- Email: [email protected]
Reticular stimulation and hippocampal theta rhythm in rats: Effects of drugs
N~uroscww. 0
Vol. 3. pp. 629-632
Pergamon Press Ltd. 1978. Printed m Great Britain.
030MS22/78/0701-0629802.0010
IBRO
RETICULAR STIMULATION AND HIPPOCAMPAL RHYTHM IN RATS: EFFECTS OF DRUGS
THETA
N. MCNAUGHTON Department of Experimental Psychology, University of Oxford, Oxford, OX1 3UD, U.K.
and E. M.
SEDGWICK
Wessex Neurological Centre, Southampton General Hospital, Southampton, SO9 4XY, U.K. Abstract-The effect of drugs on hippocampal theta rhythm induced by high frequency stimulation of the midbrain reticular formation was investigated in free-moving rats. The linearity of the relationship between the frequency of theta produced and the intensity of the stimulating current was unchanged by injections of sodium amylobarbitone; however, the frequency itself was reduced. Choline@ blockade or depletion of noradrenaline, dopamine or serotonin levels in brain did not produce such a reduction in frequency, nor did they change the linearity of the function. These results contrast with the nonlinear effects which have been found with sodium amylobarbitone when septal stimulation is used to evoke theta rhythm; and with the fact that such nonlinear effects can be reproduced by depletion of forebrain noradrenaline levels. Sodium amylobarbitone appears, therefore, to affect control of hippocampal theta rhythm by actions on two systems, only one of which is dependent on noradrenaline. The duplication of behavioural effects of the drug by lesions of the dorsal ascending noradrenergic bundle may imply that the frequency at which theta occurs is less important for the control of such behaviours than other aspects of this electrical activity.
GRAY (1970; 1972) has proposed that sodium amylobarbitone influences behaviour by altering the septal control (STUMPF,1965) of hippocampal theta rhythm in a middle frequency band (7.7 Hz in the rat). Confirmation of the physiological part of this hypothesis comes from work with septal stimulation and hippocampal recording. Low frequency stimulation of the medial septum will drive hippocampal theta rhythm (BROCKE,PEDXHE, PILLAT & DEISENHAMMER, 1959), and in the free-moving rat the threshold current required to elicit such driving is minimal at 7.7 Hz (GRAY & BALL, 1970;JAMES,MCNAUGHTON,RAWLINS, FELDON& GRAY, 1977). GRAY & BALL (1970) found that sodium amylobarbitone eliminated this minimum by raising thresholds selectively in the region of 7.7 Hz. As might be predicted from the similarities between the behavioural effects of the barbiturates, benzodiazepines and ethanol (GRAY, 1977), this effect is also produced by injections of chlordiazepoxide and ethanol (MCNAUGHTON,JAMES,STEWART,GRAY, VALERO & DREWNOWSKI,1977). This specific effect in the region of 7.7 Hz is not reproduced by manipulation of the cholinergic, serotonergic or dopaminergic systems (MCNAUGHTONet al., 1977), while it is produced by both systemic injections which deplete noradrenergic systems and neurotoxic lesions of the dorsal ascending noradrenergic bundle (GRAY, MCNAUGHTON, JAMES &
1977).
KELLY, 1975; MCNAUGHTON et al.,
The full range of hippocampal theta frequencies can also be produced by high frequency stimulation of the midbrain reticular formation (GREENdt ARDUINI, 1954), but the frequency of theta produced is monotonically related to the stimulation intensity (SAILER & STUMPF,1957). STUMPF(1965) reported that hexobarbitone reduced the frequency of such theta rhythm, while scopolamine, although it could produce total blockade of theta, did not affect frequency. He did not report any frequency-specific effects of the drugs. Septal and reticular elicitation differ, then, as to whether frequency specific effects can be observed, both in control curves and in effects produced by injections of barbiturates. One possible source of discrepancy lies in the use of free-moving rats for the septal experiments and urethane-anaesthetized rabbits for the reticular ones. Other evidence makes this less likely. JAMESet al. (1977) reported linear reticular control curves and KRAMIS, VANDERWOW & BLAND (1975) reported a reduction in frequency with pentobarbital in the free-moving rat. However, KRAMISet al. (1975) did not report the drug effect systematically with respect to frequency, and it is possible, therefore, that the barbiturates produce a change which is not linearly related to frequency. This paper reports a more detailed analysis of the linearity of the stimulation intensity-theta frequency data previously reported by JAMESet al. (1977) and investigates the effects on this function of injections
629
630
N.
