Name: recording spatially restricted oscillations in the hippocampus of behaving mice
Uploaded: 2018-07-01T14:30:00
Description: Watch this Scientific Journal Video about Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice at JoVE.com

We are grateful to Karin Winterhalter and Kerstin Semmler for technical assistance. This work was supported by the cluster of excellence BrainLinks - BrainTools (EXC 1086) of the German Research Foundation.

All methods involving living animals have been approved by the Regierungspr&#228;sidium Freiburg in accordance with the German Animal Welfare Act.
1. Preparations
<ol>
	<li>Design and build an appropriate insertion tool transiently carrying the silicon probe and the electrode connector during the process of implantation. See Figure 1 for an example custom built insertion tool.</li>
	<li>Carefully release the silicon probe and electrode connector from its packagi...

<ol>
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Increasing evidences indicate that brain oscillations in hippocampal neuronal circuits occur in discrete spatial domains10,11,16. CSD analysis drastically reduces the influence of volume conduction, a crucial prerequisite for the study of local oscillation events. With this video, we provide a guide to implanting silicon probes into the mouse hippocampus for the analysis of CSD data. We show representative examples of CSD signal...

Oscillations in the LFP are critically involved in information processing by neuronal circuits. They cover a wide spectrum of frequencies, ranging from slow waves (~1 Hz) to fast ripple oscillations (~200 Hz)1. Distinct frequency bands are associated with cognitive functions including memory, emotional processing, and navigation2,3,4,5,6,7. Current flow across neuronal membranes constitutes the largest part of the LFP signal8. Cations entering the cell (e.g. via activation of glutamatergic excitatory synapses) represent an active current sink (as charge leaves the extracellular medium). In contrast, net flow of positive charge to the extracellular medium, for instance by activation of GABAergic inhibitory synapses, depicts an active current source at that location. In neuronal dipoles, current sinks are paired with passive sources and vice versa due to compensating currents affecting membrane charge at distant sites.
The electrical field produced by remote neural processes can also result in considerable voltage deflections on a recording electrode and might thus be falsely considered as a local event. This volume conduction poses a serious challenge to the interpretation of LFP signals. CSD analysis provides information about local current sinks and sources underlying LFP signals and therefore comprises a means to reduce the impact of volume conduction8. In laminated structures like the hippocampus, one-dimensional CSD signals can be obtained by the second spatial derivative of the LFP recorded from equidistant electrodes arranged perpendicular to the laminar planes9. The advent of commercially available linear silicon probes has allowed researchers to utilize the CSD method for the study of local oscillation activity in the hippocampus. For example, it has been demonstrated that distinct gamma oscillations emerge in a layer-specific manner in the CA1 area10. Furthermore, CSD analysis has identified independent hot spots of gamma activity in the principal cell layer of the dentate gyrus11. Importantly, these findings were only apparent in local CSD but not in LFP signals. CSD analysis thus provides a powerful tool to gain insight in the microcircuit operations of the hippocampus.
In this protocol, we provide a comprehensive guide to obtain one-dimensional CSD signals with silicon probes. These methods will enable users to investigate localized oscillation events in the hippocampus of behaving mice.

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			<td>OpenEphys (www.open-ephys.org)</td>
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			<td>allows platform-independent data acquisition</td>
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			<td>Python (www.python.org)</td>
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			<td>allows platform-independent data analysis</td>
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</table>

recording spatially restricted oscillations in the hippocampus of behaving mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

Figure 1 illustrates the insertion tool used for the implantation of silicon probes. Recordings from chronically implanted silicon probes targeting the CA1 area and the granule cell layer of the dentate gyrus are shown in Figure 2. We recorded LFPs from the probe shanks during free movement in the homecage. To minimize the effect of volume conduction, the obtained signals were converted to CSD along each shank of the probe (<stro...

