Habituation of neuro-electric responses negative results

Nouropsychologia, 1968, Vol. 6, pp. 65 to 72. Pergamon Press. Printed in England

HABITUATION OF NEURO-ELECTRIC RESPONSES NEGATIVE RESULTS FREDERICK J. BREMNERand J. GUTHRIE FORD Trinity University, San Antonio, Texas (Received 20 September 1967)

Abstract--Brain waves recorded from electrodes permanently implanted in the hippocampi of white rats were spectrally analyzed in order to investigate neuro-electric changes occurring during presentation of a stimulus at regular intervals. Although the regular presentation of a stimulus is usually accompanied by the habituation of orientation responses the neuroelectric data recorded in this study did not show characteristics definable as habituation. 17 IS generally held that neuro-electric responses recorded from the brain, wane as Ss lose interest in a stimulus [1-4]. This phenomenon is referred to as habituation which is taken to mean the decrease in amplitude of some electro-physiological event recorded from the brain. It is assumed that this supposed electro-physiological habituation occurs simultaneously with the habituation of some overt behavioral response. There are two classes of neuro-electric responses for which habituation has been claimed: (a) Those responses called evoked potentials, a biphasic response of short duration and fast rise time which closely follows a sensory input in time; and (b) those responses called inherent rhythms (alpha, theta, 40Hz) which occur at their own frequency regardless of the frequency of the incoming sensory stimulus. A large majority of the investigations on neuro-electric habituation have been done using evoked potentials as data [2]. These studies have concentrated on subcortical and particularly brain stem sites in the auditory system [5]. Recently published more sophisticated analyses have demonstrated, however, that evoked potentials in brain stem auditory structures do not habituate as was earlier reported [6-9]. There still seems to be some question whether or not habituation occurs at the cortex and higher brain levels [6, 10, 11]. Because of this, it seemed worthwhile to investigate high level non-auditory structures for possible habituation effects. Due to its supposed relationship to attention mechanisms, the limbic system was chosen for this invesigation [12-15]. Moreover, rather than investigate evoked potentials, the data of choice were inherent rhythms. Like the early evoked potentials data, it has been reported that inherent rhythms in the hippocampus (6Hz theta) habituated [13, 16, 17]. Studies using computer analysis, however, have never reported the diminution to hippocampal theta during any behavioral treatment studied to date (except for the effect of LSD-25 [18-20, 14, 15]). With these seeming contradictions in hand, the following study was undertaken. METHOD Subjects Eleven adult, male Holtzman rats were implanted bilaterally with bipolar stainless steel electrodes in both hippocampi. 65

66

FREDERICKJ. BREMNERand J. GUTHR1EFORD

Apparatus The EEG responses were recorded on both a strip-chart recorder and on an FM analog tape recorder. These taped EEG responses were later spectrally analyzed [21, 22] by either an IBM 1800 or a Philco 2000 computer. The stimulus to be habituated was the extinction of an incandescent bulb illuminated by a 6V power supply. The duration and frequency of "light off period" was controlled by an electronic programmer built from DEC logic modules.

Procedure Eleven Ss were stereotaxically implanted bilaterally in the hippocampus (Bregma; post. 3ram, lat. 3ram, and --3mm from skull) as well as other sites not reported in this paper. Following a one week recovery from surgery, the Ss were habituated to a light stimulus by placing them on a 4 by 8 in. platform atop a 36 in. tower inside a 4 ft square soundproof electrically shielded chamber. The stimulus to be habituated was the extinguishing of an indirect flood light, whose intensity was 0.5 ft candles, reducing the illumination inside of the chamber to 4.0 ft candies. Since the light reflected off all the walls of the chamber, the stimulus field was very large, rendering the stimulus inescapable to the Ss. Also, the stimulus was of nearly equal intensity in all directions, thus preventing spurious variations due to changes in receptor orientation as reported by WOaDEN and MARSH [23] for auditory stimuli. The decay function of extinguishing this light was slow enough to prevent any evoked potential from occurring in the hippocampograms. Three types of habituation trials were used in this study. Four Ss were habituated by presenting 50 Cs events a day for at least eight days. Since it has been reported that habituated responses are subject to spontaneous recovery, a second group of four Ss was habituated with 100 presentations for five days. (Both of the above will be referred to as "short habituation" runs). It has also been reported that the duration of the habitation session affects the course of habituation; therefore, three of the Ss habituated at 50 presentations per day were tested for six consecutive hours with the same stimulus. This was done by using a paradigm borrowed from MARSHand WOm~EN[24] in which after the first hour, the Ss are aroused and two data samples are taken, one before arousal and one after arousal. The stimulus was programmed to extinguish every 3 sec and to remain off for 1.36 sec before automatically illuminating again. The sample times used were 0 rain, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr after the Ss were placed in the chamber. It became apparent to the authors, however, that this paradigm actually might render the Ss only one hour habituated if one counted from the last arousal. In order to prevent the possible confounding affect of this procedure a second group of 3 Ss was habituated for six consecutive hours with the same sampling times but only aroused following the sixth hour sample.

