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Comparison of changes in the EEG of freely moving rats induced by enciprazine, buspirone and diazepam
0028-3908/89 S3.00 + 0.00
Neuropharmacology Vol. 28, No. 8, pp. 799-803, 1989
Copyright 0 1989 Maxwell
Printed in Great Britain. All rights reserved
COMPARISON MOVING
PcrgamonMacmillanplc
OF CHANGES IN THE EEG OF FREELY RATS INDUCED BY ENCIPRAZINE, BUSPIRONE AND DIAZEPAM
B. NICKEL and I. SZELENYI* Department of Pharmacology, ASTA Pharma AG, WeismiillerstraDe45, D-6000 Frankfurt 1, Federal Republic of Germany (Accepted 27 February 1989)
Summary-The effect of enciprazine, buspirone and diazepam was investigated on the cortical electrical activity in freely-moving rats. Enciprazine (5 mg/kg, i.p.) and buspirone (5 mg/kg, i.p.) induced comparable changes, consisting in decreases of mean power values in delta and theta and increases in alpha and beta EEG frequency bands. Regarding only a particular area of the brain or particular frequency bands, these two compounds could not be clearly separated from each other. Changes in frequency bands induced by 0 -methoxy-phenyl-piperazine (5 mg/kg i.p.) (D 15157), the presumed main metabolite of enciprazine, were dose-related to that caused by the parent compound. The second metabolite (R,S)-l-Q(l-methoxy4-hydroxy-phenyl) piperazin-l-yl-3-(3,4,5-trimethoxyphenoxy)propan-2-ol-dihydr~~o~de (D 20092) (5 mg/kg Lp.) evoked only minimal changes in the different frequency bands of the rats. The power spectra did not significantly differ from those seen in animals treated with saline. The action of diazepam (2 mg/kg i.p.1)was characterized by decreases in alpha and delta frequency bands, accompanied by marked increases in fast beta waves. The marked frequency shifts caused by buspirone and enciprazine could clearly be differentiated from the EEG changes evoked by the minor tranquilizer, diazepam. Ke), words-pharmaco
EEG, enciprazine, buspirone, diazepam, rat.
Benzodiazepines represent the first class of drugs which are useful in the management of generalised anxiety syndromes. They have come to be widely used. Although their side-effects (e.g. abuse potential, interaction with alcohol or sedative/hypnotic drugs) are controversely discussed, there is an increasing demand for new anxiolytics without these undesired actions. Buspirone, recently introduced into therapy, is the first in a new class of compounds with a reduced sedative effect and an apparent lack of abuse potential and interaction with alcohol. Enciprazine is chemically unrelated to the benzodiazepines and bears no obvious structural resemblance to buspirone. Based on preclinical experimental data, enciprazine appears to have several advantages over benzodiazepines. Its anxiolytic effect is comparable with that of diazepine. By contrast, it possesses less sedative activity and does not affect motor coordination adversely. Furthermore, enciprazine is apparently free of abuse potential as demonstrated in animal studies (Engel, Kleemann and Jakovlev, 1981). It is well accepted that the reaction of the brain to exposure to drugs is determined by the modulation of neuronal activity caused by the interaction of the drugs with receptors on a molecular level. Therefore, monitoring the functional consequences of this interaction can be regarded as a sensitive method of following the dynamic actions of a compound (Bronzino,
*To whom correspondence
should be addressed.
1984; Herrmann, 1982; Herrmann and Irrgang, 1983; Dimpfel, Spiiler, Nickel and Tibes, 1986a; Dimpfel, Spiiler and Nickel, 1986b). Changes in the EEG enable similarities or differences among the drugs tested to be established. The aim of the present study was, therefore, to investigate the changes in the electrical activity of the brain of freely-moving rats induced by diazepam, buspirone and enciprazine. Two presumed main metabolites of enciprazine were also studied. METHODS
Male Sprague-Dawley rats (160-210 g SIV 50, Fa. Savo, Kisslegg, F.R.G) were used. Animals were housed individually and had free access to food (standard pellet diet for rats, Altromin, Lage-Lippe, F.R.G) and drinking water. They were kept under constant environmental conditions (temperature: 2123°C; relative humidity: 55-60%; reversed lightdark-rhythm, 8 : 00 a.m.-8 : 00 p.m. dark). Experimental procedure
