The authors would like to thank Dr. Christina Ginkel (German Center for Neurodegenerative Diseases, DZNE) and Dr. Robert Stark (DZNE) for their assistance with animal breeding and animal healthcare. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut f&#252;r Arzneimittel und Medizinprodukte, BfArM), Bonn, Germany.

All animal experimentation was performed according to the guidelines of the local and institutional Council on Animal Care (University of Bonn, BfArM, LANUV, Germany). In addition, all animal experimentation was carried out in accordance with superior legislation, e.g., the European Communities Council Directive of 24 November 1986 (86/609/EEC), or individual regional or national legislation. Specific effort was made to minimize the number of animals used, as well as their suffering.
1. Animal Housing and EEG Recording Conditions
<ol>
	<li>House mice in filter-top cages or use individually-ventilated cages.</li>
	<li>Transfer mice from the animal facility to ventilated cabinets in special lab rooms that are appropriate for implanted animals and telemetric recordings.</li>
	<li>Perform all animal experimentation, including EEG electrode implantation and the subsequent recordings, under standardized conditions (22 &#176;C temperature, 12 h/12 h light-dark cycle, 50-60% relative humidity, noise attenuation, etc.)18.</li>
	<li>Prior to radiofrequency transmitter implantation, house animals in groups of 3 - 4 in clear type II polycarbonate cages, with food and water ad libitum. Do not isolate individual mice, as this can cause stress and interfere with subsequent experimentation and results.</li>
	<li>Do not use open housing conditions, but use ventilated cabinets instead during experimentation and recording.</li>
</ol>
2. Radiotelemetric EEG Electrode Implantation and EEG Recordings
<ol>
	<li>Anesthetize the mice using injection narcotics, e.g., ketaminehydrochloride/xylazinehydrochloride (100/10 mg/kg, intraperitoneally, i.p.) or inhalation narcotics, e.g., isoflurane17,18.
	<ol>
		<li>For isoflurane narcosis, position the mouse into an induction chamber with 4-5% isoflurane and 0.8-1% oxygen or carbogen (5% CO2 and 95% O2).</li>
		<li>Place a silicon facemask on the nose/mouth of the animal to control the desired depth of anesthesia and avoid experimenter exposure to isoflurane using a scavenging system.</li>
		<li>Use injectable anesthetics, e.g., esketaminhydrochloride (100 mg/kg, i.p.) and xylazinehydrochloride (10 mg/kg, i.p.), if inhalation anesthesia is not available.</li>
		<li>Monitor the depth of anesthesia by checking the tail pinch reflex, foot pinch reflex, and respiration rate. Note that artificial respiration via tracheal intubation is not necessary in mice.</li>
	</ol>
	</li>
	<li>Implant the radiofrequency transmitter into a subcutaneous pouch on the back of the animal.
	<ol>
		<li>Remove the body hair from the scalp and pretreat the shaved scalp with two disinfectants, i.e., 70% ethanol and an iodine-based scrub.</li>
		<li>Using a scalpel, make a midline incision on the scalp from the forehead to the nucheal region.</li>
		<li>Starting from the nuchal incision, prepare a subcutaneous pouch on one side of the back of the animal by performing a blunt dissection using surgical scissors or a surgical probe.</li>
		<li>Insert the transmitter into the subcutaneous pouch and deposit the excess length of the flexible transmitter leads into the pouch as well.&#160;Pay special attention to preventing contamination of the surgical site and transmitter implant. Properly isolate sterile from non-sterile areas using drapes.</li>
	</ol>
	</li>
	<li>Place the experimental animal on the stereotaxic frame, e.g., a computerized 3D stereotaxic device. Fix the skull using a nose clamp and ear bars.</li>
	<li>Pretreat the skull with 0.3% H2O2 to remove further tissue from the skull and to illuminate the cranial sutures and craniometrics landmarks, bregma and lambda.</li>
	<li>Drill holes at the coordinates of choice (see step 2.6) using a high-speed neurosurgical drill in a pressure-free mode at maximum velocity. 
	NOTE: Pressure-free drilling avoids the sudden breakthrough of the drill head and damage to the cortex. For a craniotomy, a neurosurgical high-speed drill is recommended. Choose standard drill-head diameters of 0.3 - 0.5 mm, depending on the electrode diameter.</li>
	<li>Carefully select the electrode type, considering the impedance, diameter, coating, etc. 
	NOTE: Parylene-coated tungsten or stainless steel electrodes are the most common. The electrode types should be chosen according to experimental requirements. As a preemptive maneuver, sterilize electrode tips prior to implantation using 70 % ethanol.&#160;&#160;Note that the electrode coating does not allow for heat sterilization.</li>
	<li>For intrahippocampal CA1 EEG recordings, drill a hole stereotaxically at the following coordinates: bregma, -2 mm; mediolateral, 1.5 mm (right hemisphere); dorsoventral, 1.3 mm (target region: cornu ammonis (CA1) pyramidal layer). For the reference electrode, drill a hole above the cerebellar cortex at the following stereotaxic coordinates: bregma, -6.2 mm, mediolateral, 0 mm; dorsoventral, 0 mm. 