RESULTS

Short habituations F i g u r e 1 c o n t a i n s typical s p e c t o g r a m s f o r a n a n i m a l d u r i n g s h o r t h a b i t u a t i o n . E a c h s p e c t o g r a m in this s t u d y r e p r e s e n t s 10 s t i m u l u s p r e s e n t a t i o n s . A section o f E E G d a t a lasting for o n e s e c o n d ( e p o c h ) was t a k e n at the i n i t i a t i o n o f the s t i m u l u s p r e s e n t a t i o n . T h e s e 10 o n e - s e c o n d e p o c h s w e r e t h e n a b u t t e d e n d to e n d by t h e c o m p u t e r y i e l d i n g 10 sec o f data. O n e h u n d r e d lags w e r e t h e n t a k e n f r o m this a b u t t e d s a m p l e y i e l d i n g o n e s p e c t o g r a m for the 10 sec chosen. T h e e p o c h s e x a m i n e d for s h o r t h a b i t u a t i o n s as s h o w n in Fig. 1 w e r e r a n d o m l y p i c k e d f r o m t h e p o p u l a t i o n o f s t i m u l u s p r e s e n t a t i o n o n a g i v e n day. T h e r e f o r e , e a c h s p e c t o g r a m is a m e a s u r e o f t h e p e r c e n t a g e o f d a t a falling in t h e t h e t a r a n g e f o r a p a r t i c u l a r day. F i g u r e 2 g r a p h i c a l l y displays the d a t a for six o f t h e eight Ss e x p e r i e n c i n g s h o r t h a b i t u a t i o n s (no m a g n e t i c t a p e r e c o r d i n g s w e r e m a d e o n the o t h e r t w o Ss). E a c h p l o t t e d p o i n t r e p r e s e n t s the a v e r a g e p e r c e n t a g e o f p o w e r falling in the t h e t a r a n g e ( 4 - 7 H z ) f o r six Ss d u r i n g e a c h d a y p l o t t e d . T h e r e are f o u r days p l o t t e d ; t h e first t w o days a n d t h e last t w o d a y s o f h a b i t u a t i o n . A l t h o u g h all the Ss r e c e i v e d 500 h a b i t u a t i o n trials, t w o o f the Ss b e i n g r e p o r t e d w e r e h a b i t u a t e d at 100 trials a d a y w h i l e t h e o t h e r f o u r w e r e h a b i t u a t e d at 50 trials p e r day. T h e a v e r a g e p e r c e n t a g e o f d a t a in the t h e t a r a n g e ( 4 - 7 H z ) stays q u i t e c o n s t a n t . T h e d a t a in Fig. 2 i n d i c a t e t h a t h a b i t u a t i o n d o e s n o t o c c u r d u r i n g t h e s e s h o r t sessions. T a b l e 1 c o n t a i n s the results o f an analysis o f v a r i a n c e p e r f o r m e d on these d a t a [25]. T h e r e were n o significant differences.

HABITUATION OF NEURO-ELECTRICRESPONSESNEGATIVERESULTS

67

Six hour cont&uous habituation

Figure 3 shows data for six Ss habituated for six consecutive hours. Each plotted point represents the total percentage power for a particular subject at the sampling time indicated on the absissa as determined by spectral analysis. The data spectrally analyzed ssoIo EARLY CS I_ HI PP 5 0 0 M.SEC 4 0 0 PTS,

• io i f req

cps

I

.,~S. OI 0 LATE CS

n.

i

L. HIPP 5 0 0 M.SEC. 4 0 0 PTS.

oa i

~ 4 s • •

i i

freq cps

1. Examples of spectrograms for subject 010 during short habituation. - The upper spectrogram was reduced from left hippocampal EEG for 10 stimulus presentations during the first day of habituation while the lower spectrogram represents the analysis of the left hippocampal EEG for 10 stimulus presentations during the fifth day of habituation. This animal was presented with 100 stimulus events per day. FIG.

