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Effects of low doses of physostigmine on avoidance learning and EEG in two strains of mice
ELSEVIER
BEHAVIOURAL BRAIN RESEARCH Behavioural Brain Research 81 (1996) 155-161
Research report
Effects of low doses of physostigmine on avoidance learning and EEG in two strains of mice Alberto Loizzo a,,, Sergio Palazzesi a, Stefano Loizzo a, Mario Battaglia b, Mario Sansone b a Istituto Superiore di Sanitd, Viale Regina Elena 299, 00161 Roma, Italy b Istituto di Psicobiologia e Psicofarmacologia, CNR, Roma, Italy
Received 10 October 1995; revised 4 March 1996; accepted 6 March 1996
Abstract
The effects of the cholinomimetic drug, physostigmine (0, 0.01, 0.025, 0.05 and 0.1 mg/kg, i.p.), on shuttle-box avoidance learning and electroencephalographic (EEG) activity were investigated, in two separate studies, in mice belonging to the inbred C57BL/6 (C57) and DBA/2 (DBA) strains. The results of the behavioral investigation showed a consistent, significant enhancement of avoidance performance, on the whole of 5 daily training sessions, in C57 mice treated with the lowest dose (0.01 mg/kg) and in DBA mice treated with the highest doses (0.05 and 0.1 mg/kg) of the drug. Doses higher than 0.01 mg/kg, in C57 mice, and lower than 0.05 mg/kg, in DBA mice, had no significant effect. The avoidance improvements induced by physostigmine cannot be ascribed to a general behavioral activation, since the doses that increased avoidance responses did not affect or even depressed spontaneous locomotor activity. The same doses of treatment which increased avoidance responding, also induced, in the same strains, consistent enhancement of 4-7 Hz (theta) EEG band power and decrease of 7-12 Hz (alpha) band power. Results suggest that the effects induced by physostigmine on the EEG and on the shuttle-box performance of mice are related to the same neurochemical systems, and are dependent upon the interaction of the dose with specific strain sensitivity. Keywords: C57BL/6;DBA/2; Electroencephalogram;Hippocampus; Inbred strain; Mouse; Physostigmine;Shuttle-box
1. Introduction
Experimental evidence indicates that the central cholinergic system is involved integrally in cognitive function and drugs affecting this system have been shown either to enhance or to hinder performance in tests of learning and memory [3,18,24]. Some cholinomimetic agents, as the acetylcholinesterase inhibitor, physostigmine, are classified among the putative cholinomimetic cognition enhancers [44]. Physostigmine facilitates learning in laboratory animals [ 19,45], but only in some experimental tasks and in a narrow range of doses [14]. In the shuttle-box avoidance test, physostigmine exerted impairing rather than improving effects on avoidance performance in rats [16,38,41], while mice of the randomly bred CD-1 strain, receiving low doses of the drug during shuttle-box avoidance training, showed a consistent tendency to better performance [43]. * Corresponding author. Tel.: (39) (6) 4990-2882/4990-2873;Fax: (39) (6) 4440-053. 0166-4328/96/$15.00© 1996ElsevierScienceB.V. All rights reserved PII S0166-4328 (96)00057-5
Cholinergic drugs also have a profound influence on brain electrical activity [24,28]. Several studies investigated the biochemical background of cholinergic influences on electrical brain activity and rhythms, specially referring to hippocampal E E G waves, and hippocampal electrical activity has been correlated with spontaneous motor behavior in animals (cf. for example E4, 6,12,17,26,40,47, 50, 51 ]) and with the learning behavior 1-10,22,34,52]. However, information on the possible relationships between those doses of cholinergic drugs which enhance some forms of learning or performance behavior in a specific species (or strain) and the E E G effects induced by the same drug doses in the same species or strain are still scanty. In this paper, we show studies performed on the effects induced by low doses of physostigmine in mice which underwent daily shuttle-box active avoidance trials. In different groups of mice, we also studied EEG spectral power effects induced by the same doses of the drug. In order to minimize species variability, two inbred
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Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
strains of mice were used, i.e., DBA/2 (DBA) and C57BL/6 (C57), which have quite a different learning behavior [5], different hippocampal function [37] and are also different in response to cholinergic drugs [23]. In preliminary experiments, spontaneous locomotor activity was also tested in order to assess the specificity of the drug effects on learning.
saline solution (0.9% NaC1) or physostigmine sulfate (0.01, 0.025, 0.05 or 0.1 mg/kg). Each experimental group consisted of 8 subjects. The results were statistically evaluated by two-factor analyses of variance (ANOVAs), followed by Duncan's multiple range test, where appropriate. The factors were drug (5 levels) and time blocks (2 levels; repeated measures) in the activity test, or drug (5 levels) and sessions (5 levels; repeated measures) in avoidance training.
2. Materials and methods
2.2. EEG investigation The experiments reported here were conducted according to the directives of the Council of the European Communities [9].