	NOTE: The cerebellar electrode serves as a pseudoreference electrode, as the cerebellum is a rather silent brain region. The stereotaxic coordinates were derived from a standard mouse brain atlas.</li>
	<li>Prior to the insertion of the electrodes, shorten them to the required length. Mechanically clip the extracranial part of the electrode to the stainless steel helix of the transmitter. 
	NOTE: Soldering should be avoided, as this can introduce substantial noise in the system.</li>
	<li>Attach the electrode to the vertical arm of the stereotaxic device and insert the electrode according to the aforementioned stereotaxic coordinates.</li>
	<li>Fix the electrodes using glass ionomer cement and wait until the cement has fully hardened.</li>
	<li>Close the scalp using over-and-over sutures with non-absorbable 5-0 or 6-0 suture material.</li>
	<li>For postoperative pain management, administer carprofen (5 mg/kg, subcutaneously, s.c.) once a day for 4 consecutive days post-implantation.&#160;Note that carprofen should be injected prior to the initial incision (step 2.2.2).</li>
	<li>Allow the animals to recover for 10 - 14 days post-implantation before the recordings and/or injection experiments are started.</li>
</ol>
3. Spontaneous Recordings of Theta Oscillations and Pharmacological Induction
<ol>
	<li>Activate the radiofrequency transmitter using the magnetic switch. Place the animal with its home cage on the receiver plate. Perform long-term hippocampal EEG recordings for at least 24-48 h. 
	NOTE: The analysis of EEG amplitude and EEG frequency characteristics of long-term recordings provides detailed insight into the circadian dependency of theta oscillations and their association with specific behavioral and cognitive conditions/tasks. Always combine EEG recordings with video monitoring of the experimental animals.</li>
	<li>For pharmacological induction of theta oscillations, administer a single dose of urethane (800 mg/kg i.p.) or a single dose of a muscarinic receptor agonists, e.g. pilocarpine (10 mg/kg i.p.), arecoline (0.3 mg/kg i.p.) or oxotremorine (0.03 mg/kg i.p.). Pretreat mice with N-methylscopolamine (0.5 mg/kg i.p.) to avoid peripheral muscarinic reactions. Freshly dissolve all drugs in 0.9% NaCl or Ringer solution.&#160; 
	NOTE: Higher dosages of muscarinic receptor agonists might result in seizure induction in experimental animals. Also consider that the dosages given here represent landmarks that require prior dose-effect studies in the mouse line under investigation.&#160;Note that urethane is a mutagen and carcinogen that requires appropriate precautions in storage and handling.</li>
	<li>Inject atropine (50 mg/kg, i.p.) to differentiate atropine-sensitive type II from atropine-insensitive type I theta oscillations. 
	NOTE: The atropine dosage is again species- and strain-dependent and requires prior dose-effect evaluation. The optimal time point of atropine injection depends on the pharmacodynamics of muscarinic receptor agonists. For identification of type II theta, atropine injection is recommended 1 h after urethane administration.</li>
	<li>Try to avoid the subsequent application of several drugs, as systemic drug administration alters global transcriptional and translational patterns, which influence subsequent EEG recordings. Note that short-lasting theta oscillations can also be induced by sensory stimuli, such as tail- or paw-pinching.</li>
	<li>Extract/export representative EEG data sets of the pre-phase (baseline) and the post-injection phase from the total EEG recording as ASCI or TXT files, considering the pharmacokinetics of the drugs applied and the demands of the individual study protocol.</li>
</ol>
4. Validation of EEG Electrode Placement
<ol>
	<li>Euthanize animals by placing them in an incubation chamber and introduce 100% carbon dioxide. Use a fill rate of 10-30% of the chamber volume per min with carbon dioxide added to the existing air in the incubation chamber; this will result in rapid unconsciousness with minimal stress to the animals.</li>
	<li>Remove the mouse from the chamber once respiratory arrest occurs and a faded eye color persists for 2-3 min.</li>
	<li>Cut the stainless steel electrodes and explant the radiofrequency transmitter. Decapitate the mouse using scissors or a guillotine and remove the brain from the neurocranium by gentle manipulation with surgical scissors and forceps.</li>
	<li>Fix the brains in 4% paraformaldehyde in phosphate buffer saline (PBS) (pH 7.4) overnight. For cryoprotection, transfer the brains to 30% glucose and store them at 4 &#176;C until further processing. 
