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Control of the rat's circadian self-stimulation rhythm by light-dark cycles
Physiology and Behavior, Vol. 14, pp. 7 8 1 - 7 8 9 .
Brain Research Publications Inc., 1975. Printed in the U.S.A.
Control of the Rat's Circadian Self-Stimulation Rhythm by Light-Dark C y c l e s I MICHAEL TERMAN AND JIUAN S. TERMAN
Department o f Psychology, Northeastern University, Boston, MA 02115
(Received 11 October 1974) TERMAN, M. AND J. S. TERMAN. Control of the rat's circadian self-stimulation rhythm by light-clark cycles. PHYSIOL. BEHAV. 14(6) 781-789, 1975. - Rats with hypothalamic and septal electrodes were maintained in continuous test environments where bar-press responses produced brief reinforcing electrical stimulations. Long-term trends in response emission were measured under continuous exposure to light, dark and 12 hr light-dark alternations. In addition, transient behavioral adjustment to sudden 180° phase shifts in the light-dark schedule was studied. The ambient light condition was found to control the period and phase of the circadian rhythm of brain self-stimulation behavior, as quantified by Fourier analysis. The circadian period was greatest under constant light (up to 24.90 hr under dim illumination), and approximated 24.00 hr under constant dark. Successful nocturnal entrainment to 12 hr light-dark alternations was obtained, with the peak of the 24 hr Fourier fundamental occurring in the middle-to-late dark segments. Three to 11 days were required for re-entrainment to 180° light-dark phase shifts, during which the behavioral oscillation period increased to values comparable to periods under constant light. The rate of re-entrainment appeared to be proportional to illumination intensity during light segments. Circadian rhythms
Electrical brain stimulation
LD phase shifts
LD entrainment
and water. Behavioral periods slightly exceeding 24 hr indicated that the rhythm was not entrained by uncontrolled diurnal variables. The question of entrainability of the selfstimulation rhythm by light-dark (LD) cycles is important to define the operating characteristics of this circadian system in terms of its susceptibility to external control, and to discover possible functional interrelationships to more extensively studied behavioral and physiological circadian systems. (One could guess that interference by brain stimulation with inferior accessory optic tract function might affect the rate of entrainment to LD schedules [ 19,23] ; or that the unusually stereotyped behavior patterns maintained by brain stimulation might lead to a pathological state akin to behavioral stress, rendering external entrainment agents ineffective [ 27 ] .) Several experimental procedures address themselves to the question of entrainability: The circadian periods may be compared under LD schedules, constant light and constant dark. Free-running periods deviating from 24 hr should lock into strict 24 hr rhythmicity under an effective LD 12:12 cycle. Further, the behavioral oscillator must be shown to adjust dynamically by changing its phase in response to programmed phase shifts in the entraining agent
STUDIES of behavioral steady-states under the control of reinforcement contingencies have rarely taken into account circadian oscillations that may co-determine momentary response probability. The typical use of brief test sessions, run at a standard time of day (and often with trial-by-trial restrictions), may obscure the contribution of circadian variables to the behavior stream. In contrast, the biological literature is replete with examples of continuous circadian oscillations (cf. [1,9]), including neural, hormonal and metabolic rhythms that may be functionally linked to behavioral phenomena. Analyses of behavioral circadian rhythms have concentrated on gross activity measures and running wheel activity (e.g., [4,24] ), or feeding and drinking (e.g., [33]), where high rates of spontaneous emission insure long-term data production. The application of a reinforcement contingency allows experimenters to study circadian oscillations in specific behavioral units that would not otherwise show high probability in an organism's daily repertory. Terman and Terman [29] showed that the rate of the bar-press operant maintained by reinforcing brain stimulation oscillates in a circadian rhythm when rats respond under contant dim illumination with free access to food
1This research was supported in part by NASA Grant NGR 22-011-070 and HEW Grant RR07143. We owe special thanks to J. Anlinker (NASA), for advice and encouragement; J. Zimmerman and C. W. Tyler, for assistance in developing procedures for waveform analysis; J. Armington and the Northeastern Unversity Computation Center staff, for computation facilities and advice; M. Raibert, for preparation of computer graphics; H. Mahut, for assistance in histological procedures; and L. Thorington (Duro-Test Corp.), for contribution of Vita-Lite sources. Reprints may be obtained from M. Terman, Department of Psychology, 440 United Building, Northeastern University, Boston MA 02115. 781
782 [18]. Annau et al. [2] have demonstrated nocturnal activity patterns for self-stimulation, concurrent with feeding and drinking rhythms. Their main entrainment index was the percentage of responding in the dark segment of an LD 12:12 schedule. This index is sensitive to both amplitude and phase characteristics of the oscillating behavioral function, and is not a direct measure of periodicity. The nocturnal response patterns may be presumed to have been entrained, but the analysis did not document an oscillation period locked strictly to the LD period over extended test sessions, and the procedure did not demonstrate dynamic phase adjustment to LD cycle shifts. In the present experiment, the self-stimulation rhythm was examined under constant light, constant dark and a succession of LD 12:12 phase shifts, within single animals whose oscillation patterns contrasted in amplitude and phase characteristics. Possible locus specificity of the rhythmic effects was tested by the use of both hypothalamic and septal electrode placements. Fourier analysis was applied to specify phase and period information independently of amplitude (i.e., overall response output) variations. METHOD Animals
Four adult male pigmented rats (Lewis/BN stock, caesarian derived) were used. Under sodium pentobarbital anesthesia, each rat received a chronic bipolar electrode implant (Plastic Products MS 303). Following experimentation, the brains were examined histologically and electrode tip placements (cf. [ 14] ) were identified in the following areas: Rat 3E, nucleus septi lateralis; Rat 5E, fasciculus medialis prosencephali; Rat 8E, nucleus septi medialis; Rat 2G, nucleus ventromedialis (hypothalami), pars anterior. For convenience, this report labels the placements for Rats 3E and 8E as septal, and Rats 5E and 2G as hypothalamic.
