Brain Research, 103 (1976) 57 70
57
,~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
H I P P O C A M P A L T H E T A R H Y T H M . I. DEPTH PROFILES IN T H E CURARIZED RAT
JONATHAN WINSON
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted July 14th, 1975)
SUMMARY
Systemic injection of curare changes the depth profile of theta rhythm seen in the hippocampus of the freely moving rat. Under curare, dorsoventral microelectrode advancement reveals the presence of a sudden phase reversal and null occurring at the level of the stratum radiatum of CA1. Further advancement reveals the presence of an amplitude peak in the vicinity of the hippocampal fissure. In addition to the change in depth profile, curare alters the relationship between the amplitudes of the two phasereversed components of the theta rhythm. The change in theta rhythm brought about by curare outlasts the paralytic effect of the drug.
INTRODUCTION
Green e t al. 8 investigated theta rhythm in the hippocampus of the rabbit immobilized by injection of D-tubocurarine (D-TC). Using a microelectrode to penetrate the dorsal hippocampus dorsoventrally, they reported that a sharp phase reversal of the theta rhythm occurred in the stratum radiatum of CA1, the point of reversal coinciding with a null point of the amplitude of theta activity. A point of maximum theta amplitude occurred within CA1, ventral to the point of phase reversal. It was suggested that theta rhythm was generated by postsynaptic potentials in CAt pyramidal cells. These results were confirmed in a later study of the curarized rabbit 2. The phase-amplitude depth profile of the type that has just been described, consisting of a reversal of phase coincident with a null occurring in the stratum radiatum of CA1 and also an amplitude maximum located ventrally to the stratum radiatum, has been called here a type I profile (Fig. la). The present author has recently carried out an investigation of theta rhythm in the freely moving rat 2° during those behaviors in which the rhythm occurs naturally. Under these circumstances a different profile of theta activity was found, which is
58 called type II (Fig. la). The type I1 prolile is characterized by a point c,l" lnaxhllc,tn theta amplitude occurring approximately tit the level of the stratum pyramidalc c,i CA1 and a second, more ventrally located, point of maximum theta amplitude occmring at the level of the dorsal blade ot" the dentate gyrus. A slow phase shift is found to occur between the two peaks. Proceeding dorsoventrally, the shift starts in the stratum radiatum of CA1 and reaches phase reversal at the hippocampal fissure. These data suggested the existence in the freely moving rat of two generators of theta rhythm, one associated with CA~ and the other with the dentate gyrus. In the same study 2° el" hippocampal theta rhythm in the rat, depth profiles were also determined for the theta rhythms in several rats that were paralyzed by D-TC and in which the theta rhythm was produced pharmacologically. The profiles were found to be of type I. just as in the curarized rabbit. In these preliminary experiments in the curarized rat, the position of the point of maximum amplitude of the type 1 profile within the hippocampus was not determined. These data raised the possibility that the profiles of both rat and rabbit in the freely moving state were the same (type II), but that curare changed the theta-generating systems in each species so as to produce a type I profile in the curarized animal. it was also possible that a species difference existed in the generating systems of the normal rat and rabbit, the rat's giving rise to a type II profile and the rabbit's to a type I. As a step toward resolving these questions and to further elucidate the nature of the theta-generating system, experiments were carried out in the curarized rat (reported here) and in the freely moving rabbit~L The present study in the rat further investigated the effect of curare on the patterns of theta rhythm. A curarized preparation was used in which theta rhythm was elicited naturally rather than pharmacologically, and the location of the major physiological landmarks of the theta rhythm were established within the characteristic histological infrastructure of the hippocampus ~.
METHODS
A movable microelectrode device 19 was used in each of 21 male, adult SpragueDawley rats to lower a stainless steel microelectrode (tip 2-3/~m) perpendicular to the skull flat plane (plane passing through bregma and lambda and parallel to the interaural line). The device was positioned so that penetrations would pass through the dorsal blade of the dentate gyrus within a limited longitudinal portion of the dorsal hippocampus. Two fixed macroelectrodes (125/~m stainless steel wire) were implanted one above the other in the contralateral hippocampus, one dorsal to the stratum radiatum of CA1 and the other ventral to the hippocampal fissure. Ground and indifferent lead was a screw in the frontal bone. Unipolar recordings were made simultaneously from the microelectrode and the two contralateral macroelectrodes. All signals were carried through a 6-channel FET follower mounted on the rat's head to Grass (model 7) amplifiers by means of a counterbalanced lead wire and a mercury commutator. Signals were passed through 3-20 Hz bandpass filters matched lbr amplitude and phase characteristics in the 5-10 Hz range and periodically calibrated
59 (see data reduction). Signals were displayed on a storage oscillocope and recorded on polygraph paper and FM magnetic tape. Testing was carried out after a 1-week postoperative recovery period. Theta rhythms from the fixed macroelectrodes were first recorded in the freely moving animal during its initial exploration of a test cage is. The animal was then immobilized by an injection of D-TC (2 mg/kg, i.p.) and artificially respired via a face mask while a stepby-step microelectrode penetration was carried out in the dorsoventral direction. Theta rhythm was elicited by a method described below and was recorded from both micro- and macroelectrodes at each microelectrode depth. Penetrations were made in each of 6 trial animals in which a catheter in the femoral artery was used to sample blood during the experiment. These experiments were used to determine settings of respiration parameters (respiration pressure, rate, and inspiration-expiration ratio) which maintained values of blood pO~ and pCO2 within a range that is normal for the freely moving rat (pO2:87-95 mm Hg; p C O 2 : 3 1 - 3 5 mm Hg) ~,14. The parameter settings thus determined were used for the remainder of the experiments. During the 6 trial experiments, broader conditions were explored (pO2:75-99 mm Hg; pCO,_,: 19-40 mm Hg). Amplitude and phase profiles of the hippocampal theta rhythm were unaffected. At the end of each experiment, direct current was passed through the tip of the recording electrode, and the Prussian blue reaction was subsequently used to identify each electrode tip. The animal was first perfused with a solution of 1 0 ~ formalin, 4 i~ potassium ferrocyanide, and 4 ~ acetic acid, and then ferric ions were deposited. A current of 2/~A for 2 3 sec was passed through the microelectrode. The brain was removed, stored for 24 h in a solution composed of equal quantities of 40 ~ formalin and 95 ~ ethyl alcohol 16, frozen, cut into 50 /~m sections, and stained with cresyl violet stain.
Elicitation of theta rhythm In contrast to the curarized rabbit v, it was found that in the curarized rat theta rhythm was not readily evoked by sensory stimuli. Visual and auditory stimuli were completely ineffective. Brief episodes of theta rhythm could be elicited by tactile stimuli such as stroking of the animal's fur, but these were of insufficient duration and stability to allow accurate data reduction. A means was found to produce clear and long-lasting theta rhythm without electrical or pharmacological intervention. The animal was placed on an open turntable 24 in. in diameter. Air for respiration was conducted through an air-conducting bearing in the base of the device so that respiration was undisturbed when the turntable was rotated. Rotation of the table (to a speed of 15 30 rev./min) produced an immediate train of theta waves. Habituation occurred with continued turning, but the effect was reinstituted with reversal of direction. Theta rhythm was elicited in this way in all experiments. (It was found that theta rhythm could be elicited equally well by this technique in blinded rats.)