2.1. Behavioral investigation Male mice of the inbred C57 and DBA strains, obtained by Charles River (Italy) at the age of 9 weeks, were housed in standard transparent plastic cages (8 per cage) under standard animal room conditions (free access to food and water, 12-h light/dark cycle, ambient temperature of 23°C). In preliminary experiments, mice were tested for locomotor activity 6-7 days after their arrival in the laboratory. Nine to 10 days after the activity test, the animals were reassigned to different treatment groups and subjetted to avoidance training. The experiments were carried out between 09.00 and 16.00 h. The same apparatus was employed to measure locomotor activity and active avoidance [43]. The apparatus consisted of 8 automated shuttle-boxes, each one divided into two 20x10 cm compartments, connected by a 3 x 3 cm opening. To measure spontaneous locomotor activity, the lamps of the shuttle-boxes were switched off and no electric shock was applied to the grid floor. For each mouse, the number of crossings from one compartment to the other was recorded during 30 min and cumulated into two 15-min blocks. In avoidance training, a light (10 W) was switched on alternately in the two compartments and used as a conditioned stimulus (CS). The CS preceded the onset of the unconditioned stimulus (US) by 5 s and overlapped it for 25 s. The US was an electric shock (0.2 mA) applied continuously to the grid floor. The intertrial interval was 30 s. An avoidance response was recorded when the animal avoided the US by running into the dark compartment within 5 s of the onset of the CS. If animals failed to avoid the shock they could escape it by crossing during the US. Spontaneous crossings from the dark to the light compartment were punished and recorded as intertrial responses. Training consisted of one 50-trial (25 min) avoidance session per day, for 5 consecutive days. Both activity test and avoidance sessions were preceded (15 min) by intraperitoneal administration of
Groups of 4 naive DBA and C57 male mice were purchased and maintained under the same conditions as for the behavioral investigation. The animals were chronically implanted under pentobarbital anesthesia with epidural stainless-steel electrodes in the left and right anterior (A -1.3 mm anterior to bregma and lateral to sagittal suture) and posterior (P - 3 m m posterior to bregma and 2.5 mm lateral to sagittal suture) sensorimotor regions. Recording sessions were performed at least 7 days after surgery. EEG taken from the left A to P sensorimotor regions (right A electrode served as ground) of freely moving mice was preamplified on a Grass model 8-10D EEG machine, and recorded on paper and on magnetic tape (HP3964A FM analog recorder, speed 3-3/4 in. per second) in a shielded, sound-proof recording chamber for at least 60 min. The animals were then treated with the drug, and EEG was again recorded for at least 5 min at fixed intervals: 15, 30, 60 and 120 min after drug injection. Conditions and treatments were identical as for behavioral studies. Each mouse was recorded only once. During the EEG recording, an operator followed the animals behavior on video camera, and continuously reported on paper any behavioral changes using the following code: W, walking or rearing; G, grooming; I, immobile, alert; +, all other behaviors. EEG recorded from the left A to P cortex was bandpass filtered off-line (0.5-42 Hz, nominal attenuation slope 24 dB/octave), then converted to digital signal, using a sampling rate of 256 Hz and an A/D conversion precision of 13 bits (by HP-3561A signal analyzer, controlled by a HP-9816S personal computer) and sequentially stored on a disk. Once digitized, artifactfree 4-s segments corresponding to desynchronized (wakefulness) periods were selected: EEG segments recorded during various periods were recalled from disk on display and carefully compared to the EEG and behavior recorded on paper. Both the control EEG and the EEG following drug administration showed a prevalence of desynchronized pattern. Only segments which showed low-voltage fast activity or the characteristic saw-tooth shaped theta activity [26,51 ] and at the same time corresponded to W-, G- or I-related behaviors, were considered for the analysis. On the other hand,
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
those segments showing random high-voltage waves (i.e., waves with amplitude above 150% of the background theta activity), drowsiness periods and any segments related to + behavior were discarded by an operator blind to the strain and treatment [49]. Spectra were constructed from 0 to 100 Hz with 0.25-Hz steps, using standard fast Fourier transform (FFT) algorithm on Hamming-windowed segments, and stored on a disk. For each animal, 30-60 power spectral plots were thus selected in each 5 min duration time record, corresponding to the mean power spectra of about 120-240 s. Spectral data underwent further elaboration: frequency steps showing high inter-correlation were cumulated in wider frequency bands. Finally, 8 parameters were chosen, i.e., the total power of the spectrum and its standard deviation, and the power of 6 frequency bands, expressed as percentage of the same band power of predrug period. The frequency bands were: (1) 0.5-3.75 Hz; (2) 4-7 Hz; (3) 7.25-12 Hz; (4) 12.25-16 Hz; (5) 16.25-20 Hz; (6) 20.25-40 Hz (from this point on, frequency bands should be called as: 0.5-4; 4-7; 7 12; 12-16; 16-20 and 20-40 Hz). The mean frequency of the spectrum was finally computed (for references see [2,1 t,27,28, 54]). The statistical analysis was performed on single band power using the Student's t-test for paired data.
157
C57BL/6
l 0 ~..
0 (SAL)
0.01
V-lmin0-15 ]
0.025
0.05
0,1
0.025
0.05
0.1
DBA/2
~= 75
~t
0
0 (SAL)
0.01
Physostigminemg/kg Fig. 1. Effect of physostigmine on locomotor activity of C57 and DBA mice. Ordinate represent mean activity crossings in two 15-min blocks, in groups of 8 mice. Vertical bars indicate SEM. Mice received physostigmine sulfate, at the doses of 0 (saline), 0.01, 0.025, 0.05 or 0.1 mg/kg, i.p., 15 min before the activity test. *P<0.05 vs. saline.
C57BL/6
3. Results
3.1. Behavioral investigation 0 (SAL)
Results of preliminary experiments on the effects induced by physostigmine on locomotor activity are shown in Fig. 1. A two-way ANOVA, for C57 mice, showed a significant difference among treatment groups (F(4,35)=6.81, P<0.001) and between time blocks (F(1,35)=66.68, P<0.001) and a significant (F(4,35)= 2.83, P<0.05) treatment xtime interaction. In DBA mice, the difference among groups was very near to the level of significance (F(4,35)=2.57, P=0.054); the difference between time blocks (F(1,35)=144.93, P<0.001) and treatment x time interaction (F(4,35)= 5.18, P < 0.01 ) were statistically significant. Duncan's test showed that physostigmine was able to increase locomotor activity only at the lowest dose (0.01 mg/kg), in the first 15-min block in DBA mice (Fig. 1). The highest dose of the drug (0.1 mg/kg) reduced locomotion both in the DBA strain (first time block) and to a greater extent, in C57 mice (in both 15-min blocks). Fig. 2 reports, for all the experimental groups, the mean percent avoidance responses in each daily session and in the 5 sessions combined. ANOVA showed both in C57 and in DBA mice a significant difference among treatment groups (F(4,35)=2.74 and 3.12 respectively, P<0.05), and sessions (F(4,140)=45.90 and 106.45,
0.01
0.025
0.05
0.1
0.01
0.025
0.05
0.1
DBA/2 75
,~ 50
25-
0
0 (SAL)
Physostigmine mg/kg Fig. 2. Effect of physostigmine on shuttle-box avoidance acquisition in groups of 8 C57 and DBA mice. Mean percent avoidance responses on the whole of the five 50-trial daily sessions (columns) and in each session (circles). Vertical bars indicate SEM. Mice received physostigmine sulfate, i.p., 15 min before each daily session. *P < 0.05 vs. saline, for the 5 sessions combined.
respectively, P
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
158
est doses (0.05 and 0.1 mg/kg). A more detailed statistical analysis showed similar significant improvements in the first and in the second 25-trial blocks of the 5 sessions, thus indicating that avoidance facilitation by physostigmine was not a short-lasting effect.
EFFECT INDUCED BY PHYSOSTIGMINE ON 4 - 7 Hz AND 7 - 12 Hz BAND POWER DBA, 0.05 f
2 0 0 - 2.3-]
DBA, 0.1 ]
f
C57, 0.01 q
F
I
150- 2.2-~
3.2. EEG investigation Basal EEG pattern and EEG effects after physostigmine were quite analogous to those described in previous papers after acetylcholinesterase inhibitors [13,15,27,29,49,53]. Table l summarizes the effects induced by the 4 doses of physostigmine on the 6 frequency bands, plus total power and the mean frequency of the spectrum, in the two strains of mice. Each value is a mean of 4 mice, representing in percent the difference of the 4 postdrug recording periods versus the mean of their own control. Plus or minus indicate increase or decrease of the mean power value versus control (= 100%). Detailed analysis of data was further performed for C57 dose of 0.01 mg/kg, and DBA doses of 0.05 and 0.1 mg/kg, since a significant increase of shuttle-box performance was elicited in these 3 conditions (Fig. 3). A 4 - 7 H z band power ('theta' range) significant increase was the common effect, while a decrease of the 7-12 Hz band power ('alpha' range) was significant in the case of C57 (0.01 mg/kg) and DBA (0.05 mg/kg), and was borderline significant (P <0.1) in the case of DBA (0.1 mg/kg).