TERMAN AND TERMAN Since current amplitude changes often induced transients in the data, response rates were allowed to stabilize before steady-state circadian rhythm data were considered. For Rat 3E, current levels ranged from 0.30 to 0.50 mA (p-p voltage monitored by oscilloscope across a resistor in series with the animal); Rat 5E, 0.50 to 0.60 mA; Rat 8E, 0.12 to 0.20 mA; Rat 2G, 0.08 mA. Previous data [29] show that current amplitude controls daily response output, but not the periodicity of the free-running rhythm of self-stimulation behavior. The experimental contingencies (continuous reinforcement throughout all test phases) were controlled by solidstate logic (DEC K-series Flip Chips) located in a room separate from the test chambers. Responses were counted by cumulative recorders, and reduced into tabular format in 1 hr blocks for subsequent computer analysis. Fourier analyses, least-squares fits, and statistical tests were run on PDP-12, LAB-8, and CDC Cyber 70 computer systems. The graphical presentations in Fig. 2 and 3 were prepared by the CDC's plotting facility. Procedure
Table 1 summarizes the successive experimental phases for each animal, from which data are sampled in the present report. Each rat began the experiment with a sequence of light-dark schedules. Light and dark segments were 12 hr long, with light turning on either at 6 a.m. (EST) or 6 p.m. We use abbreviations denoting successive 6 hr blocks of L and D in the 24 hr day (e.g., DLLD or LDDL) to specify experimental phases involving different light-dark arrangements. When light-dark cycles were inverted (180 ° phase shifts) the dark segment was extended by 12 hr on the first transition day. Following LD entrainment tests, the animals were exposed to periods of constant dark and constant light, to verify the presence of free-running (circadian) rhythms, and to specify their periods.
Apparatus
The animals were maintained in chambers with sound attenuation, programmable light schedules, constant temperature (21°C) and relative humidity ( 4 0 - 5 0 percent). Inside each environmental control chamber was a compartment constructed of a 24 cm dia. Plexiglas cylinder, with water and Purina Lab chow available continuously via drinking tube and food cylinder mounted on the wall. A Lehigh Valley mouse lever was also mounted on the wall at the level of the rat's head. The compartment floor was constructed of wire mesh, with a large droppings tray below. (Interruptions for maintenance - food, water, cleaning - were required less than once per week, allowing virtually uninterrupted long-term sessions.) A Vita-Lite flourescent source (see Table 1) was mounted on the wall of the chamber outside the test compartment, with on-off functions controlled by an external timer. Light intensities (see Table 1) were calibrated by placing an Eppley thermopile at the rat's position facing the light source, within the test compartment. A pulley-swivel mercury commutator system [6] was attached to the ceiling of the chamber above each compartment, for delivery of reinforcing brain stimulation. Stimulations were 0.5 sec sinusoids from a constant-current stimulator. Current amplitude was set at levels maintaining longt e r m self-stimulation behavior, and required periodic increases over the many months of continuous testing.
RESULTS The entrainment effect of successive LD 12:12 phases is summarized in Fig. 1, where mean daily response proportion is plotted in 3 hr blocks, to correct for absolute response rate variations within and across animals during the test series. These steady state data are based on results for the final week of each condition. The filled bars represent daily response proportions for the 12 hr dark segment, which began alternately at 6 p.m. and 6 a.m. across successive experimental phases. The details of the response proportion profile vary across conditions, but typically response proportions peaked in the dark and dipped in the light, displaying a nocturnal activity pattern. In all cases, the repeated 180 ° LD phase shifts successfully re-entrained the behavioral rhythm, so that peak activity periods fell at times corresponding to low activity periods in the previous condition. The fine pattern of the behavioral rhythm showed some change with continued testing and light-dark inversions, for all rats but 2G: there was a tendency for the differential between active and quiet phases to become accentuated as a function of time. Rat 2G showed a comparatively constant cyclic pattern across conditions, with accelerating high response rates during dark, sometimes leading to a peak in the first 3 hr block of light. The latter finding suggests that light onset does not necessarily produce an immediate suppressive effect on responding. Rat
SELF-STIMULATION AND LIGHT-DARK CYCLES
783
TABLE 1 EXPERIMENTAL PHASES (LIGHT CONDITION)* Phase
Condition
Rat 3E
Rat 5E
Rat 8E
Rat 2G
I (DLLD)
Light on 6 a.m. Light off 6 p.m.
12-10-71 to 12-24-71
12-10-71 to 1-24-72
6-8-72 to 6-24-72
5-23-73 to 7-15-73
II (LDDL)
Light on 6 p.m. Light off 6 a.m.
12-25-71"~ to 1-16-72
1-25-72 to 2-15-72
6-25-72 to 7-25-72
7-16-73 to 8-9-73
III (DLLD)
Light on 6 a.m. Light off 6 p.m.
1-17-72 to 2-1-72
2-16-72 to 3-5-72
7-26-72 to 8-14-72
8-10-73 to 9-7-73
IV (LDDL)
Light on 6 p.m. Light off 6 a.m.
2-2-72 to 2-16-72
_
_
9-8-73 to 10-8-73
V (DDDD)
Constant dark
6-18-72 to 7-22-72
3-6-72 to 5-15-72
9-2-72 to 9-25-72
10-9-73 to 11-27-73
VI (LLLL)
Constant light
5-25-72 to 6-16-72
5-16-72 to 6-7-72
10-17-72 to 11-6-72
11-28-73 to 1-16-74
*Vita-Lite fluorescents served as the light sources, approximating the solar spectrum more closely than conventional lamps. Previous research [31] has demonstrated successful entrainment of deep-body temperature with this lamp. For light segments in the entrainment tests (Phases I to IV) Rats 8E and 2G received 15 #W/cm 2 illumination, and Rats 3E and 5E received 390 /~W/cm2. When the animals were tested under constant light (Phase VI), illumination was set at 15 tzW/cm~ in all cases. Use of the dimmer illumination was in response to reports of retinal damage due to constant exposure to bright light (of. [20] ). All light was absent during Phase V (constant dark) and the dark segments of LD cycles. ~'On the first day of Phases II, III and IV, the duration of the dark segment was extended to accomplish the 180° light-dark phase shift. Thus, the 3-day transition pattern between Phases I to II and III to IV was DLLD-DDDL-LDDL; for Phases II to III, LDDL-LDDD-DLLD. 2G's record differs from the others also in that the contrast in proportions between light and dark segments was not so great. Some of the data suggest a relative on-off activity pattern (e.g., Rat 5E, Phase II; Rat 3E, Phase III), though response proportions never fell to zero for an entire 3 hr block. However, the general profile was a gradual transition in response proportions from active to quiet periods and vice versa. To examine the transient behavioral adjustment during LD phase shifts, and free-running drift patterns in constant light and dark conditions, hourly response counts were divided into ranked quartiles in Figs. 2 and 3. The quartile plots indicate at a glance how tightly the periods of high and low responding are correlated with the light-dark schedule, the spread of the oscillation pattern (dense vs. loose packing of symbols) in each condition, and the rapidity and orderliness of response phase shifts when the light-dark cycle is inverted or eliminated. Note that the quartile data are not sensitive to transient or long-term changes in overall daily response output, since the quartile criteria are adjusted each day. In the light-dark conditions, Rat 3E showed responding
