Article Figures & Data
Figures
- Figure 1.
Activation and expression of the transgene. Targeting of the optic fibers. A, The mice expressing archaerhodopsin in the hippocampal CA1 cells were obtained by crossing the homozygous transgenic Tg29 (T29-1) strain expressing the Cre recombinase in the targeted cells with the homozygous Ai35 strain carrying an archaerhodopsin-EGFP transgene at the Rosa26 locus that can be activated by the Cre recombinase B, Dark-field image of a paraformaldehyde-fixed, unstained section showing the targeting of the optic fiber into the stratum oriens. The trace left by the fiber (four-point star) is visible above the stratum pyramidale (arrowhead). C, The CA1 area of the hippocampus, the four-point star indicates the startum radiatum and the startum pyramidale is visible between the two arrows. Of note, the perisomatic region of the pyramidal cells contains little of the exogenous protein while their dendrites light up, consistently with the membrane targeted expression. The strong green signal in the CA1 is derived from the apical and the basal dendrites of the pyramidal neurons in the stratum pyramidale. D, Comparison of the expression in CA1 and CA3. The large arrow points to the CA2 on the right hand side of which the CA1 shows strong expression. On the left hand side the cells of the CA3 show minimal expression with the stratum lucidum lighting up (small arrowhead) as a result of strong expression in the granule cells of the dentate gyrus. E, A close-up on the lower blade of the dentate gyrus, where membrane localization can be suspected even when using traditional light microscopy (similar membrane localization was seen in the CA1 region when the Arch-EGFP transgene was activated only in a few CA1 cells in a different genetic intercross) The two arrowheads point to two cells with a suspected stretch of Arch-EGFP rich plasma membrane. F, The CA1 fibers terminating in the subiculum (large arrowhead) and the stratum moleculare of the dentate gyrus (small arrowhead) express strongly the gene construct, and this can be compared to the extremely low level of expression in the subcortical structures (four-point star). Scale bars: 100 µm (C, D) 200 µm (E, F), and 500 µm (B). C–F, EGFP fluorescence is visualized using light microscopy
- Figure 2.
Designing the microdrive. A, Top view of the pieces, from left to right: left shuttles, bottom part, right shuttles, middle part, and lid. The red arrowhead points to the annulus (inside the socket for the ferrule) that avoids the ferrule being pressed into body of the microdrive. Main dimensions are indicated in millimeters. B, Occipital view of the same pieces as in A, in the same order. C, Bottom view of the same pieces as in A, in the same order. The arrowhead points to the locking piece that slides into the diced groove of the ferrule and is locked in position by two insect pins to avoid the ferrule being torn out when the mating sleeves are pulled off to disconnect the animal from the laser.
- Figure 3.
Integrating the optic system into the microdrive. A, The assembled bottom and middle parts without insect pins, viewed from right side of the animal. Two locking pieces on the left side of the animal are slid into the grooves of the ferrules, the other two are simply indicated in their final position. B, Same assembly as in A, viewed from the front. C, Same assembly as in A, viewed from the top. Only the left occipital optic fiber is shown in full length, guided between the shuttles into the desired electrode hole. D, Fully assembled microdrive (for sake of clarity, insect pins are not shown, and the lid appears in pink). E, Dimensions of the metallic optic ferrule (gray) with an integrated PMMA optic fiber (blue). F, Photo of a microdrive with four optic fibers protruding from the electrode holes, three of them having polyimide tubing (yellow) in final position, while the tubing on the forth fiber is being mounted. Insect pins are visible on both sides. Note the conical tip of the PMMA fibers.
- Figure 4.
Building the microdrive. A, Bottom part (black), shuttles (white), middle part (black) held together by the insect pins. The optic system is fully integrated at this stage, but the ferrules are not visible since the assembly is viewed from the bottom. The shuttle screws are not yet in position (that would hold back the shuttles inside the middle part). B, The steel cannulae are already inserted, the microdrive is fully closed, the blunted ends of the insect pins are bent and cut away and the polyimide tubing is mounted onto the optic fibers. C, The microdrive is being loaded with tetrodes. Four individual microwires are guided through the desired holes of the lid, the corresponding tetrode is visible on the right hand side, coming out of its cannula. D, The drive is placed in a vice (blue), the sharp end of the insect pins is bent and then cut away. The contactor pins are being pushed into position, the tetrode wires and the thicker ground wires are visible as coming out through their holes in the lid. A loop formed by the same ground wires, guided out through two holes in the middle part is shown by a red arrowhead. Ground wires from the occipital skull screws will be soldered to this loop during implantation and the loop will be buried in cement. E, The cut surface of tetrodes is being gold plated by directing current through each channel individually while the tetrodes are dipped in Au(III) solution. F, The light transmission of the drive (at stage B of this figure) is verified by plugging it directly to the laser source via the four daughter cables that will be used for recording as well. This procedure can also be completed with a fully finished drive (at stage E of this figure) and the amount of light transmitted by each fiber can also be measured individually.