Rats were prepared for implantation of the set of electrodes, under general anesthesia (chloral hydrate, 300mg/kg, i.p.), by exposure of the skull and trepanation of 6 holes during atraumatic fixation in a stereotactic device (David Kopf Instruments, Boston, U.S.A.). Target positions of the 4 cortical electrodes were based on coordinates given by the atlas of 799
800
B. NICKELand I. SZELENYI
Zilles (1985). Two electrodes were placed over the left hemisphere and the two other electrodes at the same position over the right hemisphere (limb area, occipital cortex area). The lateral position was 4.5 and 5.5 mm; the caudal position was 1.5 and 4.3 mm. Attachment of the electrode set (bipolar concentric stainless steel from Rhodes Medical Instruments, New York, U.S.A.) was achieved by cementing it to 2 stainless steel screws, drilled into the bone, by means of dental acrylic cement @‘alavita, Kulzer GmbH, Wehrheim, F.R.G.). After surgery the animals were allowed to recover for 2 weeks. After the beginning of their active phase (8 : 00 a.m.) the fasted (18 hr) animals were transferred to the recording room in a sound-insulated box, localized in a Faraday cage and allowed to adapt for 30 min. Thereafter 2 periods of 15 min were recorded as reference periods. The particular drug was given in a volume of lOml/kg by intra~~toneal injection, followed by a period of 5 min before the electroencephalographic (EEG) recording was restarted. Effects of drugs were observed for the next 165 min, in 11 successive periods, each lasting 15 min. Each of the following drugs was tested in 6 experiments in a total of 6 animals: physiological saline (0.9% w/v), enciprazine (5 mg/kg i.p.) diazepam (2 mg/kg i.p.), buspirone (5 mg/kg i.p.) D 15157 (U-methoxy-phenylpiperazine) (5mg/kg i.p.) and D 20092 (R,S)-1-4(2-methoxy-4-hydroxy-phenyl)piperazin-l-yl-3-(3,4,5trimethoxy-phenoxy)propan-2-ol-dihydrochloride (5 mg/kg i.p.). The animals were allowed washout periods of at least 7 days between experiments. The EEG was analyzed by Fourier analysis as originally suggested by Dietsch (1932). The EEG signals were digitized with a sample rate of 512 Hz and a resolution of 12 bits. Four successive samples were averaged to give an effective sampling rate of 128 Hz. After rejection of artefacts, elimination of d.c.-terms from the signals and windowing power density spectra were sampled online for periods of 4 set by fast Fourier transformation on a PDP 1l/23 (Digital Equipment, Boston, U.S.A.) and averaged on each channel over time blocks of 15 min. The power spectra were cut into 6 frequency bands, in congruence with clinical standards. Delta activity was defined from 1.5 to 3.5 Hz, theta activity from 3.7 to 7.5 Hz, alpha, from 7.8 to 10 Hz, alpha* from 10.3 to 13.5Hz, beta, from 13.8 to 20.0 and beta, reached from 20.3 to 30.0Hz. Each frequency band from every area of brain was evaluated separately in percentage of the mean value, measured during the first 2 pre-drug recording periods of 15 min. For more details see Nickel and Zerrahn (1987).
DELTA ACTIVITY (1.5 3.5Ht) Encipmzln lm@kg
Lp.
BUSfllfOll* Sm@/kgi.p.
Diuopam
2mo1koiP.
0 15157 5m&kfji.p.
THETA ACTIVITY (3.7 - 7*5&l I O-
5.
- lo-15-2O-
-25-J ALPHA ACTIVITY f 7.8 -
13SHz)
40
30 20 10 0 -
10
-20 -30 BETA ACTIVITY (13.8 - 30.0 ttz) .*
30. 20-
Fig. 1. Time-dependent EEG changes in the delta (1.53.5 Hz), theta (3.7-7.5 Hz), alpha (7.8-13.5 Hz) and beta (13.8-30.0 Hz) frequency bands of freely moving rats after intraperitoneal injection of enciprazine (5 mg/kg), buspirone (Smg/kg) diazepam (Zmg/kg) and D 15157 (5mg/kg). Asterisks indicate that the EEG frequency changes after admjnistration of drug differed signi~~ntly from the control value of the untreated rat (*P c 0.05; **P < 0.01).
Drugs Enciprazine as the hydrochloride, and its two metabolites D 15157: 0-methoxy-phenyl-piperazine and D 20092: (R,S)-I-4-(2-methoxy-4-hydroxyphenyl)-piperazin- l-yi-3-(3,4,5-t~methoxyphenoxy)propan-2-ol-dihydro~hloride were synthesized by
the Department of Chemistry, ASTA Pharma AG, Frankfurt, F.R.G. Diazepam was obtained from Pfannenschmidt, Hamburg, F.R.G. Buspirone was purchased from B~stol-Myers, Evansville, U.S.A.
