Closed-loop optogenetic intervention in mice

Abstract

Optogenetic interventions offer novel ways of probing, in a temporally specific manner, the roles of specific cell types in neuronal network functions of awake, behaving animals. Despite the unique potential for temporally specific optogenetic intervention in disease states, a major hurdle in its broad application to unpredictable brain states in a laboratory setting is constructing a real-time responsive system. We recently created a closed-loop system for stopping spontaneous seizures in chronically epileptic mice by using optogenetic intervention. This system performs with a very high sensitivity and specificity, and the strategy is not only relevant to epilepsy but also can also be used to react to diverse brain states in real time, with optogenetic or other interventions. The protocol presented here is highly modular and requires variable amounts of time to perform. We describe the basic construction of a complete system, and we include our downloadable custom closed-loop detection software, which can be used for this purpose.

Figure 1. Generating implantable optical fibers

Insert the unstripped fiber into the appropriately sized fiber stripping tool (a). The stripped fiber (b) should then be cleaved off using the diamond scribe (c) and cut into short (10–15mm) segments which will be placed into the ferrule (shown together in d, small tick marks on the ruler are 1mm apart). Place the ferrule into an alligator clip or clamp (e) and note that there is a smaller convex end which will be polished, and a larger, concave end into which the epoxy and fiber will be loaded, and from which the implantable end of the fiber will emerge. Use a syringe to fill the larger, concave end of the ferrule until a bead of epoxy emerges from the other end (f). Next, gently pick up one segment of stripped fiber and insert it into the same end of the ferrule (g), being careful not to nick or shatter the fiber, and leaving as much fiber behind as you may want to implant (note that fibers can be further cleaved later). After allowing the epoxy to cure at least 24 hours, cleave the fiber emerging from the end to be polished as close to the epoxy bead as possible (h). Prepare the polishing surface with the 5μm polishing paper, and place the fiber into the polishing disk. Use very gentle, gradual pressure to polish the cleaved end in figure 8s, being careful not to break the fiber and working across the paper until the fiber and ferrule are flat with the polishing disk (i). Examine the end of the fiber in the polishing disk under a dissecting scope (j). Note that after the first polish, while no dark areas or cracks are visible in the fiber, fine scratches still appear on the ferrule and fiber core. A fully polished fiber (k) should appear quite shiny and free of scratches, particularly in the fiber core. If any cracks are visible after the last polishing step (l), the fiber should be re-polished, starting again from the 5μm polishing sheet, making sure to polish beyond all cracks before continuing.

Figure 2. Assembling optrodes for implantation

To attach an implantable optical fiber (the product of the steps shown in Figure 1, shown here in panel a, with polished and implantable ends labeled), which has been cleaved to the desired length, to a bipolar PlasticsOne electrode (also shown in panel a), bend the electrode to be almost parallel with the base of the pedestal, and again at 90 degrees to run alongside the optical fiber. Measure and cut the electrode (b) such that it will be just shorter than the optical fiber (c). Use a fine thread or suture material to tie the fiber and the electrode together (d–f). Use a drop of glue on a syringe tip or fine forceps to secure the fiber to the electrode (g), taking care not to get any glue on the bare wire ends of the electrodes. Trim the ends of the thread or suture material (h), and allow the optrode to dry (i) before further bending the electrode into the final desired orientation for implanting.

Figure 3. Implanting the optical fiber and electrode headstage

The implanted headstage consists of the fully assembled optrode, any additional implantable optical fibers, screws, and a small plastic ring which will hold the dental cement (a). Expose the skull, remove all fascia in the area in which you will place the implant, and note the locations of bregma and lambda (b). Note that this animal was previously injected in the left hippocampus with kainate to induce epilepsy, so there is a prior craniotomy present in the skull to the left of the sagittal suture. Measure bregma (green circles, c) and lambda (blue circles, d), and adjust the mouth bar as necessary to ensure that these points are at the same z position. Next, measure and mark the desired anterior-posterior and medial-lateral position for the optrode (e) as well as any additional optical fibers. It may also be helpful to mark the locations for the screws for planning purposes. Once the locations of the headstage components have been determined, fit the plastic ring to the shape of the skull (f) and ensure that all components will fit. Drill the holes in the skull, ensuring that those for the screws are just smaller than the thread size (g). Use firm pressure to screw the screws into the holes (h), keeping in mind that secure screws generated by the teeth of the screw biting into the edges of the craniotomy are key to a successful implant. Note that the screws are positioned here in such a way as to distribute any force from the tethers across more than one skull bone and suture (i). Use glue on the base of the plastic ring to fix the ring to the skull, and pull the edges of the incision around the ring into the glue (j). Next, insert the first optical fiber into the stereotaxic adapter, measure the location of bregma and navigate to the correct anterior-posterior and medial-lateral position. Use pointed forceps or small scissors to break through the dura to allow the optrode to pass into the brain (k). Place gel glue at the base of the ferrule, avoiding getting glue on the optical fiber itself, and lower the optrode into the brain (l). Carefully release the optical fiber from the stereotaxic adapter and place any other optical fibers, being careful not to knock over previously placed implanted fibers (m). Mix dental cement until it is slightly thickened, but still pourable, and pour it into the implant, being careful not to get any cement on the upper sides of the optical fibers or electrode pedestal (n). Allow the cement to harden and release the final implantable optical fiber from the stereotaxic adapter (o) to complete the procedure. All procedures in live animals were performed with the approval of the UC Irvine Animal Care and Use Committee.

