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
2. Radiotelemetric EEG Electrode Implantation and EEG Recordings
3. Spontaneous Recordings of Theta Oscillations and Pharmacological Induction
4. Validation of EEG Electrode Placement
5. Data Acquisition
6. EEG Data Analysis
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 under control conditions. If the animal is not in a spontaneous "theta state," the intrahippocampal EEG is often characterized by large-irregular-amplitude (LIA) activity. Administration of muscarinic receptor agonists (e.g., arecoline, pilocarpine, or urethane) results in highly-organized theta oscillations that can be blocked by atropine (50 mg/kg, i.p., Figure 1).
In order to quantify highly-organized theta oscillations with the appropriate time resolution, the theta detection tool was used to classify 2.5 s EEG epochs as either theta-negative or theta-positive (Figure 2). Based on this classification, it is possible to quantify the total duration of theta oscillations under spontaneous conditions or specific behavioral and cognitive tasks.
In order to analyze a 30-min EEG segment (as for pharmacological urethane/atropine theta dissection), we first perform a time-frequency analysis for a frequency range of 0.2-12 Hz, which displays the amplitude (mV) in a color-coded fashion (Figure 3 A). As becomes obvious in Figure 3 A, high-amplitude theta activity, which is confirmed by a visual inspection of the EEG (white arrows), is accompanied by a low amplitude in the delta frequency range. Then, the maximum amplitudes of the theta (3.5-8.5 Hz) and delta (2-3.4 Hz) frequency ranges are plotted (Figure 3 B). Systematic correlation studies revealed that the ratio of maximum theta amplitude to maximum delta amplitude exceeding 1.5, indicating highly-organized theta oscillations (Figure 3 C).
Figure 4 demonstrates how urethane can induce hippocampal theta oscillations (white circles in Figure 4 II). Urethane is a multi-target drug that can trigger type II theta due its agonistic action on muscarinic receptors. Following an atropine injection (Figure 4 III), these type II theta oscillations (atropine-sensitive theta oscillations) are abolished. It is important to consider that the muscarinic receptor agonists, in addition to atropine, have individual pharmacokinetic properties that affect the time characteristics of theta occurrence and theta blockade. It should be noted that atropine-insensitive type I theta remains unaffected by muscarinic receptor antagonists.
A summary of the whole theta detection and quantification tool is depicted in Figure 5. It results in the calculation of the amplitude, frequency, and sum/mean theta duration. In contrast to previously-described techniques, it makes use of a wavelet-based approach with high precision. The analytical tool described here has several fields of application. Theta oscillations are generated in the septohippocampal system and are often impaired by neurodegenerative processes, e.g., in Alzheimer's disease. Numerous mouse models of Alzheimer's disease have been described that vary in homology, isomorphism, and predictability. Some of these models were reported to exhibit a reduction in theta activity, whereas others were shown to display an increase in theta activity, the reason for which remains to be determined. We successfully applied the theta detection tool described here to characterize altered theta oscillatory architecture in the 5XFAD model of Alzheimer's disease8. However, it might also be applied in epilepsy research and neuropsychiatric diseases.
Figure 1: Theta Oscillations in C57Bl/6 Mice. Radiotelemetric intrahippocampal CA1 recording under spontaneous conditions (I) and following urethane injection (800 mg/kg, i.p., II). Following urethane injection, highly-organized theta oscillations become visible, which can be blocked by atropine (50 mg/kg, i.p.). This figure has been modified from reference20, with permission. Please click here to view a larger version of this figure.
Figure 2: A Wavelet-based Analysis of a Deep CA1 EEG Recording from a C57Bl/6 Mouse. (A and B) Two 2.5-s EEG epochs are depicted, visually classified as non-theta and theta segments, respectively. (C and D) Time-frequency analysis of the CA1 EEG segments displayed in A and B in the range of 0.2-12 Hz, with the amplitude being color-coded. The time-frequency analysis in C exhibits irregular, fluctuating theta architecture regarding frequencies and time, whereas a segment with highly synchronized theta oscillations is characterized by a regular, non-fluctuating high amplitude theta of a nearly constant frequency of 6 Hz. The ratio of maximum theta to maximum delta amplitude is 1.25 in C and 4.67 in D, clearly classifying B as a theta oscillation EEG segment. This figure was reprinted from Reference 20, with permission. Please click here to view a larger version of this figure.
Figure 3: A Wavelet-based Theta Detection Tool. (A) Time-frequency analysis of a 30 min EEG segment (not shown) that has been recorded following urethane administration. The complex Morlet wavelet-based analysis was performed in the range of 0.2-12 Hz, with the amplitude (mV) being color-coded. (B) This image displays the maximum amplitude of the theta frequency band (3.5-8.5 Hz, green) and the upper delta band (2-3.4 Hz, red) for the 30-min EEG segment. (C) This figure illustrates the ratio of the maximum theta amplitude (green in B) and the maximum delta amplitude (red in B). Note that highly-synchronized theta oscillations correlate with suprathreshold ratios in C. This figure was reprinted from Reference 20, with permission. Please click here to view a larger version of this figure.