M~+.!AI~GHTON
of sodium amylobarbitone and the effects of manipulation of cholinergic, scrotonergic, dopatninergic and noradrenergic systems.
EXPERIMENTAL
PROCEDURES
The subjects were five male Wistar rats, chronically implanted with bipolar reticular stimulating electrodes and contralaterat bipolar recording electrodes in the dorsomedial subiculum. The surgical, stimulating and recording techniques and the histological location of the electrodes have been described previously (JAMESt~l ~1.. 1977). The reticular formation was stimulated at 100 Hz with 0.5 ms pulse width and 2 s train duration with a constant current stimulator. All five animals showed induction of theta rhythm at stimulating currents between IO and 40 @. There was no obvious relation between threshold current, initial frequency of theta produced, or rate of change of theta frequency (JAMESet ai., 1977), and so the same stimulating current intensities could not be used with all animals. The procedure used was to vary the intensity of stimulation by a constant interval (10, 20 or 40bA depending on the animal), so as to provide readings at a minimum offourintervals over the range tested. This range was normally between the lowest current which showed induced theta rhythm, usually at about 5 Hz, and the highest which did not produce either vigorous body turning or an escape attempt. In the case of animals tested under sodium amylobarbitone, the highest current intensity used was one considerably higher than that used in the predrug test, but this did not usually produce any motor activity. Drug tests employed the same intensity interval as that used in predrug tests. Several readings were taken at each value of the current intensity by systematically increasing and decreasing the current until at least ten readings had been taken. Analysis was performed on paper records taken at a speed of 15mm/s (see Fig. 1). The frequency of theta rhythm was estimated (k 0.5 Hz) by wave counting and the mean amplitude over the stimulating period was measured with a calibrated gauge ( k 3”,,).
I
and E. M.
SEDGWICK
All drug tests were preceded and followed by undrugged tests. but not always on the same day. Animals were used for more than one drug treatment, but these were given to different rats in different orders and control values were tested as normal on several days before administration of a drug. Tests with cc-methyl-p-;yrosine and p-chlorophenylalanine were performed after tests with other drugs had been completed. The drugs used were: sodium amylobarbitone (Lilly) injected in saline (15 or 20 mg,/kg. I ml/kg) 10 min before recording started; ~-methyl-p-tyrosine methyl ester HCi (Sigma) injected in saline (100 mgjml) 6.5 h before recording; scopolamine HBr (BDH) injected in saline (0.9 mgiml) 10 min before recording; and p-chloro-phenylalanine methyl ester HCl (Sigma) injected in saline (SOmg/ml) at a dose of iOOmg/kg/day for 3 days before recording. All drugs were administered intraperitoneaily. RESULTS Some especially clear raw data obtained from one rat are shown in Fig. 1. On the left are shown re-
sponses observed at increasing current intensities. On the right are shown the individual values obtained in one undrugged session. As can be seen here, and in the individual curves previously reported (JAMES et al., 1977), the frequency of theta is linearly related to stimulation intensity in undrugged animals. To quantify this relationship and to test its validity after drug treatment, regression lines were fitted to all of the individual drug and nondrug data for both frequency and amplitude of theta. If a function other than a straight line describes such data better, then the values obtained at any one current intensity will vary less from their own mean than from the value predicted by the regression line. The relative size of these two sources of v~iation {within intensity mean square, deviations from linearity mean square) may he tested with an F-ratio (&Ix, 1974, p. 218) This
120 PA
10
_-__-__--.-
~T~~“LAT,~~
80
CURRENT
120
-
VA
STIMULUS 100 HZ
OSM!XC
FIG. 1. Control data from an individual rat receiving different intensities (100 Hz, 0.5 ms, 2 s train). Left: Raw EEG records. The marker indicates the lation. Right: Graph of theta frequencies elicited during a single session: single with the same value *, The line drawn is the regression line fitted
of reticular stimulation entire duration of stimureadings i, two readings to these data.
