Article Figures & Data
Figures
- Fig. 1.
Apparatus for optical imaging. The slice preparation was continuously perfused with low [Mg] ACSF in a submerge chamber on the stage of a modified Leitz Orthoplan microscope (Wetzlar, Germany). The chamber and the perfusing solution were heated at 26°C. To reduce the vibration noise for optical recordings, we mounted the microscope on an air table and paused the perfusion during optical recording trials. We made absorption measurements using transmission illumination. Light from a halogen tungsten filament bulb (12 V; 100 W) was collected and illuminated the preparation through a 705 ± 60 nm filter. The image was formed by a 2.8× (NA, 0.05) objective. With this optical magnification, the six laminae of the cortex were projected to five rows of photodetectors (lower right diagram), and a 4-mm-wide cortical section was monitored by ∼50 neighboring detectors. RF, Rhinal fissure; WM, white matter.
- Fig. 2.
Epoch oscillations measured with an LFP electrode.A, When a cortical slice is perfused in low [Mg] ACSF, epochs of oscillations spontaneously occur. This recording is made by an electrode placed in cortical layer II, after 2 hr of perfusion in low [Mg] ACSF. B, An expanded display shows that an epoch started with a spontaneous synchronous spike and 7–10 Hz oscillations gradually emerged a few seconds later. In different preparations the epochs consisted of 3–200 cycles of oscillation. In all preparations tested (n > 50), all oscillations appeared in spontaneous epochs, and each epoch started with a synchronous spike. However, in seven preparations, there were only one or two oscillation cycles in each epoch. The number of oscillation cycles in each epoch appeared to increase the longer the interepoch intervals were.
- Fig. 3.
Comparison of electrical (LFP) and voltage-sensitive dye signals. A, During oscillations, the LFP signals and optical signals from neighboring sites were highly correlated. The recording sites were ∼0.5 mm apart, and there was a small phase difference between the different locations.B, The cross-correlation between LFP signals and the two optical detectors was high during the oscillations.
- Fig. 4.
Multiple site recording of the oscillations.A, A section of cortical slice (Sm Iarea) imaged by ∼50 neighboring photodetectors and one LFP electrode (in layer II). The imaging field was ∼4.5 mm in diameter.RF, Rhinal fissure; WM, white matter.B, Selected recording traces from the sites (solid squares in A). Both electrical and optical signals were low-pass filtered at 300 Hz, and the amplitude of the traces has been normalized to emphasize the phase relationships. C, The power spectra of the data in B. The gray trace is the average of all individual FFTs, in which the major frequency peak was 6.2 Hz (at 26°C). A minor frequency peak (∼10.8 Hz) does not have a systematic representation in the raw data shown in B. This minor peak was also seen in in vivo LFP recordings (Nicolelis et al., 1995). This recording trial contained 16,000 frames of voltage-sensitive dye images. Subsequent figures (see Figs. 5-8) were made from selected sections of these same data.
- Fig. 5.
Auto- and cross-correlations. A, Cross-correlations. I, The amplitude of the correlations between the signals did not decrease significantly with distance. Thesolid and dotted traces are correlations between two detectors 800 and 3200 μm apart, respectively.II, Signals from the same column (detectors 2, 3) have a somewhat larger correlation (dotted trace) than do the signals from the same layer but different columns (detectors 1, 2;solid trace). B, Autocorrelations of oscillation signals at five different locations (filled rectangles). At different locations, phase variations appear in outside peaks. The overall frequency was 6.2 Hz.
- Fig. 6.
Variations in the cycle period at different locations. The cycle-to-cycle periodicity has sudden changes in some cycles (at cycles 7–10 in this epoch). At different locations the changes are in different directions. These kinds of sudden changes were very common. We examined 105 epochs from four preparations, and the sudden changes were found in 72 epochs.
- Fig. 7.
Propagating activation during the oscillations.A, The recording arrangement for the data inB and C. RF, Rhinal fissure. B, Raw data from the LFP electrode and six optical detectors. Three sections from these data (I,II, III) are displayed in thepseudocolor images in C.C, Pseudocolor images from the three selected sections I, II, andIII in B displayed at 10 msec/frame. In the images warm colors represent a larger depolarization. I, During each cycle of oscillation, one dynamic ensemble emerged and traveled across the field of view. Three dynamic ensembles appeared in this section and movedupward across the field of view. When they moved past the LFP electrode, a cycle of oscillation was detected on the electrode (black trace in B).II, Later (0.5 sec), two dynamic ensembles appeared simultaneously (at the top and bottom of the field of view) and collided near the center. Two sequential collision events occurred during period II. These collisions generated the dramatic change in oscillation frequency seen in Figure 6. Activity was quickly annihilated after the collisions. III, After the collisions in periodII, the propagation direction of the ensembles was reversed. The dynamic ensembles emerged from the top of the field of view and traveled downward. In all eight preparations imaged, the oscillations appeared as propagating activation—dynamic ensembles. The physical size of dynamic ensembles was estimated by the isopotential contour lines in thepseudocolor display and was ∼0.5 mm wide in the tangential direction.