- Figure 5.
The structure of a recording day. The preprobe was 10 min long and with the mouse moving around on the cheeseboard without rewards being presented. There were four learning trials in both (1st and 2nd) learning blocks and three rewards were presented in all of them, always in the same three positions within a recording day. During the 1st block, signposts placed right next to the baited holes also guided the animals. The optogenetic intervention (80% laser power) during the 3-h-long sleeps was either SWR-blockade (upper row) or control intervention (lower row). The postprobe was 10 min long, testing the memory retention of the mice, therefore no rewards were presented. The postsleep was used to assess the reactivation of the CA1 pyramidal neurons. The final 30-min-long sleep with regular laser pulses (at 50% laser power) served as a technical control to measure the efficiency of the optogenetic inhibition at single cell level.
- Figure 6.
Elements and efficiency of the pulley system. A, Elements of the pulley system: a counterweight was used to balance the weight of the headstage preamplifier (visible at the head of the mouse), the rotatory joint transmitting the laser light (blue arrowhead), the optic cables (purple arrowhead), and the recording cable (green arrowhead). The two pulleys could turn around a common axis in the horizontal plane (red arrow). B, Tracking data from a learning trial, showing the position of the animal until the moment of reentering the start-box that is located at the bottom of the figure (the area of the start-box is not included). C, Tracking data from a postprobe (duration: 10 min) showing that the movement of the animal nearly completely covers the cheeseboard.
- Figure 7.
Efficiency of the SWR-blockade. A, B, Detector signal and delayed light pulses in the control condition. Raw signal from one tetrode (green), bandpass filtered differential signal (for the details of signal processing, see Kovács et al., 2016) fed into the detector (magenta), the 200-ms-long detector signal (red), and delayed signal driving the laser (yellow) is shown. The first delayed signal visible on the left hand side belongs to a previous detector pulse not appearing in the figure. During the 1st 20 min of the 1st 3-h-long middle sleep of a control day, 319 SWR events were detected online (established by counting the detector pulses), out of which nine (2.8%) were undetected by an offline algorithm that found 14 additional SWR events (4.4%). C, D, Detector signal in the SWR blockade condition. Color coding is the same as for panel A and B. The signal coming from the ripple detector is directly driving the laser here, therefore no delayed signal is used. During the 1st 20 min of the 1st 3-h-long middle sleep of a control day, 332 SWR events were detected online (established by counting the detector pulses) out of which 298 (89.7%) were undetected by the offline algorithm, a high percentage to be attributed to the fact that these events themselves were nearly fully destroyed by the light pulses. The offline algorithm detected 11 additional events (3.3%) that were undetected by online algorithm during the intervention. A–D, Note the field responses that are generated by the light pulses and are visible as strong positive deflection of the LFP. Also note the destruction of the oscillation close to the onset of the detector pulse in C, D, and the persisting oscillation in A, B.
- Figure 8.
Stability and optogenetic inhibition at single neuron level. A, Interspike interval distributions (temporal autocorrelograms) and firing rates of five selected cells: putative pyramidal cells from the septal pole of the right CA1 (1), the septal pole of the left CA1 (2), the temporal pole of the right CA1 (3), the temporal pole of the left CA1 (4), and a putative interneuron from the CA1 (5). The first and the last hundred waveforms measured during the whole period in B are overlaid and shown as left and right insets, respectively, for each of the five neurons. B, Isolated spikes from the same five cells as in panel A during the entire recording day. The structure of the behavioral paradigm has a clear effect on the firing of the cells. To illustrate this effect, the bar at the bottom of the panel identifies the elements of the paradigm: presleep (red), preprobe (light blue), 1st block of learning trials (yellow), 1st sleep with intervention (purple), 2nd block of learning trials (light green), 2nd sleep with intervention (magenta), postprobe (dark blue), postsleep (orange), final sleep for the assessment of the inhibition (light green). C, Optogenetic inhibition of the five presented cells (same ones as in panels A, B) during the final 30-min-long sleep shown in panel B. During this sleep, regular, 500-ms-long laser pulses were delivered every 4th second. In the upper half of the panel, spike rasters cover the 28.6-min-long middle period of the sleep, in the lower half of the panel, these rasters are summed up to create a histogram; p values derived from the Wilcoxon signed rank sum test is indicated above each raster.
- Figure 9.
Behavioral performance in the cheeseboard task. A, B, Overall time spent on control days (A) and on SWR-blockade days (B) until reaching all the three goal locations. Mean values are shown for three recording days. C, D, Overall mean distance traveled on control days (C) and on SWR-blockade days (D) to reach all the three goal locations. A–D, The preprobe (PrP) and the postprobe (PoP) are both indicated (cross), L1–L8 stand for the eight learning trials (filled circles) with rewards provided. Error bars representing the SEM are included to give an insight into the variability but are derived from only three data points.
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