4. Discussion
In agreement with previous findings [43], the present study showed that some doses of physostigmine improved shuttle-box avoidance acquisition in mice. Avoidance facilitation did not seem to be related to a general psychomotor activation, because physostigmine
0
i
1 2 5 - 2.1-~
1 0 0 - 2.0
]
0
I
1.9-] 75-
1.8-]
5 0 - 1.7 -] 11513016011201 11513o 16o 112oI 11513016011201 TIME COURSE IN MINUTES AFTER DRUG _o
4-7Hz
c
7 - 1 2 Hz Fig. 3. Effects i n d u c e d b y p h y s o s t i g m i n e o n the p o w e r o f 4 7 a n d 7 - 1 2 H z f r e q u e n c y b a n d s in 3 g r o u p s of m i c e (4 a n i m a l s p e r g r o u p ) : D B A t r e a t e d w i t h 0.05 a n d 0.1 m g / k g , a n d C 5 7 t r e a t e d with 0.01. Abscissa: t i m e c o u r s e in m i n u t e s a f t e r d r u g , in the 3 g r o u p s . O r d i n a t e : p e r c e n t o f the p o w e r f o l l o w i n g d r u g , versus c o n t r o l p o w e r of the s a m e b a n d c o n s i d e r e d as 100%; values are referred to linear a n d log values. N o t e t h a t in D B A m i c e t r e a t e d w i t h 0.1 m g / k g , a n i n c r e a s e o f 7 - 1 2 H z b a n d p o w e r o v e r 100% of c o n t r o l values is r e c o r d e d 120 m i n after injection.
increased avoidance responses at doses that had either no effect or reduced spontaneous locomotor activity. Conversely, it is possible that, in some instances, as in
Table 1 Effects i n d u c e d b y p h y s o s t i g m i n e o n E E G p o w e r a n d t h e m e a n f r e q u e n c y of the s p e c t r u m in m i c e Frequency band (Hz)
0-4 4-7 7-12 12-16 16 20 20-40 TP MF
C57
DBA
Dose (mg/kg)
Dose (mg/kg)
0.01
0.025
0.05
0.1
0.01
0.025
0.05
0.1
+ 54 ~ + 67 a - 30 c + 11 a + 12 + 122 b + 12 - 11 b
+ 1 +10 -24 ¢ - 8 + 7 + 93" --17 c - 3
- 2 + 6 -32 a -20 c -26 b + 85 c -27 a - 5
+ 52 + 15 - 19 - 29" - 15 + 105 ~ + 2 - 12
-
--6.5
-18 b + 23 a - 1 9 ~'
-12 + 34 a -17
-
-
T P , t o t a l p o w e r ; M F , m e a n f r e q u e n c y . N u m b e r s r e p r e s e n t % c h a n g e f r o m p r e d r u g values. a P<0.05; bP<0.02; eP<0.01.
2
+ 14 a +16
+ 23 +32
-
+24
l
-3 + 1 + 5 -2
a
+12 +3 +16 ~ +5
6
-4 -3 - 9 +2
2
+5 -3 + 1 +3
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
the case of the C57 mice receiving the highest doses of physostigrnine, a depressant component in the action of the drug might have overshadowed its potential avoidance improving properties. Previous findings [1] demonstrated that physostigmine reduces exploratory activity both in C57 and DBA mouse strains. In the present study, C57 mice appeared to be more susceptible than DBA mice to the locomotor depressant action of the acetylcholinesterase inhibitor: on the contrary, behavioral depression induced by the muscarinic receptor agonist oxotremorine was stronger in the DBA strain [8,33,36]. Thus, it seems that the two strains exhibit a different behavioral response to cholinergic activation depending on whether such activation is produced by a direct (oxotremorine) or indirect (physostigmine) stimulation of muscarinic receptors. The mild depressant action of the acetylcholinesterase inhibitor, physostigmine, exhibited by DBA mice, might be related to the higher brain levels of acetylcholinesterase activity displayed by this mouse strain [21,32]. Effects induced by cholinergic drugs on the EEG of animals have been extensively described (cf. Introduction). The present data on the quantitative EEG effects induced by physostigmine show that in DBA mice, all doses of the drug induce enhancement of power in 4-7 Hz (theta range) frequency band, while only the two stronger doses (0.05 and 0.1 mg/kg) induce depression of 7-12 Hz (alpha range) band power (Table 1). Previous papers from our and other laboratories showed that higher doses of physostigmine induce further reduction of 7-12 Hz band power [13,49]. EEG effects analogous to the ones induced in DBA by stronger doses (0.05 and 0.1 mg/kg) in present investigation, are evidenced in C57 treated with smaller doses (0.01 and 0.025mg/kg), while the stronger doses (0.05 and 0.1 mg/kg) induce in C57 depression in power of intermediate frequency bands (%12; 12-16 and 16-20 FT) and enhancement in power of very high frequencies (20-40 Hz). In a previous paper, it has been shown that rhythmical slow activity in the frequency range of about 4-12 Hz was associated with fast rhythms of 20-70 Hz [40]. In the present investigation, doses which induced enhancement of shuttle-box performance in C57 (0.01 mg/kg) and DBA mice (0.05 and 0.1 mg/kg) induced consistent enhancement in power of 4-7 Hz EEG band and consistent decrement of 7-12 Hz band (only borderline significant for DBA treated with 0.1). No other effect shared among these 3 groups of mice was observed. It is apparent that the stimulating effect shown by physostigmine on avoidance behavior of our mice has a narrow optimal dosage, i.e., 0.01 mg/kg in C57 and 0.05 or 0.1 mg/kg in DBA. The same doses were able to induce enhancement in power of 4-7 Hz band and decrement of 7-12 Hz band, and both effects are evidently induced if the behavioral effect is desired. The 4-7 Hz band, corresponding approximately to the
159
'type 2 theta' band recorded in the hippocampus of various animal species, is allegedly dependent on muscarinic mechanisms [4,35], and was hypothesized to occur in the unrestrained immobile animal during sensory processing, but only when the animal is in a high state of arousal [39,42]. On the other hand, continuing work is needed to elucidate the role of GABA, serotonin or adrenergic mechanisms for the genesis of the 7-12 Hz band, which corresponds approximately to the 'type 1 theta' (movement-related) band [20,25,51]. Indirect influence induced by cholinomimetic drugs can also not be excluded in this case. In our mice, along with an enhancement of the 4-7 Hz band power, a decrement of the 7-12 Hz band also seems necessary in order to attain an enhancement in the shuttle-box performance. The 7-12 Hz band power in our experiments follows an inverted U-shaped trend in DBA mice: lower doses of physostigmine induce enhancement of the power, while with higher doses a decrement of the power ensues, and the decrement is inversely related to the drug dose [49]. Beyond a certain limit, however a general depression of EEG is attained, i.e., with the dose of 0.2-0.4 mg/kg and over. It is always difficult to compare behavioral data to neurophysiological data. Moreover, information drawn from an analysis of spectral power components related to the 4-7 and 7-12 Hz bands in mice is not exactly correspondent to the 'theta 2' and 'theta 1' frequency components described in the literature for the rat hippocampus. EEG was recorded in mice using epidural electrodes, which are located above the hippocampus and are receptive to the electric signals generated in the limbic circuits and diffused through the mouse