160
631
Drug effects on hippocampal theta rhythm
amyloborbltone
AMPT
scopolamine
> = 5001
1
1
f;j/
kx” b--_
p 0
PC PA
50
100
0
50
100
STIMULATING
0 CURRENT
50
-
100
0
50
100
CIA
FIG. 2. Frequency and amplitude of theta rhythm elicited by increasing intensities of reticular stimulation (100 Hz, 0.5 ms) before (0) and after (0) treatment with sodium amylobarbitone (15 mg/kg and
20 mg/kg), a-methyl-p-tyrosine (AMPT: 100 mg/kg), scopolamine (0.9 mg/kg), and p-chloro-phenylalanine (PCPA: 3 x lOOmg/kg). The lines represent the regression lines obtained under the different conditions and are drawn with mean slope (across subjects) through the mean intercept (across subjects). The points plotted are arbitrary.
test performed on over 100 sets of data yielded only 6 F-ratios which on individual tests proved significant at the 5% level or better. Since 1 of these was at the 1% level (1 expected from random variation in a population this size), and a further 1 at the 2.5% level and 4 at the 5% level, there is no reason to reject the hypothesis of linearity in control or drugged data. The slope (B) and intercept (A) coefficients for such regression lines [Frequency = A + B (Stimulating Current)] are normally distributed (ZAR, 1974) and may thus be submitted to parametric statistics. Since it was impossible to match the stimulating intervals across animals and thereby derive mean frequencies of theta produced, the individual regression coefficients were used as raw data for subsequent statistical comparisons between groups. Figure 2 shows the effects of the drugs used as lines of mean slope (across subjects) drawn through the mean intercept. These are representative of the individual regression lines (cf. Fig. 1). Sodium amylobarbitone (15 or 20 mg/kg) produced a reduction in the mean slope of the current intensitytheta frequency function which analysis of variance showed to be significant (F = 63.0, d.jI = 1,4, P < O.OOS),while there was no change in the mean intercept (F = 3.8, d.J = 1,4). There was no difference in the size of the effect produced by the two different doses (F = 3.6, d$ = 1,4). A similar change appeared to be produced on the amplitude of theta, but this was less pronounced and did not achieve significance (Slope.: F = 6.8, d$ = 1,4; intercept: F = 0.5, d.J = 174). a-Methyl-p-tyrosine at the dose (lOOmg/kg) used by MCNAIJGHTONet al. (1977). oroduced a slight
reduction in the intercept of the frequency-intensity function which proved significant with Student’s t-test for paired comparisons (t = 5.516, d$ = 3, P < 0.02) but it did not affect the slope significantly (t = 2.041, d$ = 3) nor did it have effects on amplitude. Scopolamine did not produce changes in theta frequency in doses up to 0.9 mg/kg. At 0.9 mg/kg the apparent changes in slope (t = 1.317, d.$ = 2) and intercept (t = 0.956, d$ = 2) are not reliable. However, at and above this dose production of theta was intermittent. The same is true of the amplitude data (slope: t = 3.891, d.J = 2, P < 0.1; intercept: t = 1.126, d$ = 2). p-Chloro-phenylalanine (3 x 100 mg/kg/day) produced no effect on frequency (slope: t = 0.121, d$ = 2; intercept: t = 0.248, d$ = 2) or amplitude (slope: t = 0.707, d$ = 2; intercept: t = 2.226, d$ = 2). DISCUSSION The linearity of the relationship between intensity of stimulation in the reticular formation and the frequency of theta rhythm elicited in the hippocampus of the free moving rat (Jm et al., 1977) was unchanged by injections of sodium amylobarbitone, scopolamine, a-methyl-ptyrosine or p-chloro-phenylalanine. As has been previously reported for the acute rabbit (STUMPF,1965) and free-moving rat (KRAMIS et al., 1975) barbiturate, in this case sodium amylobarbitone, reduced the frequency of theta observed. This frequency reduction was not produced by choline& blockade or by depletion of noradrenaline, dopamine or serotonin levels. As reported by STUMPF(1965) for the acute rabbit, injections of scopolamine produced no change in the
632
N. MCNAIXHTOI\:and F. M. SEDGWKK
frequency of theta rhythm up to a dose which produced blockade. The intermittent nature of the blockade observed is consistent with the data of Vanderwolf (VANDERWOLF, 1975; VAKIXRWOL.F. KRAMIS, GILLESPIE & BLANI), 1975; KRAMIS ct ul., 1975) on atropine resistant and nonresistant theta rhythm. However, neither with scopolamine, nor with a small number of experiments performed with atropine. did this blockade occur selectively with low frequencies of theta, as might be expected from Vanderwolfs conelusions. [Blockade with atropine appeared at approximately 20 mg,kg as opposed to 0.9 mgjkg for scopolamine. STUMPF (1965) has reported a similar potency ratio.] The critical findings of this work are: the linearity of the stimulation intensity-theta frequency function is unchanged by sodium amylobarbitone; and interference with noradrenaline synthesis does not repro-
duce the effect of the barbiturate on theta frequency. The results thus confirm the suggestion that the reticular and septal stimulation methods disclose effects of barbiturates on two neurochemically dissimilar systerns involved in the control of different aspects of theta rhythm. Given the duplication of the effects of sodium amylobarbitone on behaviour by lesions of the dorsal ascending noradrenergic bundle, at least in tasks involving nonreward (MASON & IVERSEN. 1975; OWN. BOARI)ER& GRAY, 1977) it seems possible that the frequency at which theta rhythm occurs may be unimportant for these tasks and some other feature of the control may be critical. .l&owled~rmmrs N. MTNAI.GH.~ONwas supported by a Grant to Dr J. A. Gray from the British Medical Research Council. We thank Eli Lilly & Co. Ltd. for supplies of sodium amylobarbitone.
REFERENCES BRDCKEF., PETSCHEH., PILLATB. & DEISENHAMMER E. (1959) Die Beeinflussung der ‘Hippocampus-arousal-Reaktion’ beim Kaninchen durch elektrische Reizung im Septum. @grrs Arch. yes. Physiol. 269, 319.-338. GRAY J. A. (1970) Sodium amobarbital, the hippocampal theta rhythm and the partial reinforcement extinction et&t. Psychol. Rev. 11, 465480. GRAY J. A. (1972) The structure of the emotions and the limbic system. In Physiology, Emotion and Psychosomatic Zllness (eds PORTERR. & KNIGHTJ.), pp. 87-130. Associated Scientific Publishers, Amsterdam. GRAY J. A. (1977) Drug effects on fear and frustration: possible sites of action of minor tranquillisers. In Handbook of Psychopharmacology (eds IVERSENL., IVERSENS. D. & SNYDERS.), Vol. 8, pp. 433- 529. Plenum Press, New York. GRAY J. A. & BALLG. G. (1970) Frequency specific relation between hippocampal theta rhythm. behaviour and amobarbital action. Science, N.Y. 168, 1246-l 248. GRAY J. A., MCNAUGHTONN., JAMESD. T. D. & KELLY P. H. (1975) ElTect of minor tranquillisers on hippocampal theta rhythm mimicked by depletion of forebrain noredrenaline. Nature, Land. 258, 424-425. GREEN J. S. & ARDUINIA. (1954) Hippocampal electrical activity in arousal. J. Neurophysiol. 