Figure 4: Wavelet-based Analysis of Pharmacologically-induced, Highly-organized Theta Oscillations. Representative 30-min EEG segments (not shown) are analyzed in the frequency range of 0-12 Hz with the amplitude (mV) being color-coded. A urethane injection at 800 mg/kg, i.p., resulted in the fragmented occurrence of highly-organized theta oscillations, with a predominant frequency of about 6 Hz (white circles). Following an atropine injection at 50 mg/kg, i.p., these theta oscillations are abolished. This figure was reprinted from Reference 20, with permission. Please click here to view a larger version of this figure.
Figure 5: Flow Diagram Illustrating the Quantification of Highly-organized Theta Oscillations Recorded from the Murine CA1. Type II theta oscillations can be analyzed using a control recording (phase), a post-injection (e.g., urethane, arecoline, or pilocarpine) phase, and a post-atropine phase (A1). 30 min EEG segments (A2) from each phase are time-frequency analyzed in the range from 0.2-12 Hz using a wavelet-based approach (B1 and B2). Next, theta segment detection is initiated (C1), giving a closer look at the time-frequency characteristics of theta range (3.5-8.5 Hz, C2) and the upper delta range (2-3.4 Hz, C3) for EEG epochs that are 2.5 s each (C4 and C5). Subsequently, the amplitude is analyzed over the theta and delta frequency range depicting the maximum values (C6 and C7). If the maximum amplitude of theta/delta exceeds 1.5, the 2.5 s EEG segment is classified as an epoch of highly-organized theta oscillation (C8), with a defined amplitude and frequency (D1-D3). This theta detection tool allows for the quantification of theta oscillation architecture (E1). Please click here to view a larger version of this figure.
Carprofen (Rimadyl VET – Injektionslösung) | Pfizer | PZN 0110208208 | 20ml |
binocular surgical magnification microscope | Zeiss Stemi 2000 | 0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000 | |
Dexpanthenole (Bepanthen Wund- und Heilsalbe) | Bayer | PZN: 1578818 | |
drapes (sterile) | Hartmann | PZN 0366787 | |
70% ethanol | Carl Roth | 9065.5 | |
0.3% / 3% hydrogene peroxide solution | Sigma | 95321 | 30% stock solution |
gloves (sterile) | Unigloves | 1570 | |
dental glas ionomer cement | KentDental /NORDENTA | 957 321 | |
heat-based surgical instrument sterilizer | F.S.T. | 18000-50 | |
high-speed dental drill | Adeor | SI-1708 | |
Inhalation narcotic system (isoflurane) | Harvard Apparatus GmbH | 34-1352, 10-1340, 34-0422, 34-1041, 34-0401, 34-1067, 72-3044, 34-0426, 34-0387, 34-0415, 69-0230 | |
Isoflurane | Baxter 250 ml | PZN 6497131 | |
Ketamine | Pfizer | PZN 07506004 | |
Lactated Ringer's solution (sterile) | Braun | L7502 | |
Nissl staining solution | Armin Baack | BAA31712159 | |
pads (sterile) | ReWa Krankenhausbedarf | 2003/01 | |
Steel and tungsten electrodes parylene coated | FHC Inc., USA | UEWLGESEANND | |
stereotaxic frame | Neurostar | 51730M | ordered at Stoelting |
(Stereo Drive-New Motorized Stereotaxic) | |||
tapes (sterile) | BSN medical GmbH & Co. KG | 626225 | |
TA10ETA-F20 | DSI | 270-0042-001X | Radiofrequency transmitter 3.9 g, 1.9 cc, input voltage range ± 2.5 mV, channel bandwidth (B) 1-200 Hz, nominal sampling rate (f) 1000 Hz (f = 5B) temperature operating range 34-41 °C warranted battery life 4 months |
TL11M2-F20EET | DSI | 270-0124-001X | Radiofrequency transmitter 3.9 g, 1.9 cc, input voltage range ± 1.25 mV, channel bandwidth (B) 1-50 Hz, nominal sampling rate (f) 250 Hz (f = 5B) temperature operating range 34-41 °C warranted battery life 1.5 months |
Vibroslicer 5000 MZ | Electron Microscopy Sciences | 5000-005 | |
Xylazine (Rompun) | Bayer | PZN: 1320422 | |
Matlab | Mathworks Inc. | programming, computing and visualization software | |
SPSS | IBM | statistical analysis software |
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'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 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'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 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'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.