- Fig. 8.
Details of the propagation of dynamic ensembles.A, The front edge contours of three dynamic ensembles are shown every 2 msec. The wide gaps between thecontour lines suggest a sudden increase in propagating velocity. I, II, Two consecutive dynamic ensembles jumped at similar (but not identical) locations.III, A dynamic ensemble that emerged 1 sec later had quite different jump locations. B, The propagation velocity of the three dynamic ensembles (measured at cortical layer II) is shown. The overall velocities are 36, 29, and 21 mm/sec, composed of jumps (arrows) and slow propagation. We have examined the front edge contours from 10 dynamic ensembles from three preparations; the dynamically distributed jumps were seen in all 10.
- Fig. 9.
Dynamic ensembles are different from epileptiform events. A, Left, Middle, The epileptiform event was 10 times larger in dye signal amplitude (705 nm absorption) than were the dynamic ensembles (which are seen as an oscillation in one detector). Intrinsic optical signal also appears at 705 nm, but with a much slower temporal course. We used high-pass filters (both analog and digital, with the RC time constant of 0.1 sec) to emphasize the voltage-sensitive dye signals in these twopanels. Right, The intrinsic optical signal of an epoch is shown. The signal is much larger and slower than are those on the left and in the middle. These were the same data shown on the left but displayed with a minimum high-pass filter and different time and gain scales. We examined three preparations in white light in which voltage-sensitive dye signals were undetectable. In these conditions the intrinsic optical signals were the same as that shown on theright. B, Top row, An epileptiform event was evoked by an electrical stimulus (Stim) at the center of the field of view (see diagram on the right). This event expanded to the entire field of view in ∼30 msec. Bottom Row, A dynamic ensemble emerged from the same cortical area (same field of view, 7 sec later) and remained cohesive for at least 80 msec. The two rows are displayed with differentpseudocolor scales to show the propagation of both events.
- Fig. 10.
Dynamic ensembles evoked in normal ACSF. A, Schematic diagram of a single-pole electrode placed above the slice is shown. This stimulation method can evoke a low density of activated neurons distributed in a large area including several cortical layers. The intensity and the size of the area can be independently controlled by adjusting stimulation voltage and the distance between the electrode and the slice. B, Long-lasting response in local field potential evoked by weak electrical shocks is shown. In this preparation an electrode was placed on top of layer IV, ∼80 μm above the preparation. The interstimulus interval was 30 sec. Trace I, With stimulus intensity of 7 V (duration = 0.05 msec, for all stimuli), just above the threshold of the passive LFP response, a passive response (short and more synchronized) was evoked. Traces II,III, With intensity reduced to 3 V, long-lasting activity was evoked. The long-lasting responses were all-or-none events. In this preparation, 98% (n = 120) of 3 V stimuli evoked a long-lasting response. When the stimulus was increased to 5.5 V, ∼60% of the stimuli failed to evoke a long-lasting response. At 7 V, all the stimuli (n = 20) evoked the passive response (trace I). Traces II and III are consecutive trials, illustrating the variation of the long-lasting response. C, The long-lasting response was spatially confined (not expanding) and propagating, similar to dynamic ensembles (Fig. 7). A weak electrical shock (∼10 V × 0.05 msec) from an electrode placed 100 μm above the preparation [at the upper right corner of the image field (see diagram on the left)] induced activity over a large area including all layers (Stim image on the left). Seven milliseconds later, a long-lasting, propagating activation emerged next to the area activated by the stimulus (second image from theleft). This activity had an amplitude that was similar to that of the dynamic ensembles observed during 7–10 Hz oscillations (e.g., Fig. 7). The activity remained cohesive for ∼40 msec and propagated downward in the image field. The slice was perfused in normal ACSF, and the optical signal had a small amplitude compared with that of an epileptiform spike. Note the propagation velocity was ∼6–10 mm/sec (n = 10, measured from 2 preparations), significantly slower than the propagation during oscillations. Stim, Stimulus.