in the upper 2 quartiles almost exclusively in the dark segment, once the steady-state was attained after each lightdark cycle inversion. The onset of high activity was quite abrupt (cf. Fig. 1), and there was occasional spilling of high response rates into the first hour of light, especially after 1 - 2 2 - 7 2 . Symbols for the lower 2 quartiles are scattered fairly evenly throughout the light segments, and an occasional high-rate symbol also appears during quiet periods, reflecting transient response accelerations. The transition data at the start of each light-dark inversion are of special interest: the oscillation period abruptly increased beyond 24 hr, and the region of highest density symbols shifted to later hours over successive days, mapping the animal's adjustment to the new entrainment schedule. Note that the behavioral rhythmicity did not disappear during phase adjustment; it appeared similar to the stabilized entrainment rhythm except for its period. During the transition, which required about a week for establishment of the new entrained pattern, considerable responding occurred in the new light segment, which corresponded in clock time to the old dark segment. Thus, while the animal converged on its final steady-state nocturnal activity pattern, light did not
784
TERMAN AND TERMAN RAT 5 E
SEPTAL
RAT 3E SEPTAL 1-3-72
)-[
1-18-72
L
D
2L D
2-3-72
:--AM-4-- p M -~
RAT BE SEPTAL 5-2-72
Z 0 fOE 0 0 n.o. 03 Z 0 tl. 03 bJ
6-1
5-18-72
>_J C3
6AM
~AM-+- PM--I
12N
6PM
12M
6AM
12N
6PM
12M
2 - D A Y SPAN (HOURS) RAT 5E HYPOTHALAMIC Z
1/18 - 1/24/72
2/9-2/15/72
2/28-3/5/72
I'-0 n 0 0.. ~J o') Z 0 co L~J r~
~- A M -"-*- PM ~
R A T 2G H Y P O T H A L A M I C Z k-(3g 0 0 Q_
Z O co
FIG. 2. Ranked quartile plot of daily response output for Rat 3E, for 2 LI) 12:12 phase shifts ( 1 - 2 - 7 2 to 2 - 1 6 - 7 2 ) , and for steadystate samples in constant light ( 6 - 1 - 7 2 to 6 - t 6 - 7 2 ) and constant dark ( 6 - 1 7 - 7 2 to 7 - 2 - 7 2 ) . Each day's hourly response counts are divided into ranked quartiles, with symbol size proportional to activity level: f'dled squares, most active 6 hr; rectangles, second highest 6 hr; horizontal lines, third highest 6 hr; empty spaces, lowest 6 hr. The abscissa spans 2 days, facilitating representation of non-24 hr circadian oscillations, where the region of maximal symbol density is seen to drift across days during entrainment phase shifts and constant light conditions. Thus, with successive days on the ordinate, each day's data are presented twice: once at the right of the abscissa, and then one line below at the left. Vertical lines at 6 a.m. and 6 p.m. denote the time of light-dark transitions. Data for 3 days are omitted in this record, resulting in 24 hr blank spaces. On 1 - 8 - 7 2 , brain stimulation intensity was raised in order to increase baseline response output, and the entrained rhythm was briefly disrupted. On 1 - 1 4 - 7 2 , the electrode lead disengaged from the skull cap, resulting temporarily in extinction. (Note that the rhythm immediately recaptured its previous phase on the following day, when stimulation was resumed.) On 2 - 6 - 7 2 , failure of the recording apparatus obliterated the record; the animal was unaffected. The blank space between the final entrainment data and the constant light condition indicates a hiatus of several months between data samples.
>-
~AM--~--PM ~
FIG. 1. Daily response proportions (3 hr blocks) for successive 180 ° LI) 12:12 phase shifts. Data are averaged over 7 day steady-state samples at the end of each entrainment phase. Filled bars denote dark segments within the LI) 12:12 cycle; open bars denote light segments.
suppress responding directly. Under c o n s t a n t light (Fig. 2, starting 6 - 1 - 7 2 ) the oscillation pattern was maintained in a steady-state w i t h a p e r i o d i c i t y greater than 2 4 hr. However, the d e n s i t y o f s y m b o l packing in the quartile plot appears m u c h l o o s e r than under the preceding e n t r a i n m e n t c o n d i t i o n s , indicating less discrete peaking in the daily behavior pattern. As
SELF-STIMULATION AND LIGHT-DARK CYCLES during the transition period at the start of an inverted lightdark condition, the region of highest density symbols shifts to the right over successive days of constant light, indicating that peak responding occurred later every day. If each behavioral oscillation is viewed as one subjective day for the animal under constant conditions, it can be seen that approximately 12 objective hours were lost in 3 weeks of running under constant light. On 6 - 1 7 - 7 2 , Rat 3E was transferred suddenly to constant dark. The quartile plot shows that the region of highest density symbols immediately shifts to the right, and remains at approximately the same position as on the final day of constant light (8 p.m. to 8 a.m.). The loose density of symbols continues to indicate greater spread in the behavioral oscillation than under the former entrainment conditions, though the periods for both entrainment and constant dark approximated 24 hr. Thus, constant light had the effect of slowing the circadian oscillation (in accordance with Aschoff's rule for nocturnal animals [21]), with constant dark maintaining a period indistinguishable from 24 hr. Figure 3 shows a corresponding set of data in quartile format for Rat 2G. The response patterns differed in detail from those of Rat 3E (Fig. 2) in that the region of the highest quartile responding was concentrated around the time of the dark-to-light transition and was not scattered evenly thoughout the dark segment. A gradient of increasing symbol density from early to late hours of dark is apparent, describing a daily gradual rate oscillation pattern, which contrasts to Rat 3E's relative on-off pattern (see also the one-week histogram summaries in Fig. 1). Note that, despite the peak in light, a greater total response output occurred in the dark segment, which would produce a standard nocturnal light:dark activity ratio (cf. [2,33]). Rat 2G showed a transient lengthening of the oscillation p e r i o d at the start of an inverted light-dark cycle ( 8 - 1 0 - 7 3 ) , enabling the behavioral phase shift to be completed in about one week, similar to Rat 3E (Fig. 2). For Rat 2G the constant dark condition preceded constant light, and the results were similar to those of Rat 3E with the opposite test order. The daily distribution of responses increased under constant conditions, with a wider spread in the high-activity period and less consistent time of peak responding. Under constant dark, the oscillation period appeared close to 24 hr. When constant light was introduced on 1 1 - 2 8 - 7 3 , the oscillation period immediately lengthened: within approximately 3 weeks the highactivity period shifted by 12 hr, closely matching the drift rate for Rat 3E under constant light (Fig. 2). The finding of reliable daily oscillations of operant response output suggests the presence of a continuous periodic process whose parameters (phase, period, amplitude) may be specified by Fourier analysis. Under LD 12:12 entrainment conditions, the data illustrated above suggest a 24 hr behavioral period, which would be represented by a high-amplitude 24 hr fundamental in Fourier analysis (for computational procedures, see [7, 8]). In Fig. 4, the fit of this fundamental waveform to a 7-day averaged steady-state data sample for Rat 8E (Phase I)is illustrated. The close agreement between data and the fundamental show that most of the variance in this sample can be accounted for by a simple 24 hr sinusoid. Indeed, examination of amplitudes of the 2nd, 3rd, and 4th harmonics revealed no component that would significantly improve the Fourier data fit beyond that shown in Fig. 4. (Occasional high-amplitude 2nd and 3rd harmonics appeared in
785
RAT 2G H Y P O T H A L A M I C 7-?_5-73
B-II-73
1-13-7:5
1-29-75
hAM
IZN 6PM IZM 6AM 12N 2 - D A Y SPAN (HOURS)
6PM
12M
FIG. 3. Ranked quartile plot of daily response output for Rat 2G, for one LD 12:12 phase shift (7-24-73 to 9-7-73), and for steady-state samples in constant dark (11-12-73 to 11-27-73) and constant light (11-28-73 to 12-15-73). The graphical procedure is explained in Fig. 2.