Data reduction The construction of a phase and amplitude profile of theta rhythm requires the
60 determination of tile phase relationship between the signal at each point of advancement of the microelectrode and that recorded simultaneously at a reference macroelectrode in tixed position, and the computation of the ratio of the amplitudes oi lhe two signals. Normalization through the use of the amplitude ratio is necessary to correct for differences in amplitude from one test episode to another, which may be due to variations in the overall theta rhythm as opposed to differences that are due to lhe position of the microelectrode in the brain. Since variations in the overall theta rhythm are reflected at the fixed reference electrode, the use of the amplitude ratio of the microelectrode signal to the fixed reference electrode signal serves to correct lOr these overall variations. Signals from the microelectrode and two fixed reference ma~'roelectrodes were simultaneously recorded during the experiment on magnetic tape and were subsequently analyzed by a PDP 15 computer. As a first step, analogue to digital conversion of'the theta signals was carried out on a LINC 8 computer. Theta voltages were converted to numerical data at I msec intervals and the numerical data were stored digitally on magnetic tape for later analysis by the PDP 15 computer. During the process of analogue to digital conversion, a visual read-out was provided on the L I N C 8 consisting of successive standing displays of 0.5 sec segments of the signals (3-4 cycles). Each display appeared as a stationary oscilloscope image until the data were either stored on the digital magnetic tape for later processing or rejected, The purpose of this procedure was to insure that only clear theta signals were analyzed. (a) PDa.re. Phase was determined by a cross-correlational technique 1,1. The PDP 15 considered a 200 msec segment of the signal at the microelectrode (approximately 1-1.5 cyclesl. It computed a series of Pearson cross-correlation coefficients tr) between this segment of the microelectrode signal and one of the two simultaneously occurring reference signals as follows. The reference signal was displaced with respect to the microelectrode signal in successive steps of 1 msec so that the displacement time varied between -50 and 50 msec, and a value of r between the 200 msec segment of" the microelectrode signal and the displaced reference signal was computed at each displacement time. Fig. l b illustrates this process. The upper trace shows 200 msec of the microelectrode signal extending from A to B while the lower trace shows the simultaneous reference signal. Schematically, the PDP 15 shifted the reference signal to the right by 50 msec so that the point A was above C and B was above D and computed the value of r between the microelectrode (A to B) and the reference (C to D) signals. The reference signal was then shifted I msec to the left and a new r was computed. This process was continued step by step until the reference signal was shifted so that A corresponded to C ~ 100 msec. The maximum value of r for this seiies of computations was then determined and stored in memory along with the number and direction of 1 msec steps needed to go from the real position of the two signals in time to the displaced position that yielded maximum r. The number of milliseconds and direction of displacement needed to bring the two signals l'rom the real time position to the position of maximum correlation defined the lead or lag of one signal with respect to the other. Fig. Ic illustrates the method of computation of lag or lead. In all, 50 or more cycles of theta rhythm were analyzed at each depth of the microelectrode. The final print-out displayed the computed lag or lead in milliseconds
61 TypeI
Type
150~-200 msec-~
(3 or
)
pyr
tad
lactool ht
I~-
mol ~ gron
300 msec
"I
D
Amplitude Amplltude
•
d
I'l
.
13
n
24
n÷l
rOE -I right
o
left
ref. signol, msec. Fig. I. a: depth profiles of theta rhythm. Left: or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; lac-mol, stratum lacunosum-moleculare of CA1; h.f., hippocampal fissure; tool. and gran., strata moleculare and granulosum of dorsal blade of dentate gyrus. Center: type I profile seen in curarized rabbit and curarized-eserinized rat. Sudden phase reversal and null at A, peak B ventral to phase-reversal region. Peak, shown here at hippocampal fissure, was reported to occur within CA~ in curarized rabbit 2,8. Right : type II profile seen in freely moving rat z°. Peak C in or near stratum pyramidale, slowly developing phase reversal without null from C to hippocampal fissure. Higher peak D in dorsal blade of the dentate gyrus, b: upper trace: microelectrode signal. Lower trace: simultaneous reference signal. Schematically the PDP 15 shifts the reference signal to the right by 50 msec so that point A lies above C and B lies above D, and calculates r between a 200 msec segment of the microelectrode signal (A to B) and the reference signal (C to D) using all 200 pairs of numerical values available on the digital tape. The value of r is stored in memory. Here r is negative and is shown as point E on the plot of r v e r s u s signal displacement in Fig. lc. The reference signal is now shifted I msec to the left and the process repeated. This continues step-by-step until A corresponds to C ? 100 msec. c: abscissa: displacement of reference signal with respect to microelectrode signal (msec); ordinate: value of r. From the values stored in memory, the PDP 15 determines the maximum value of r (shown as point F) and the corresponding displacement p corresponding to the maximum r. The microelectrode signal leads the reference signal by p milliseconds, d: computation of r amp. Upper and lower records are two theta signals recorded simultaneously. The Pearson crosscorrelation coefficient r amp is computed from successive peak-to-peak theta amplitudes as follows : I-1 paired with 2-2, 3-3 paired with 4-4 . . . . . n-n paired with (n l)-(n ÷ 1).
o f t h e m i c r o e l e c t r o d e s i g n a l w i t h r e s p e c t t o t h e r e f e r e n c e s~gnal, t h e p e r i o d o f t h e t h e t a wave (derived from the amplitude calculation below), and the maximum r for each of t h e 200 m s e c s e g m e n t s a n a l y z e d , as well as t h e a v e r a g e s a n d s t a n d a r d e r r o r s o f t h e m e a n ( S . E . M . ) o f t h e s e q u a n t i t i e s f o r all s e g m e n t s a n a l y z e d a t a p a r t i c u l a r m i c r o e l e c t r o d e d e p t h . T h e a v e r a g e v a l u e o f m a x i m u m r w a s u s u a l l y o f t h e o r d e r o f 0.95. T h e r e s o l u t i o n o f t h e m e t h o d w a s 1 m s e c o r a p p r o x i m a t e l y 2.5 ° o f p h a s e f o r a t h e t a r h y t h m f r e q u e n c y o f 7 Hz. I n t h i s s t u d y , m i c r o e l e c t r o d e s i g n a l s w e r e a l w a y s a p p r o x i m a t e l y in p h a s e w i t h the reference electrode signals chosen for comparison, and pairs of reference electrodes
62 were so placed that the signals from tllem were always approximately phase-re~.e~sed with respect to one anothel' (see Fig. 3 and Results). In determining phase relations between the phase-reversed reference electrode signals, the polarity oi" one signal was reversed on the computer, account being taken of the reversal in the Iinal re:,uh. (b) Amplitude pro,tih'. At each microelectrode depth, peak-to-peak amplitudes of individual theta cycles were computed, and the amplitudes recorded at the micn~electrode were divided by the amplitudes of corresponding cycles recorded at an appropriate (see Fig. 3 and Results) reference electrode. The average of these indixidual ratios and their S.E.M. at each microelectrode depth were printed out, and these were used to plot the amplitude profile (amplitude ratio versus depth}. For use in converting time displacements in milliseconds to phase angles (above), periods of theta cycles were computed from the times between the occurrence of successive amplitude peaks. The final amplitude profile was converted to a microvolt scale by multiplying the amplitude ratio of each point by a t'actor F such that the peak voltage shown on the profile was the actual voltage measured at the corresponding point in the brain, iF Vp/Ap where Vp = average peak voltage recorded at point of maximum ratio, Ap ratio at point of maximum ratio.) Between 50 and 150 cycles of theta were analyzed at each microelectrode depth. (c) Amplitude modulation. The theta rhythm is an approximately sinusoidal signal with considerable frequency and amplitude modulation. As a comparative measure of the time course of amplitude modulation of two theta signals, an amplitude cross-correlation coefficient (r amp) was computed. Corresponding theta waves of the two signals were considered for at least 60 successive cycles and the successive pairs of peak-to-peak amplitudes were cross-correlated. Fig. l d. illustrates this computation. On occasion, the signals from one electrode were fed into all 3 amplifier-filter channels simultaneously. Sinusoidal test signals at 7 Hz were also inserted in the same way. These data were processed along with normal signals to check for differences between channels and possible computer errors. RESULTS
Fixed electrodes As in the previous study of the theta rhythm profile in the freely moving rat "0, two macroelectrodes were implanted in the hippocampus to serve as references. One reference electrode was placed dorsal to the pyramidal layer of CAt and the other ventral to the hippocampal fissure. As reported before 20, it was found that in the freely moving animal theta rhythms at the two electrodes were approximately phasereversed and the amplitude modulations of the two signals were dissimilar. Fig. 2a shows a typical example of signals from an electrode pair recorded while the animal was moving. The polarity of the signal at electrode 2 was reversed during recording to facilitate comparison. The phase angle between the two signals, as shown with the signal from electrode 2 reversed, is 4.2" ~ 5.1 ° (S.E.M.) (electrode 2 leads electrode 1), and successive pairs of corresponding amplitudes are uncorrelated (r amp . . . . . 0.22). The relationship between the signals displayed a different quality after treatment with
63 (a) Freely moving Negotive elect. 2 /.El 2
Elect. i
(b) C u r a r i z e d
Negotive
elect. 2
~kA,/~/~;~,t,~,,.A.j~k~/¢?.~l..v~%/~',./~A.