Changes
in pharmaco-EEG
by anxiolytics
801
of the EEG frequency bands in freely-moving rats after intraperitoneal injection of enciprazine, buspirone, diazepam, D 15157 and D 20092 in percentage, related to the control value for the same animal
Table 1. Changes
Delta
Theta 3.7-7.5 Hz
Dose mg/kg
Minutes after
Compounds
i.p.
treatment
W)
W)
W)
W)
Enciprazine
5
15 30 45 60 75
-II’ -13** -12” -11** -6
-6 -8 - 16” -9 -4
+4 +12* + 18** +16** +7*
+8* +21** +24** + 14” +6
-6 - 14.’ - 12** -8’ -5
+4 + IS** +16** +9* +6
Buspirone
5
15 30 45 60 75
- 17** - 19’. -21:’ -16’ -7
-4 -12; - 18** -12; -6
f2 i-9* + 16” +14* +6
+4 + 18** +27** +21** i-9*
-4 -16** -14’ -9’ -7
+6 +22.* +24’* + 19** +8
Diazepam
2
15 30 45 60 75
-8 -9f -7 -6 -4
-3 -9’ -8* -7 -2
-4 - 17** - 19** -12* -6
-6 -8’ -9* -71 -6
+4 fll’ +14* +9* f7
+9* + 27*’ f25” +19** fl2’
D 15157
5
15 30 45 60 75
-6 -141; -11’ -9’ -9’
i2 -9’ -9’ -8’ -6
+4 +12* + 14** +12* +10*
+2 -4 -6 -3 -4
-2 -6 -8* -7f -3
-2 +9* +1i* + 101 i9’
D 20092
5
15 30 45 60 75
-2 -6 -2 -3 -4
-4 -7 +2 -4 -6
+6 +8’ +2 +4 +4
+2 -3 -2 +3 +4
-2 f4 f4 -3 -4
-4 t6 +5 +4 -3
I .5-3.5 Hz
Alpha, 7.8-10.0 Hz
Alpha, 10.3-13.5 Hz
Asterisks indxate that the EEG frequency changes after administration of drug differed significantly
rat (*P 8: 0.05;
Beta, 13.8-20.0 Hz W)
Beta, 20.3-30.0 Hz W)
from the control value of the untreated
**P < 0.01)
All drugs used were dissolved in physiological saline and given intraperitoneally in a volume of lOml/kg body weight. Statistics
Indications of significance were calculated with the paired t-test and the differences relate to the baseline values. RESULTS
Both the non-benzodiazepine anxiolytic compounds, enciprazine and buspirone, induced comparable changes, consisting in decreases of mean power values in delta and theta and increases in alpha and fast beta EEG frequency bands (Fig. 1). The changes of the EEG patterns were very similar over all four areas of the brain. The first signs of EEG changes, caused by enciprazine or buspirone, were observed 15 min after the intraperitoneal injection. The changes in frequency lasted for 75 min, with a peak between 30 and 45 min after application. The EEG changes observed in the different frequency bands (Table 1) of the freely moving rats was more marked after application of buspirone (5 mg/kg, i.p.) than after enciprazine (5 mg/kg, i.p.). Thus, for a particular area of brain or particular frequency band these 2 compounds could not be clearly separated from each other. The action of diazepam was characterized by decreases in the alpha, and delta or theta frequency bands but marked increases in the fast beta waves in all areas of the brain (Fig. 1). Similar to the two other
anxiolytic test-compounds, enciprazine and buspirone, the onset of the EEG changes was 15 min after intraperitoneal injection of diazepam. These effects could also be observed over 75 min. But opposite to buspirone and enciprazine, the main changes of the mean power values was observed 15-30 min after the injection of 2 mg/kg of diazepam. In a dose of 5 mg/kg (ip.), D 15157 (O-methoxyphenyl-piperazine) strongly caused the same changes in the EEG observed after the injection of enciprazine. Changes in the frequency bands when D 20092 (R,S)1-4-(2-methoxy-4-hydroxy-phenyl)-piperazin-l-yl-3(3,4,5-trimethoxyphenoxypropan-2-ol-dihydrochlo~de) was given in a dose of 5 mg/kg (i.p.) were minimal and did not differ significantly from those observed in animals treated with saline.
