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.2004 Jul 28;24(30):6703-14.
doi: 10.1523/JNEUROSCI.0307-04.2004.

Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones

Affiliations

Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones

Maja Djurisic et al. J Neurosci..

Abstract

To obtain a more complete description of individual neurons, it is necessary to complement the electrical patch pipette measurements with technologies that permit a massive parallel recording from many sites on neuronal processes. This can be achieved by using voltage imaging with intracellular dyes. With this approach, we investigated the functional structure of a mitral cell, the principal output neuron in the rat olfactory bulb. The most significant finding concerns the characteristics of EPSPs at the synaptic sites and surprisingly small attenuation along the trunk of the primary dendrite. Also, the experiments were performed to determine the number, location, and stability of spike trigger zones, the excitability of terminal dendritic branches, and the pattern and nature of spike initiation and propagation in the primary and secondary dendrites. The results show that optical data can be used to deduce the amplitude and shape of the EPSPs evoked by olfactory nerve stimulation at the site of origin (glomerular tuft) and to determine its attenuation along the entire length of the primary dendrite. This attenuation corresponds to an unusually large mean apparent "length constant" of the primary dendrite. Furthermore, the images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. Finally, secondary dendrites clearly support the active propagation of action potentials.

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Figures

Figure 2.
Figure 2.
Spatial resolution and sensitivity of voltage imaging.A, Low resolution (top) and high resolution image (bottom) of the terminal dendritic tuft.B, Recordings of action potential signals from four individual locations on the dendritic tuft in single-trial measurements (no averaging).C, The signal-to-noise ratio with spatial averaging of four pixels fromB. D, The signal-to-noise ratio with spatial averaging of the entire tuft area.E, An evoked EPSP recorded from 4 individual locations on the dendritic tuft; 20 trials were averaged.F, Spatial average of four pixels shown inE. G, Spatial average of the entire tuft area.
Figure 4.
Figure 4.
Voltage imaging of the spike initiation and propagation pattern in a mitral cell stained with voltage-sensitive dye. The two consecutive spikes evoked by a single stimulus have different initiation sites and propagation patterns.A, A composite image of a mitral cell obtained with a conventional high-resolution CCD camera. Soma, primary dendrite (d′), terminal dendritic tuft, and a secondary dendrite (d″) are clearly visible in this fluorescence measurement.B, Single frame recording of the same cell obtained with a fast low resolution (80 × 80 pixels) CCD camera used for voltage imaging.C, Optical recording of action potentials evoked by olfactory nerve stimulation (single volley, 40 μA, 0.1 msec) obtained simultaneously from three different regions (B, red, blue, and green rectangles).D, The propagation of the first of the two spikes shown inC; expanded time scale. The earliest action potential (red trace) was initiated in the tuft and propagated toward the soma (blue trace) and secondary dendrite (green trace). The spike occurred with a delay of 2 msec in the distal part of secondary dendrite, ∼300 μm away from the soma. The propagation delay and the constant shape of the spike indicate active propagation. Action potential signals are scaled to the same height.E, The recordings from two selected locations [tuft (red) and soma (blue)] on the expanded time scale used to determine the propagation velocity of the first spike.F, Color-coded representation of the data showing spatial and temporal dynamics of the first synaptically evoked spike fromC. The peak of the action potential, in this and all subsequent figures, is shown in red. Consecutive frames represent data points around the peak of the action potential separated by 0.37 msec. The first action potential was clearly initiated in the tuft (red arrow) and propagated to the soma.G, The recordings from two selected locations [tuft (red) and soma (blue)] on the expanded time scale used to determine the propagation velocity of the second spike.H, Color-coded representation of the data showing spatial and temporal dynamics of the second synaptically evoked spike fromC. The second spike was initiated in the soma (red arrow) andpropagated to the tuft.
Figure 3.
Figure 3.
Voltage imaging of the EPSP from the terminal tuft and along the primary dendrite.A, High- and low-resolution image of a mitral cell.B, Single-trial optical recordings from 10 different regions on the primary and oblique dendrites showing signals corresponding to an evoked action potential. The colored rectangles indicate pixels averaged to obtain traces 1-10. The amplitude of the action potential measured from the soma by a patch electrode was 93 mV (resting potential-to-peak). The amplitude of optical signals (ΔF/F) corresponding to a spike of constant size varied with the location (1-10) because of unequal sensitivity of the optical measurement from different locations. The sensitivity profile, determined from action potential measurements, provides a calibration for the conversion of optical signals to membrane potential changes. In the measurements shown, the calibration factors (from the tuft to the most proximal part of the primary dendrite) are 1; 0.74; 0.56; 0.63; 0.53; 0.38; 0.40; 0.32 and 0.47 for the left and 0.59 for the right oblique branch.C, Calibration of optical signals (ΔF/F) in terms of membrane potential (in millivolts). All traces represent the average output of the same group of 35 pixels that receive light from the dendritic tuft (red rectangles inA). Twenty trials were averaged to improve the signal-to-noise ratio. Trace 1 shows an optical signal corresponding to an action potential (red trace) of 93 mV used as a calibration standard. Trace 2 is a