	NOTE: Paraformaldehyde is considered hazardous by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). Take the necessary precautions: use personal protective equipment, ensure appropriate ventilation, and avoid dust formation. In addition, remove sources of ignition and take precautionary measures against static discharges. Paraformaldehyde should not be released into the environment.</li>
	<li>Mount the extracted brain on the tissue holder of a cryostat using an adhesive and cut the brains into 40 - 75 &#181;m coronal slices. Mount the slices onto glass slides and stain them with Nissl blue using standard histological procedure; this procedure will visualize the branch channel that reflects the previous electrode position. Note that it is also possible to cut coronal slices from the native brain using a vibroslicer</li>
	<li>Incorporate only those animals that meet correct EEG electrode placement criteria; for the CA1 region, the tip of the deep electrode should be localized inside the CA1 pyramidal layer.</li>
</ol>
5. Data Acquisition
<ol>
	<li>Record CA1 intrahippocampal EEGs with an appropriate sampling rate with no a priori filter cut-off. 
	NOTE: The sampling rate, which is transmitter-specific, determines the upper frequency limit of the EEG analysis.</li>
	<li>Process the recorded data with an analysis software. Program time-frequency analyses and calculations with custom-made procedures for the adequate control of analysis methods (Figure 5)20.</li>
	<li>Cut the recorded EEG into sections with a length of 1 h each. Use fast computer processors, since computation time is high. Additionally, make use of software that can parallelize computing on multiple kernels20.</li>
</ol>
6. EEG Data Analysis
<ol>
	<li>Analyze data segments using a complex Morlet wavelet to calculate both the frequency and the amplitude of oscillations. 
	NOTE: This wavelet (e.g.,&#936;(x)=(&#960;b)(&#8722;1/2) exp(2i&#960;cx) exp(&#8722;x2/b), where b is the bandwidth parameter, c the center frequency, and i the imaginary unit) has often been applied in literature to study EEG data, as it guarantees optimal resolution in both frequency and time23,24.</li>
	<li>Use a bandwidth parameter and center frequency setting that particularly weights the frequency resolution to distinguish the frequency differences on the 0.1-Hz level while still not neglecting a sufficient time resolution. 
	NOTE: Neuronal processes in the gamma band are short-lasting25, and this can also hold true for theta rhythms. Thus, the analytical approach must consider adequate temporal resolution.</li>
	<li>Analyze the EEG data in the frequency range of 0.2 - 12 Hz, with a step-size of 0.1 Hz, thus including the typical delta, theta, and alpha frequency ranges.</li>
	<li>Set up an automated, complex analytical tool for the elaboration of the theta frequency architecture, which can replace a standard visual inspection of theta oscillations; this procedure is called the theta detection method (TDM).</li>
	<li>Compute the quotient of the maximum amplitude in the theta frequency range (3.5-8.5 Hz) and the maximum amplitude in the upper delta frequency range (2-3.4 Hz) for time windows of 2.5 s each. 
	NOTE: The value of this quotient serves as a measure to decide if a theta oscillation occurred. The definition of the theta frequency range can vary depending on the functional state and neuroanatomical circuitry/system to be analyzed.</li>
	<li>Classify a segment as a &#34;theta oscillatory epoch,&#34; if the computed ratio during this segment is above 1.5. 
	NOTE: This guarantees that the maximum theta amplitude is at least 50% higher than the amplitude in the upper delta band during the related 2.5-s EEG segment. Note that the ratio might need adaptation depending on the line and/or species used. An interval of 2.5 s represents a minimal duration for a theta oscillation, prevents false-positive detections of certain noisy epochs, and lies within the range definitions of other publications19,26. The upper delta frequency range serves as a control frequency band because physiologically-relevant delta activity appears during non-theta epochs, for example, during slow-wave sleep, which is highly dampened during theta activity.</li>
	<li>Repeat this procedure for every 1 h section; therefore, every section consists of 1,440 EEG segments with lengths of 2.5 s each.</li>
	<li>Statistically evaluate the data of the identified theta oscillation epochs.</li>
	<li>Compute the statistics of the total duration times of detected theta epochs; distinct or predefined groups; cycles, such as light/dark; and others parameters. 
	NOTE: Statistics may include t-test, ANOVA, or MANOVA, depending on the variable, the number of groups, the conditions, etc.</li>
	<li>Calculate the statistics of the amplitude of the detected theta epochs, but only in the theta frequency range (3.5 - 8.5 Hz).</li>
	<li>Evaluate the statistics of the frequency of the detected theta epochs, but only in the theta frequency range (3.5 - 8.5 Hz). 
	NOTE: The theta frequency of a theta epoch is defined as the frequency belonging to the maximum theta amplitude of a theta epoch.</li>
</ol>