thin cortical layers. EEGs recorded in our mice reveal, therefore, not only EEG waves typical of the hippocampal range (theta and alpha ranges), but also spectral components - - particularly in the higher frequencies - - generated in the cortex. On the other hand, in our experience, the EEG recorded in inbred strains of mice usually maintain a good stationarity, and is particularly suitable for the spectral analysis and for statistical elaboration of data. However, some possible sources of misinterpretation must be discussed. One of the points is that in our behavioral studies, physostigmine effects were measured while mice were undergoing shuttle-box performance (drug-environment interaction). On the other hand, our EEG studies were performed while mice were undisturbed in their cages. During the latter experiments, therefore, environmental condition lacked stimulations due to the shuttle-box device, and this might have led to a different EEG effect. A previous paper [30] showed that rat hippocampal theta waves (in this case, in the range of 6-8 Hz) measured by means of a Grey Walter spectral analyzer either after saline injection followed by shuttle-box training, or after pentamethylenetetrazol
160
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
alone (PTZ, 10mg/kg, i.p.) were not different from control periods. However, the interaction of the two conditions (PTZ injection plus shuttle-box) induced strong enhancement of the 6-8 Hz rhythm, presumably through a non-specific mechanism. In recent data obtained in our laboratories (unpublished) the rat hippocampal EEG recorded in quiet conditions after physostigmine, showed distribution of power in EEG bands partly different from EEG recorded in the rat during forced walking performed in a slowly rotating Plexiglas drum, after physostigmine injection. An analogous interaction on biochemical effects induced by physostigmine in the presence and absence of sensorimotor stimulation was described by Someani et al. [46], on rats chronically treated with a small dose of the drug (0.07 mg/kg/day). Further experiments are therefore required to verify whether EEG effects elicited after drug in a quiet condition are comparable to EEG effects obtained in a drug plus psychomotor stimulation condition, as was the case of our mice in the shuttle-box. There are other possible confounding factors. For example, it is known that physostigmine induces hypothermia, probably as a central muscarinic response [-31], and this may, in turn, induce some alterations in energy utilization at the neuronal level, e.g., hypothermia induces diminution of mean frequency and voltage of cortical EEG in cats [48]. Therefore, we should remember that the EEG effect of physostigmine in a quiet condition may be influenced by a number of factors different from those intervening in a psychomotor stimulating condition (such as shuttle-box avoidance). In conclusion, we describe, for the first time, spectral EEG variation induced in two inbred strains of mice which appears to behave similar to shuttle-box changes, within a range of physostigmine doses. This does not yet mean that the two phenomena are strictly related, and further investigations to be performed also with EEG recordings in the animal during shuttle-box training (e.g., with a method analogous to the one previously described in another animal species [7]) may contribute to the further understanding of EEG to behavior relationship.
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Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161 EEG 'theta' in hippocampal formation slices: evidence for a third generator of theta in CA3 area, Brain Res., 451 (1988) 33-42. [26] Leung, L.-W.S., Lopes da Silva, F.H. and Wadman, W.J., Spectral characteristics of the hippocampal EEG in the freely moving rat, Electroenceph. Clin. Neurophysiol., 54 (1982)203-219. 1-27] Loizzo, A., Correlati elettroencefalografici deU'azione di farmaci sul topo libero non anestetizzato, Studi Sassaresi (Suppl. Medicina), 45 (1969) 110-125. [ 28 ] Loizzo, A. and Longo, V., EEG effects of cholinergic and anticholinergic drugs. In: A. Remond (Ed.), Handbook of Electroencephalography and Clinical Neurophysiology, Vol.7C, Elsevier, Amsterdam, 1977, pp. 7-22. [29] Loizzo, A., Zapponi, G.A., Ortolani, E., Longo, V., Palazzesi, S., Electroencephalographic investigation of the effects of organophosphorus pesticides and their metabolites on the central nervous system. In: H. Ott (Ed.), Environment and the Quality of Life. Commission of the European Communities, EUR 5870 DE/EN/ FR, 1978. [30] Longo, V. and Loizzo, A., Effects of drugs on the hippocampal theta-rhythm. Possible relationship to learning and memory processes. In: Pharmacology and Future of Man, Proc. 5th Int. Congr. Pharmacology, San Francisco, Vol. 4, 1973, pp. 46-54. 1,31] Maayani, S., Egozi, Y., Pinchasi, I. and Sokolosky, M., On the interaction of drugs with the cholinergic nervous system, Biochem. Pharmacol., 27 (1978) 203-211. [32] Mandel, P., Ayid, G., Hermetet, J.C. and Ebel, A., Correlation between choline acetyltransferase activity and learning ability in different mice strains and their offspring, Brain Res., 72 (1974) 65-70. [33] Marks, M.J., Patinkin, D.M., Artman, L.D., Burch, J.B. and Collins, A.C., Genetic influences on cholinergic drug response, Pharmacol. Biochem. Behav., 15 (1981) 271-279. 1,34] Morris, R.G.M., Garrud, P., Rawlins, J.N.P. and O'Keefe, J., Place-navigation impaired in rats with hippocampal lesions, Nature, 297 (1982) 681-683. [35] Olpe, H.R., Klebs, K., Kung, E., Campiche, P., Glatt, R., Ortman, R., D'Amato, F., Pozza, M.F. and Mondator, C., Cholinomimetics induce theta rhythm and reduce hyppocampal pyramidal cell excitability, Eur. J. Pharmacol., 142 (1987) 275-283. [36] Pavone, F. and Fagioli, S., Serotoninergic influence on cholinergic-induced analgesia: difference in two inbred strains of mice. Brain Res., 577 (1992) 347 350. [37] Paylor, R., Baskall, L. and Wehner, J.M., Behavioral dissociations between C57/BL6 and DBA/2 mice on learning and memory tasks: a hippocampal-dysfunction hypothesis, Psychobiology, 21 (1993) 11 26. [38] Rech, R.H., Effects of cholinergic drugs on poor performance of rats in a shuttle-box, Psychopharmacologia, 12 (1968) 371-383. [39] Riekkinen, P., Buzsaki, G., Riekkinen, P., Jr., Soininen, M. and
161