60 50

40

~_~3o ~..U) IX

2O CS ALONE SHORT HABITUATION

10

I

2

// DAYS

1

2

F1G. 2. Summary plot of the average percentage power in the theta range (4-7 Hz) for six Ss during the first two and the last two days of short habituation. The arrows indicate the first standard deviation above and below the mean.

were similar to that described above except that the stimulus presentations chosen were consecutive rather than random as for short habituations. That is, one spectrogram was generated for 10 consecutive stimulus presentations during each sampling period (0, ¼ hr, ½ hr, 1 hr, 2 hr, etc.). Visual inspection of Fig. 3 shows the percentage of power of neuroelectric responses in the theta range wax and wane. Therefore a further statistical analysis

68

FREDERICK J. BREMNER and J. GUTHRIE FORD Table 1. Analysis of variance of short term habituations

Source

Ss

df

Between subjects

960

5

Within subjects

560

MS

F

1.51

Days

175

3

58.3

Error

581

15

38.7

1716

23

Total

was performed to determine any consistent pattern in the data. Table 2 shows the results of an analysis of variance performed on these Ss. One animal was omitted from the analysis of variance because it did not meet the preanalysis criterion of at least 30 per cent theta during at least one sample; this would indicate a poor implant with low signal to noise ratio. !

SIX HOUR HABITUATION

6oi 50

~40

~o3o oxo 20

016o._

~

~

\

/\

\

,'B,

C1~16

10

0.0 .25 .50

1.0

2.0 3.0 4.0 5.0

6.0

HOURS

FIG. 3. Summary of data for six Ss during six hours of continuous habituation. Each plotted point represents the total percentage of power in the theta range (4-7 Hz) at the time period indicated.

The data analyzed were percentage of power at each of the theta range frequencies (4-7 Hz) for each of the samples taken (the variable labelled "Frequency"). The sample periods were zero, ¼ hr, ½ hr, and 1 hr and then at the end of each succeeding hour until the completion of the six hour session (the variable labelled "Period"). Because two of the five Ss being reported were alerted after each sample, a comparison between those Ss alerted after each sample and those not alerted until after the sixth hour sample was made (the "Treatment" variable in the table). Since frequencies were found to be significantly different, a trend analysis was performed on this variable yielding a significant linear trend. In fact, each sample spectrally analyzed had more power at six to seven Hertz than at lower frequencies. Although "Frequencies" by "Treatment" by sampling "Period" was

HABITUATION OF NEURO-ELECTRIC

69

RESPONSES NEGATIVE RESULTS

f o u n d to be significant, one is always sceptical o f the reliability o f second o r d e r interactions, p a r t i c u l a r l y when no i n t e r p r e t a t i o n has been assigned to t h e m p r i o r to the analysis.

Separate analysis of first five stimulus presentations Even t h o u g h the variable labelled " P e r i o d " in the a b o v e analysis o f variance was f o u n d to be non-significant, a separate analysis o f the first five trials was u n d e r t a k e n since it has been r e p o r t e d that considerable h a b i t u a t i o n takes place d u r i n g the first few trials [5]. However, the results o f a completely r a n d o m i s e d design c o n d u c t e d on four Ss c o m p a r i n g the spectra o f the first five stimulus presentations with the second five stimulus p r e s e n t a t i o n s showed no significant difference. Table 2. Analysis of variance of long term habituations Source

Ss

df

665.17

4

Treatment

145.83

1

145.83

Error

519.34

3

173.11

2509.40

310

45.25

8

5.66

0.57

Frequency

929.10

16

54.85

13.20"

FXP

142.22

48

2.96

0.74

PXT

39.03

8

4.88

0.49

FXT

47.11

6

7.85

0.67

278.03

48

5.79

1.44"

1028.66

186

P X Sc

240.26

24

10.01

F X Sc

211.08

18

11.73

P X F X Sc

577.32

144

4.01

3174.57

314

Between subjects

Within subjects Period

FXTXP Error

Total

MS

F

0.84

* 0.05.

Effect of alerting the Ss Since it h a d been d e t e r m i n e d to c o m p a r e the six h o u r alerted and the six h o u r nonalerted samples if there was no significant difference between the two types o f six h o u r h a b i t u a t i o n runs (with the results o f Table 2 in hand) such an analysis was conducted. T a b l e 3 presents the results o f an analysis o f variance testing the d a t a recorded d u r i n g

70

FREDERICK J. BREMNER a n d J. GUTHRIE FORD

alerted and non-alerted samples taken at the end of the sixth hour. The two treatments are alerted and non-alerted, while Frequencies are the power at each of the theta range frequencies. The significant difference in frequencies means that there is not equal power at all frequencies. Table 3. Analysis of variance comparing 6th hour and 6th hour alerted Source

Ss

df

MS

F

Between subjects

71.85

3

Within subjects

168.87

28

Treatment

0.78

1

0.78

0.20

Frequency

76.85

3

25.62

6.57*

TXF

9.39

3

3.31

0.85

Error

81.90

21

240.72

31

Total * 0.05.