+•o-
300u~ t~ o~ z O ~ o3 200t~ tv
RAT BE SEPTAL
;",,
',, ,,o
,,
;'
•
D'"
X+"a"a'
,0o-
. . . . .
+
. . . .
TIME
N. . . . .
in
61,M
. . . .
M
HOURS
FIG. 4. Waveform resynthesis for a 7 day steady-state sample under LD 12:12 (Phase I). Triangles represent averaged hourly response totals. Dots represent amplitude of the 24 hr Fourier fundamental.
786 other data samples, but were not consistently related to any procedure. One effect of such harmonic components is to shift the Fourier waveform into closer agreement with the behavioral data. However, such harmonics do not affect determinations of the circadian period.) Notable deviations from the fundamental sinusoid appear in Fig. 4 as transient response accelerations in the second hour of dark (7 p.m.), the last hour of dark (5 a.m.) and the first hour of light (6 a.m.). Such accelerations are typical of the steady-state entrainment data across animals, and may reflect secondary effects of the light-dark transitions that are distinct from the circadian process per se. The phase angle of the peak of the Fourier fundamental ($) provides a convenient statistical estimator of the moment of peak activity in the 24 hr day. Under LD 12:12 entrainment conditions ~b does not change systematically over days. During LD phase shifts, and under constant illumination conditions, the daily changes in ~b index the rate of the behavioral drift, allowing precise specification of periods deviating from 24.00 hr. Figure 5 shows ~b plotted over successive days across a sequence of light-dark schedules, for Rat 2G. The time of day and corresponding phase angles are replicated on the ordinate to enable following behavioral phase transitions across the daily boundary. U n d e r steady-state entrainment conditions, the peak occurred toward the end of each day's 12 hr dark segment, with an occasional day's peak early in light (cf. Fig. 3). Some variability in the position of the peak over successive days is evident, although the steady-state data show no consistent drift in phase angle over days, indexing a precisely timed 24 hr behavioral cycle. When the light-dark cycle was inverted (starting at 8 - 1 0 - 7 3 and 9 - 8 - 7 3 ) , the peak of the rhythm began to shift to a later hour over days, as suggested earlier in the response quartile plots (Figs. 2 and 3). The slope of these transient data specifies the drift rate exhibited by the animal as it adjusts its behavioral cycle to the new illumination condition. For both phase transitions illustrated, the analysis shows that the 180 ° shift in behavior required 9 - 1 0 days following the light-dark inversion. A summary of the number of days required for the 180 ° behavioral transition across all subjects in Phases I to IV is shown in Table 2. The two rats receiving bright light for daytime segments (3E and 5E) required from 3 to 7 days, while the two rats receiving dim light (8E and 2G) required 7 to 11 days, suggesting that greater light intensity controls more rapid behavioral phase adjustment to a new light-dark cycle. The time of the peak of the Fourier fundamental is shown for all animals across entrainment conditions, in Fig. 6. With the exception of Rats 5E and 8E under LDDL, two independent determinations (Phases I and III, Phases II and IV) of the peak were made for each condition. In all cases, the fundamental peak occurred in the middle-to-late period of the dark segment, with only two determinations out of 14 before 6 hr of dark had elapsed (Rat 5E, DLLD, 10.76 PM; and LDDL, 11.46 AM). Repeated determinations for a given condition showed close agreement, with a maximum discrepancy of 1.43 hr (Rat 5E, DLLD). Individual animals showed peaks at different times relative to dark onset. From earliest to latest, the ordering was Rat 5E, Rat 8E, Rat 3E, Rat 2G. These results do not suggest that the locus of electrode placement or illumination intensity during light segments determines the time of peak of the fundamental, since the ordering of animals is mixed with respect to these variables.
TERMAN AND TERMAN @
HR
RAT
2G
HYPOTHALAMIC
o4 •
06.
0 ~:;~!:Y'/, j"
24.
~ ] Wz(
!
;
"I
oem
-
r-as-Ta
a-~o
~-a
10-4
DAYS FIG. 5. Phase angle ($) and corresponding clock time of daily peak in the Fourier fundamental, over three LD 12:12 phase shifts for Rat 2G. Rapid drifting in the daily peak coincides with the start of each 180°LD 12:12 phase shift. Under steady-state entrainment conditions the peaks tend to fall in the late portion of the dark segment. TABLE 2 NUMBER OF DAYS REQUIRED FOR 180° LD RE-ENTRAINMENT 15 uW/cm: Phase Transition I -
Rat 8E
390 uW/cm2
Rat 2G
Rat 3E
Rat 5E
II
7
9
5
7
II - I I I
9
10
6
5
-
11
3
-
III -
IV
PHASE I • PHASE III o
DLLD
PHASE II • PHASE IV
LDDL
12M
'~
6PM
o i
6AM
12 M
3E
8E
5E
2G
w
i
I
3E
8E
5E
2G
RAT
FIG. 6. Time of day of peak of the Fourier fundamental, for steadystate entrainment samples across DLLD and LDDL conditions (Phases I to IV for Rats 3E and 2G; Phases I to III for Rats 5E and 8E).