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H., Bland, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+hippocampal+rhythmical+slow+activity+in+both+the+rabbit+and+the+rat:+relations+to+behavior+and+effects+of+atropine,+diethyl+ether,+urethane,+and+pentobarbital.">Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital.</a> Exp Neurol. 49, 58-85 (1975).</li><li>Lu, B. L., Shin, J., Ichikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+classification+of+single-trial+EEG+signals+using+a+min-max+modular+neural+network.">Massively parallel classification of single-trial EEG signals using a min-max modular neural network.</a> IEEE Trans Biomed Eng. 51, 551-558 (2004).</li><li>Robinson, T. E., Kramis, R. C., Vanderwolf, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+cerebral+activation+during+active+sleep:+relations+to+behavior.">Two types of cerebral activation during active sleep: relations to behavior.</a> Brain Res. 124, 544-549 (1977).</li></ol>

The authors have nothing to disclose.

Theta activity is of central relevance in systemic neurophysiology. It can be observed in various brain regions, particularly in the hippocampus, where it is related to specific behavioral and cognitive states. In addition, hippocampal theta can be pharmacologically differentiated into atropine-sensitive type II and atropine-insensitive type I theta. Type I is thought to be related to locomotion, such as walking or running27,28,29</su...

Theta activity in the brain is related to different cognitive and functional states, including arousal, attention, voluntary movement, exploratory behavior, attention behavior, learning and memory, somatosensory integration, and rapid eye movement (REM) sleep1,2. Principally, theta activity as a rhythmic entity can be generated in various cerebral regions and is highly organized and synchronized as theta oscillations. Below, we will focus on the analysis and quantification of theta activity/oscillations that are generated within the septohippocampal system3,4. Within the septum, GABAergic, glutamatergic, and cholinergic neurons project to the hippocampus and contribute to the initiation and maintenance of theta oscillatory behavior. There is an ongoing discussion on whether hippocampal theta oscillations are initiated in the septum, i.e., the septal pacemaker-hippocampal follower model, (extrahippocampal theory) or intrinsically within the hippocampus (intrahippocampal theory)5,6,7.
Regardless of their origin, hippocampal theta oscillations have been in the focus of interest for years, particularly in transgenic mouse models. These models allow for the implantation of deep EEG electrodes and for the recording of hippocampal theta oscillations under specific cognitive and behavioral tasks8. Hippocampal theta oscillations are heterogenous in nature. Based on the so-called dualistic theory of theta oscillations, one can differentiate between atropine-sensitive type II theta and atropine-insensitive type I theta9,10,11. The latter can typically be induced by muscarinic M1/M3 receptor agonists, e.g., arecoline, pilocarpine, and urethane. However, urethane is a multi-target drug that, besides muscarinic activation, also exerts complex effects on other ion channel entities. For type II theta, the muscarinic pathway includes the activation of M1/M3 and a subsequent Gq/11 (G&#945;)-mediated activation of phospholipase C &#946;1/4 (PLC&#946;1/4), inositol trisphosphate (InsP3), diacylglycerole (DAG), Ca2+, and protein kinase C (PKC). The role of PLC&#946;1 and PLC&#946;4 in thetagenesis has been validated in knock-out studies using PLC&#946;1-/- and PLC&#946;4-/- mice exhibiting a complete loss or significant attenuation of theta oscillation12,13,14. Additional M1, M3, and M5 downstream targets (channels/currents) of the muscarinic signaling cascade include various conductances, such as M-type K+ channel (KM) via voltage-dependent K+ channel (Kv7); slow after hyperpolarization K+ channel (KsAHP); leak K+ channel (Kleak), probably via TWIK-related acid-sensitive K+ channel (TASK1/3); cation current (ICAT), probably via Na+ leak channel (NALCN); and Ih via hyperpolarization and cyclic nucleotide gated channels (HCN). In addition, M2/M4 acetylcholine receptors (AChRs) were reported to interfere with inward rectifier K+ channel 3.1 (Kir3.1) and inward rectifier K+ channel 3.2 (Kir3.2)15.