Partanen, J., The cholinergic systems and EEG slow waves, Electroenceph. Clin. Neurophysiol., 78 (1991) 89 96. [40] Robinson, T.E., Hippocampal rhythmic slow activity (RSA; theta): a critical analysis of selected studies and discussion of possible species-differences, Brain Res. Rev., 2 (1980) 69 101. 1-41] Rosic, N. and Bigmami, G., Depression of two-way avoidance learning and enhancement of passive avoidance learning by small doses of physostigmine, Neuropharmacology, 9 (1970) 311-316. 1,42] Sainsbury, R., Heynen, A. and Montaya, C.P., Behavioral correlates of hyppocampal type 2 theta in the rat. Physiol. Behav., 39 (1987) 513-519. [43] Sansone, M., Castellano, C., Palazzesi, S., Battaglia, M. and Ammassari-Teule, M., Effects of oxiracetam, physostigrnine and their combination on active and passive avoidance learning in mice, Pharmacol. Biochem. Behav., 44 (1993) 451-455. [44] Sarter, M., Taking stock of cognition enhancers, Trends Pharmacol. Sci., 12 (1991) 456 461. [45] Sarter, M., Hagan, J. and Dudchenko, P., Behavioral screening for cognition enhancers: from indiscriminate to valid testing: Part II, Psychopharmacology, 107 (1992) 461-473. [46] Someani, S.M., Babu, S.R., Arneric, S.P. and Dube, S.N., Effect of cholinesterase inhibitor and exercise on choline acetyltransferase and acetylcholinesterase in rat brain regions, Pharmaeol. Biochem. Behav., 39 (1991) 337-343. [47] Stewart, M. and Fox, S.E., Do septal neurones pace the hippocampal theta rhythm? Trends Neurol. Sci., 13 t1990) 163-168. [48] ten Cate, J., Horsten, G.P.M. and Koopman, L.J., The influence of the body temperature on the EEG of the cat, Electroenceph. Clin. Neurophysiol., 1 (1949) 231-235. [49] Valente, P., Palazzesi, S., Zapponi, G.A. and Loizzo, A., Diphasic EEG effects induced by anticholinesterasic agents in mice, Bull. Environ. Contain. Toxicol., 43 (1989) 670-675. 1,50] Vanderwolf, C.H. and Leung, L.-W. S, Brain electrical activity in relation to behavior. In A.A. Boulton and G.B. Baker (Eds.), Neuromethods, Vol. 1, Humana Press, Clifton, N.J., 1985, pp. 305-341. [51] Vanderwolf, C.H. and Robinson, T.E., Reticulo-cortical activity and behavior: a critique of the arousal theory and a new synthesis, Behav. Brain Sci., 4 (1981) 459 514. [52] Winson, J., Loss of hippocampal theta rhythm results in spatial memory deficit in the rat, Science, 201 (19781 160 163. [ 53 ] Zapponi, G.A., Lindsey, C.J., Loizzo, A., Electroencephalographic analysis of subacute effects of methyl-parathion in the mouse. In: D. Otto (Ed.), Multidisciplinary Perspectives in Event-Related Brain Potential Research. U.S. Environmental Protection Agency, EPA-600/9-77-043, Dec. 1978, pp. 481-484. [54] Zapponi, G.A., Ceccarelli, P. and Ortolani, E., Alcuni metodi di analisi multivariata per la semplificazione dell' interpretazione delI'EEG. Applicazione agli spettri di potenza. Riv. ltal. Elettroencef. Neurofisiol. Clin., 2 (19791 393 400.
BEHAVIOURAL BRAIN RESEARCH Behavioural Brain Research 81 (1996) 155-161
Research report
Effects of low doses of physostigmine on avoidance learning and EEG in two strains of mice Alberto Loizzo a,,, Sergio Palazzesi a, Stefano Loizzo a, Mario Battaglia b, Mario Sansone b a Istituto Superiore di Sanitd, Viale Regina Elena 299, 00161 Roma, Italy b Istituto di Psicobiologia e Psicofarmacologia, CNR, Roma, Italy
Received 10 October 1995; revised 4 March 1996; accepted 6 March 1996
Abstract
The effects of the cholinomimetic drug, physostigmine (0, 0.01, 0.025, 0.05 and 0.1 mg/kg, i.p.), on shuttle-box avoidance learning and electroencephalographic (EEG) activity were investigated, in two separate studies, in mice belonging to the inbred C57BL/6 (C57) and DBA/2 (DBA) strains. The results of the behavioral investigation showed a consistent, significant enhancement of avoidance performance, on the whole of 5 daily training sessions, in C57 mice treated with the lowest dose (0.01 mg/kg) and in DBA mice treated with the highest doses (0.05 and 0.1 mg/kg) of the drug. Doses higher than 0.01 mg/kg, in C57 mice, and lower than 0.05 mg/kg, in DBA mice, had no significant effect. The avoidance improvements induced by physostigmine cannot be ascribed to a general behavioral activation, since the doses that increased avoidance responses did not affect or even depressed spontaneous locomotor activity. The same doses of treatment which increased avoidance responding, also induced, in the same strains, consistent enhancement of 4-7 Hz (theta) EEG band power and decrease of 7-12 Hz (alpha) band power. Results suggest that the effects induced by physostigmine on the EEG and on the shuttle-box performance of mice are related to the same neurochemical systems, and are dependent upon the interaction of the dose with specific strain sensitivity. Keywords: C57BL/6;DBA/2; Electroencephalogram;Hippocampus; Inbred strain; Mouse; Physostigmine;Shuttle-box
1. Introduction
Experimental evidence indicates that the central cholinergic system is involved integrally in cognitive function and drugs affecting this system have been shown either to enhance or to hinder performance in tests of learning and memory [3,18,24]. Some cholinomimetic agents, as the acetylcholinesterase inhibitor, physostigmine, are classified among the putative cholinomimetic cognition enhancers [44]. Physostigmine facilitates learning in laboratory animals [ 19,45], but only in some experimental tasks and in a narrow range of doses [14]. In the shuttle-box avoidance test, physostigmine exerted impairing rather than improving effects on avoidance performance in rats [16,38,41], while mice of the randomly bred CD-1 strain, receiving low doses of the drug during shuttle-box avoidance training, showed a consistent tendency to better performance [43]. * Corresponding author. Tel.: (39) (6) 4990-2882/4990-2873;Fax: (39) (6) 4440-053. 0166-4328/96/$15.00© 1996ElsevierScienceB.V. All rights reserved PII S0166-4328 (96)00057-5
Cholinergic drugs also have a profound influence on brain electrical activity [24,28]. Several studies investigated the biochemical background of cholinergic influences on electrical brain activity and rhythms, specially referring to hippocampal E E G waves, and hippocampal electrical activity has been correlated with spontaneous motor behavior in animals (cf. for example E4, 6,12,17,26,40,47, 50, 51 ]) and with the learning behavior 1-10,22,34,52]. However, information on the possible relationships between those doses of cholinergic drugs which enhance some forms of learning or performance behavior in a specific species (or strain) and the E E G effects induced by the same drug doses in the same species or strain are still scanty. In this paper, we show studies performed on the effects induced by low doses of physostigmine in mice which underwent daily shuttle-box active avoidance trials. In different groups of mice, we also studied EEG spectral power effects induced by the same doses of the drug. In order to minimize species variability, two inbred
156
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
strains of mice were used, i.e., DBA/2 (DBA) and C57BL/6 (C57), which have quite a different learning behavior [5], different hippocampal function [37] and are also different in response to cholinergic drugs [23]. In preliminary experiments, spontaneous locomotor activity was also tested in order to assess the specificity of the drug effects on learning.
