• Abstract
• Table of Contents
• Materials
• Figures
• Literature Cited

Abstract

Oscillations in hippocampal local field potentials (LFP) reflect the coordinated, rhythmic activity of constituent interneuronal and principal cell populations. Quantifying changes in oscillatory patterns and power therefore provides a powerful metric through which to infer mechanisms and functions of hippocampal network activity at the mesoscopic level, bridging single?neuron studies to behavioral assays of hippocampal function. Here, complementary protocols that enable mechanistic analyses of oscillation generation in vitro (in slices and a whole hippocampal preparation) and functional analyses of hippocampal circuits in behaving mice are described. Used together, these protocols provide a comprehensive view of hippocampal phenotypes in mouse models, highlighting oscillatory biomarkers of hippocampal function and dysfunction. Curr. Protoc. Mouse Biol. 2:273?294 © 2012 by John Wiley & Sons, Inc.

Keywords: theta rhythm; gamma rhythm; electrophysiology; LFP; slice; in vitro; in vivo

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Table of Contents

• Introduction
• Basic Protocol 1: Producing Hippocampal Slices
• Basic Protocol 2: Pharmacological Induction of Gamma Oscillations
• Support Protocol 1: Recording Spontaneous Theta Oscillations in an In Vitro Mouse Whole Hippocampus Preparation
• Basic Protocol 3: Recording of Local Field Potentials in Mouse Hippocampus In Vivo
• Reagents and Solutions
• Commentary
• Literature Cited
• Figures

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Materials

GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures

• Figure 1. Hippocampal isolation and generation of transverse slices. (A ) The brain hemisphere (arrow) is placed on its frontal cortex and a spatula is used to support the medial dorsal cortex. (B ) A second spatula is used to gently peel away the brainstem and thalamus. (C ) The hemisphere following B. The exposed hippocampus is outlined in black. (D ) Isolate the hippocampus (outlined in black) by sliding a spatula between the extreme dorsal surface of the hippocampus and the surrounding cortex. (E ) The isolated hippocampus (arrow). (F ) Place the hippocampal isolate (arrow) into a shallow vessel containing ice‐cold cutting solution and gently bubble with carbogen. Repeat A through F on the second brain hemisphere. (G ) Use a large spatula to lift the hippocampus from the cutting solution. CA1/CA3 should be facing downwards and the dentate gyrus/subiculum facing vertically upwards. (H –I ) Position the spatula above the flat surface of an agar block. Rotate 180° and place the hippocampus onto the agar block with the dentate gyrus/subiculum facing downwards. (J ‐K ) Repeat H through I for the second hippocampal isolate. The hippocampus should be positioned on the block such that the extreme dorsal and ventral ends of the two hippocampi are side by side (arrow). Use a small piece of filter paper to remove excess cutting solution. (L ) Use a razor blade to make a vertical cut through the extreme ventral end of the hippocampi and agar block. (M ) Place a dab of superglue on the microtome stage. (N ) Glue the cut end of the hippocampi and agar block to the microtome stage. Try to avoid getting glue on the hippocampal tissue. Holding the stage vertically should help with this. (O ) Place the stage into the vibrating microtome and cover with ice cold cutting solution.

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• Figure 2. Kainate‐induced gamma oscillations in transverse hippocampal slices. (A ) Example spectrogram showing the development of gamma (30 to 100 Hz) oscillations in CA3 following the application of kainate (100 nM, red line) to a transverse hippocampal slice. (B ) Example of steady‐state gamma activity in (A) 80 min after kainate application. The raw trace has been filtered between 1 and 150 Hz. (C ) Pooled data from six transverse hipppocampal slices. The figure plots the time‐course of gamma oscillation generation after application of 100 nM kainate (arrow). Peak gamma band power (filled squares) and frequency (open circles) are plotted.

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• Figure 3. Spontaneous theta oscillations in the intact hippocampal isolate. (A ) Example of spontaneous theta (3 to 12 Hz) oscillations recorded in CA1 of the intact hippocampus. The raw trace has been filtered between 1 and 20 Hz. (B ) Spectrogram of steady‐state, spontaneous theta activity in CA1 over a 10‐min recording epoch. Note that in this example the peak frequency was at the lower end of the theta band as the hippocampal isolate was superfused with low K+ (3 mM) containing aCSF. As demonstrated by Goutagny et al. (), the frequency of spontaneous hippocampal theta activity increases with higher K+ concentration in the perfusate. (C ) Fourier generated power spectrum of the recording epoch in B.

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• Figure 4. Schematic of array assembly. (A ) Top‐down view of holes drilled in a 2‐mm thick Delrin platform, arranged according to stereotaxic coordinates; this example would target frontal cortex and hippocampus bilaterally. Lengths of 2‐mm of 23‐G cannulae (gray) are glued into the holes, and a connector appropriate for the headstage preamplifier is mounted in the center. (B ) Lengths of 30‐G cannulae (gray) are inserted and glued into the 23‐G holders, and (C ) lengths of nichrome wire loaded into the 30‐G and connected to the electrode interface (e.g., Neuralynx EIB‐18). After checking the electrical connection, the wire is glued in place at the top end of the 30‐G cannulae. (D ) Silver wire for the ground and reference connections is attached and nichrome electrode wire is then trimmed to the required lengths for implantation.

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• Figure 5. Overview of typical mouse LFP recording setup. (A ) Photograph showing recording arena (ra), fine‐wire tether (fwt) connected to pulleys (p) and held balanced by counterweights (cw) to allow free movement. Overhead camera (c) monitors behavior. (B ) Implanted adult mouse connected to Neuralynx recording hardware via EIB‐18 and HS18‐LED.

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• Figure 6. Examples of LFP data and spectral analysis. (A ) 2 sec of CA1 LFP recorded during active exploration of an open field environment. Top trace shows 0.1 to 475 Hz wideband LFP, with 5 to 10 Hz (theta, middle trace) and 60 to 100 Hz (gamma, lower trace) band‐pass filtered data shown below. Note characteristic correlation between gamma amplitude and theta phase. (B ) CA1 LFP power spectra from eight adult C57BL6 mice exploring a novel environment. Thin lines show spectra for individuals, thick line shows group median. (C ) Running speed over the course of a recording session from a single animal placed in a series of different environments at times shown by arrowheads. Speed is derived from LED‐based positional tracking; inset shows raw tracking data from a single oval open field environment. (D ) Spectrogram taken from same recording session as C, showing characteristic theta‐band power at 5 to 10 Hz. Note increases in theta power coinciding with increased running speed as mouse enters different environments.

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