DISCUSSlON
The possibility of recording EEG potentials with chronically-implanted electrodes, from freely moving rats enabled the processing of nearly artefact-free signals by the computer. Moreover, the quantitative EEG has not only been shown to be a valuable tool in the analysis of actions of drugs in the human (Ktinkel, Luba and Niethardt, 1976; Fink, 1977; Itil, 1982; Herrmann and Irrgang, 1983), but has gained considerable interest also for drug-monitoring purposes in various animals (Fairchild, Jenden, Michey and Yale, 1980; Depoortere and Granger, 1982; Krijzer, van den Molen, Oorschot and Vollmer,
802
B. NICKELand I. SZELENYI
1983; Glatt, Duerst, Miiller and Demieville, 1983; Yamamoto, 1985). The EEG frequency changes in rats after the injection of enciprazine or buspirone, e.g. a decrease of the mean power values in the delta and theta and increases of the alpha and beta EEG frequency bands, could not be clearly separated from each other but were distinct from the changes in the EEG power spectrum induced by diazepam, which was characterized by a decrease in the alpha and delta frequency bands, accompanied by marked increases in the fast beta waves. These differences in the EEG changes caused by buspirone or diazepam could also be observed in humans (Bond and Lader, 1981). Nearly the same pattern of changes after administration of diazepam, e.g. a reduced activity in the lower frequency bands and a massive increase in the higher frequencies, has been described for a comparable dose of diazepam in the rat by Glatt et al, (1983). Such an increase in beta power has not only been observed in animals (Yamamoto, 1985) but also after the application of diazepam in the human (Bond and Lader, 1981). Even the decrease in delta and alpha frequencies has been reported for recordings from certain locations of the human scalp (Buchsbaum, Hazlett, Sicotte, Stein, Wu and Zetin, 1985; Bond and Lader, 1981). These characteristics persisted during chronic application. However, this pattern of changes
is not confined
only to diazepam
but may
be
generalized for the benzodiazepines, since Borbely, Mattmann, Loepfe, Strauch and Lehmann (1985) reported essentially the same changes after giving flunitrazepam, flurazepam and triazolam to human
subjects. The conformities of the EEG frequency changes in rats, after injection
of buspirone
or enciprazine
and
the differences in comparison to diazepam, correspond with a number of other pharmacological results. In the opposite way to diazepam, both non-benzodiazepine anxiolytic compounds, buspirone and enciprazine fail to possess sedative actions and do not affect motor coordination adversely. Furthermore, enciprazine and buspirone are apparently free of abuse potential and interactions with alcohol or barbiturates as demonstrated in corresponding animals studies (Nickel, 1987). Although buspirone and enciprazine have different chemical structures, both compounds demonstrated nearly the same pharmacological activity and similar frequency changes in the EEG of freely-moving rats. The different pharmacological changes with diazepam, in comparison to buspirone and enciprazine, were also manifested in clearly different EEG changes. Changes in EEG induced by O-methoxy-phenylpiperazine (D 15157) strongly resembled those seen with enciprazine. It may, therefore, be assumed that this metabolite represents one of the active metabolites of the parent compound. Corroborating this assumption, D 15157 showed in the fighting test in mice, the same antiaggressive activity as that seen
after injection of the parent compound, enciprazine. Also, no dose-related differences between enciprazine and D 15157 were observed. Changes in EEG, after giving D 20092 (5 mg/kg i.p.), were minimal and did not differ significantly from those seen in animals treated with saline. It is likely that the metabolite D 20092 does not contribute to the anxiolytic activity of enciprazine. Also, in the fighting test in mice D 20092
produced no antiaggressive activity even when given in large doses. In summa~, enciprazine and buspirone, with respect to their action on the electrical activity in several areas of the brain, could be separated from the EEG frequency changes produced by diazepam. Further experiments using drugs as pharmacological tools should allow more detailed interpretations of the changes in single frequency bands to be made in terms of biochemical and/or ~havioural correlates. REFERENCES Bond A. J. and Lader H. (1981) Comparative effects of diazepam and buspirone on subjective feelings, psychological tests and the EEG. Inr. Pharmacopsychiat. 16: 212-220.
Borbely A. A., Mattmann P., Loepfe M., Strauch J. and Lehmann D. (1985) Effect of benzodiazepine hypnotics on all-night sleep EEG spectra. Human Neurobiol. 4:
189-195. Bronzino J. D. (1984) Quantitative analysis of the EEG-general concepts and animals studies. IEEE Trans. biomed. 31: 85&856.
Buchsbaum M. S., Hazlett E., Sicotte N., Stein M,, Wu J. and Zetin M. (1985) Topographic EEG changes with ~nz~iazepine administration in generalized anxiety disorders. Bioi. Psychiat. 20: 832-842. Depoortere H. and Granger, P. K. (1982) Comparative EEG study of some hypnotics (chlormethiazole, flunitrazepam, zopiclone). 6th Eur. Congr. Sleep Res., pp. 285-287, Ziirich. Dietsch G. (1932) Fourier-Analyse von Elektroencephalogrammen des Menschen. Ppiigrs Arch. ges. Physiol. 230: 106-I 12. Dimpfel W., Spiiler M., Nickel B. and Tibes, U. (1986a) Fingerprints of central stimulatory drug effects by means of quantitative radioelectroencephalography in- the rat (tele-stereo EEG). Neuromvchobioloav 15: 101-108. Dimpfel W., Spiil& M. &l Nickel-B. (1986b) Radioelectroencephalography (Tele-Stereo-EEG) in the rat as a pharmacological model to differentiate the central action of flupirtine from that of opiates, diazepam and phenobarbital. ~europsycbobio~ogy 16: 163-168. Engel I., Kleemann A. and Jakovlev V. (1981) Enciprazine. Drugs of :he Future 6: 278-279. Fairchild M. D., Jenden D., Michey M. R. and Yale C. (1980) The quantitative measurement of changes in EEG frequency spectra produced in the cat by sedative hypnotics and neuroleptics. Electroenceph. clin. Neurophysiol. 49: 382-390.