saline solution (0.9% NaC1) or physostigmine sulfate (0.01, 0.025, 0.05 or 0.1 mg/kg). Each experimental group consisted of 8 subjects. The results were statistically evaluated by two-factor analyses of variance (ANOVAs), followed by Duncan's multiple range test, where appropriate. The factors were drug (5 levels) and time blocks (2 levels; repeated measures) in the activity test, or drug (5 levels) and sessions (5 levels; repeated measures) in avoidance training.
2. Materials and methods
2.2. EEG investigation The experiments reported here were conducted according to the directives of the Council of the European Communities [9].
2.1. Behavioral investigation Male mice of the inbred C57 and DBA strains, obtained by Charles River (Italy) at the age of 9 weeks, were housed in standard transparent plastic cages (8 per cage) under standard animal room conditions (free access to food and water, 12-h light/dark cycle, ambient temperature of 23°C). In preliminary experiments, mice were tested for locomotor activity 6-7 days after their arrival in the laboratory. Nine to 10 days after the activity test, the animals were reassigned to different treatment groups and subjetted to avoidance training. The experiments were carried out between 09.00 and 16.00 h. The same apparatus was employed to measure locomotor activity and active avoidance [43]. The apparatus consisted of 8 automated shuttle-boxes, each one divided into two 20x10 cm compartments, connected by a 3 x 3 cm opening. To measure spontaneous locomotor activity, the lamps of the shuttle-boxes were switched off and no electric shock was applied to the grid floor. For each mouse, the number of crossings from one compartment to the other was recorded during 30 min and cumulated into two 15-min blocks. In avoidance training, a light (10 W) was switched on alternately in the two compartments and used as a conditioned stimulus (CS). The CS preceded the onset of the unconditioned stimulus (US) by 5 s and overlapped it for 25 s. The US was an electric shock (0.2 mA) applied continuously to the grid floor. The intertrial interval was 30 s. An avoidance response was recorded when the animal avoided the US by running into the dark compartment within 5 s of the onset of the CS. If animals failed to avoid the shock they could escape it by crossing during the US. Spontaneous crossings from the dark to the light compartment were punished and recorded as intertrial responses. Training consisted of one 50-trial (25 min) avoidance session per day, for 5 consecutive days. Both activity test and avoidance sessions were preceded (15 min) by intraperitoneal administration of
Groups of 4 naive DBA and C57 male mice were purchased and maintained under the same conditions as for the behavioral investigation. The animals were chronically implanted under pentobarbital anesthesia with epidural stainless-steel electrodes in the left and right anterior (A -1.3 mm anterior to bregma and lateral to sagittal suture) and posterior (P - 3 m m posterior to bregma and 2.5 mm lateral to sagittal suture) sensorimotor regions. Recording sessions were performed at least 7 days after surgery. EEG taken from the left A to P sensorimotor regions (right A electrode served as ground) of freely moving mice was preamplified on a Grass model 8-10D EEG machine, and recorded on paper and on magnetic tape (HP3964A FM analog recorder, speed 3-3/4 in. per second) in a shielded, sound-proof recording chamber for at least 60 min. The animals were then treated with the drug, and EEG was again recorded for at least 5 min at fixed intervals: 15, 30, 60 and 120 min after drug injection. Conditions and treatments were identical as for behavioral studies. Each mouse was recorded only once. During the EEG recording, an operator followed the animals behavior on video camera, and continuously reported on paper any behavioral changes using the following code: W, walking or rearing; G, grooming; I, immobile, alert; +, all other behaviors. EEG recorded from the left A to P cortex was bandpass filtered off-line (0.5-42 Hz, nominal attenuation slope 24 dB/octave), then converted to digital signal, using a sampling rate of 256 Hz and an A/D conversion precision of 13 bits (by HP-3561A signal analyzer, controlled by a HP-9816S personal computer) and sequentially stored on a disk. Once digitized, artifactfree 4-s segments corresponding to desynchronized (wakefulness) periods were selected: EEG segments recorded during various periods were recalled from disk on display and carefully compared to the EEG and behavior recorded on paper. Both the control EEG and the EEG following drug administration showed a prevalence of desynchronized pattern. Only segments which showed low-voltage fast activity or the characteristic saw-tooth shaped theta activity [26,51 ] and at the same time corresponded to W-, G- or I-related behaviors, were considered for the analysis. On the other hand,
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
those segments showing random high-voltage waves (i.e., waves with amplitude above 150% of the background theta activity), drowsiness periods and any segments related to + behavior were discarded by an operator blind to the strain and treatment [49]. Spectra were constructed from 0 to 100 Hz with 0.25-Hz steps, using standard fast Fourier transform (FFT) algorithm on Hamming-windowed segments, and stored on a disk. For each animal, 30-60 power spectral plots were thus selected in each 5 min duration time record, corresponding to the mean power spectra of about 120-240 s. Spectral data underwent further elaboration: frequency steps showing high inter-correlation were cumulated in wider frequency bands. Finally, 8 parameters were chosen, i.e., the total power of the spectrum and its standard deviation, and the power of 6 frequency bands, expressed as percentage of the same band power of predrug period. The frequency bands were: (1) 0.5-3.75 Hz; (2) 4-7 Hz; (3) 7.25-12 Hz; (4) 12.25-16 Hz; (5) 16.25-20 Hz; (6) 20.25-40 Hz (from this point on, frequency bands should be called as: 0.5-4; 4-7; 7 12; 12-16; 16-20 and 20-40 Hz). The mean frequency of the spectrum was finally computed (for references see [2,1 t,27,28, 54]). The statistical analysis was performed on single band power using the Student's t-test for paired data.
157
C57BL/6
l 0 ~..
0 (SAL)
0.01
V-lmin0-15 ]
0.025
0.05
0,1
0.025
0.05
0.1
DBA/2
~= 75
~t
0
0 (SAL)
0.01
Physostigminemg/kg Fig. 1. Effect of physostigmine on locomotor activity of C57 and DBA mice. Ordinate represent mean activity crossings in two 15-min blocks, in groups of 8 mice. Vertical bars indicate SEM. Mice received physostigmine sulfate, at the doses of 0 (saline), 0.01, 0.025, 0.05 or 0.1 mg/kg, i.p., 15 min before the activity test. *P<0.05 vs. saline.