17, 533-557. JAMESD. T. D., MCNAUGHTONN., RAWLINSJ. N. P., FELDONJ. & GRAY J. A. (1977) Septal driving of hippocampal theta rhythm as a function of frequency in the free-moving rat. Neuroscience 2, 1007-1017. KRAMISR., VANDERWOLF C. H. & BLANDB. H. (1975) Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behaviour and effects of atropine, diethyl ether. urethane and pentobarbital. Expl Neural. 49, 58-85. MCNAUGHTONN., JAMESD. T. D., STEWARTJ., GRAY J. A.. VALEROI. & DREWN~WSKIA. (1977) Septal driving of hippocampal theta rhythm as a function of frequency in the male rat: effects of drugs. Neuroscience 2, 1019-1027. MASON S. T. & IVERSENS. D. (1975) Learning in the absence of forebrain noradrenaline. Nature, Land. 258, 422424. OWEN S., BOARDERM. R. & GRAY J. A. (1977) The effect of depletion of forebrain noradrenaline on the runway behaviour of rats. Expl Bruin Res. 28, R 32-R 33. SAILERS. & STUMPFC. (1957) Beeinflussbarkeit der rhinencephalen TLtigkeit des Kaninchens. Arch. exp. Path. Pharmak. 231, 63.-77. STUMPFC. (196.5) Drug action on the electrical activity of the hippocampus. Int. Rev. Neurobiol. 8, 77-137. VANDERWOLF C. H. (1975) Neocortical and hippocampal activation in relation to behaviour: Effects of atropine, eserine, phenothiazines and amphetamine. J. camp. physiol. Psychol. 88, 3W323. VANDERWOLF C. H., KRAMISR., GILLESPIEL. A. & BLAND B. H. (1975) Hippocampal rhythmic slow activity and neocortical low-voltage fast activity: relations to behaviour. In The Hippocampus (eds ISAACSONR. L. & PRIBRAM K. H.), Vol. 2, pp. 101-128. Plenum Press, New York. ZAR J. H. (1974) Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, N.J. (Accepted 27 February
1978)
Vol. 3. pp. 629-632
Pergamon Press Ltd. 1978. Printed m Great Britain.
030MS22/78/0701-0629802.0010
IBRO
RETICULAR STIMULATION AND HIPPOCAMPAL RHYTHM IN RATS: EFFECTS OF DRUGS
THETA
N. MCNAUGHTON Department of Experimental Psychology, University of Oxford, Oxford, OX1 3UD, U.K.
and E. M.
SEDGWICK
Wessex Neurological Centre, Southampton General Hospital, Southampton, SO9 4XY, U.K. Abstract-The effect of drugs on hippocampal theta rhythm induced by high frequency stimulation of the midbrain reticular formation was investigated in free-moving rats. The linearity of the relationship between the frequency of theta produced and the intensity of the stimulating current was unchanged by injections of sodium amylobarbitone; however, the frequency itself was reduced. Choline@ blockade or depletion of noradrenaline, dopamine or serotonin levels in brain did not produce such a reduction in frequency, nor did they change the linearity of the function. These results contrast with the nonlinear effects which have been found with sodium amylobarbitone when septal stimulation is used to evoke theta rhythm; and with the fact that such nonlinear effects can be reproduced by depletion of forebrain noradrenaline levels. Sodium amylobarbitone appears, therefore, to affect control of hippocampal theta rhythm by actions on two systems, only one of which is dependent on noradrenaline. The duplication of behavioural effects of the drug by lesions of the dorsal ascending noradrenergic bundle may imply that the frequency at which theta occurs is less important for the control of such behaviours than other aspects of this electrical activity.