SELF-STIMULATION AND LIGHT-DARK CYCLES
787 TABLE 3
PERIOD (r IN HR) AND 95 PERCENT CONFIDENCE INTERVAL UNDER LIGHT-ENTRAINED AND FREE-RUNNING CONDITIONS Septal
Hypothalamic
Phase
Rat 3E
Rat 8E
Rat 5E
Rat 2G
I (DLLD)
23.99 -+ 0.26
24.08 -+ 0.11
24.01 -+ 0.07
24.04 -+ 0.08
III (DLLD)
23.98 -+ 0.41
24.06 -+ 0.13
24.07 -+ 0.30
24.00 -+ 0.10
II (LDDL)
23.98 -+ 0.13
23.97 -+ 0.15
24.04 -+ 0.21
24.09 -+ 0.12
IV (LDDL)
24.07 -+ 0.22
V (DDDD)
23.94 -+ 0.12
23.13 -+ 0.10"
24.01 +- 0.05
23.96 -+ 0.03 t
VI (LLLL)
24.82 -+ 0.11~
24.90 -+ 0.17~
24.58 -+ 0.10~t
24.48 -+ 0.13~
24.05 -+ 0.12
*p<0.02 tp<0.01 ~tp<0.002 Quantitative estimates of the period (r) of the behavioral rhythm under free-running or entrainment conditions can be obtained by computing the slope of the least-squares fit line though successive daily ¢ values in a given condition. Successful entrainment to an LD 12:12 schedule is indicated if a 2-tailed regression test [ 11 ] on the data points around the least-squares f i t does not support the hypothesis that the slope differs from zero, where ~- = 24.00 hr. Table 3 summarizes r's for all four animals under the entrainment regimes (DLLD and LDDL) as well as constant dark and constant light. Data samples for ~- determinations were based on steady-state performance in each condition, omitting transient data at the start of each LD phase shift. An obtained r not significantly different from 24 hr may reflect successful entrainment to the light-dark cycle (as was found in all cases for Phases I - I V ) , or an absence of drift of the free-running rhythm under constant conditions (as was found for Rats 3E and 5E in Phase V, constant dark). The 24 hr periodicity of all entrainment data following LD phase shifts shows that the relatively low intensity light (15 tzW/cm2) used for Rats 8E and 2G was able to entrain the behavior rhythm as well as the higher intensity light (390 uW/cm 2) used for Rats 3E and 5E. Significant drifting from 24 hr periodicity was found for Rats 8E and 2G under constant dark, and for all animals under constant light. The range of obtained r's all exceeded 24 hr under constant light (24.48 to 24.90 hr, mean = 24.70 hr) and, in agreement with Aschoff's generalization for nocturnal animals [21], r's for constant light were always greater than 7% for constant dark. DISCUSSION In our earlier study [29], brain self-stimulation rates were shown to oscillate with circadian periodicity in con-
stant light. The present data further define the dynamics of the circadian system by successful entrainment of the freerunning oscillator to strict 24 hr periodicity under imposed light-dark cycles. Successive LD phase shifts allowed an analysis of the transient adjustment of the baseline oscillation to new environmental conditions. Comparison of freerunning rates under constant light and constant dark demonstrated the sensitivity of the circadian system to the presence and absence of ambient illumination. In general, these results indicate that the brain self-stimulation rhythm follows rules of circadian organization similar to other popularly measured motor activity and physiological variables in the rodent (cf. [9] ). The 12 hr on-off entrainment procedures in the present experiment revealed successful control over the free-running rhythm by light intensities ranging from 15 uW/cm 2 (fairly dim to the human eye) to 390 ~W/cm 2 (extremely bright). Steady-state entrainment patterns were similar under both bright and dim illumination levels, although the 180 ° phase transitions appeared to show more rapid reentrainment under bright light. The behavioral phase adjustment to a 12 hr LD shift was not immediate: the oscillation periods increased above 24 hr for as long as 11 days (cf. Table 2), until peak activity stabilized in the new dark segment. Thus, the function of light and dark is not simply to turn the behavior off and on, but to guide the phase of a baseline rhythm which would persist and free-drift under constant illumination conditions. The 180 ° light inversion paradigm may require the longest duration of any possible rhythmic re-entrainment, since the former peak of the fundamental circadian process must be replaced by its most extreme opposite, the trough of the sinusoid. Several procedures may be applied to expedite the rate of entrainment to new LD schedules: (a) Table 2 suggests that bright illumination during light segments is optimal; (b) graded dawn-dusk
788
TERMAN AND TERMAN
light transitions [ 13] may provide stronger control by the LD cycle than sudden on-off light transitions; and (c) a succession of LD phase shifts less than 12 hr [ 16] may be used to "fade in" a full 12 hr shift with maximum speed. The operant response output showed graded probabilities throughout the day. Some responding usually occurred during quiet periods, and the data suggested a gradual acceleration in hourly output (although not necessarily momentary rate) preceding the most active period, followed by a gradual deceleration. Such a pattern contrasts with the often-observed sudden transition patterns in behavioral activity studies (e.g., [3, 21, 24] ) which suggest a circadian square wave oscillator. In those studies, the time of activity onset is a satisfactory index for determining the periodicity of oscillation across days. However, our operant measure as well as many circadian physiological functions (e.g., [10]) show gradual transitions, suggestive of a sine wave oscillator. In these cases, one cannot specify a discrete moment of activity onset to index the oscillation period and phase. Instead, estimation of the time of peak activity in the cycle may be substituted as the anchor point for such calculations. An intriguing result of the constant dark condition (Phase V) was that all of the animals showed free-running rhythms very close to 24.00 hr (Table 3). The behavioral oscillation period (7") for Rats 3E and 5E did not differ significantly from the r's obtained under the LD entrainment regimes, although the time of the peak of the Fourier fundamental was not anchored to any programmed external synchronizer. Such results suggest that the function of constant light (which always produces r's greater than 24.00 hr) is to slow a circadian clock which would match the solar period in the dark. A reliable deviation from strict 24 hr rhythmicity is determined by a statistical test of slope difference between the linear fit to the fundamental phase angle (~b) across days, and zero slope. With greater day-today variability in ~b, it is increasingly difficult to demonstrate a significant, albeit minimal, free-running drift away from 24.00 hr in constant dark. Different experimental procedures may help to reduce such variability in future experiments. For example, Zimmerman and Terman [32] successfully reduced the variability of 4) by lowering the temporal density of reinforcing brain stimulation below that available under continuous reinforcement, using a differential-reinforcement-of-low-ratesprocedure [ 28]. The function of light in controlling behavioral circadian oscillation is a problem for further experimental investigation. We have demonstrated the effectiveness of the LD cycle as synchronizer of the 24 hr operant rhythm, but how does it work? In addition to its entrainment function, light may serve to suppress responding (perhaps with a delay after onset) below an otherwise higher baseline, or dark may serve to facilitate responding above an otherwise lower baseline. To investigate these possibilities, comparisons of