C57BL/6
3. Results
3.1. Behavioral investigation 0 (SAL)
Results of preliminary experiments on the effects induced by physostigmine on locomotor activity are shown in Fig. 1. A two-way ANOVA, for C57 mice, showed a significant difference among treatment groups (F(4,35)=6.81, P<0.001) and between time blocks (F(1,35)=66.68, P<0.001) and a significant (F(4,35)= 2.83, P<0.05) treatment xtime interaction. In DBA mice, the difference among groups was very near to the level of significance (F(4,35)=2.57, P=0.054); the difference between time blocks (F(1,35)=144.93, P<0.001) and treatment x time interaction (F(4,35)= 5.18, P < 0.01 ) were statistically significant. Duncan's test showed that physostigmine was able to increase locomotor activity only at the lowest dose (0.01 mg/kg), in the first 15-min block in DBA mice (Fig. 1). The highest dose of the drug (0.1 mg/kg) reduced locomotion both in the DBA strain (first time block) and to a greater extent, in C57 mice (in both 15-min blocks). Fig. 2 reports, for all the experimental groups, the mean percent avoidance responses in each daily session and in the 5 sessions combined. ANOVA showed both in C57 and in DBA mice a significant difference among treatment groups (F(4,35)=2.74 and 3.12 respectively, P<0.05), and sessions (F(4,140)=45.90 and 106.45,
0.01
0.025
0.05
0.1
0.01
0.025
0.05
0.1
DBA/2 75
,~ 50
25-
0
0 (SAL)
Physostigmine mg/kg Fig. 2. Effect of physostigmine on shuttle-box avoidance acquisition in groups of 8 C57 and DBA mice. Mean percent avoidance responses on the whole of the five 50-trial daily sessions (columns) and in each session (circles). Vertical bars indicate SEM. Mice received physostigmine sulfate, i.p., 15 min before each daily session. *P < 0.05 vs. saline, for the 5 sessions combined.
respectively, P
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
158
est doses (0.05 and 0.1 mg/kg). A more detailed statistical analysis showed similar significant improvements in the first and in the second 25-trial blocks of the 5 sessions, thus indicating that avoidance facilitation by physostigmine was not a short-lasting effect.
EFFECT INDUCED BY PHYSOSTIGMINE ON 4 - 7 Hz AND 7 - 12 Hz BAND POWER DBA, 0.05 f
2 0 0 - 2.3-]
DBA, 0.1 ]
f
C57, 0.01 q
F
I
150- 2.2-~
3.2. EEG investigation Basal EEG pattern and EEG effects after physostigmine were quite analogous to those described in previous papers after acetylcholinesterase inhibitors [13,15,27,29,49,53]. Table l summarizes the effects induced by the 4 doses of physostigmine on the 6 frequency bands, plus total power and the mean frequency of the spectrum, in the two strains of mice. Each value is a mean of 4 mice, representing in percent the difference of the 4 postdrug recording periods versus the mean of their own control. Plus or minus indicate increase or decrease of the mean power value versus control (= 100%). Detailed analysis of data was further performed for C57 dose of 0.01 mg/kg, and DBA doses of 0.05 and 0.1 mg/kg, since a significant increase of shuttle-box performance was elicited in these 3 conditions (Fig. 3). A 4 - 7 H z band power ('theta' range) significant increase was the common effect, while a decrease of the 7-12 Hz band power ('alpha' range) was significant in the case of C57 (0.01 mg/kg) and DBA (0.05 mg/kg), and was borderline significant (P <0.1) in the case of DBA (0.1 mg/kg).
4. Discussion
In agreement with previous findings [43], the present study showed that some doses of physostigmine improved shuttle-box avoidance acquisition in mice. Avoidance facilitation did not seem to be related to a general psychomotor activation, because physostigmine
0
i
1 2 5 - 2.1-~
1 0 0 - 2.0
]
0
I
1.9-] 75-
1.8-]
5 0 - 1.7 -] 11513016011201 11513o 16o 112oI 11513016011201 TIME COURSE IN MINUTES AFTER DRUG _o
4-7Hz
c
7 - 1 2 Hz Fig. 3. Effects i n d u c e d b y p h y s o s t i g m i n e o n the p o w e r o f 4 7 a n d 7 - 1 2 H z f r e q u e n c y b a n d s in 3 g r o u p s of m i c e (4 a n i m a l s p e r g r o u p ) : D B A t r e a t e d w i t h 0.05 a n d 0.1 m g / k g , a n d C 5 7 t r e a t e d with 0.01. Abscissa: t i m e c o u r s e in m i n u t e s a f t e r d r u g , in the 3 g r o u p s . O r d i n a t e : p e r c e n t o f the p o w e r f o l l o w i n g d r u g , versus c o n t r o l p o w e r of the s a m e b a n d c o n s i d e r e d as 100%; values are referred to linear a n d log values. N o t e t h a t in D B A m i c e t r e a t e d w i t h 0.1 m g / k g , a n i n c r e a s e o f 7 - 1 2 H z b a n d p o w e r o v e r 100% of c o n t r o l values is r e c o r d e d 120 m i n after injection.
increased avoidance responses at doses that had either no effect or reduced spontaneous locomotor activity. Conversely, it is possible that, in some instances, as in
Table 1 Effects i n d u c e d b y p h y s o s t i g m i n e o n E E G p o w e r a n d t h e m e a n f r e q u e n c y of the s p e c t r u m in m i c e Frequency band (Hz)
0-4 4-7 7-12 12-16 16 20 20-40 TP MF
C57
DBA
Dose (mg/kg)
Dose (mg/kg)
0.01
0.025
0.05
0.1
0.01
0.025
0.05
0.1
+ 54 ~ + 67 a - 30 c + 11 a + 12 + 122 b + 12 - 11 b
+ 1 +10 -24 ¢ - 8 + 7 + 93" --17 c - 3
- 2 + 6 -32 a -20 c -26 b + 85 c -27 a - 5
+ 52 + 15 - 19 - 29" - 15 + 105 ~ + 2 - 12
-
--6.5
-18 b + 23 a - 1 9 ~'
-12 + 34 a -17
-
-
T P , t o t a l p o w e r ; M F , m e a n f r e q u e n c y . N u m b e r s r e p r e s e n t % c h a n g e f r o m p r e d r u g values. a P<0.05; bP<0.02; eP<0.01.