1480 ,u,v .1.150/N
E,ect. 1
1 sec
Fig. 2. Signals from one pair of fixed electrodes whose locations are shown at left. In this and subsequent figures, location designations refer to K6nig and Klippel's atlas 9. Rat is freely moving in (a) and curarized in (b). Voltage scale applies to (a) and (b). Signals from electrode 2 and electrode I are phase-reversed both before and after injection of curare. Polarity of electrode 2 signal was reversed here for ease of comparison. In the presence of curare, amplitude similarity is increased. Further de-
tails in text. curare. Fig. 2b shows this change in relationship. The phase angle between the signals remained virtually unchanged, electrode 2 leading the negative of electrode 1 by 7.8 ° ± 1.6°, but the amplitude modulations were now very similar, as were the higher harmonic components. The amplitude correlation coefficient was +0.52. The signals from pairs of reference electrodes in 6 animals were analyzed, and all showed the same phenomenon. In the moving state, the signals were approximately phase-reversed and the amplitudes were uncorrelated or showed some negative correlation (range of r amp: - - 0.22 to --0.36). There was no significant change of phase relationship after curare but the amplitude modulations in each case took on a degree of positive correlation (r amp: +0.30-+0.58). Positive correlation was significantly greater after curare than before in each animal (P < 0.01). Phase reversal and null Fig. 3 shows signals recorded in a typical microelectrode penetration in the curarized rat. The signals from the two fixed electrodes were approximately phasereversed. (The average deviation from phase reversal for all fixed electrode recordings made during the penetration was 5.7 ° ~ 2.1°.) As the microelectrode was lowered through the neocortex, a theta rhythm was detected dorsal to the corpus callosum. The signal was approximately in phase with the signal from the dorsal reference electrode and, further, the individual shapes of corresponding waves and the time course of amplitude modulations of the two signals were very similar (upper two records of Fig. 3a). For convenience of discussion, signals with this relationship are termed isomorphic 2°. As the dorsoventral penetration continued into the stratum radiatum, the theta rhythm abruptly disappeared and was replaced by a null signal (Fig. 3b). The null signal was present for a dorsoventral distance of about 90/~m, below which the theta
64 {o)
t ool /
?
i
f(\F; J
!
electrode l 3430kL
A 4110#
(c)
(b)
,
, ql
'
~
%
e
~
f
,
[
5oo ~v
I
J,
I sec
Fig. 3. Records from penetration. Electrode positions shown in upper left. In this case, fixed reference electrodes and microelectrode penetration are at different rostrocaudal locations. Upper record is from dorsal reference electrode, middle record from microelectrode, and lower record from ventral reference electrode. Voltage scales for electrodes 2 and 1 are indicated in (c) for all records. Amplifier gain for microelectrode signal is halved in (c). Signals from electrodes 2 and 1 are phase-reversed. Microelectrode is dorsal to stratum radiatum in (a); signal is isomorphic with electrode2. Microelectrode is in proximal stratum radiatum in (b) and records a null signal, in (c) microelectrode is in ventral stratum radiatum, signal is isomorphic with electrode 1.
rhythm reappeared, now isomorphic with the signal from the ventral macroelectrode (Fig. 3c) and therefore approximately phase-reversed from the signal that had been detected by the microelectrode above the null. Deeper penetrations showed that isomorphism with the ventral reference electrode was maintained into the dorsal thalamus. (As in previous work 9°, for maximum accuracy in the computation of anaplitude ratios and phases, reference signals were used that were isomorphic with signals recorded from the microelectrode.) As indicated, the signals from dorsal and ventral reference electrodes were phase-reversed within 6 °. The average signals seen by the microelectrode above and below the null were also phase-reversed within a few degrees. Signals recorded at a single rostrocaudal position in the hippocampus were generally either in phase or phase-reversed within 10% When electrodes were in different rostrocaudal placements, however, somewhat greater deviations were found. In the experiment depicted in Fig. 3, for example, the microelectrode was not inserted at a rostrocaudal level identical to that o f the fixed electrodes but at a level approximately 700/~m caudal to them. In Fig. 3a the signal from the microelectrode lags the signal from electrode 2 by 12.2 1.9 msec (about 30°). The situation is similar below the point of phase reversal. In Fig. 3c, the signal from the microelectrode lags the signal from electrode 1 by 13.1 7 msec. Within the rostrocaudal limits A4110 #m to A3180 ,um (ref. 9), the maxi-
65
[
Fig. 4. Penetration to determine location of null and phase reversal. Microelectrode was advanced to ventral limit of null region, withdrawn 60/era to the dorsal limit, and ferric ions were deposited. In three such preparations null was located 30 150/~m below pyramidal layer.
mum time displacement encountered between isomorphic signals was 14.3 msec. Penetrations in this experiment were designed to fall within the mediolateral limits of the granule cell layer of dorsal blade of the dentate gyrus. A clear null extending through a dorsoventral span of 50-110 #m was found in all such traverses. Three penetrations were terminated at the null in order to determine unequivocally the dorsoventral position of the null. All three experiments showed the null to be in the proximal stratum radiatum, extending approximately from 30 to 150/~m below the pyramidal layer. Fig. 4 shows the termination of one of these penetrations. In this case, after the null was detected, the microelectrode was advanced 60/~m until the signal began to show renewed theta activity. The microelectrode was then withdrawn 60 pro, the experiment terminated, and ferric ions were deposited at this position. Two traverses were more medial than the others, passing just medial to the crest of the granule cell layer of the dentate gyrus. In these exceptional cases, instead of a null, a region of irregular signals extending about 250/~m dorsoventrally was found, above and below which the phase of the theta rhythm was reversed.
Amplitude profile Fig. 5 shows a typical type I amplitude profile in a curarized rat, with a more or less constant amplitude of theta rhythm for positions dorsal to the point of phase reversal and a sharp peak ventral to it. Five experiments were performed to determine the location of the amplitude peak in the hippocampus. In these cases, recordings were made every 44 #m in the vicinity of the peak, and the penetration terminated within 88/~m of its occurrence. Fig. 6 shows the results of one such experiment. The penetration was terminated 44 #m ventral to the peak (lower right panel) and the point of termination proved to be just ventral to the hippocampal fissure (lower left panel). Peak positions in the brain were determined by extrapolating back on the histological
66 800 ,~543C,~.
,,0 700 o
F~
~ebec~rode~/
A "l
zL 600
d £
-~ 500 (3)
o 400 (.~
E -~ 3OO .-,.~_ Q.
200
E < 100
electrode 2
'
electrode 1
-I
Null
[
I
I
I
I
I
0
I
2
3
4
5
352 H. Intervals Fig. 5. Amplitude profile: normalized amplitude of theta rhythm v e r s u s depth in the brain. Left to right: dorsoventral penetration of microelectrode in units of 352 #m (one turn ofmicroelectrodeadvancing screw). Zero chosen for abscissa is arbitrary. Ordinates are normalized theta amp|itudes. Vertical bars are S.E.M. Line above abscissa indicates the relationship of microelectrode signal to reference electrode signal. Amplitude profile is type 1.
sections of this and the other four penetrations from the positions of the electrode tips through the short distances derived from the amplitude plots. Allowing a possible error of as much as half the test point spacing in the position of the peak on the amplitude plots and taking into account possible errors introduced by the finite size of the Prussian blue markings, it was nevertheless found that all peaks were located within a span running from 70 # m above the hippocampal fissure to 50 # m below it.
Time-course of effect of curare on theta profile The finding here of a type I profile in the curarized rat taken with the previous report that the profile in the freely moving animal was type II offered the opportunity to follow the time-course of the effect of curare on the theta profile. A penetration was made in the curarized rat to the null point and the microelectrode was locked in place. This point is indicated as A in Fig. l a. The rat was then allowed to recover from the effect of the curare. It was to be expected that a substantial theta rhythm would be recorded from this location in stratum radiatum once the profile had returned to type II (Fig. l a) and the time-course of the effect of curare on profile could be gauged by the appearance of this theta rhythm. This procedure was carried out in 5 animals. Fig. 7 shows a typical result. The upper record is from a reference electrode in stratum oriens, the lower record is from a microelectrode lowered to the null position in stratum radiatum. Fig. 7a shows the null signal approximately 2 h after injection of curare and penetra-
67
1000 o "G 900 ¢M
tb
800
E 700
._-=_ E
lotion
I
600 i
I
I
[
I
I
i
I
I
i
I
Depth, 44p. intervals Fig. 6. Experiment to determine location of peak. Lower right: plot of normalized amplitude of theta rhythm v e r s u s depth in brain. Penetration in the dorsoventral direction is represented from left to right on the abscissa. Electrode was advanced in 44 p m steps and the penetration was terminated 44 /~m ventral to the peak. Upper photograph shows location of penetration. Enlargement at lower left shows position of electrode tip close to hippocampal fissure at termination.
tion to the null point, which was basically identical to the record originally obtained upon reaching the null point approximately 15 rain after injection. At the time of the recording of Fig. 7a, the animal was still being artificially respired but was displaying the first signs of recovery of voluntary muscular activity. Fig. 7b was recorded 45 rain later. The animal was sufficiently active to free itself from the face mask and was breathing unaided. Eight minutes later (Fig. 7c) the animal was moving in its cage with no effects of paralysis evident in its behavior. The signal at the microelectrode exhibited sporadic bursts of theta rhythm, although the theta rhythm recorded at the reference electrode was regular and clear. The signal at the microelectrode continued to gain in amplitude and regularity. Fig. 7d, recorded 30 min after (c) shows a signal at the microelectrode approximately equal to the reference signal in amplitude and leading it by about 50° as would be expected in the stratum radiatum of the freely moving animal 2o.