GRAY (1970; 1972) has proposed that sodium amylobarbitone influences behaviour by altering the septal control (STUMPF,1965) of hippocampal theta rhythm in a middle frequency band (7.7 Hz in the rat). Confirmation of the physiological part of this hypothesis comes from work with septal stimulation and hippocampal recording. Low frequency stimulation of the medial septum will drive hippocampal theta rhythm (BROCKE,PEDXHE, PILLAT & DEISENHAMMER, 1959), and in the free-moving rat the threshold current required to elicit such driving is minimal at 7.7 Hz (GRAY & BALL, 1970;JAMES,MCNAUGHTON,RAWLINS, FELDON& GRAY, 1977). GRAY & BALL (1970) found that sodium amylobarbitone eliminated this minimum by raising thresholds selectively in the region of 7.7 Hz. As might be predicted from the similarities between the behavioural effects of the barbiturates, benzodiazepines and ethanol (GRAY, 1977), this effect is also produced by injections of chlordiazepoxide and ethanol (MCNAUGHTON,JAMES,STEWART,GRAY, VALERO & DREWNOWSKI,1977). This specific effect in the region of 7.7 Hz is not reproduced by manipulation of the cholinergic, serotonergic or dopaminergic systems (MCNAUGHTONet al., 1977), while it is produced by both systemic injections which deplete noradrenergic systems and neurotoxic lesions of the dorsal ascending noradrenergic bundle (GRAY, MCNAUGHTON, JAMES &
1977).
KELLY, 1975; MCNAUGHTON et al.,
The full range of hippocampal theta frequencies can also be produced by high frequency stimulation of the midbrain reticular formation (GREENdt ARDUINI, 1954), but the frequency of theta produced is monotonically related to the stimulation intensity (SAILER & STUMPF,1957). STUMPF(1965) reported that hexobarbitone reduced the frequency of such theta rhythm, while scopolamine, although it could produce total blockade of theta, did not affect frequency. He did not report any frequency-specific effects of the drugs. Septal and reticular elicitation differ, then, as to whether frequency specific effects can be observed, both in control curves and in effects produced by injections of barbiturates. One possible source of discrepancy lies in the use of free-moving rats for the septal experiments and urethane-anaesthetized rabbits for the reticular ones. Other evidence makes this less likely. JAMESet al. (1977) reported linear reticular control curves and KRAMIS, VANDERWOW & BLAND (1975) reported a reduction in frequency with pentobarbital in the free-moving rat. However, KRAMISet al. (1975) did not report the drug effect systematically with respect to frequency, and it is possible, therefore, that the barbiturates produce a change which is not linearly related to frequency. This paper reports a more detailed analysis of the linearity of the stimulation intensity-theta frequency data previously reported by JAMESet al. (1977) and investigates the effects on this function of injections
629
630
N.
M~+.!AI~GHTON
of sodium amylobarbitone and the effects of manipulation of cholinergic, scrotonergic, dopatninergic and noradrenergic systems.
EXPERIMENTAL
PROCEDURES
The subjects were five male Wistar rats, chronically implanted with bipolar reticular stimulating electrodes and contralaterat bipolar recording electrodes in the dorsomedial subiculum. The surgical, stimulating and recording techniques and the histological location of the electrodes have been described previously (JAMESt~l ~1.. 1977). The reticular formation was stimulated at 100 Hz with 0.5 ms pulse width and 2 s train duration with a constant current stimulator. All five animals showed induction of theta rhythm at stimulating currents between IO and 40 @. There was no obvious relation between threshold current, initial frequency of theta produced, or rate of change of theta frequency (JAMESet ai., 1977), and so the same stimulating current intensities could not be used with all animals. The procedure used was to vary the intensity of stimulation by a constant interval (10, 20 or 40bA depending on the animal), so as to provide readings at a minimum offourintervals over the range tested. This range was normally between the lowest current which showed induced theta rhythm, usually at about 5 Hz, and the highest which did not produce either vigorous body turning or an escape attempt. In the case of animals tested under sodium amylobarbitone, the highest current intensity used was one considerably higher than that used in the predrug test, but this did not usually produce any motor activity. Drug tests employed the same intensity interval as that used in predrug tests. Several readings were taken at each value of the current intensity by systematically increasing and decreasing the current until at least ten readings had been taken. Analysis was performed on paper records taken at a speed of 15mm/s (see Fig. 1). The frequency of theta rhythm was estimated (k 0.5 Hz) by wave counting and the mean amplitude over the stimulating period was measured with a calibrated gauge ( k 3”,,).