subthreshold EPSP signal evoked by olfactory nerve stimulation and calibrated to be 9 mV in amplitude at the site of origin (tuft). Trace 3 shows a threshold EPSP signal recorded from the tuft after the action potential was blocked by intracellular application of QX-314. In the measurement shown in trace 3, the stimulus delivered to the olfactory nerve was identical to the one applied in the measurement shown in trace 1. The EPSP signal in trace 3 (also superimposed over trace 1 as a black trace) overlaps closely with the local response preceding a spike in trace 1, indicating that the spike is eliminated by QX-314, whereas the synaptic potential was unchanged.D, The amplitude of EPSP signals on a voltage scale at 10 recording sites. The calibration of optical signals shows that EPSP at location 8 (red), only 15 μm away from soma, is 13.2 mV in amplitude. Electrical recording from the soma (D, trace 11) was similar (12.5 mV). Traces 9 and 10 are signals from left and right oblique dendritic branches.E, The peak amplitude of EPSP plotted as a function of distance along the primary dendrite measured from the site of origin (terminal tuft).F, The shape of EPSP as a function of distance from the site of origin (tuft). Red trace, Optical recording from the tuft. Blue trace, Electrical recording from the soma. The EPSP has a slower rate of rise in the soma compared with the tuft.G, Action potentials generated by numerical simulation. Trace 1, The control action potential in the primary dendrite. Trace 2, The action potential in the tuft with fully active dendritic branches. Trace 3, The action potential in the tuft with fully passive dendritic branches.
Figure 1.
Figure 1.
Pharmacological effects and photodynamic damage.A, The resting membrane potential and action potential size and shape remained constant from the start (black) to the end of the 54 min staining period (gray). The resting membrane potential was -54mV. Whole-cell recording from the soma with the patch pipette was used to deposit the dye.B, The 1st (black) and 30th (gray) 200 msec optical recording trial of the evoked spike signal in the dendritic tuft. No photodynamic damage was detected after a total of 6 sec exposure to high-intensity excitation light.
Figure 5.
Figure 5.
Multiple spike trigger zones in two mitral cells (E, F). Color-coded representation of changes in fluorescence light intensity corresponding to action potentials evoked by olfactory nerve stimulation of different intensity. The peak of the action potential is shown in red. Individual frames are separated by 0.37 msec. Red arrow indicates the position of the trigger zone.A, A strong stimulus (60 μA, 0.1 msec) initiated the spike in the dendritic tuft.B, A weak stimulus (25 μA, 0.1 msec) initiated the spike in the soma-axon region.C, A medium stimulus (30 μA, 0.1 msec) initiated the spike in the trunk of the primary dendrite.D, A medium stimulus (40 μA, 0.1 msec) initiated the spike in the trunk of the primary dendrite of another mitral cell.
Figure 6.
Figure 6.
Feedback inhibition in secondary dendrites shifts the site of spike initiation.A, High-resolution composite image of a mitral cell stained with the voltage-sensitive dye.B, F, The initiation and propagation of an action potential evoked by a single volley to the olfactory nerve (60 μA, 0.1 msec) recorded optically from the tuft (red) and close to the soma (blue). The recordings from two different locations were compared on an expanded time scale in control solution (B) and with GABA-A receptors blocked with picrotoxin and bicuculline (F). Action potentials started in the soma-axon region under both conditions.D, H, The single volley to the olfactory nerve was preceded by an action potential evoked by direct stimulation of the soma to induce feedback inhibition in secondary dendrites. Under control conditions, the initiation of the subsequent spike by olfactory nerve stimulation was shifted to the tuft (D). After GABA-A receptors were blocked, the initiation of the spike evoked by ON stimulation was unchanged (H).C, E, G, I, Color-coded representation of the data shown inB, D, F, andH for the spike evoked by ON stimulation.C andE illustrate the shift in the initiation site evoked by preceding activity under control conditions.G andI illustrate that the trigger zone shift was absent after feedback inhibition was removed by blocking GABA-A receptors.
Figure 7.
Figure 7.
Secondary dendrites support active propagation of action potentials.A, Fluorescence image of a mitral cell with two parallel branches of a secondary dendrite. A frame obtained with the CCD camera used for voltage imaging is shown below the high-resolution image.B, An action potential was evoked in the tuft of the primary dendrite, by a single volley delivered via a stimulating electrode in the olfactory nerve layer (S-1; 80 μA, 0.1 msec), or in the distal region of the secondary dendrite by direct stimulation with a stimulating electrode positioned next to the dendrite (S-2; 40 μA, 0.1 msec). The site of spike initiation was determined by comparing optical signals from two selected regions (red and black rectangles superimposed on the CCD image of the cell inA). Signals were scaled to the same height. The spike initiated in the tuft (S-1) propagated from the somatic region into the secondary dendrites. The action potentials initiated distally in secondary dendrites propagated in the opposite direction.C, Color-coded representation of the same data as shown inB. Spatial and temporal dynamics of evoked signals clearly show the two patterns of propagation described above. The propagation velocity is markedly higher for impulses traveling away from the soma.
Figure 8.
Figure 8.
Secondary dendrites support active propagation of action potentials. Action potential signals from selected regions on secondary dendrites (1-3) are compared for a spike evoked by depolarizing the soma under control conditions (black traces) and for a spike-like waveform signal (red traces) produced in the soma by voltage-clamp commands under conditions that block sodium channels (TTX).
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