the shapes of the circadian waveform (including amplitude characteristics, phase, presence of harmonics, local rates of response acceleration and deceleration) are necessary under LD, constant light and constant dark conditions. Such an analysis proved unfeasible in the present experiment, where baseline shifts in overall response output over the many months of testing would confound the comparisons. Procedures are necessary that will allow behavior sampling within shorter time periods, where rates are stable and adjustments in reinforcer magnitude are not required to maintain the baseline. Light onset may have different (non-sensory) physiological effects than light offset, as has been shown for pineal activity [30]. Such effects may be reflected in the fine grain of operant activity, either as local rate transients or as prolonged influences on the circadian waveform. Figure 1 suggests that response acceleration in dark often was not symmetrical with the deceleration in light. Procedures employing different ratios of light-to-dark time, brief light probes during dark segments (and vice versa), and different light intensities may help to clarify the function of light in determining parameters of the circadian waveform. The demonstration of successful entrainment of operant behavior to the LD 12:12 cycle is but a first step toward such an analysis. Measurement of concurrent physiological rhythms such as deep body temperature [ 12,31] and noradrenergic activity [ 15 ] may reveal light-sensitive functions that affect behavior rates on the oscillating baseline. It may be instructive to monitor behavior patterns concurrent with the operant measure, such as ingestion, grooming and sleeping [2, 26, 33], since they may show circadian periodicities that correlate or interact with brain self-stimulation. Taken together, these measures form a group of highly probable units in an animal's repertory, yet each is incompatible with the others. When the animal does not self-stimulate, there is the opportunity to engage in the other behaviors, and one might expect to find specific classes that show peak probability when operant output is lowest. Available evidence suggests that ingestive activity follows a circadian rhythm quite closely phased with brain self-stimulation [2,29], and informal observations suggest that bouts of grooming and sleeping occur throughout the 24 hr day. Direct tests are required to determine if one rhythmic behavioral class entrains - or obscures - others. It is possible that all effortful behaviors (i.e., excluding sleep) oscillate together, as subsets of a general activity rhythm (cf. [2,22]). The reinforcement contingency may be viewed as selecting a subset of activity which will predominate in a given environment and in a given rhythmic state (cf. [25]). As such, we need not presently posit that some aspect of reinforcement per se oscillates with circadian rhythmicity (e.g., the detection threshold for brain stimulation [ 5] ), though this may also be the case.
REFERENCES 1. Altman, P. H. and D. S. Dittmer, Eds. Environmental Biology. • Bethesda: Federation of American Societies for Experimental Biology, 1966. 2. Annau, Z., R. Heffner and G. F. Koob. Electrical self-stimulation of single and multiple loci: long term observations. Physiol. Behav. 13: 281-290, 1974. 3. Aschoff, J. Exogenous and endogenous components in circadian rhythms. Cold Spring Harb. Symp. quant. Biol. 25: 11-28, 1960.
4. Aschoff, J., J. Figala and E. PiSppel. Circadian rhythms of locomotor activity in the golden hamster (Mesocricetus Auratus) measured with two different techniques. J. comp. physiol. Psychol. 85: 20-28, 1973. 5. Bass, R. W. Detection of electrical brain stimulation at hypothalamic and septal sites. J. comp. physiol. Psychol. 87: 458-465, 1974.
SELF-STIMULATION AND LIGHT-DARK CYCLES 6. Berkley, M. A. and J. W. Kling. A small animal coupler with vertical movement compensation. Physiol. Behav. 2: 3 1 5 - 3 1 6 , 1967. 7. Bevington, P. Data Reduction and Error Analysis for the Physical Sciences. New York: McGraw Hill, 1969. 8. Brown, B. M. The Mathematical Theory o f Linear Systems. London: Chapman and Hall, 1965. 9. Frisch, L., Ed. Biological clocks. Cold Spring Harb. Symp. quant. Biol. 25: 1960. 10. Halberg, F., E. Halberg, C. P. Barnum and J. J. Bittner. Physiologic 24-hour periodicity in human beings and mice, the lighting regimen and daily routine. In: Photoperiodism and Related Phenomena in Plants and Animals, edited by A. R. Withrow, Washington, D. C.: AAAS, 1959, pp. 803-878. 11. Hays, W. and R. L. Winkler. Statistics: Probability, Inference, and Decision. New York: Holt, Rinehart and Winston, 1971. 12. lllingworth, G. and M. Terman. Phase locked loop: an application in temperature telemetry and a method for its evaluation. Physiol. Behav. 13: 3 3 5 - 3 3 8 , 1974. 13. Kavanau, J. L. Twilight transitions and biological rhythmicity. Nature 194: 1293-1295, 1962. 14. K6nig, J. F. R. and R. A. Klippel. The Rat Brain. Baltimore: Williams and Wilkins, 1963. 15. Margules, D. L., M. J. Lewis, J. A. Dragovich and A. S. Margules. Hypothalamic norepinephrine circadian rhythms and the control of feeding behavior. Science 178: 6 4 0 - 6 4 2 , 1972. 16. McGuire, R. A. Radiotelemetry study of body temperature rhythms in the rat. Unpublished Ph.D. thesis, M.I.T., 1973. 17. McGuire, R. A., W. M. Rand and R. J. Wurtman. Entrainment of the body temperature rhythm in rats: effect of color and intensity of environmental light. Science 181: 9 5 6 - 9 5 7 , 1973. 18. Menaker, M. and A. Eskin. Circadian clock in photoperiodic time measurement: a test of the Biinning hypothesis. Science 157: 1182-1184, 1967. 19. Moore, R. Y., A. Heller, R. J. Wurtman and J. Axelrod. Visual pathway mediating pineal response to environmental light. Science 155: 2 2 0 - 2 2 3 , 1967. 20. Noell, W. K., V. S. Walker, B. S. Kang and S. Berman. Retinal damage by light in rats. Invest. Ophthalmol. 5: 4 5 0 - 4 7 3 , 1966.
