EEG signals were recorded from frontal cortex, hippocampus, striatum and reticular formation of freely moving rats from inside a totally copper shielded room. Signals were wirelessly transmitted by a radio-telemetric system (Rhema Labortechnik, Hofheim, Germany, using 40 Megahertz as carrier frequency) and were amplified and processed as described earlier to give power spectra with a resolution of 0.25 Hz [8] . In short, after automatic artefact rejection signals were collected in sweeps of 4 s duration and Fast Fourier transformed using a Hanning window. Sampling frequency was 512 Hz. Four values were averaged to give a final sampling frequency of 128 Hz, well above the Nyquist frequency. The resulting electrical power spectra were divided into 8 specially defined frequency ranges (delta: 1.50 - 4.00 Hz; theta: 4.25 - 6.75 Hz; alpha1: 7.00 - 9.50 Hz; alpha2: 9.75 - 12.25 Hz; beta1a: 12.50 - 15.00 Hz; beta1b: 15.25 - 17.75 Hz; beta2: 18.00 - 34.25 Hz; gamma: 34.50 - 81.00 Hz). Spectra were averaged in steps of 3 minutes each and displayed on-line. In an off-line procedure spectra were averaged to give longer periods for further analysis and data presentation. All dosages of caffeine and its combination with Zynamite® were tested by oral acute administration. Dosage of Zynamite was chosen on the base of earlier experiments. Solutions were prepared fresh for each experimental day and administered orally by gavage after 45 minutes of pre-drug Vehicle recording. Vehicle was water. Extracts are listed in Table 1.

Nine adult male Fisher 344 rats (5 months of age and day-night converted, weight about 350 - 400 g, provided by Charles River Laboratories, D-97633, Sulzfeld) remained from a preceding study with allowance from the local legal authority “Regierungspräsidum Giessen” and were used in this experimental series. Animals were day-night reversed (12h/12h). The principles of good laboratory animal care were followed in all trials. Animals were implanted with

electrodes into the brain and were given two weeks for recovery from surgery. After this, the wireless transmitter was plugged in for adaptation and control experiments. Unfortunately, 3 animals dropped the implant after performance of the placebo series. After testing the two combinations one further animal dropped the implant thus allowing to test the final single dose of caffeine in only 5 animals. During the recording rats were not restricted and could move freely but did not have food available (chewing would have produced too many artefacts).

Rats were implanted as reported earlier [8] . A crossover design with at least three days of drug holidays in between the administrations was used. Controls were performed by oral administration of 1.0 ml/kg of vehicle (0.9% NaCl) and the mixture Zynamite® (25 mg/kg) and Caffeine (0.25 and 0.50 mg/kg) as well as Caffeine (0.50 mg/kg). Data were averaged from 5 - 9 animals. Data are expressed as mean values ± S.E.M. Statistics were calculated by means of the Wilcoxon, Mann, Whitney U-test. The animals were dosed orally at a constant volume of 1.0 ml/kg per kg body weight. The dosage administered to each animal was determined every day by the weight of that animal at the time of administration. Wilcoxon, Mann, Whitney U-test was used throughout all experimental data for comparison to results obtained by vehicle administration at the particular timing.

Hippocampus slices were prepared as reported earlier [8] . In short, after daily administration of trial medication for one week 4 animals were sacrificed under deep anesthesia and the hippocampus was quickly taken out and chopped by a vibrating razor blade to give 6 slices of about 400 µ thickness per animal. After adaptation to artificial cerebrospinal fluid for one hour, slices were transferred to an incubation chamber [9] and superfused with cerebrospinal fluid at 35˚C [10] . Stimulation of the Schaffer Collaterals by single stimuli or theta burst stimuli resulted in the induction of a population spike or long-term potentiation, respectively [5] . Amplitudes of the population spikes were recorded after stimulation every 10 minutes to prevent potentiation mechanisms. Each animal had been treated for one week with each one of the preparations. Slices were taken one day after the last treatment. Excitability of the hippocampus slices was checked after administration of 25 mg/kg of Zynamite®, a combination with 0.5 mg/kg of caffeine and 0.5 mg/kg of caffeine alone. Rats were treated with each of the preparations daily for one week before the hippocampus was taken out at the next day. An example of the amplitude of the population spike after single shock stimulation and theta burst stimulation is given in Figure 1 for the animal treated with placebo (upper part of the image) and the combination of Zynamite® with caffeine (lower part of the image). Stimulation of the Schaffer Collaterals was performed only every 10 minutes in order to prevent physiological potentiation mechanisms.

Oral administration of the vehicle (0.9% NaCl) only resulted in very minor changes of spectral power within the four brain areas. During the second and third to the fifth hour tiny decreases of power emerged in the frontal cortex and hippocampus. With respect to gamma power some tiny increases were observed. A complete time course is given in Figure 2.