DISCUSSION It is apparent from the above that a habituation of neuro-electric response hypothesis, is hard pressed to explain the data reported. Nevertheless, there are several points needing further discussion. The results of this paper should not be taken to mean that overt behavioral responses do not habituate. On the contrary, observations of the Ss during the habituation trials showed that head and body orientation did wane and in many cases, the Ss went to sleep. Moreover, it is this very phenomenon which brings the present data into conflict with HERN~NDEZ-PE6N[5] and GRASTY,~Net al. [13]. When present data are pooled with other reports yielding negative evidence of neuro-electric habituation, the model used to explain the previously reported neuro-electric habituation cited above is called into question. That is, it was assumed by some investigators that the nervous system was capable of shutting itself off at the lowest levels. As DEUTSCH [ll] has so nicely pointed out, this would be extremely inefficient as a survival mechanism since the cortex would not be informed of sensory events once habituated. If, at some future date, these previously habituated stimuli should become "meaningful" for survival, the organisms would be ill informed of impending danger. The basis of this model for "self-shut-off" at down stream nuclei was the work of GALAMBOS [26]. It is interesting to note in this regard that while GALAMBOSreported neuro-anatomical structures for such "shut-off" mechanisms, he was never able to observe them working in the awakeunmanipulated animal [27]. It seems to the present authors the data reported in this study on inherent rhythms would strongly suggest that all sensory inputs are relayed to higher structures. The fact that some investigators [13, 16] have failed to report this is attributed

HABITUATION OF NEURO-ELECTRIC RESPONSES NEGATIVE RESULTS

71

to the fact that their d a t a analysis was by visual inspection while the d a t a o f the present study was m a t h e m a t i c a l l y analyzed by computer. I n short, the present d a t a w o u l d not s u p p o r t the GRASTYXN et al. [ 13] c o n t e n t i o n that theta r h y t h m is a correlate o f the orienting reflex and should therefore, h a b i t u a t e when the overt orienting responses habituate. Since the c o n t e n t i o n that u p o n repeated presentation o f a stimulus any associated neuroelectric responses will decrease in size, if n o t entirely disappear, is n o w being called into question, the authors w o u l d like to offer an alternative e x p l a n a t i o n within the b r o a d f r a m e w o r k o f a n attention hypothesis. R a t h e r t h a n a s s u m i n g that the neuro-electric code for attention is the presence or absence o f a neuro-electric response, let us assume t h a t the code is m o r e subtle, b u t nonetheless a signature o f the neuro-electric response depicting its i m p o r t a n c e to the organism. In the case o f inherent rhythms, like 4-7 Hz, h i p p o c a m p a l theta, it is suggested that the code m i g h t be a slight shift in the p o w e r o f certain frequencies. It has been r e p o r t e d t h a t during b o t h instrumental [19] a n d classical c o n d i t i o n i n g such shifts do occur [15]. In short, during h a b i t u a t i o n , spectrograms tend to be b r o a d a n d less p e a k e d than spectrograms t a k e n f r o m the h i p p o c a m p u s during learning. M o r e o v e r , these differences in spectra, t h o u g h subtle, are significantly different statistically [15]. Acknowledgment--This investigation was supported in part by U.S. Public Health Service Research Grant

NIMH 11618 and in part by the Trinity University Faculty Research and Development Committee.