789 21. Pittendrigh, C. S. Circadian rhythms and the circadian organization of living systems. Cold Spring Harb. Syrup. quant. Biol. 25: 159-182, 1960. 22. Prescott, R. G. W. Diurnal activity cycles and intracranial selfstimulation in the rat. J. comp. physiol. Psychol. 64: 3 4 6 - 3 4 9 , 1967. 23. Quay, W. B. Physiological significance of the pineal during adaptation to shifts in photoperiod. Physiol. Behav. 5: 3 5 3 - 3 6 0 , 1970. 24. Richter, C. P. Biological Clocks in Medicine and Psychiatry. Springfield: C. T. Thomas, 1965. 25. Staddon, J. E. R. and Simmelhag, V. L. The "superstition" experiment: a re-examination of its implications for the principles of adaptive behavior. Psychol. Rev. 78: 3 - 4 3 , 1971. 26. Steiner, S. S. and S. J. Ellman. Relation between REM sleep and intracranial self-stimulation. Science 1 7 7 : 1 1 2 2 - 1 1 2 4 , 1972. 27. Stroebel, C. F. Correlates of disturbed behavior in the rhesus monkey. In: Orcadian Rhythms in Nonhuman Primates, edited by F. H. Rohles. Basel: S. Karger, 1969. 28. Terman, J. S. The control of interresponse time probabilities by the magnitude of reinforcing brain stimulation. Physiol. Behav. 12: 219-229, 1974. 29. Terman, M. and J. S. Terman. Circadian rhythm of brain selfstimulation behavior. Science 168: 1242-1244, 1970. 30. Wurtman, R. J., J. Axelrod and D. E. Kelly. The h'neal. New York: Academic Press, 1968. 31. Zigmond, M. J., D. L. Holmquest and R. J. Wurtman. Telemetric measurement of effects of light and drugs on diurnal body temperature rhythms. Proc. Fourth Ann. Conf. Pharmac. 5: 279-287, 1970. 32. Zimmerman, J. and J. S. Terman. 24-hour periodicity of intracranial self-stimulation behavior under low-rate schedules of reinforcement. Paper presented at Eastern Psychological Association, Washington D.C., 1973. 33. Zucker, I. Light-dark rhythms in rat eating and drinking behavior. Physiol. Behav. 6: 115-126, 1971.
Brain Research Publications Inc., 1975. Printed in the U.S.A.
Control of the Rat's Circadian Self-Stimulation Rhythm by Light-Dark C y c l e s I MICHAEL TERMAN AND JIUAN S. TERMAN
Department o f Psychology, Northeastern University, Boston, MA 02115
(Received 11 October 1974) TERMAN, M. AND J. S. TERMAN. Control of the rat's circadian self-stimulation rhythm by light-clark cycles. PHYSIOL. BEHAV. 14(6) 781-789, 1975. - Rats with hypothalamic and septal electrodes were maintained in continuous test environments where bar-press responses produced brief reinforcing electrical stimulations. Long-term trends in response emission were measured under continuous exposure to light, dark and 12 hr light-dark alternations. In addition, transient behavioral adjustment to sudden 180° phase shifts in the light-dark schedule was studied. The ambient light condition was found to control the period and phase of the circadian rhythm of brain self-stimulation behavior, as quantified by Fourier analysis. The circadian period was greatest under constant light (up to 24.90 hr under dim illumination), and approximated 24.00 hr under constant dark. Successful nocturnal entrainment to 12 hr light-dark alternations was obtained, with the peak of the 24 hr Fourier fundamental occurring in the middle-to-late dark segments. Three to 11 days were required for re-entrainment to 180° light-dark phase shifts, during which the behavioral oscillation period increased to values comparable to periods under constant light. The rate of re-entrainment appeared to be proportional to illumination intensity during light segments. Circadian rhythms
Electrical brain stimulation
LD phase shifts
LD entrainment
and water. Behavioral periods slightly exceeding 24 hr indicated that the rhythm was not entrained by uncontrolled diurnal variables. The question of entrainability of the selfstimulation rhythm by light-dark (LD) cycles is important to define the operating characteristics of this circadian system in terms of its susceptibility to external control, and to discover possible functional interrelationships to more extensively studied behavioral and physiological circadian systems. (One could guess that interference by brain stimulation with inferior accessory optic tract function might affect the rate of entrainment to LD schedules [ 19,23] ; or that the unusually stereotyped behavior patterns maintained by brain stimulation might lead to a pathological state akin to behavioral stress, rendering external entrainment agents ineffective [ 27 ] .) Several experimental procedures address themselves to the question of entrainability: The circadian periods may be compared under LD schedules, constant light and constant dark. Free-running periods deviating from 24 hr should lock into strict 24 hr rhythmicity under an effective LD 12:12 cycle. Further, the behavioral oscillator must be shown to adjust dynamically by changing its phase in response to programmed phase shifts in the entraining agent
STUDIES of behavioral steady-states under the control of reinforcement contingencies have rarely taken into account circadian oscillations that may co-determine momentary response probability. The typical use of brief test sessions, run at a standard time of day (and often with trial-by-trial restrictions), may obscure the contribution of circadian variables to the behavior stream. In contrast, the biological literature is replete with examples of continuous circadian oscillations (cf. [1,9]), including neural, hormonal and metabolic rhythms that may be functionally linked to behavioral phenomena. Analyses of behavioral circadian rhythms have concentrated on gross activity measures and running wheel activity (e.g., [4,24] ), or feeding and drinking (e.g., [33]), where high rates of spontaneous emission insure long-term data production. The application of a reinforcement contingency allows experimenters to study circadian oscillations in specific behavioral units that would not otherwise show high probability in an organism's daily repertory. Terman and Terman [29] showed that the rate of the bar-press operant maintained by reinforcing brain stimulation oscillates in a circadian rhythm when rats respond under contant dim illumination with free access to food
1This research was supported in part by NASA Grant NGR 22-011-070 and HEW Grant RR07143. We owe special thanks to J. Anlinker (NASA), for advice and encouragement; J. Zimmerman and C. W. Tyler, for assistance in developing procedures for waveform analysis; J. Armington and the Northeastern Unversity Computation Center staff, for computation facilities and advice; M. Raibert, for preparation of computer graphics; H. Mahut, for assistance in histological procedures; and L. Thorington (Duro-Test Corp.), for contribution of Vita-Lite sources. Reprints may be obtained from M. Terman, Department of Psychology, 440 United Building, Northeastern University, Boston MA 02115. 781
782 [18]. Annau et al. [2] have demonstrated nocturnal activity patterns for self-stimulation, concurrent with feeding and drinking rhythms. Their main entrainment index was the percentage of responding in the dark segment of an LD 12:12 schedule. This index is sensitive to both amplitude and phase characteristics of the oscillating behavioral function, and is not a direct measure of periodicity. The nocturnal response patterns may be presumed to have been entrained, but the analysis did not document an oscillation period locked strictly to the LD period over extended test sessions, and the procedure did not demonstrate dynamic phase adjustment to LD cycle shifts. In the present experiment, the self-stimulation rhythm was examined under constant light, constant dark and a succession of LD 12:12 phase shifts, within single animals whose oscillation patterns contrasted in amplitude and phase characteristics. Possible locus specificity of the rhythmic effects was tested by the use of both hypothalamic and septal electrode placements. Fourier analysis was applied to specify phase and period information independently of amplitude (i.e., overall response output) variations. METHOD Animals
Four adult male pigmented rats (Lewis/BN stock, caesarian derived) were used. Under sodium pentobarbital anesthesia, each rat received a chronic bipolar electrode implant (Plastic Products MS 303). Following experimentation, the brains were examined histologically and electrode tip placements (cf. [ 14] ) were identified in the following areas: Rat 3E, nucleus septi lateralis; Rat 5E, fasciculus medialis prosencephali; Rat 8E, nucleus septi medialis; Rat 2G, nucleus ventromedialis (hypothalami), pars anterior. For convenience, this report labels the placements for Rats 3E and 8E as septal, and Rats 5E and 2G as hypothalamic.