68 (a)
(b)
(c)
(d)
I 420/~V ~l~I~l~l~ ~~~ I ~~ iO o ~ v~l ~ 1 sec Fig. 7. Records from curarized rat during recovery. Upper records are from fixed reference electrode
in stratum oriens. Lower record is from microelectrode which was inserted into the curarized animal and advanced to the null position. Voltage scales in (d) apply to all records. In (a) animal had been immobilized for 2 h and was showing first signs of voluntary movement. Null signal was essentially the same as that observed when the electrode was fixed in position, in (b), recorded 45 rain later, animal was breathing unaided. In (c), recorded 8 rain after (b), animal was moving with no behavioral effects of curare evident. Although fixed electrode shows a continuous theta rhythm, microelectrode rhythm is clearly of a different type. Signals in (d) were recorded 30 min after (c), by which time the microelectrode signal had reverted to clear theta rhythm. DISCUSSION
The present experiment confirms earlier results obtained in the freely moving rat and in the curarized rat on profiles o f theta rhythm, and indicates that systemically introduced curare affects the neural system responsible for the generation o f theta r h y t h m in the rat hippocampus. The effect is detected as a change from type II profile in the freely moving state to a type I profile under curare, in which there is an amplitude peak in the vicinity o f the hippocampal fissure. In addition to the profile change, curare brings about a closer synchrony o f the two phase-reversed components o f theta rhythm (dorsal and ventral) as is evidenced by the increased correspondence in amplitude modulation that occurs after injection. In a previous report '>0it was suggested that the type lI profile reflected the activity o f two coupled generators, one in the dentate gyrus and a second in the overlying CAj layer. The existence o f two generators o f this nature has recently been demonstrated in the lightly anesthetized rabbitL In the rabbit the profile was type 1, the peak being ill the dentate gyrus 4. The same profile with the peak in the dentate gyrus is reported in the freely moving rabbit in the following paper 2a. It is not known whether or not the profile f o u n d in the present experiment results from the activity o f both dentate and CA~ generators. It is possible that the peak actually lies slightly dorsal to the fissure and that it reflects activity only in the apical dendrites o f CA1. The position o f the peak so close to the fissure may also indicate that a dentate c o m p o n e n t is present. The profile found here in the curarized rat is very similar to that reported in the curarized rabbit. Both are type l with a phase reversal and null in stratum radiatum
69 a n d with a m o r e ventral peak. In the c u r a r i z e d r a b b i t the p e a k was r e p o r t e d to be in distal region o f the apical dendrites o f CA1, b u t its precise l o c a l i z a t i o n was n o t a t t e m p t ed s. It m a y be t h a t the profiles in the c u r a r i z e d rat a n d r a b b i t are the same, each different from the profile in the freely m o v i n g animal 4,2°,21. A study localizing the p e a k in the c u r a r i z e d r a b b i t profile is required to resolve this point. The d i s p a r i t y in time o f m u s c u l a r a n d profile recovery from the effects o f curare suggests that the effect on theta profile is not a consequence o f paralysis. Rather, the change b r o u g h t a b o u t in the t h e t a - g e n e r a t i n g system w o u l d seem to require specific central intervention either within the h i p p o c a m p u s or elsewhere in the neuronal netw o r k associated with the theta r h y t h m t L Central effects o f systemic injection o f curare have been r e p o r t e d t'5. These have consisted o f the reversible b l o c k a d e o f electrically i n d u c e d evoked potentials in the neocortex o f the cat after intravenous a d m i n i s t r a t i o n o f curare. The medial septum has been identified as a p a c e m a k e r o f t h e t a r h y t h m iv and the medial s e p t a l - h i p p o c a m p a l p r o j e c t i o n is believed to be cholinergic 5,1°,1:~. A n i o n t o p h o r e t i c study ~ has shown that, a l t h o u g h most acetylcholine-sensitive cells in the h i p p o c a m p u s a p p e a r to be muscarinic, there are cells that are selectively b l o c k e d by curare. F u r t h e r clarification o f the role o f curare in modifiying the patterns o f theta r h y t h m awaits the identification o f the p a r t i c u l a r h i p p o c a m p a l neurons and circuits involved in theta r h y t h m generation. ACKNOWLEDGEMENTS This study was s u p p o r t e d by grants f r o m the N a t i o n a l A s s o c i a t i o n for M e n t a l Health, M e r c k a n d Co., a n d the G r a n t F o u n d a t i o n . T h e a u t h o r t h a n k s Dr. Charles A b z u g for his critical reading o f the manuscript.
REFERENCES I APOSTOL, G., AND CREUTZFELDT, O. D., Cross correlation between the activity of septal units and hippocampal EEG during arousal, Brain Research, 67 (1974) 65-75. 2 ARTEMENKO, D. P., Role of hippocampal neurons in theta-wave generation, Nierafiziologiya, 4
(1972) 531-539. 3 BISCOE, T. J., AND STRAUGHAN, D. W., Micro-electrophoretic studies of neurones in the cat hippocampus, J. Physiol. (Land.), 183 (1966) 341-359.
4 BLAND, B. H., ANDERSEN,P., AND GANES,T., Two generators of hippocampal theta activity in rabbits, Brain Research, 94 (1975) 199 218. 5 DUDAR,J. D., The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus, Brain Research, 83 (1975) 123 133. 6 EISSENBERG,E., Intermittent positive pressure in the curarized rat: implication for cardiovascular conditioning, Physiol. Behav., in press. 7 GREEN, J. D., AND ARDUINI, A. A., Hippocampal electrical activity in arousal, J. Neurophysial., 17 (1954) 533-557. 8 GREEN, J. D., MAXWELL, D. S., SCHINDLER, W. J., AND STUMPF, C., Rabbit EEG 'theta" rhythrn: its anatomical source and relation to activity in single neurons, J. Neurophysiol., 23 (1960) 403 420. 9 K/3NIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Kreiger, Huntington, N.Y., 1970, 162 pp. 10 LEWIS, P. R., AND SHUTE, C. C. D., The cholinergic limbic system: projection to hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-539.
70 I I LORENTE DI~ NO, R., Studies on the structure of the cerebral cortex. 11. Conti~3uation of lhe ,itidy of the ammonic system, J. l'~ychol. Nemol. ~'Lpz. ), 46 (1934) 113-177. 12 McLENNA~,, H., At;D M iLL~;R. J. J.. The hippocampal control of neuronal discharges in the ,,cpttim of the rat, J. Physiol. fLoral., 237 (1974) 607 624. 13 MosKo, S., LVNC:H, G., AxlJ COrMAN, C. W.+ The distribution of septal projections to the hippocampus of the rat, J. comp. Neuro[., 152 (1973) 163 174. 14 NATHAN, M. A., AND REIS, D. J., Hypoxemia, atelectasis, and the elevation of arterial pressure and heart rate in paralyzed artificially ventilated rat, L(/b Sci., 16 (1975) 1103--1120. 15 PURPURA, O. P., AND GRUNDFEST, H., Nature of dendritic potentials and synaptic mechanisms in cerebral cortex of cat, J. Neuroplo,siol., 19 (1956) 573-595. 16 SCHEmEL, M. E., AND S(THEmH_, A. B., Histological localization of microelectrode placement itl brain by ferrocyanide and silver staining, Stain Technol., 31 (1955) 1-5. 17 STUMPF, C., Drug action on the electrical activity of the hippocampus, Int. Rev. Xeurobiol., 8 (I 965) 77-138. 18 VANDERWOLF,C. H., Hippocampal electrical activity and voluntary movement in the rat, Eh, ctroencepk, clit#. Neurophysiol., 26 (1969) 407- 418. 19 WINSON, J., A compact movable microelectrode assembly for recording from the freely-moving rat, Electroenceph. olin. Neurophysiol., 35 (1973) 215-217. 20 W[NSON, J., Patterns of hippocampal theta rhythm in the freely moving rat, Electroenceph. olin. Neurophysiol., 36 (1974) 291 30l. 21 WINSON, J., Hippocampal theta rhythm. 11. Depth profiles in the freely moving rabbit, Brain Research, 103 (1976) 71 .79.