Fink M. (1977) Quantitative EEG analysis and psychopha~acology. In: Remand, EEG Info~atics. A Didactic Review of Methods and Applications of EEG Data Processing, pp.” 301-318. Else&r/North-Holland, Amsterdam. Glatt A.. Duerst T.. Miiller B. and Demieville H. fl9831 EEG dvaluation ok drug effects in the rat. Neuropiycho: biology 9: 163-166. Hertmann W. (1982) Development and critical evaluation of an objective procedure for the el~troencephalographi~ classi~cation of psychotropic drugs. In: EEG in Drug
Changes in pharmaco-EEG Research (Hermann W., Ed.), pp. 249-351. Fischer, Stuttgart. Herrmann W. and Irrgang V. (1983) An absolute must in clinicopharmacological research: pharmaco-electroencephalography, its possibilities and limitations. Pfiarmaeopsychiatry 16: 140-142. Itil T. M. (1982) The significance of quantitative pharmacoEEG in the discovery and classification of psychotropic drugs. In: EEG in Drug Research (Hermann W., Ed.), pp. 131-I 57. Fischer, Stuttgart. Krijzer F., van den Molen R. J., Van Oorschot R. and Volhner F. (1983) Effects of antidepressants on the EEG of the rat. Neuropsyehobiology 9: 167-173. Kiinkel H., Luba A. and Niethardt P. (1976) Topographic
by anxiolytics
803
and psychosomatic aspects of spectral EEG analysis of drug effects. In: Q~~i~ati~ Analytic Studies its EpiIepsy (Kellaway P., Ed.), pp. 207-223. Raven Press, New York. Nickel B. (1987) Profile of enciprazine, ASTA Pharma AG, Frankfurt, internal report. Nickel B. and Zerrahn H. (1987) Pharmaco-electroencephalography in the rat as a method for chara~te~tion of different types of analgesics. Posrgrud. Med. J. 63: Suppl. 3, 45-47. Yamamoto J. (1985) Characteristics of the cortical and hippocampal EEG power spectra of rabbits during normal behavioral states and after drug ad~nistrations of CNS acting drugs. Jap. J. Pharmac. 37: 227-234. Zilles K. (1985) The Cortex of the Rat. Springer, Berlin,
Neuropharmacology Vol. 28, No. 8, pp. 799-803, 1989
Copyright 0 1989 Maxwell
Printed in Great Britain. All rights reserved
COMPARISON MOVING
PcrgamonMacmillanplc
OF CHANGES IN THE EEG OF FREELY RATS INDUCED BY ENCIPRAZINE, BUSPIRONE AND DIAZEPAM
B. NICKEL and I. SZELENYI* Department of Pharmacology, ASTA Pharma AG, WeismiillerstraDe45, D-6000 Frankfurt 1, Federal Republic of Germany (Accepted 27 February 1989)
Summary-The effect of enciprazine, buspirone and diazepam was investigated on the cortical electrical activity in freely-moving rats. Enciprazine (5 mg/kg, i.p.) and buspirone (5 mg/kg, i.p.) induced comparable changes, consisting in decreases of mean power values in delta and theta and increases in alpha and beta EEG frequency bands. Regarding only a particular area of the brain or particular frequency bands, these two compounds could not be clearly separated from each other. Changes in frequency bands induced by 0 -methoxy-phenyl-piperazine (5 mg/kg i.p.) (D 15157), the presumed main metabolite of enciprazine, were dose-related to that caused by the parent compound. The second metabolite (R,S)-l-Q(l-methoxy4-hydroxy-phenyl) piperazin-l-yl-3-(3,4,5-trimethoxyphenoxy)propan-2-ol-dihydr~~o~de (D 20092) (5 mg/kg Lp.) evoked only minimal changes in the different frequency bands of the rats. The power spectra did not significantly differ from those seen in animals treated with saline. The action of diazepam (2 mg/kg i.p.1)was characterized by decreases in alpha and delta frequency bands, accompanied by marked increases in fast beta waves. The marked frequency shifts caused by buspirone and enciprazine could clearly be differentiated from the EEG changes evoked by the minor tranquilizer, diazepam. Ke), words-pharmaco
EEG, enciprazine, buspirone, diazepam, rat.
Benzodiazepines represent the first class of drugs which are useful in the management of generalised anxiety syndromes. They have come to be widely used. Although their side-effects (e.g. abuse potential, interaction with alcohol or sedative/hypnotic drugs) are controversely discussed, there is an increasing demand for new anxiolytics without these undesired actions. Buspirone, recently introduced into therapy, is the first in a new class of compounds with a reduced sedative effect and an apparent lack of abuse potential and interaction with alcohol. Enciprazine is chemically unrelated to the benzodiazepines and bears no obvious structural resemblance to buspirone. Based on preclinical experimental data, enciprazine appears to have several advantages over benzodiazepines. Its anxiolytic effect is comparable with that of diazepine. By contrast, it possesses less sedative activity and does not affect motor coordination adversely. Furthermore, enciprazine is apparently free of abuse potential as demonstrated in animal studies (Engel, Kleemann and Jakovlev, 1981). It is well accepted that the reaction of the brain to exposure to drugs is determined by the modulation of neuronal activity caused by the interaction of the drugs with receptors on a molecular level. Therefore, monitoring the functional consequences of this interaction can be regarded as a sensitive method of following the dynamic actions of a compound (Bronzino,
*To whom correspondence
should be addressed.