2
+ 14 a +16
+ 23 +32
-
+24
l
-3 + 1 + 5 -2
a
+12 +3 +16 ~ +5
6
-4 -3 - 9 +2
2
+5 -3 + 1 +3
Alberto Loizzo et al./Behavioural Brain Research 81 (1996) 155-161
the case of the C57 mice receiving the highest doses of physostigrnine, a depressant component in the action of the drug might have overshadowed its potential avoidance improving properties. Previous findings [1] demonstrated that physostigmine reduces exploratory activity both in C57 and DBA mouse strains. In the present study, C57 mice appeared to be more susceptible than DBA mice to the locomotor depressant action of the acetylcholinesterase inhibitor: on the contrary, behavioral depression induced by the muscarinic receptor agonist oxotremorine was stronger in the DBA strain [8,33,36]. Thus, it seems that the two strains exhibit a different behavioral response to cholinergic activation depending on whether such activation is produced by a direct (oxotremorine) or indirect (physostigmine) stimulation of muscarinic receptors. The mild depressant action of the acetylcholinesterase inhibitor, physostigmine, exhibited by DBA mice, might be related to the higher brain levels of acetylcholinesterase activity displayed by this mouse strain [21,32]. Effects induced by cholinergic drugs on the EEG of animals have been extensively described (cf. Introduction). The present data on the quantitative EEG effects induced by physostigmine show that in DBA mice, all doses of the drug induce enhancement of power in 4-7 Hz (theta range) frequency band, while only the two stronger doses (0.05 and 0.1 mg/kg) induce depression of 7-12 Hz (alpha range) band power (Table 1). Previous papers from our and other laboratories showed that higher doses of physostigmine induce further reduction of 7-12 Hz band power [13,49]. EEG effects analogous to the ones induced in DBA by stronger doses (0.05 and 0.1 mg/kg) in present investigation, are evidenced in C57 treated with smaller doses (0.01 and 0.025mg/kg), while the stronger doses (0.05 and 0.1 mg/kg) induce in C57 depression in power of intermediate frequency bands (%12; 12-16 and 16-20 FT) and enhancement in power of very high frequencies (20-40 Hz). In a previous paper, it has been shown that rhythmical slow activity in the frequency range of about 4-12 Hz was associated with fast rhythms of 20-70 Hz [40]. In the present investigation, doses which induced enhancement of shuttle-box performance in C57 (0.01 mg/kg) and DBA mice (0.05 and 0.1 mg/kg) induced consistent enhancement in power of 4-7 Hz EEG band and consistent decrement of 7-12 Hz band (only borderline significant for DBA treated with 0.1). No other effect shared among these 3 groups of mice was observed. It is apparent that the stimulating effect shown by physostigmine on avoidance behavior of our mice has a narrow optimal dosage, i.e., 0.01 mg/kg in C57 and 0.05 or 0.1 mg/kg in DBA. The same doses were able to induce enhancement in power of 4-7 Hz band and decrement of 7-12 Hz band, and both effects are evidently induced if the behavioral effect is desired. The 4-7 Hz band, corresponding approximately to the
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'type 2 theta' band recorded in the hippocampus of various animal species, is allegedly dependent on muscarinic mechanisms [4,35], and was hypothesized to occur in the unrestrained immobile animal during sensory processing, but only when the animal is in a high state of arousal [39,42]. On the other hand, continuing work is needed to elucidate the role of GABA, serotonin or adrenergic mechanisms for the genesis of the 7-12 Hz band, which corresponds approximately to the 'type 1 theta' (movement-related) band [20,25,51]. Indirect influence induced by cholinomimetic drugs can also not be excluded in this case. In our mice, along with an enhancement of the 4-7 Hz band power, a decrement of the 7-12 Hz band also seems necessary in order to attain an enhancement in the shuttle-box performance. The 7-12 Hz band power in our experiments follows an inverted U-shaped trend in DBA mice: lower doses of physostigmine induce enhancement of the power, while with higher doses a decrement of the power ensues, and the decrement is inversely related to the drug dose [49]. Beyond a certain limit, however a general depression of EEG is attained, i.e., with the dose of 0.2-0.4 mg/kg and over. It is always difficult to compare behavioral data to neurophysiological data. Moreover, information drawn from an analysis of spectral power components related to the 4-7 and 7-12 Hz bands in mice is not exactly correspondent to the 'theta 2' and 'theta 1' frequency components described in the literature for the rat hippocampus. EEG was recorded in mice using epidural electrodes, which are located above the hippocampus and are receptive to the electric signals generated in the limbic circuits and diffused through the mouse thin cortical layers. EEGs recorded in our mice reveal, therefore, not only EEG waves typical of the hippocampal range (theta and alpha ranges), but also spectral components - - particularly in the higher frequencies - - generated in the cortex. On the other hand, in our experience, the EEG recorded in inbred strains of mice usually maintain a good stationarity, and is particularly suitable for the spectral analysis and for statistical elaboration of data. However, some possible sources of misinterpretation must be discussed. One of the points is that in our behavioral studies, physostigmine effects were measured while mice were undergoing shuttle-box performance (drug-environment interaction). On the other hand, our EEG studies were performed while mice were undisturbed in their cages. During the latter experiments, therefore, environmental condition lacked stimulations due to the shuttle-box device, and this might have led to a different EEG effect. A previous paper [30] showed that rat hippocampal theta waves (in this case, in the range of 6-8 Hz) measured by means of a Grey Walter spectral analyzer either after saline injection followed by shuttle-box training, or after pentamethylenetetrazol
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alone (PTZ, 10mg/kg, i.p.) were not different from control periods. However, the interaction of the two conditions (PTZ injection plus shuttle-box) induced strong enhancement of the 6-8 Hz rhythm, presumably through a non-specific mechanism. In recent data obtained in our laboratories (unpublished) the rat hippocampal EEG recorded in quiet conditions after physostigmine, showed distribution of power in EEG bands partly different from EEG recorded in the rat during forced walking performed in a slowly rotating Plexiglas drum, after physostigmine injection. An analogous interaction on biochemical effects induced by physostigmine in the presence and absence of sensorimotor stimulation was described by Someani et al. [46], on rats chronically treated with a small dose of the drug (0.07 mg/kg/day). Further experiments are therefore required to verify whether EEG effects elicited after drug in a quiet condition are comparable to EEG effects obtained in a drug plus psychomotor stimulation condition, as was the case of our mice in the shuttle-box. There are other possible confounding factors. For example, it is known that physostigmine induces hypothermia, probably as a central muscarinic response [-31], and this may, in turn, induce some alterations in energy utilization at the neuronal level, e.g., hypothermia induces diminution of mean frequency and voltage of cortical EEG in cats [48]. Therefore, we should remember that the EEG effect of physostigmine in a quiet condition may be influenced by a number of factors different from those intervening in a psychomotor stimulating condition (such as shuttle-box avoidance). In conclusion, we describe, for the first time, spectral EEG variation induced in two inbred strains of mice which appears to behave similar to shuttle-box changes, within a range of physostigmine doses. This does not yet mean that the two phenomena are strictly related, and further investigations to be performed also with EEG recordings in the animal during shuttle-box training (e.g., with a method analogous to the one previously described in another animal species [7]) may contribute to the further understanding of EEG to behavior relationship.
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