I
and E. M.
SEDGWICK
All drug tests were preceded and followed by undrugged tests. but not always on the same day. Animals were used for more than one drug treatment, but these were given to different rats in different orders and control values were tested as normal on several days before administration of a drug. Tests with cc-methyl-p-;yrosine and p-chlorophenylalanine were performed after tests with other drugs had been completed. The drugs used were: sodium amylobarbitone (Lilly) injected in saline (15 or 20 mg,/kg. I ml/kg) 10 min before recording started; ~-methyl-p-tyrosine methyl ester HCi (Sigma) injected in saline (100 mgjml) 6.5 h before recording; scopolamine HBr (BDH) injected in saline (0.9 mgiml) 10 min before recording; and p-chloro-phenylalanine methyl ester HCl (Sigma) injected in saline (SOmg/ml) at a dose of iOOmg/kg/day for 3 days before recording. All drugs were administered intraperitoneaily. RESULTS Some especially clear raw data obtained from one rat are shown in Fig. 1. On the left are shown re-
sponses observed at increasing current intensities. On the right are shown the individual values obtained in one undrugged session. As can be seen here, and in the individual curves previously reported (JAMES et al., 1977), the frequency of theta is linearly related to stimulation intensity in undrugged animals. To quantify this relationship and to test its validity after drug treatment, regression lines were fitted to all of the individual drug and nondrug data for both frequency and amplitude of theta. If a function other than a straight line describes such data better, then the values obtained at any one current intensity will vary less from their own mean than from the value predicted by the regression line. The relative size of these two sources of v~iation {within intensity mean square, deviations from linearity mean square) may he tested with an F-ratio (&Ix, 1974, p. 218) This
120 PA
10
_-__-__--.-
~T~~“LAT,~~
80
CURRENT
120
-
VA
STIMULUS 100 HZ
OSM!XC
FIG. 1. Control data from an individual rat receiving different intensities (100 Hz, 0.5 ms, 2 s train). Left: Raw EEG records. The marker indicates the lation. Right: Graph of theta frequencies elicited during a single session: single with the same value *, The line drawn is the regression line fitted
of reticular stimulation entire duration of stimureadings i, two readings to these data.
160
631
Drug effects on hippocampal theta rhythm
amyloborbltone
AMPT
scopolamine
> = 5001
1
1
f;j/
kx” b--_
p 0
PC PA
50
100
0
50
100
STIMULATING
0 CURRENT
50
-
100
0
50
100
CIA
FIG. 2. Frequency and amplitude of theta rhythm elicited by increasing intensities of reticular stimulation (100 Hz, 0.5 ms) before (0) and after (0) treatment with sodium amylobarbitone (15 mg/kg and
20 mg/kg), a-methyl-p-tyrosine (AMPT: 100 mg/kg), scopolamine (0.9 mg/kg), and p-chloro-phenylalanine (PCPA: 3 x lOOmg/kg). The lines represent the regression lines obtained under the different conditions and are drawn with mean slope (across subjects) through the mean intercept (across subjects). The points plotted are arbitrary.