57
,~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
H I P P O C A M P A L T H E T A R H Y T H M . I. DEPTH PROFILES IN T H E CURARIZED RAT
JONATHAN WINSON
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted July 14th, 1975)
SUMMARY
Systemic injection of curare changes the depth profile of theta rhythm seen in the hippocampus of the freely moving rat. Under curare, dorsoventral microelectrode advancement reveals the presence of a sudden phase reversal and null occurring at the level of the stratum radiatum of CA1. Further advancement reveals the presence of an amplitude peak in the vicinity of the hippocampal fissure. In addition to the change in depth profile, curare alters the relationship between the amplitudes of the two phasereversed components of the theta rhythm. The change in theta rhythm brought about by curare outlasts the paralytic effect of the drug.
INTRODUCTION
Green e t al. 8 investigated theta rhythm in the hippocampus of the rabbit immobilized by injection of D-tubocurarine (D-TC). Using a microelectrode to penetrate the dorsal hippocampus dorsoventrally, they reported that a sharp phase reversal of the theta rhythm occurred in the stratum radiatum of CA1, the point of reversal coinciding with a null point of the amplitude of theta activity. A point of maximum theta amplitude occurred within CA1, ventral to the point of phase reversal. It was suggested that theta rhythm was generated by postsynaptic potentials in CAt pyramidal cells. These results were confirmed in a later study of the curarized rabbit 2. The phase-amplitude depth profile of the type that has just been described, consisting of a reversal of phase coincident with a null occurring in the stratum radiatum of CA1 and also an amplitude maximum located ventrally to the stratum radiatum, has been called here a type I profile (Fig. la). The present author has recently carried out an investigation of theta rhythm in the freely moving rat 2° during those behaviors in which the rhythm occurs naturally. Under these circumstances a different profile of theta activity was found, which is
58 called type II (Fig. la). The type I1 prolile is characterized by a point c,l" lnaxhllc,tn theta amplitude occurring approximately tit the level of the stratum pyramidalc c,i CA1 and a second, more ventrally located, point of maximum theta amplitude occmring at the level of the dorsal blade ot" the dentate gyrus. A slow phase shift is found to occur between the two peaks. Proceeding dorsoventrally, the shift starts in the stratum radiatum of CA1 and reaches phase reversal at the hippocampal fissure. These data suggested the existence in the freely moving rat of two generators of theta rhythm, one associated with CA~ and the other with the dentate gyrus. In the same study 2° el" hippocampal theta rhythm in the rat, depth profiles were also determined for the theta rhythms in several rats that were paralyzed by D-TC and in which the theta rhythm was produced pharmacologically. The profiles were found to be of type I. just as in the curarized rabbit. In these preliminary experiments in the curarized rat, the position of the point of maximum amplitude of the type 1 profile within the hippocampus was not determined. These data raised the possibility that the profiles of both rat and rabbit in the freely moving state were the same (type II), but that curare changed the theta-generating systems in each species so as to produce a type I profile in the curarized animal. it was also possible that a species difference existed in the generating systems of the normal rat and rabbit, the rat's giving rise to a type II profile and the rabbit's to a type I. As a step toward resolving these questions and to further elucidate the nature of the theta-generating system, experiments were carried out in the curarized rat (reported here) and in the freely moving rabbit~L The present study in the rat further investigated the effect of curare on the patterns of theta rhythm. A curarized preparation was used in which theta rhythm was elicited naturally rather than pharmacologically, and the location of the major physiological landmarks of the theta rhythm were established within the characteristic histological infrastructure of the hippocampus ~.
METHODS
A movable microelectrode device 19 was used in each of 21 male, adult SpragueDawley rats to lower a stainless steel microelectrode (tip 2-3/~m) perpendicular to the skull flat plane (plane passing through bregma and lambda and parallel to the interaural line). The device was positioned so that penetrations would pass through the dorsal blade of the dentate gyrus within a limited longitudinal portion of the dorsal hippocampus. Two fixed macroelectrodes (125/~m stainless steel wire) were implanted one above the other in the contralateral hippocampus, one dorsal to the stratum radiatum of CA1 and the other ventral to the hippocampal fissure. Ground and indifferent lead was a screw in the frontal bone. Unipolar recordings were made simultaneously from the microelectrode and the two contralateral macroelectrodes. All signals were carried through a 6-channel FET follower mounted on the rat's head to Grass (model 7) amplifiers by means of a counterbalanced lead wire and a mercury commutator. Signals were passed through 3-20 Hz bandpass filters matched lbr amplitude and phase characteristics in the 5-10 Hz range and periodically calibrated
59 (see data reduction). Signals were displayed on a storage oscillocope and recorded on polygraph paper and FM magnetic tape. Testing was carried out after a 1-week postoperative recovery period. Theta rhythms from the fixed macroelectrodes were first recorded in the freely moving animal during its initial exploration of a test cage is. The animal was then immobilized by an injection of D-TC (2 mg/kg, i.p.) and artificially respired via a face mask while a stepby-step microelectrode penetration was carried out in the dorsoventral direction. Theta rhythm was elicited by a method described below and was recorded from both micro- and macroelectrodes at each microelectrode depth. Penetrations were made in each of 6 trial animals in which a catheter in the femoral artery was used to sample blood during the experiment. These experiments were used to determine settings of respiration parameters (respiration pressure, rate, and inspiration-expiration ratio) which maintained values of blood pO~ and pCO2 within a range that is normal for the freely moving rat (pO2:87-95 mm Hg; p C O 2 : 3 1 - 3 5 mm Hg) ~,14. The parameter settings thus determined were used for the remainder of the experiments. During the 6 trial experiments, broader conditions were explored (pO2:75-99 mm Hg; pCO,_,: 19-40 mm Hg). Amplitude and phase profiles of the hippocampal theta rhythm were unaffected. At the end of each experiment, direct current was passed through the tip of the recording electrode, and the Prussian blue reaction was subsequently used to identify each electrode tip. The animal was first perfused with a solution of 1 0 ~ formalin, 4 i~ potassium ferrocyanide, and 4 ~ acetic acid, and then ferric ions were deposited. A current of 2/~A for 2 3 sec was passed through the microelectrode. The brain was removed, stored for 24 h in a solution composed of equal quantities of 40 ~ formalin and 95 ~ ethyl alcohol 16, frozen, cut into 50 /~m sections, and stained with cresyl violet stain.
Elicitation of theta rhythm In contrast to the curarized rabbit v, it was found that in the curarized rat theta rhythm was not readily evoked by sensory stimuli. Visual and auditory stimuli were completely ineffective. Brief episodes of theta rhythm could be elicited by tactile stimuli such as stroking of the animal's fur, but these were of insufficient duration and stability to allow accurate data reduction. A means was found to produce clear and long-lasting theta rhythm without electrical or pharmacological intervention. The animal was placed on an open turntable 24 in. in diameter. Air for respiration was conducted through an air-conducting bearing in the base of the device so that respiration was undisturbed when the turntable was rotated. Rotation of the table (to a speed of 15 30 rev./min) produced an immediate train of theta waves. Habituation occurred with continued turning, but the effect was reinstituted with reversal of direction. Theta rhythm was elicited in this way in all experiments. (It was found that theta rhythm could be elicited equally well by this technique in blinded rats.)