REFERENCES 1. ISAACSON,R. L. (Editor) Basic Readings in Neuropsychology. Harper & Row, New York, 1964. 2. THOMPSON,R. F. Foundations of Physiological Psychology, pp. 501-513. Harper & Row, New York, 1967. 3. MAGOUN,H. W. The Waking Brain 111. Charles C. Thomas, Springfield, 1964. 4. GROSSMAN,S. P. A Textbook of Physiological Psychology, p. 646. John Wiley, New York, 1967. 5. HERN/~NDEZ-PErN,R. and STERMAN,M.B. Brain Functions, Annual Review of Psychology, p. 363, 1966. 6. WORDEN,F. G. and MARSH,J. T. Amplitude changes of auditory potentials evoked at cochlear nucleus during acoustic habituation. Electroenceph. clin. NeurophysioL 15, 866-881, 1963. 7. THOMPSON,R. F. and SPENCER, W. A. Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychol. Rev. 17, 16-43, 1966. 8. HORN, G. and HILL R. M. Habituation of the response to sensory stimuli of neurons in the brain stem of rabbits. Nature 202, 296-298, 1964. 9. HORN, G. In Advances in the Study of Behavior, D. S. LEHRMANet al. (Editor). Academic Press, New York, 1965. 10. WINTERS,W. D. Comparison of the average cortical and subcortical evoked response to clicks during various stages of wakefulness, slow wave sleep and rhombencephalic sleep. Electroenceph. clin. NeurophysioL 17, 234-245, 1964. 11. DEUTSCH,J. A. and DEUTSCH, D. Attention: Some theoretical considerations. Psych. Rev. 70, 80-91, 1963. 12. GREEN, J. D. and ARDUINI, A. A. Hippocampal electrical activity in arousal. J. NeurophysioL 17, 553-557, 1954. 13. GRASTYJ~N,E., LISSXK,K., MADARASZ,I. and DONHOFFER,H. Hippocampal electrical activity during the development of conditionel reflexes. Electroenceph. clin. NeurophysioL 11, 409-430, 1959. 14. BREMNER,F. J. Hippocampal activity during avoidance behavior in the rat. J. cutup, physioL PsychoL 58, 16-22, 1964. 15. BREMNER,F. J. Hippocampal correlates of classical conditioning. J. cornp. PhysioL PsychoL, in press, 1968. 16. SACHS,E., WEINGARTEN,M. and KLEIN, N. W., JR. Effects of chlordiazepoxide on the acquisition of avoidance learning and its transfer to the normal state and other drug conditions. Psychopharmacologia 9, 17-30, 1966. 17. TORII, S. and WIKLER,A. Effects of atropine on electrical activity of hippocampus and cerebralcorte in cat. Psychopharmacologia. 9, 189-204, 1966. 18. ADEY,W. R. and DUNLOP, C. W. The action of certain cyclohexamines on hippocampal system during approach performance in the eat. J. Pharmac. exp. Ther. 130, 418-426, 1960.

72

FREDERICK J. BREMNER and J. GUTHRIE FORD

19. Ar~EY, W. R., DUNLOP, C. W. and HENDR1X C, E. Hippocampal slow waves. A.M.A. Archs. Neurol. 3, 74-90, 1960. 20. TRAVIS,R. P. The effects of septal lesions on hippocampal activity. Ala. J. Med. Sets. 1, 399403, 1964. 21. WALTER,D. O. Spectral analysis for electroencephalograms: mathematical determination of neurophysiological relationships from records of limited duration. ExpL Neural. 8, 155-181, 1963. 22. BLACKMAN,R. B. and TUKEY, J. W. The Measurement o f Power Spectra. Dover Publication, New York, 1958. 23. WORDEN, F. G. and MARSH, J. T. Amplitude changes of auditory potentials evoked at cochlear nucleus during acoustic habituation. Electroenceph. clin. Neurophysiol. 15, 866-882, 1963. 24. MARSH, J. T. and WORDEN F. G. Personal communication, 1964. 25. WALKER, H. W. and LEV, J. Statistical Inference. Henry Holt, New York, 1953. 26. GALAMBOS,R. Suppression of auditory nerve activity by stimulation of efferent fibers to the cochlea. J. Neurophysiol. 19, 424-437, 1956. 27. GALAMBOS,R. Personal communication, 1966.

R6sum6--Les ondes c6r6brales enregistr6es ~tpartir d'61ectrodes implant6es de fagon permanente dans les hippocampes de rats blancs 6taient soumises ~t l'analyse spectrale dans le but de rechercher les modifications neuro-61ectriques survenant pendant la pr6sentation h intervalle r6gulier d'un stimulus. Bien que la pr6sentation r6guli6re d'un stimulus soit accompagn6e en g6n6ral par l'habituation des r6ponses d'orientation, les donn6es neuro-61ectriques recueillies dans cette 6tude ne pr6sentent pas les caract6ristiques de celles reconnues ~t l'habituation. Zusammenfassung--Es wurden Hirnstr6me yon dauerimplantierten Elektroden aus dem Hippocampus weisser Ratten abgeleitet und mit der Absicht spektral analysiert, neuroelektrische Anderungen festzustellen, die sich w~ihrend eines Reizes in regelmfissigen Abstfinden zeigen. Obgleich bet einer regul~ren Reizung gew6hnlich Zeichen yon Orientierrungsantworten auftreten, ergaben die abgeleiteten neuroelektrischen Daten im Verlaufe dieser Untersuchung keine charaktertischen definierbaren Merkmale einer Habituation.