1984; Herrmann, 1982; Herrmann and Irrgang, 1983; Dimpfel, Spiiler, Nickel and Tibes, 1986a; Dimpfel, Spiiler and Nickel, 1986b). Changes in the EEG enable similarities or differences among the drugs tested to be established. The aim of the present study was, therefore, to investigate the changes in the electrical activity of the brain of freely-moving rats induced by diazepam, buspirone and enciprazine. Two presumed main metabolites of enciprazine were also studied. METHODS
Male Sprague-Dawley rats (160-210 g SIV 50, Fa. Savo, Kisslegg, F.R.G) were used. Animals were housed individually and had free access to food (standard pellet diet for rats, Altromin, Lage-Lippe, F.R.G) and drinking water. They were kept under constant environmental conditions (temperature: 2123°C; relative humidity: 55-60%; reversed lightdark-rhythm, 8 : 00 a.m.-8 : 00 p.m. dark). Experimental procedure
Rats were prepared for implantation of the set of electrodes, under general anesthesia (chloral hydrate, 300mg/kg, i.p.), by exposure of the skull and trepanation of 6 holes during atraumatic fixation in a stereotactic device (David Kopf Instruments, Boston, U.S.A.). Target positions of the 4 cortical electrodes were based on coordinates given by the atlas of 799
800
B. NICKELand I. SZELENYI
Zilles (1985). Two electrodes were placed over the left hemisphere and the two other electrodes at the same position over the right hemisphere (limb area, occipital cortex area). The lateral position was 4.5 and 5.5 mm; the caudal position was 1.5 and 4.3 mm. Attachment of the electrode set (bipolar concentric stainless steel from Rhodes Medical Instruments, New York, U.S.A.) was achieved by cementing it to 2 stainless steel screws, drilled into the bone, by means of dental acrylic cement @‘alavita, Kulzer GmbH, Wehrheim, F.R.G.). After surgery the animals were allowed to recover for 2 weeks. After the beginning of their active phase (8 : 00 a.m.) the fasted (18 hr) animals were transferred to the recording room in a sound-insulated box, localized in a Faraday cage and allowed to adapt for 30 min. Thereafter 2 periods of 15 min were recorded as reference periods. The particular drug was given in a volume of lOml/kg by intra~~toneal injection, followed by a period of 5 min before the electroencephalographic (EEG) recording was restarted. Effects of drugs were observed for the next 165 min, in 11 successive periods, each lasting 15 min. Each of the following drugs was tested in 6 experiments in a total of 6 animals: physiological saline (0.9% w/v), enciprazine (5 mg/kg i.p.) diazepam (2 mg/kg i.p.), buspirone (5 mg/kg i.p.) D 15157 (U-methoxy-phenylpiperazine) (5mg/kg i.p.) and D 20092 (R,S)-1-4(2-methoxy-4-hydroxy-phenyl)piperazin-l-yl-3-(3,4,5trimethoxy-phenoxy)propan-2-ol-dihydrochloride (5 mg/kg i.p.). The animals were allowed washout periods of at least 7 days between experiments. The EEG was analyzed by Fourier analysis as originally suggested by Dietsch (1932). The EEG signals were digitized with a sample rate of 512 Hz and a resolution of 12 bits. Four successive samples were averaged to give an effective sampling rate of 128 Hz. After rejection of artefacts, elimination of d.c.-terms from the signals and windowing power density spectra were sampled online for periods of 4 set by fast Fourier transformation on a PDP 1l/23 (Digital Equipment, Boston, U.S.A.) and averaged on each channel over time blocks of 15 min. The power spectra were cut into 6 frequency bands, in congruence with clinical standards. Delta activity was defined from 1.5 to 3.5 Hz, theta activity from 3.7 to 7.5 Hz, alpha, from 7.8 to 10 Hz, alpha* from 10.3 to 13.5Hz, beta, from 13.8 to 20.0 and beta, reached from 20.3 to 30.0Hz. Each frequency band from every area of brain was evaluated separately in percentage of the mean value, measured during the first 2 pre-drug recording periods of 15 min. For more details see Nickel and Zerrahn (1987).
DELTA ACTIVITY (1.5 3.5Ht) Encipmzln lm@kg
Lp.
BUSfllfOll* Sm@/kgi.p.
Diuopam
2mo1koiP.
0 15157 5m&kfji.p.
THETA ACTIVITY (3.7 - 7*5&l I O-
5.