Currently, commercially-available analytical software allows for fast FFT-based frequency analysis, e.g., analysis of power (P, mV2) or power spectrum density (PSD, mV2/Hz). Power or power spectrum density (PSD) analysis of the theta frequency range only gives a global overview of its activity. However, in order to get a detailed insight into cognitive and behavior-related theta activity, the analysis of highly-organized theta oscillations is mandatory. The assessment of highly-organized theta oscillations is of central importance in the field of neurodegenerative and neuropsychiatric diseases. Most experimental disease studies are carried out in transgenic mouse models using highly-sophisticated neurosurgical approaches to record epidural surface and deep intracerebral EEGs. These techniques include both tether systems16 and radiotelemetric setups17,18. Theta oscillations can be recorded as spontaneous and behavior-related theta oscillations under long-term recording conditions. Additionally, theta oscillations can be recorded following pharmacological induction but also following the exposure of animals to behavioral or cognitive tasks or to sensory stimuli, such as tail pinching.
Early approaches to characterize theta oscillations were described by Csicsvari et al.19. The authors designed a semi-automated tool for short-term theta analysis (15 - 50 min) that is not suitable for long-time EEG recordings. Our method, described here, allows for the analysis of long-term EEG recording &#62; 48 h20. Csicsvari et al.10 also referred to the theta-delta ratio, but no threshold for the determination of highly-organized theta oscillations is provided. The delta and theta range definitions match our frequency range definitions. As it is not explicitly mentioned, we presume that an FFT-based method is used by Csicsvari et al. to calculate the power of the theta-delta frequency bands. This again clearly differs from our method, since we calculate wavelet-based amplitudes on a large number of frequency scales (frequency steps &#916;(f) = 0.05 Hz), resulting in much higher precision. The duration of the individually-analyzed EEG epoch is similar to our definition.
Klausberger et al.21 also make use of theta-delta ratios for the analysis of long-term EEG recordings. However, there are three major differences compared to our approach: i) the EEG epoch duration is much longer, i.e., at least 6 s; ii) the theta-delta ratio is set to 4, which is much higher than our threshold, and is related to different frequency range definitions; and iii) the power definition is likely to be based on an FFT approach, which lacks high precision, particularly for very short time windows (2 s, i.e., 5 cycles for oscillations with a frequency of 2.5 Hz). In such cases, a wavelet-based procedure is more recommendable. A study by Caplan et al.22 solely calculated theta power while ignoring the theta-delta power ratio. Thus, the Caplan approach22 cannot differentiate between cognitive theta-rich processes accompanied by a high or low delta.
In the following protocol, we will present our analytical wavelet-based approach to reliably analyze highly-organized theta oscillations in hippocampal EEG recordings from mice. Since this procedure works automatically, it can be applied to large data sets and long-term EEG measurements.