Oral administration of 25 mg/kg of Zynamite® resulted in a statistically significant attenuation of all classic frequencies during the first hour after administration. Strongest effects were documented with respect to alpha2 and beta1 spectral frequencies. Mainly frontal cortex and striatum were involved. During the third hour after administration significant increases of delta power were seen (p < 0.05). Within the striatum a non-significant attenuation of alpha1 and beta power was recognized throughout the whole recording period of 5 hours (Figure 3).

Oral administration of Caffeine (0.50 mg/kg) resulted in a statistically conspicuous attenuation (p < 0.1) of delta power in the frontal cortex. In the hippocampus statistically significant attenuation of delta, theta and beta power was

recognized. In the striatum a minor but statistically conspicuous decrease of delta waves was seen. From the second hour after administration on, increases of delta power emerged for the rest of the recording period in the frontal cortex and reticular formation. During the last hour increases of alpha2 power were observed except for the striatum. A whole time course is given in Figure 4.

Oral administration of the combination of Zynamite® (25 mg/kg) + Caffeine (0.25 mg/kg) resulted in a statistically significant attenuation of all frequencies except for delta spectral power in comparison to placebo during the first hour after administration. Strongest attenuation was seen with respect to alpha1 and beta1 power in all brain regions. The alpha1 attenuation lasted into the second hour after administration, within the striatum into the last hour. During the third hour an increase of delta power emerged in the frontal cortex and hippocampus and somewhat less in the reticular formation. At the same time significant increases of alpha2 power were documented during the last 2 hours after administration. A total time course is given in Figure 5.

Oral administration of the mixture of Zynamite® (25 mg/kg) with Caffeine (0.50 mg/kg) resulted in a statistically significant attenuation of spectral power with respect to all frequencies except for gamma power in comparison to placebo during the first two hours after administration. Effects were strongest in frontal cortex and hippocampus. The attenuation lasted until the third hour after administration, in the hippocampus and striatum until the end of the recording period. A total time course is given in Figure 6. When directly comparing the

effects of Zynamite® and caffeine during the first hour after administration a close similarity between both becomes visible. Both combinations induce a much stronger attenuation of nearly all frequencies. Strongest difference to the mono-preparation consists in a large decrease of alpha1 spectral frequencies indicating a different mechanism of action (Figure 7). This alpha1 attenuation also dominates the effect of Zynamite® and caffeine during the second hour after administration. Likewise, the combination of Zynamite® with the low dose of caffeine shows a strong attenuation with respect to alpha1 waves (Figure 8). A massive attenuation of all frequencies is observed in the presence of the combination of Zynamite® (25 mg/kg) with the higher dose of caffeine (0.5 mg/kg). This indicates a synergistic action, which can be demonstrated even more clearly when looking at the frequency changes in the hippocampus during the first 2 hours after administration (Figure 9).

A valuable methodology for comparison of frequency changes induced by different preparations has been described in numerous publications. Feeding all 24 variables (4 brain areas times 6 frequency ranges) into linear discriminant analysis the similarity of the action of Zynamite® and a low dose of caffeine is confirmed by projection of both in close neighborhood and showing the identical blue colour (Figure 10). The result of the combination is shifted more towards the plots for Paullinia and Methylphenidate indicating a different mechanism of

action for the combination than for the mono-preparations. Details of administration of the preparation are listed in Table 2.

Analysis of motion only revealed a statistically significant transient increase in the presence of the combination of Zynamite® with the lower dose of caffeine during the first hour. Caffeine alone and the combination of Zynamite® with the higher dose of caffeine induced higher motion during the first hour after administration, but did not reach statistical significance (Table 3).

Time courses of the recordings from the 4 animals revealed very stable results over 2 hours. Population spike amplitudes after administration of 0.5 mg/kg of caffeine revealed no difference to placebo during single shock stimulation (blue line in Figure 11), but revealed clearly higher amplitudes during theta burst stimulation (green line in Figure 11).

The daily administration of 25 mg/kg of Zynamite® resulted in definitely higher amplitudes of the population spike already during single shock stimulation as documented in Figure 11. Theta burst stimulation induced not only massive higher amplitudes than after Placebo administration, but also clearly higher amplitudes in comparison to administration of caffeine (red line in Figure 11).

Finally, recording of population spike amplitudes after daily administration of the combination of 25 mg/kg Zynamite® with 0.5 mg/kg of caffeine revealed highest values during single shock stimulation as well during theta burst stimulation (dark blue line in Figure 11). The mean average values of three measurements (shadowed area in Figure 11) are documented with statistical significances in Figure 12.

Regarding now the numerical differences of the population spike amplitudes during single shock stimulation an over-additive effect becomes visible when comparing the effect of caffeine and Zynamite® alone with the combination. With respect to theta burst stimulation at least an additive effect is obvious (Table 4).