- lo-15-2O-
-25-J ALPHA ACTIVITY f 7.8 -
13SHz)
40
30 20 10 0 -
10
-20 -30 BETA ACTIVITY (13.8 - 30.0 ttz) .*
30. 20-
Fig. 1. Time-dependent EEG changes in the delta (1.53.5 Hz), theta (3.7-7.5 Hz), alpha (7.8-13.5 Hz) and beta (13.8-30.0 Hz) frequency bands of freely moving rats after intraperitoneal injection of enciprazine (5 mg/kg), buspirone (Smg/kg) diazepam (Zmg/kg) and D 15157 (5mg/kg). Asterisks indicate that the EEG frequency changes after admjnistration of drug differed signi~~ntly from the control value of the untreated rat (*P c 0.05; **P < 0.01).
Drugs Enciprazine as the hydrochloride, and its two metabolites D 15157: 0-methoxy-phenyl-piperazine and D 20092: (R,S)-I-4-(2-methoxy-4-hydroxyphenyl)-piperazin- l-yi-3-(3,4,5-t~methoxyphenoxy)propan-2-ol-dihydro~hloride were synthesized by
the Department of Chemistry, ASTA Pharma AG, Frankfurt, F.R.G. Diazepam was obtained from Pfannenschmidt, Hamburg, F.R.G. Buspirone was purchased from B~stol-Myers, Evansville, U.S.A.
Changes
in pharmaco-EEG
by anxiolytics
801
of the EEG frequency bands in freely-moving rats after intraperitoneal injection of enciprazine, buspirone, diazepam, D 15157 and D 20092 in percentage, related to the control value for the same animal
Table 1. Changes
Delta
Theta 3.7-7.5 Hz
Dose mg/kg
Minutes after
Compounds
i.p.
treatment
W)
W)
W)
W)
Enciprazine
5
15 30 45 60 75
-II’ -13** -12” -11** -6
-6 -8 - 16” -9 -4
+4 +12* + 18** +16** +7*
+8* +21** +24** + 14” +6
-6 - 14.’ - 12** -8’ -5
+4 + IS** +16** +9* +6
Buspirone
5
15 30 45 60 75
- 17** - 19’. -21:’ -16’ -7
-4 -12; - 18** -12; -6
f2 i-9* + 16” +14* +6
+4 + 18** +27** +21** i-9*
-4 -16** -14’ -9’ -7
+6 +22.* +24’* + 19** +8
Diazepam
2
15 30 45 60 75
-8 -9f -7 -6 -4
-3 -9’ -8* -7 -2
-4 - 17** - 19** -12* -6
-6 -8’ -9* -71 -6
+4 fll’ +14* +9* f7
+9* + 27*’ f25” +19** fl2’
D 15157
5
15 30 45 60 75
-6 -141; -11’ -9’ -9’
i2 -9’ -9’ -8’ -6
+4 +12* + 14** +12* +10*
+2 -4 -6 -3 -4
-2 -6 -8* -7f -3
-2 +9* +1i* + 101 i9’
D 20092
5
15 30 45 60 75
-2 -6 -2 -3 -4
-4 -7 +2 -4 -6
+6 +8’ +2 +4 +4
+2 -3 -2 +3 +4
-2 f4 f4 -3 -4
-4 t6 +5 +4 -3
I .5-3.5 Hz
Alpha, 7.8-10.0 Hz
Alpha, 10.3-13.5 Hz
Asterisks indxate that the EEG frequency changes after administration of drug differed significantly
rat (*P 8: 0.05;
Beta, 13.8-20.0 Hz W)
Beta, 20.3-30.0 Hz W)
from the control value of the untreated
**P < 0.01)
All drugs used were dissolved in physiological saline and given intraperitoneally in a volume of lOml/kg body weight. Statistics
Indications of significance were calculated with the paired t-test and the differences relate to the baseline values. RESULTS
Both the non-benzodiazepine anxiolytic compounds, enciprazine and buspirone, induced comparable changes, consisting in decreases of mean power values in delta and theta and increases in alpha and fast beta EEG frequency bands (Fig. 1). The changes of the EEG patterns were very similar over all four areas of the brain. The first signs of EEG changes, caused by enciprazine or buspirone, were observed 15 min after the intraperitoneal injection. The changes in frequency lasted for 75 min, with a peak between 30 and 45 min after application. The EEG changes observed in the different frequency bands (Table 1) of the freely moving rats was more marked after application of buspirone (5 mg/kg, i.p.) than after enciprazine (5 mg/kg, i.p.). Thus, for a particular area of brain or particular frequency band these 2 compounds could not be clearly separated from each other. The action of diazepam was characterized by decreases in the alpha, and delta or theta frequency bands but marked increases in the fast beta waves in all areas of the brain (Fig. 1). Similar to the two other
anxiolytic test-compounds, enciprazine and buspirone, the onset of the EEG changes was 15 min after intraperitoneal injection of diazepam. These effects could also be observed over 75 min. But opposite to buspirone and enciprazine, the main changes of the mean power values was observed 15-30 min after the injection of 2 mg/kg of diazepam. In a dose of 5 mg/kg (ip.), D 15157 (O-methoxyphenyl-piperazine) strongly caused the same changes in the EEG observed after the injection of enciprazine. Changes in the frequency bands when D 20092 (R,S)1-4-(2-methoxy-4-hydroxy-phenyl)-piperazin-l-yl-3(3,4,5-trimethoxyphenoxypropan-2-ol-dihydrochlo~de) was given in a dose of 5 mg/kg (i.p.) were minimal and did not differ significantly from those observed in animals treated with saline.