Figure 4. Equipment setup

An example of the hardware setup for real-time optogenetic intervention is shown. Implanted mice (only 2 of the possible 8 cages are shown here for simplicity) are tethered for both electrical recording (green cables) and laser stimulation (blue and red cables). Electrical and optical commutators are positioned above the cages to allow the animal to freely move about the cage. The cables have enough slack that the animal can freely enter all areas of the cage. The electrical signal is then passed on to the amplifier, and the output of the amplifier is routed to the appropriate channel of the digitizer. The digitizer is connected by USB cable (black) to the computer running the custom recording software. Once an event is detected and light triggered by the software (see Figure 5), the digitizer output (purple cables) send a TTL signal to the laser, and the laser will then transmit the signal back to the mouse (both a blue and a red laser are shown here as examples). A camera connected to the computer by USB should be positioned in such a way as to capture the desired cages. Up to 4 cameras can be used at one time. It may be helpful to cover the shelving in a wire mesh to reduce electrical noise from the environment.

Figure 5. Summary of Detection Algorithms

A number of features of the signal can be used to trigger detection, summarized here and detailed in the accompanying User Guide (Supplementary Manual), as well as in the supplementary Material of Krook-Magnuson et al., 2013. In this figure, all words in color are user-defined parameters. Portions of this figure have been modified from Krook-Magnuson et al., 2013. Spikes: Spike detection uses a user-defined amplitude threshold (green) based on a user-adjustable slow integrator (red, also used in calculating Power properties). Additionally, minimum and maximum spike width (maroon arrows) at a specific relative height, and spike distance (orange) are user defined. Once the number of valid spikes in a user specified sliding time window crosses a user defined threshold (# of spikes threshold, blue), the program will begin to calculate the regularity of the spikes --the inverse of the coefficient of variation (CV, orange). When the user-defined ratio threshold based on this calculation is reached, the Spikes condition will trigger. This is done separately for positive (pos) and negative (neg) spike directions. Frequency: An example power spectrum of the frequencies present during a seizure is shown. The user can examine this spectrum to determine the specific ranges of frequencies that change most during the signal of interest (shown here as f1 to f2, in maroon, and f3 to f4, in blue). The signal is filtered using a Fourier transform (FFT) and the ratio of the power of the signal in the two user specified bands of frequencies is calculated. When the value of this calculation is either above or below a user-specified threshold (turquoise), the Frequency Band Ratio condition will trigger. Power: The power of the signal is examined in one of three ways. The properties of the slow (red) and fast (brown) integrators (i.e., how fast the signal changes in response to a change in the signal power) are user-specified by changing the leading and falling edge time constants (τ). Top: The user-defined threshold (purple) scaling of the slow integrator is compared with the amplitude correlation to determine triggering (see User Guide (Supplementary Manual) for more details, including equations) of the Amplitude Correlation condition. Middle: By comparing the values of the slow and fast integrators, the speed at which the signal changes can be used to exclude sudden movement artifacts using the Fast/Slow exclusion threshold (pink). Bottom: When the Fast integrator is below a user specified threshold (gold), triggering can occur. Coastline: The path length between each sample of the signal is determined and integrated in a user-specified manner over time. When the value exceeds a user-specified threshold, the Coastline condition is triggered.

Figure 6. On-line seizure detection and fiber tract location

a–c. The detection software should be tuned for each animal, to avoid false positives (red vertical line) while detecting true events (green vertical line). Even in cases of rhythmic artifact, due to for example scratching, good event detection can be achieved. a. If detection criteria are too broad, false positives can result. In this example, the movement artifact (expanded in the bottom trace), has wide spikes. As a result of poor, broad, tuning, these spikes are included (as indicated by the dots riding each spike). b. Poor tuning can also result in false negatives. In the example shown, the threshold for spike detection is set too high to detect the true event (bottom panel, horizontal lines indicate thresholds). Note, as detailed in the accompanying User Manual, that the threshold value entered by the user is not the same as the simple amplitude of the signal, and the user should inspect the plot to ensure the threshold set is appropriate to detect the events of interest. c. Once appropriately tuned, false negatives and false positives are avoided. Exclusion criteria, such as the Fast Integrator (bottom panel), can additionally be used to improve specificity. When the Fast Integrator value exceeds the user set threshold (horizontal black bar), detection is suppressed. Thin vertical red lines indicate where the Fast Integrator values return to below the threshold. d. Once tuned, on-line seizure detection (vertical green lines indicate event detection) can trigger light delivery (indicated by horizontal orange lines) to a percent of events, in a random fashion. In mice expressing the inhibitory opsin halorhodopsin in excitatory cells, light delivery to the hippocampus rapidly stops seizures. e. Post hoc analysis confirms the location of an optical fiber (200μm in diameter) implanted to deliver light to the hippocampus. Bright field image of unstained tissue. Scale bars d: 100μV, 5s. e: 200μm. Panel d modified from Krook-Magnuson et al., 2013.