test performed on over 100 sets of data yielded only 6 F-ratios which on individual tests proved significant at the 5% level or better. Since 1 of these was at the 1% level (1 expected from random variation in a population this size), and a further 1 at the 2.5% level and 4 at the 5% level, there is no reason to reject the hypothesis of linearity in control or drugged data. The slope (B) and intercept (A) coefficients for such regression lines [Frequency = A + B (Stimulating Current)] are normally distributed (ZAR, 1974) and may thus be submitted to parametric statistics. Since it was impossible to match the stimulating intervals across animals and thereby derive mean frequencies of theta produced, the individual regression coefficients were used as raw data for subsequent statistical comparisons between groups. Figure 2 shows the effects of the drugs used as lines of mean slope (across subjects) drawn through the mean intercept. These are representative of the individual regression lines (cf. Fig. 1). Sodium amylobarbitone (15 or 20 mg/kg) produced a reduction in the mean slope of the current intensitytheta frequency function which analysis of variance showed to be significant (F = 63.0, d.jI = 1,4, P < O.OOS),while there was no change in the mean intercept (F = 3.8, d.J = 1,4). There was no difference in the size of the effect produced by the two different doses (F = 3.6, d$ = 1,4). A similar change appeared to be produced on the amplitude of theta, but this was less pronounced and did not achieve significance (Slope.: F = 6.8, d$ = 1,4; intercept: F = 0.5, d.J = 174). a-Methyl-p-tyrosine at the dose (lOOmg/kg) used by MCNAIJGHTONet al. (1977). oroduced a slight
reduction in the intercept of the frequency-intensity function which proved significant with Student’s t-test for paired comparisons (t = 5.516, d$ = 3, P < 0.02) but it did not affect the slope significantly (t = 2.041, d$ = 3) nor did it have effects on amplitude. Scopolamine did not produce changes in theta frequency in doses up to 0.9 mg/kg. At 0.9 mg/kg the apparent changes in slope (t = 1.317, d.$ = 2) and intercept (t = 0.956, d$ = 2) are not reliable. However, at and above this dose production of theta was intermittent. The same is true of the amplitude data (slope: t = 3.891, d.J = 2, P < 0.1; intercept: t = 1.126, d$ = 2). p-Chloro-phenylalanine (3 x 100 mg/kg/day) produced no effect on frequency (slope: t = 0.121, d$ = 2; intercept: t = 0.248, d$ = 2) or amplitude (slope: t = 0.707, d$ = 2; intercept: t = 2.226, d$ = 2). DISCUSSION The linearity of the relationship between intensity of stimulation in the reticular formation and the frequency of theta rhythm elicited in the hippocampus of the free moving rat (Jm et al., 1977) was unchanged by injections of sodium amylobarbitone, scopolamine, a-methyl-ptyrosine or p-chloro-phenylalanine. As has been previously reported for the acute rabbit (STUMPF,1965) and free-moving rat (KRAMIS et al., 1975) barbiturate, in this case sodium amylobarbitone, reduced the frequency of theta observed. This frequency reduction was not produced by choline& blockade or by depletion of noradrenaline, dopamine or serotonin levels. As reported by STUMPF(1965) for the acute rabbit, injections of scopolamine produced no change in the
632
N. MCNAIXHTOI\:and F. M. SEDGWKK
frequency of theta rhythm up to a dose which produced blockade. The intermittent nature of the blockade observed is consistent with the data of Vanderwolf (VANDERWOLF, 1975; VAKIXRWOL.F. KRAMIS, GILLESPIE & BLANI), 1975; KRAMIS ct ul., 1975) on atropine resistant and nonresistant theta rhythm. However, neither with scopolamine, nor with a small number of experiments performed with atropine. did this blockade occur selectively with low frequencies of theta, as might be expected from Vanderwolfs conelusions. [Blockade with atropine appeared at approximately 20 mg,kg as opposed to 0.9 mgjkg for scopolamine. STUMPF (1965) has reported a similar potency ratio.] The critical findings of this work are: the linearity of the stimulation intensity-theta frequency function is unchanged by sodium amylobarbitone; and interference with noradrenaline synthesis does not repro-
duce the effect of the barbiturate on theta frequency. The results thus confirm the suggestion that the reticular and septal stimulation methods disclose effects of barbiturates on two neurochemically dissimilar systerns involved in the control of different aspects of theta rhythm. Given the duplication of the effects of sodium amylobarbitone on behaviour by lesions of the dorsal ascending noradrenergic bundle, at least in tasks involving nonreward (MASON & IVERSEN. 1975; OWN. BOARI)ER& GRAY, 1977) it seems possible that the frequency at which theta rhythm occurs may be unimportant for these tasks and some other feature of the control may be critical. .l&owled~rmmrs N. MTNAI.GH.~ONwas supported by a Grant to Dr J. A. Gray from the British Medical Research Council. We thank Eli Lilly & Co. Ltd. for supplies of sodium amylobarbitone.
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