Data reduction The construction of a phase and amplitude profile of theta rhythm requires the
60 determination of tile phase relationship between the signal at each point of advancement of the microelectrode and that recorded simultaneously at a reference macroelectrode in tixed position, and the computation of the ratio of the amplitudes oi lhe two signals. Normalization through the use of the amplitude ratio is necessary to correct for differences in amplitude from one test episode to another, which may be due to variations in the overall theta rhythm as opposed to differences that are due to lhe position of the microelectrode in the brain. Since variations in the overall theta rhythm are reflected at the fixed reference electrode, the use of the amplitude ratio of the microelectrode signal to the fixed reference electrode signal serves to correct lOr these overall variations. Signals from the microelectrode and two fixed reference ma~'roelectrodes were simultaneously recorded during the experiment on magnetic tape and were subsequently analyzed by a PDP 15 computer. As a first step, analogue to digital conversion of'the theta signals was carried out on a LINC 8 computer. Theta voltages were converted to numerical data at I msec intervals and the numerical data were stored digitally on magnetic tape for later analysis by the PDP 15 computer. During the process of analogue to digital conversion, a visual read-out was provided on the L I N C 8 consisting of successive standing displays of 0.5 sec segments of the signals (3-4 cycles). Each display appeared as a stationary oscilloscope image until the data were either stored on the digital magnetic tape for later processing or rejected, The purpose of this procedure was to insure that only clear theta signals were analyzed. (a) PDa.re. Phase was determined by a cross-correlational technique 1,1. The PDP 15 considered a 200 msec segment of the signal at the microelectrode (approximately 1-1.5 cyclesl. It computed a series of Pearson cross-correlation coefficients tr) between this segment of the microelectrode signal and one of the two simultaneously occurring reference signals as follows. The reference signal was displaced with respect to the microelectrode signal in successive steps of 1 msec so that the displacement time varied between -50 and 50 msec, and a value of r between the 200 msec segment of" the microelectrode signal and the displaced reference signal was computed at each displacement time. Fig. l b illustrates this process. The upper trace shows 200 msec of the microelectrode signal extending from A to B while the lower trace shows the simultaneous reference signal. Schematically, the PDP 15 shifted the reference signal to the right by 50 msec so that the point A was above C and B was above D and computed the value of r between the microelectrode (A to B) and the reference (C to D) signals. The reference signal was then shifted I msec to the left and a new r was computed. This process was continued step by step until the reference signal was shifted so that A corresponded to C ~ 100 msec. The maximum value of r for this seiies of computations was then determined and stored in memory along with the number and direction of 1 msec steps needed to go from the real position of the two signals in time to the displaced position that yielded maximum r. The number of milliseconds and direction of displacement needed to bring the two signals l'rom the real time position to the position of maximum correlation defined the lead or lag of one signal with respect to the other. Fig. Ic illustrates the method of computation of lag or lead. In all, 50 or more cycles of theta rhythm were analyzed at each depth of the microelectrode. The final print-out displayed the computed lag or lead in milliseconds
61 TypeI
Type
150~-200 msec-~
(3 or
)
pyr
tad
lactool ht
I~-
mol ~ gron
300 msec
"I
D
Amplitude Amplltude
•
d
I'l
.
13
n
24
n÷l
rOE -I right
o
left
ref. signol, msec. Fig. I. a: depth profiles of theta rhythm. Left: or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; lac-mol, stratum lacunosum-moleculare of CA1; h.f., hippocampal fissure; tool. and gran., strata moleculare and granulosum of dorsal blade of dentate gyrus. Center: type I profile seen in curarized rabbit and curarized-eserinized rat. Sudden phase reversal and null at A, peak B ventral to phase-reversal region. Peak, shown here at hippocampal fissure, was reported to occur within CA~ in curarized rabbit 2,8. Right : type II profile seen in freely moving rat z°. Peak C in or near stratum pyramidale, slowly developing phase reversal without null from C to hippocampal fissure. Higher peak D in dorsal blade of the dentate gyrus, b: upper trace: microelectrode signal. Lower trace: simultaneous reference signal. Schematically the PDP 15 shifts the reference signal to the right by 50 msec so that point A lies above C and B lies above D, and calculates r between a 200 msec segment of the microelectrode signal (A to B) and the reference signal (C to D) using all 200 pairs of numerical values available on the digital tape. The value of r is stored in memory. Here r is negative and is shown as point E on the plot of r v e r s u s signal displacement in Fig. lc. The reference signal is now shifted I msec to the left and the process repeated. This continues step-by-step until A corresponds to C ? 100 msec. c: abscissa: displacement of reference signal with respect to microelectrode signal (msec); ordinate: value of r. From the values stored in memory, the PDP 15 determines the maximum value of r (shown as point F) and the corresponding displacement p corresponding to the maximum r. The microelectrode signal leads the reference signal by p milliseconds, d: computation of r amp. Upper and lower records are two theta signals recorded simultaneously. The Pearson crosscorrelation coefficient r amp is computed from successive peak-to-peak theta amplitudes as follows : I-1 paired with 2-2, 3-3 paired with 4-4 . . . . . n-n paired with (n l)-(n ÷ 1).
o f t h e m i c r o e l e c t r o d e s i g n a l w i t h r e s p e c t t o t h e r e f e r e n c e s~gnal, t h e p e r i o d o f t h e t h e t a wave (derived from the amplitude calculation below), and the maximum r for each of t h e 200 m s e c s e g m e n t s a n a l y z e d , as well as t h e a v e r a g e s a n d s t a n d a r d e r r o r s o f t h e m e a n ( S . E . M . ) o f t h e s e q u a n t i t i e s f o r all s e g m e n t s a n a l y z e d a t a p a r t i c u l a r m i c r o e l e c t r o d e d e p t h . T h e a v e r a g e v a l u e o f m a x i m u m r w a s u s u a l l y o f t h e o r d e r o f 0.95. T h e r e s o l u t i o n o f t h e m e t h o d w a s 1 m s e c o r a p p r o x i m a t e l y 2.5 ° o f p h a s e f o r a t h e t a r h y t h m f r e q u e n c y o f 7 Hz. I n t h i s s t u d y , m i c r o e l e c t r o d e s i g n a l s w e r e a l w a y s a p p r o x i m a t e l y in p h a s e w i t h the reference electrode signals chosen for comparison, and pairs of reference electrodes
62 were so placed that the signals from tllem were always approximately phase-re~.e~sed with respect to one anothel' (see Fig. 3 and Results). In determining phase relations between the phase-reversed reference electrode signals, the polarity oi" one signal was reversed on the computer, account being taken of the reversal in the Iinal re:,uh. (b) Amplitude pro,tih'. At each microelectrode depth, peak-to-peak amplitudes of individual theta cycles were computed, and the amplitudes recorded at the micn~electrode were divided by the amplitudes of corresponding cycles recorded at an appropriate (see Fig. 3 and Results) reference electrode. The average of these indixidual ratios and their S.E.M. at each microelectrode depth were printed out, and these were used to plot the amplitude profile (amplitude ratio versus depth}. For use in converting time displacements in milliseconds to phase angles (above), periods of theta cycles were computed from the times between the occurrence of successive amplitude peaks. The final amplitude profile was converted to a microvolt scale by multiplying the amplitude ratio of each point by a t'actor F such that the peak voltage shown on the profile was the actual voltage measured at the corresponding point in the brain, iF Vp/Ap where Vp = average peak voltage recorded at point of maximum ratio, Ap ratio at point of maximum ratio.) Between 50 and 150 cycles of theta were analyzed at each microelectrode depth. (c) Amplitude modulation. The theta rhythm is an approximately sinusoidal signal with considerable frequency and amplitude modulation. As a comparative measure of the time course of amplitude modulation of two theta signals, an amplitude cross-correlation coefficient (r amp) was computed. Corresponding theta waves of the two signals were considered for at least 60 successive cycles and the successive pairs of peak-to-peak amplitudes were cross-correlated. Fig. l d. illustrates this computation. On occasion, the signals from one electrode were fed into all 3 amplifier-filter channels simultaneously. Sinusoidal test signals at 7 Hz were also inserted in the same way. These data were processed along with normal signals to check for differences between channels and possible computer errors. RESULTS
Fixed electrodes As in the previous study of the theta rhythm profile in the freely moving rat "0, two macroelectrodes were implanted in the hippocampus to serve as references. One reference electrode was placed dorsal to the pyramidal layer of CAt and the other ventral to the hippocampal fissure. As reported before 20, it was found that in the freely moving animal theta rhythms at the two electrodes were approximately phasereversed and the amplitude modulations of the two signals were dissimilar. Fig. 2a shows a typical example of signals from an electrode pair recorded while the animal was moving. The polarity of the signal at electrode 2 was reversed during recording to facilitate comparison. The phase angle between the two signals, as shown with the signal from electrode 2 reversed, is 4.2" ~ 5.1 ° (S.E.M.) (electrode 2 leads electrode 1), and successive pairs of corresponding amplitudes are uncorrelated (r amp . . . . . 0.22). The relationship between the signals displayed a different quality after treatment with
63 (a) Freely moving Negotive elect. 2 /.El 2
Elect. i
(b) C u r a r i z e d
Negotive
elect. 2
~kA,/~/~;~,t,~,,.A.j~k~/¢?.~l..v~%/~',./~A.