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Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

This method can help answer key questions in the neuroscience field such as how neuronal network activities are organized on fine temporal and spatial scales. The main advantage of this technique is that using linear silicon probes current data analysis allows the reconstruction of local oscillations that cannot be easily detected in conventional field potential recordings. To begin this procedure, sterilize the surgical instruments with a hot bead sterilizer, then, wipe all the working surfaces with 70%ethanol.Next, mount the anesthetized animal in a stereotaxic frame by gently inserting ear bars into the ear canal. Once the head of the mouse is stabilized by the ear bars, place a mouthpiece over the snout for continuous isoflurane delivery, then apply ointment to the eyes to prevent drying out. Afterward, place a heating pad underneath the mouse and inject buprenorphine subcutaneously to ensure postoperative analgesia.Subsequently, shave its head and disinfect the skin with 70%ethanol. Using surgical scissors, make an incision on the skin along the midline of the skull and open the skin using surgical clamps. Align the head of the animal with the aid of a stereotaxic alignment tool to level bregma and lambda.There should be less than 50 micrometers of height offset between bregma and lambda. Furthermore, level the head along the medial-lateral axis by measuring the depth at defined distance from bregma on both left and right sides. Adjust the head if it is tilted.Clean the head with 3%hydrogen peroxide and dry it with sterile cotton wipes. Determine the location of the craniotomy relative to bregma using an appropriate stereotaxic atlas. Using a 0.9 millimeter drill head, drill two screw holes in the bone over the cerebellum to place the ground and reference screws.Additionally, drill one to three holes for anchoring screws to stabilize the implant. The location of anchor screws will depend on the location of the craniotomy. Insert the screws in the bone using a suitable screwdriver.Take care not to penetrate into the brain. Now, perform the craniotomy by slowly thinning the skull with the drill in a rectangular area around the implantation side. Frequently moisten the bone with sterilized phosphate buffer.Gently pierce the remaining thinned skull with a 27 gauge injection needle and remove it with a pair of tweezers. Next, carefully pierce the dura mater with a 27 gauge injection needle. Using a pair of tweezers, form a small hook by bending the tip of the needle and use this hook to remove the dura.Then, apply phosphate buffer to prevent the brain surface from drying out. Afterward, mount the electrode insertion tool on a stereotaxic holder and zero the probe on bregma. Move the probe to the stereotaxic coordinates over the craniotomy and slowly penetrate the brain surface.Make sure the probe shafts do not bend and avoid implanting through blood vessels. Then, slowly lower the probe until about 200 micrometers above the desired depth. Next, cover the craniotomy and shanks of the silicon probe with sterilized Vaseline for protection.Subsequently, apply dental cement to fix the base of the probe to the anchoring screw in the skull. Right after cement application, slowly move the probe to the target depth. Advance the last 200 micrometers after applying the cement to reduce lateral movement of the probe and ensure minimal tissue damage in the target area.After the cement has cured, release the probe from the insertion tool by melting the wax with a cauterizer. Then, release the connector board from the insertion device and position it at a suitable place on the skull using a crocodile clamp. Fix the connector board to the skull using dental cement.In the case of probe implantation in the hippocampus, place the connector board on the contralateral parietal bone. Then, solder the ground and reference wires of the connector board to the wires attached to the two screws over the cerebellum. Trim the protective cover to the correct height and place it over the silicon probe.Afterward, fix the cover of the connector board and skull using dental cement, avoiding the skin around the exposed skull. After the surgery, apply appropriate analgesic treatment to the mouse for at least two days. Single-house the mouse to prevent damage to the implant.Recordings from chronically implanted silicon probes targeting the CA1 area are shown here. Current source density analysis is applied along the individual shanks of the probe. In this example, a single sharp wave ripple event leads to a prominent current sink in stratum radiatum of CA1 and in the second example, two distinct sites in the granule cell layer of the dentate gyrus were recorded.The high gamma band was isolated by applying a 60 to 80 hertz band pass filter, revealing local gamma bursts on one of the two recording sites located in the granule cell layer. Note that the local gamma oscillation activity can only be detected after conversion of the recorded signals to CSD. CSD, but not LFP signals, identify the periods of phase and amplitude asynchrony between both recording sites.This segment shows a brief epoch of synchronized gamma activity. The traces here illustrate phase difference and amplitude difference index for LFP and CSD traces. After watching this video, you should have a good understanding of how to implant silicon probes in the hippocampus.

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