<table><tbody><tr><td>Carprofen (Rimadyl VET - Injektionsl&amp;ouml;sung)</td><td>Pfizer</td><td>PZN 0110208208</td><td>20ml</td></tr><tr><td>binocular surgical magnification microscope</td><td>Zeiss Stemi 2000</td><td>0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000</td><td></td></tr><tr><td>Dexpanthenole (Bepanthen Wund- und Heilsalbe)</td><td>Bayer</td><td>PZN: 1578818</td><td></td></tr><tr><td>drapes (sterile)</td><td>Hartmann</td><td>PZN 0366787</td><td></td></tr><tr><td>70% ethanol</td><td>Carl Roth</td><td>9065.5</td><td></td></tr><tr><td>0.3% / 3% hydrogene peroxide solution</td><td>Sigma</td><td>95321</td><td>30% stock solution</td></tr><tr><td>gloves (sterile)</td><td>Unigloves</td><td>1570</td><td></td></tr><tr><td>dental glas ionomer cement</td><td>KentDental /NORDENTA</td><td>957 321</td><td></td></tr><tr><td>heat-based surgical instrument sterilizer</td><td>F.S.T.</td><td>18000-50</td><td></td></tr><tr><td>high-speed dental drill</td><td>Adeor</td><td>SI-1708</td><td></td></tr><tr><td>Inhalation narcotic system (isoflurane)</td><td>Harvard Apparatus GmbH</td><td>34-1352, 10-1340, 34-0422, 34-1041, 34-0401, 34-1067, 72-3044, 34-0426, 34-0387, 34-0415, 69-0230</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter 250 ml</td><td>PZN 6497131</td><td></td></tr><tr><td>Ketamine</td><td>Pfizer</td><td>PZN 07506004</td><td></td></tr><tr><td>Lactated Ringer&#039;s solution (sterile)</td><td>Braun</td><td>L7502</td><td></td></tr><tr><td>Nissl staining solution</td><td>Armin Baack</td><td>BAA31712159</td><td></td></tr><tr><td>pads (sterile)</td><td>ReWa Krankenhausbedarf</td><td>2003/01</td><td></td></tr><tr><td>Steel and tungsten electrodes parylene coated</td><td>FHC Inc., USA</td><td>UEWLGESEANND</td><td></td></tr><tr><td>stereotaxic frame</td><td>Neurostar</td><td>51730M</td><td>ordered at Stoelting</td></tr><tr><td>(Stereo Drive-New Motorized Stereotaxic)</td><td></td><td></td><td></td></tr><tr><td>tapes (sterile)</td><td>BSN medical GmbH &amp;amp; Co. KG</td><td>626225</td><td></td></tr><tr><td>TA10ETA-F20</td><td>DSI</td><td>270-0042-001X</td><td>Radiofrequency transmitter 3.9 g, 1.9 cc, input voltage range &amp;plusmn; 2.5 mV, channel bandwidth (B) 1 - 200 Hz, nominal sampling rate (f) 1,000 Hz (f = 5B) temperature operating range 34 - 41 &amp;deg;C warranted battery life 4 months</td></tr><tr><td>TL11M2-F20EET</td><td>DSI</td><td>270-0124-001X</td><td>Radiofrequency transmitter 3.9 g, 1.9 cc, input voltage range &amp;plusmn; 1.25 mV, channel bandwidth (B) 1 - 50 Hz, nominal sampling rate (f) 250 Hz (f = 5B) temperature operating range 34 - 41 &amp;deg;C warranted battery life 1.5 months</td></tr><tr><td>Vibroslicer 5000 MZ</td><td>Electron Microscopy Sciences</td><td>5000-005</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer</td><td>PZN: 1320422</td><td></td></tr><tr><td>Matlab</td><td>Mathworks Inc.</td><td></td><td>programming, computing and visualization software</td></tr><tr><td>SPSS</td><td>IBM</td><td></td><td>statistical analysis software</td></tr></tbody></table>

automatic detection of highly organized theta oscillations in the murine eeg

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity can be recorded in a wide range of central nervous system (CNS) regions. Here, we present an analysis of theta oscillations from the murine hippocampus. Such oscillations can occur during different behavioral and cognitive states. It is highly recommended to analyze theta oscillations under both spontaneous long-term, task-related short-term, and pharmacologically-induced conditions. 
Figure 1 illustrates a representative intrahippocampal CA1 recording...