1480 ,u,v .1.150/N
E,ect. 1
1 sec
Fig. 2. Signals from one pair of fixed electrodes whose locations are shown at left. In this and subsequent figures, location designations refer to K6nig and Klippel's atlas 9. Rat is freely moving in (a) and curarized in (b). Voltage scale applies to (a) and (b). Signals from electrode 2 and electrode I are phase-reversed both before and after injection of curare. Polarity of electrode 2 signal was reversed here for ease of comparison. In the presence of curare, amplitude similarity is increased. Further de-
tails in text. curare. Fig. 2b shows this change in relationship. The phase angle between the signals remained virtually unchanged, electrode 2 leading the negative of electrode 1 by 7.8 ° ± 1.6°, but the amplitude modulations were now very similar, as were the higher harmonic components. The amplitude correlation coefficient was +0.52. The signals from pairs of reference electrodes in 6 animals were analyzed, and all showed the same phenomenon. In the moving state, the signals were approximately phase-reversed and the amplitudes were uncorrelated or showed some negative correlation (range of r amp: - - 0.22 to --0.36). There was no significant change of phase relationship after curare but the amplitude modulations in each case took on a degree of positive correlation (r amp: +0.30-+0.58). Positive correlation was significantly greater after curare than before in each animal (P < 0.01). Phase reversal and null Fig. 3 shows signals recorded in a typical microelectrode penetration in the curarized rat. The signals from the two fixed electrodes were approximately phasereversed. (The average deviation from phase reversal for all fixed electrode recordings made during the penetration was 5.7 ° ~ 2.1°.) As the microelectrode was lowered through the neocortex, a theta rhythm was detected dorsal to the corpus callosum. The signal was approximately in phase with the signal from the dorsal reference electrode and, further, the individual shapes of corresponding waves and the time course of amplitude modulations of the two signals were very similar (upper two records of Fig. 3a). For convenience of discussion, signals with this relationship are termed isomorphic 2°. As the dorsoventral penetration continued into the stratum radiatum, the theta rhythm abruptly disappeared and was replaced by a null signal (Fig. 3b). The null signal was present for a dorsoventral distance of about 90/~m, below which the theta
64 {o)
t ool /
?
i
f(\F; J
!
electrode l 3430kL
A 4110#
(c)
(b)
,
, ql
'
~
%
e
~
f
,
[
5oo ~v
I
J,
I sec
Fig. 3. Records from penetration. Electrode positions shown in upper left. In this case, fixed reference electrodes and microelectrode penetration are at different rostrocaudal locations. Upper record is from dorsal reference electrode, middle record from microelectrode, and lower record from ventral reference electrode. Voltage scales for electrodes 2 and 1 are indicated in (c) for all records. Amplifier gain for microelectrode signal is halved in (c). Signals from electrodes 2 and 1 are phase-reversed. Microelectrode is dorsal to stratum radiatum in (a); signal is isomorphic with electrode2. Microelectrode is in proximal stratum radiatum in (b) and records a null signal, in (c) microelectrode is in ventral stratum radiatum, signal is isomorphic with electrode 1.
rhythm reappeared, now isomorphic with the signal from the ventral macroelectrode (Fig. 3c) and therefore approximately phase-reversed from the signal that had been detected by the microelectrode above the null. Deeper penetrations showed that isomorphism with the ventral reference electrode was maintained into the dorsal thalamus. (As in previous work 9°, for maximum accuracy in the computation of anaplitude ratios and phases, reference signals were used that were isomorphic with signals recorded from the microelectrode.) As indicated, the signals from dorsal and ventral reference electrodes were phase-reversed within 6 °. The average signals seen by the microelectrode above and below the null were also phase-reversed within a few degrees. Signals recorded at a single rostrocaudal position in the hippocampus were generally either in phase or phase-reversed within 10% When electrodes were in different rostrocaudal placements, however, somewhat greater deviations were found. In the experiment depicted in Fig. 3, for example, the microelectrode was not inserted at a rostrocaudal level identical to that o f the fixed electrodes but at a level approximately 700/~m caudal to them. In Fig. 3a the signal from the microelectrode lags the signal from electrode 2 by 12.2 1.9 msec (about 30°). The situation is similar below the point of phase reversal. In Fig. 3c, the signal from the microelectrode lags the signal from electrode 1 by 13.1 7 msec. Within the rostrocaudal limits A4110 #m to A3180 ,um (ref. 9), the maxi-
65
[
Fig. 4. Penetration to determine location of null and phase reversal. Microelectrode was advanced to ventral limit of null region, withdrawn 60/era to the dorsal limit, and ferric ions were deposited. In three such preparations null was located 30 150/~m below pyramidal layer.
mum time displacement encountered between isomorphic signals was 14.3 msec. Penetrations in this experiment were designed to fall within the mediolateral limits of the granule cell layer of dorsal blade of the dentate gyrus. A clear null extending through a dorsoventral span of 50-110 #m was found in all such traverses. Three penetrations were terminated at the null in order to determine unequivocally the dorsoventral position of the null. All three experiments showed the null to be in the proximal stratum radiatum, extending approximately from 30 to 150/~m below the pyramidal layer. Fig. 4 shows the termination of one of these penetrations. In this case, after the null was detected, the microelectrode was advanced 60/~m until the signal began to show renewed theta activity. The microelectrode was then withdrawn 60 pro, the experiment terminated, and ferric ions were deposited at this position. Two traverses were more medial than the others, passing just medial to the crest of the granule cell layer of the dentate gyrus. In these exceptional cases, instead of a null, a region of irregular signals extending about 250/~m dorsoventrally was found, above and below which the phase of the theta rhythm was reversed.
Amplitude profile Fig. 5 shows a typical type I amplitude profile in a curarized rat, with a more or less constant amplitude of theta rhythm for positions dorsal to the point of phase reversal and a sharp peak ventral to it. Five experiments were performed to determine the location of the amplitude peak in the hippocampus. In these cases, recordings were made every 44 #m in the vicinity of the peak, and the penetration terminated within 88/~m of its occurrence. Fig. 6 shows the results of one such experiment. The penetration was terminated 44 #m ventral to the peak (lower right panel) and the point of termination proved to be just ventral to the hippocampal fissure (lower left panel). Peak positions in the brain were determined by extrapolating back on the histological
66 800 ,~543C,~.
,,0 700 o
F~
~ebec~rode~/
A "l
zL 600
d £
-~ 500 (3)
o 400 (.~
E -~ 3OO .-,.~_ Q.
200
E < 100
electrode 2
'
electrode 1
-I
Null
[
I
I
I
I
I
0
I
2
3
4
5
352 H. Intervals Fig. 5. Amplitude profile: normalized amplitude of theta rhythm v e r s u s depth in the brain. Left to right: dorsoventral penetration of microelectrode in units of 352 #m (one turn ofmicroelectrodeadvancing screw). Zero chosen for abscissa is arbitrary. Ordinates are normalized theta amp|itudes. Vertical bars are S.E.M. Line above abscissa indicates the relationship of microelectrode signal to reference electrode signal. Amplitude profile is type 1.
sections of this and the other four penetrations from the positions of the electrode tips through the short distances derived from the amplitude plots. Allowing a possible error of as much as half the test point spacing in the position of the peak on the amplitude plots and taking into account possible errors introduced by the finite size of the Prussian blue markings, it was nevertheless found that all peaks were located within a span running from 70 # m above the hippocampal fissure to 50 # m below it.
Time-course of effect of curare on theta profile The finding here of a type I profile in the curarized rat taken with the previous report that the profile in the freely moving animal was type II offered the opportunity to follow the time-course of the effect of curare on the theta profile. A penetration was made in the curarized rat to the null point and the microelectrode was locked in place. This point is indicated as A in Fig. l a. The rat was then allowed to recover from the effect of the curare. It was to be expected that a substantial theta rhythm would be recorded from this location in stratum radiatum once the profile had returned to type II (Fig. l a) and the time-course of the effect of curare on profile could be gauged by the appearance of this theta rhythm. This procedure was carried out in 5 animals. Fig. 7 shows a typical result. The upper record is from a reference electrode in stratum oriens, the lower record is from a microelectrode lowered to the null position in stratum radiatum. Fig. 7a shows the null signal approximately 2 h after injection of curare and penetra-
67
1000 o "G 900 ¢M
tb
800
E 700
._-=_ E
lotion
I
600 i
I
I
[
I
I
i
I
I
i
I
Depth, 44p. intervals Fig. 6. Experiment to determine location of peak. Lower right: plot of normalized amplitude of theta rhythm v e r s u s depth in brain. Penetration in the dorsoventral direction is represented from left to right on the abscissa. Electrode was advanced in 44 p m steps and the penetration was terminated 44 /~m ventral to the peak. Upper photograph shows location of penetration. Enlargement at lower left shows position of electrode tip close to hippocampal fissure at termination.
tion to the null point, which was basically identical to the record originally obtained upon reaching the null point approximately 15 rain after injection. At the time of the recording of Fig. 7a, the animal was still being artificially respired but was displaying the first signs of recovery of voluntary muscular activity. Fig. 7b was recorded 45 rain later. The animal was sufficiently active to free itself from the face mask and was breathing unaided. Eight minutes later (Fig. 7c) the animal was moving in its cage with no effects of paralysis evident in its behavior. The signal at the microelectrode exhibited sporadic bursts of theta rhythm, although the theta rhythm recorded at the reference electrode was regular and clear. The signal at the microelectrode continued to gain in amplitude and regularity. Fig. 7d, recorded 30 min after (c) shows a signal at the microelectrode approximately equal to the reference signal in amplitude and leading it by about 50° as would be expected in the stratum radiatum of the freely moving animal 2o.