DISCUSSlON
The possibility of recording EEG potentials with chronically-implanted electrodes, from freely moving rats enabled the processing of nearly artefact-free signals by the computer. Moreover, the quantitative EEG has not only been shown to be a valuable tool in the analysis of actions of drugs in the human (Ktinkel, Luba and Niethardt, 1976; Fink, 1977; Itil, 1982; Herrmann and Irrgang, 1983), but has gained considerable interest also for drug-monitoring purposes in various animals (Fairchild, Jenden, Michey and Yale, 1980; Depoortere and Granger, 1982; Krijzer, van den Molen, Oorschot and Vollmer,
802
B. NICKELand I. SZELENYI
1983; Glatt, Duerst, Miiller and Demieville, 1983; Yamamoto, 1985). The EEG frequency changes in rats after the injection of enciprazine or buspirone, e.g. a decrease of the mean power values in the delta and theta and increases of the alpha and beta EEG frequency bands, could not be clearly separated from each other but were distinct from the changes in the EEG power spectrum induced by diazepam, which was characterized by a decrease in the alpha and delta frequency bands, accompanied by marked increases in the fast beta waves. These differences in the EEG changes caused by buspirone or diazepam could also be observed in humans (Bond and Lader, 1981). Nearly the same pattern of changes after administration of diazepam, e.g. a reduced activity in the lower frequency bands and a massive increase in the higher frequencies, has been described for a comparable dose of diazepam in the rat by Glatt et al, (1983). Such an increase in beta power has not only been observed in animals (Yamamoto, 1985) but also after the application of diazepam in the human (Bond and Lader, 1981). Even the decrease in delta and alpha frequencies has been reported for recordings from certain locations of the human scalp (Buchsbaum, Hazlett, Sicotte, Stein, Wu and Zetin, 1985; Bond and Lader, 1981). These characteristics persisted during chronic application. However, this pattern of changes
is not confined
only to diazepam
but may
be
generalized for the benzodiazepines, since Borbely, Mattmann, Loepfe, Strauch and Lehmann (1985) reported essentially the same changes after giving flunitrazepam, flurazepam and triazolam to human
subjects. The conformities of the EEG frequency changes in rats, after injection
of buspirone
or enciprazine
and
the differences in comparison to diazepam, correspond with a number of other pharmacological results. In the opposite way to diazepam, both non-benzodiazepine anxiolytic compounds, buspirone and enciprazine fail to possess sedative actions and do not affect motor coordination adversely. Furthermore, enciprazine and buspirone are apparently free of abuse potential and interactions with alcohol or barbiturates as demonstrated in corresponding animals studies (Nickel, 1987). Although buspirone and enciprazine have different chemical structures, both compounds demonstrated nearly the same pharmacological activity and similar frequency changes in the EEG of freely-moving rats. The different pharmacological changes with diazepam, in comparison to buspirone and enciprazine, were also manifested in clearly different EEG changes. Changes in EEG induced by O-methoxy-phenylpiperazine (D 15157) strongly resembled those seen with enciprazine. It may, therefore, be assumed that this metabolite represents one of the active metabolites of the parent compound. Corroborating this assumption, D 15157 showed in the fighting test in mice, the same antiaggressive activity as that seen
after injection of the parent compound, enciprazine. Also, no dose-related differences between enciprazine and D 15157 were observed. Changes in EEG, after giving D 20092 (5 mg/kg i.p.), were minimal and did not differ significantly from those seen in animals treated with saline. It is likely that the metabolite D 20092 does not contribute to the anxiolytic activity of enciprazine. Also, in the fighting test in mice D 20092
produced no antiaggressive activity even when given in large doses. In summa~, enciprazine and buspirone, with respect to their action on the electrical activity in several areas of the brain, could be separated from the EEG frequency changes produced by diazepam. Further experiments using drugs as pharmacological tools should allow more detailed interpretations of the changes in single frequency bands to be made in terms of biochemical and/or ~havioural correlates. REFERENCES Bond A. J. and Lader H. (1981) Comparative effects of diazepam and buspirone on subjective feelings, psychological tests and the EEG. Inr. Pharmacopsychiat. 16: 212-220.
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