Watch this Scientific Journal Video about Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG at JoVE.com

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

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Research

JoVE Journal

Behavior

9.1K Views.  University of Cologne. Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Video: Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

EEG Mu Rhythm in Typical and Atypical Development

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal resolution, EEG can be used to examine the neural response related to specific behaviors, states, or external stimuli. An example of this utility is the assessment of the mirror neuron system (MNS) in humans through the examination of the EEG mu rhythm. The EEG mu rhythm, oscillatory activity in the 8-12 Hz frequency range recorded from centrally located electrodes, is suppressed when an individual executes, or simply observes, goal directed actions. As such, it has been proposed to reflect activity of the MNS. It has been theorized that dysfunction in the mirror neuron system (MNS) plays a contributing role in the social deficits of autism spectrum disorder (ASD). The MNS can then be noninvasively examined in clinical populations by using EEG mu rhythm attenuation as an index for its activity. The described protocol provides an avenue to examine social cognitive functions theoretically linked to the MNS in individuals with typical and atypical development, such as ASD.&#160;

Assessment of the EEG mu rhythm provides a unique methodology for examining brain activity and when combined with behaviorally based assays, can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations.

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal ...

Medicine

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Neuroscience

Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency Oscillations (HFOs) have emerged as potential presurgical biomarkers for the identification of the EZ in addition to Interictal Epileptiform Discharges (IEDs) and ictal activity. Although they are promising to localize the EZ, they are not yet suited for the diagnosis or monitoring of epilepsy in clinical practice. Primary barriers remain: the lack of a formal and global definition for HFOs; the consequent heterogeneity of methodological approaches used for their study; and the practical difficulties to detect and localize them noninvasively from scalp recordings. Here, we present a methodology for the recording, detection, and localization of interictal HFOs from pediatric patients with refractory epilepsy. We report representative data of HFOs detected noninvasively from interictal scalp EEG and MEG from two children undergoing surgery.
The underlying generators of HFOs were localized by solving the inverse problem and their localization was compared to the Seizure Onset Zone (SOZ) as this was defined by the epileptologists. For both patients, Interictal Epileptogenic Discharges (IEDs) and HFOs were localized with source imaging at concordant locations. For one patient, intracranial EEG (iEEG) data were also available. For this patient, we found that the HFOs localization was concordant between noninvasive and invasive methods. The comparison of iEEG with the results from scalp recordings served to validate these findings. To our best knowledge, this is the first study that presents the source localization of scalp HFOs from simultaneous EEG and MEG recordings comparing the results with invasive recordings. These findings suggest that HFOs can be reliably detected and localized noninvasively with scalp EEG and MEG. We conclude that the noninvasive localization of interictal HFOs could significantly improve the presurgical evaluation for pediatric patients with epilepsy.

High Frequency Oscillations (HFOs) have emerged as presurgical biomarkers for the identification of the epileptogenic zone in pediatric patients with medically refractory epilepsy. A methodology for the noninvasive recording, detection, and localization of HFOs with simultaneous scalp electroencephalography (EEG) and magnetoencephalography (MEG) is presented.

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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.

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 ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Computer-based Multitaper Spectrogram Program for Electroencephalographic Data

Current web resources provide limited, user friendly tools to compute spectrograms for visualizing and quantifying electroencephalographic (EEG) data. This paper describes a Windows-based, open source code for creating EEG multitaper spectrograms. The compiled program is accessible to Windows users without software licensing. For Macintosh users, the program is limited to those with a MATLAB software license. The program is illustrated via EEG spectrograms that vary as a function of states of sleep and wakefulness, and opiate-induced alterations in those states. The EEGs of C57BL/6J mice were wirelessly recorded for 4 h after intraperitoneal injection of saline (vehicle control) and antinociceptive doses of morphine, buprenorphine, and fentanyl. Spectrograms showed that buprenorphine and morphine caused similar changes in EEG power at 1&#8722;3 Hz and 8&#8722;9 Hz. Spectrograms after administration of fentanyl revealed maximal average power bands at 3 Hz and 7 Hz. The spectrograms unmasked differential opiate effects on EEG frequency and power. These computer-based methods are generalizable across drug classes and can be readily modified to quantify and display a wide range of rhythmic biological signals.

This protocol provides an open source, compiled MATLAB program that generates multitaper spectrograms for electroencephalographic data.

Current web resources provide limited, user friendly tools to compute spectrograms for visualizing and quantifying electroencephalographic (EEG) data. ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://app.jove.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp (2018)
This video demonstrates a protocol to modulate theta oscillations in a genetically engineered mouse model with light-sensitive ion channels in its GABAergic medial septum neurons. By delivering light pulses to the hippocampus, the protocol aims to synchronize neuronal activity and generate theta oscillations, which are recorded as local field potentials.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
PV-Cre ...