dendritic spikes (+; *P < 0.05). (f) Summary graph of the average current threshold for the initiation of axosomatic spikes under control conditions and after local application of 1 μM TTX to the dendrite (*P < 0.05). In e and f, circles and lines represent data from individual experiments and bars indicate mean ± s.e.m.