68 (a)
(b)
(c)
(d)
I 420/~V ~l~I~l~l~ ~~~ I ~~ iO o ~ v~l ~ 1 sec Fig. 7. Records from curarized rat during recovery. Upper records are from fixed reference electrode
in stratum oriens. Lower record is from microelectrode which was inserted into the curarized animal and advanced to the null position. Voltage scales in (d) apply to all records. In (a) animal had been immobilized for 2 h and was showing first signs of voluntary movement. Null signal was essentially the same as that observed when the electrode was fixed in position, in (b), recorded 45 rain later, animal was breathing unaided. In (c), recorded 8 rain after (b), animal was moving with no behavioral effects of curare evident. Although fixed electrode shows a continuous theta rhythm, microelectrode rhythm is clearly of a different type. Signals in (d) were recorded 30 min after (c), by which time the microelectrode signal had reverted to clear theta rhythm. DISCUSSION
The present experiment confirms earlier results obtained in the freely moving rat and in the curarized rat on profiles o f theta rhythm, and indicates that systemically introduced curare affects the neural system responsible for the generation o f theta r h y t h m in the rat hippocampus. The effect is detected as a change from type II profile in the freely moving state to a type I profile under curare, in which there is an amplitude peak in the vicinity o f the hippocampal fissure. In addition to the profile change, curare brings about a closer synchrony o f the two phase-reversed components o f theta rhythm (dorsal and ventral) as is evidenced by the increased correspondence in amplitude modulation that occurs after injection. In a previous report '>0it was suggested that the type lI profile reflected the activity o f two coupled generators, one in the dentate gyrus and a second in the overlying CAj layer. The existence o f two generators o f this nature has recently been demonstrated in the lightly anesthetized rabbitL In the rabbit the profile was type 1, the peak being ill the dentate gyrus 4. The same profile with the peak in the dentate gyrus is reported in the freely moving rabbit in the following paper 2a. It is not known whether or not the profile f o u n d in the present experiment results from the activity o f both dentate and CA~ generators. It is possible that the peak actually lies slightly dorsal to the fissure and that it reflects activity only in the apical dendrites o f CA1. The position o f the peak so close to the fissure may also indicate that a dentate c o m p o n e n t is present. The profile found here in the curarized rat is very similar to that reported in the curarized rabbit. Both are type l with a phase reversal and null in stratum radiatum
69 a n d with a m o r e ventral peak. In the c u r a r i z e d r a b b i t the p e a k was r e p o r t e d to be in distal region o f the apical dendrites o f CA1, b u t its precise l o c a l i z a t i o n was n o t a t t e m p t ed s. It m a y be t h a t the profiles in the c u r a r i z e d rat a n d r a b b i t are the same, each different from the profile in the freely m o v i n g animal 4,2°,21. A study localizing the p e a k in the c u r a r i z e d r a b b i t profile is required to resolve this point. The d i s p a r i t y in time o f m u s c u l a r a n d profile recovery from the effects o f curare suggests that the effect on theta profile is not a consequence o f paralysis. Rather, the change b r o u g h t a b o u t in the t h e t a - g e n e r a t i n g system w o u l d seem to require specific central intervention either within the h i p p o c a m p u s or elsewhere in the neuronal netw o r k associated with the theta r h y t h m t L Central effects o f systemic injection o f curare have been r e p o r t e d t'5. These have consisted o f the reversible b l o c k a d e o f electrically i n d u c e d evoked potentials in the neocortex o f the cat after intravenous a d m i n i s t r a t i o n o f curare. The medial septum has been identified as a p a c e m a k e r o f t h e t a r h y t h m iv and the medial s e p t a l - h i p p o c a m p a l p r o j e c t i o n is believed to be cholinergic 5,1°,1:~. A n i o n t o p h o r e t i c study ~ has shown that, a l t h o u g h most acetylcholine-sensitive cells in the h i p p o c a m p u s a p p e a r to be muscarinic, there are cells that are selectively b l o c k e d by curare. F u r t h e r clarification o f the role o f curare in modifiying the patterns o f theta r h y t h m awaits the identification o f the p a r t i c u l a r h i p p o c a m p a l neurons and circuits involved in theta r h y t h m generation. ACKNOWLEDGEMENTS This study was s u p p o r t e d by grants f r o m the N a t i o n a l A s s o c i a t i o n for M e n t a l Health, M e r c k a n d Co., a n d the G r a n t F o u n d a t i o n . T h e a u t h o r t h a n k s Dr. Charles A b z u g for his critical reading o f the manuscript.
REFERENCES I APOSTOL, G., AND CREUTZFELDT, O. D., Cross correlation between the activity of septal units and hippocampal EEG during arousal, Brain Research, 67 (1974) 65-75. 2 ARTEMENKO, D. P., Role of hippocampal neurons in theta-wave generation, Nierafiziologiya, 4
(1972) 531-539. 3 BISCOE, T. J., AND STRAUGHAN, D. W., Micro-electrophoretic studies of neurones in the cat hippocampus, J. Physiol. (Land.), 183 (1966) 341-359.
4 BLAND, B. H., ANDERSEN,P., AND GANES,T., Two generators of hippocampal theta activity in rabbits, Brain Research, 94 (1975) 199 218. 5 DUDAR,J. D., The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus, Brain Research, 83 (1975) 123 133. 6 EISSENBERG,E., Intermittent positive pressure in the curarized rat: implication for cardiovascular conditioning, Physiol. Behav., in press. 7 GREEN, J. D., AND ARDUINI, A. A., Hippocampal electrical activity in arousal, J. Neurophysial., 17 (1954) 533-557. 8 GREEN, J. D., MAXWELL, D. S., SCHINDLER, W. J., AND STUMPF, C., Rabbit EEG 'theta" rhythrn: its anatomical source and relation to activity in single neurons, J. Neurophysiol., 23 (1960) 403 420. 9 K/3NIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Kreiger, Huntington, N.Y., 1970, 162 pp. 10 LEWIS, P. R., AND SHUTE, C. C. D., The cholinergic limbic system: projection to hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-539.
70 I I LORENTE DI~ NO, R., Studies on the structure of the cerebral cortex. 11. Conti~3uation of lhe ,itidy of the ammonic system, J. l'~ychol. Nemol. ~'Lpz. ), 46 (1934) 113-177. 12 McLENNA~,, H., At;D M iLL~;R. J. J.. The hippocampal control of neuronal discharges in the ,,cpttim of the rat, J. Physiol. fLoral., 237 (1974) 607 624. 13 MosKo, S., LVNC:H, G., AxlJ COrMAN, C. W.+ The distribution of septal projections to the hippocampus of the rat, J. comp. Neuro[., 152 (1973) 163 174. 14 NATHAN, M. A., AND REIS, D. J., Hypoxemia, atelectasis, and the elevation of arterial pressure and heart rate in paralyzed artificially ventilated rat, L(/b Sci., 16 (1975) 1103--1120. 15 PURPURA, O. P., AND GRUNDFEST, H., Nature of dendritic potentials and synaptic mechanisms in cerebral cortex of cat, J. Neuroplo,siol., 19 (1956) 573-595. 16 SCHEmEL, M. E., AND S(THEmH_, A. B., Histological localization of microelectrode placement itl brain by ferrocyanide and silver staining, Stain Technol., 31 (1955) 1-5. 17 STUMPF, C., Drug action on the electrical activity of the hippocampus, Int. Rev. Xeurobiol., 8 (I 965) 77-138. 18 VANDERWOLF,C. H., Hippocampal electrical activity and voluntary movement in the rat, Eh, ctroencepk, clit#. Neurophysiol., 26 (1969) 407- 418. 19 WINSON, J., A compact movable microelectrode assembly for recording from the freely-moving rat, Electroenceph. olin. Neurophysiol., 35 (1973) 215-217. 20 W[NSON, J., Patterns of hippocampal theta rhythm in the freely moving rat, Electroenceph. olin. Neurophysiol., 36 (1974) 291 30l. 21 WINSON, J., Hippocampal theta rhythm. 11. Depth profiles in the freely moving rabbit, Brain Research, 103 (1976) 71 .79.