TERMAN AND TERMAN Since current amplitude changes often induced transients in the data, response rates were allowed to stabilize before steady-state circadian rhythm data were considered. For Rat 3E, current levels ranged from 0.30 to 0.50 mA (p-p voltage monitored by oscilloscope across a resistor in series with the animal); Rat 5E, 0.50 to 0.60 mA; Rat 8E, 0.12 to 0.20 mA; Rat 2G, 0.08 mA. Previous data [29] show that current amplitude controls daily response output, but not the periodicity of the free-running rhythm of self-stimulation behavior. The experimental contingencies (continuous reinforcement throughout all test phases) were controlled by solidstate logic (DEC K-series Flip Chips) located in a room separate from the test chambers. Responses were counted by cumulative recorders, and reduced into tabular format in 1 hr blocks for subsequent computer analysis. Fourier analyses, least-squares fits, and statistical tests were run on PDP-12, LAB-8, and CDC Cyber 70 computer systems. The graphical presentations in Fig. 2 and 3 were prepared by the CDC's plotting facility. Procedure
Table 1 summarizes the successive experimental phases for each animal, from which data are sampled in the present report. Each rat began the experiment with a sequence of light-dark schedules. Light and dark segments were 12 hr long, with light turning on either at 6 a.m. (EST) or 6 p.m. We use abbreviations denoting successive 6 hr blocks of L and D in the 24 hr day (e.g., DLLD or LDDL) to specify experimental phases involving different light-dark arrangements. When light-dark cycles were inverted (180 ° phase shifts) the dark segment was extended by 12 hr on the first transition day. Following LD entrainment tests, the animals were exposed to periods of constant dark and constant light, to verify the presence of free-running (circadian) rhythms, and to specify their periods.
Apparatus
The animals were maintained in chambers with sound attenuation, programmable light schedules, constant temperature (21°C) and relative humidity ( 4 0 - 5 0 percent). Inside each environmental control chamber was a compartment constructed of a 24 cm dia. Plexiglas cylinder, with water and Purina Lab chow available continuously via drinking tube and food cylinder mounted on the wall. A Lehigh Valley mouse lever was also mounted on the wall at the level of the rat's head. The compartment floor was constructed of wire mesh, with a large droppings tray below. (Interruptions for maintenance - food, water, cleaning - were required less than once per week, allowing virtually uninterrupted long-term sessions.) A Vita-Lite flourescent source (see Table 1) was mounted on the wall of the chamber outside the test compartment, with on-off functions controlled by an external timer. Light intensities (see Table 1) were calibrated by placing an Eppley thermopile at the rat's position facing the light source, within the test compartment. A pulley-swivel mercury commutator system [6] was attached to the ceiling of the chamber above each compartment, for delivery of reinforcing brain stimulation. Stimulations were 0.5 sec sinusoids from a constant-current stimulator. Current amplitude was set at levels maintaining longt e r m self-stimulation behavior, and required periodic increases over the many months of continuous testing.
RESULTS The entrainment effect of successive LD 12:12 phases is summarized in Fig. 1, where mean daily response proportion is plotted in 3 hr blocks, to correct for absolute response rate variations within and across animals during the test series. These steady state data are based on results for the final week of each condition. The filled bars represent daily response proportions for the 12 hr dark segment, which began alternately at 6 p.m. and 6 a.m. across successive experimental phases. The details of the response proportion profile vary across conditions, but typically response proportions peaked in the dark and dipped in the light, displaying a nocturnal activity pattern. In all cases, the repeated 180 ° LD phase shifts successfully re-entrained the behavioral rhythm, so that peak activity periods fell at times corresponding to low activity periods in the previous condition. The fine pattern of the behavioral rhythm showed some change with continued testing and light-dark inversions, for all rats but 2G: there was a tendency for the differential between active and quiet phases to become accentuated as a function of time. Rat 2G showed a comparatively constant cyclic pattern across conditions, with accelerating high response rates during dark, sometimes leading to a peak in the first 3 hr block of light. The latter finding suggests that light onset does not necessarily produce an immediate suppressive effect on responding. Rat
SELF-STIMULATION AND LIGHT-DARK CYCLES
783
TABLE 1 EXPERIMENTAL PHASES (LIGHT CONDITION)* Phase
Condition
Rat 3E
Rat 5E
Rat 8E
Rat 2G
I (DLLD)
Light on 6 a.m. Light off 6 p.m.
12-10-71 to 12-24-71
12-10-71 to 1-24-72
6-8-72 to 6-24-72
5-23-73 to 7-15-73
II (LDDL)
Light on 6 p.m. Light off 6 a.m.
12-25-71"~ to 1-16-72
1-25-72 to 2-15-72
6-25-72 to 7-25-72
7-16-73 to 8-9-73
III (DLLD)
Light on 6 a.m. Light off 6 p.m.
1-17-72 to 2-1-72
2-16-72 to 3-5-72
7-26-72 to 8-14-72
8-10-73 to 9-7-73
IV (LDDL)
Light on 6 p.m. Light off 6 a.m.
2-2-72 to 2-16-72
_
_
9-8-73 to 10-8-73
V (DDDD)
Constant dark
6-18-72 to 7-22-72
3-6-72 to 5-15-72
9-2-72 to 9-25-72
10-9-73 to 11-27-73
VI (LLLL)
Constant light
5-25-72 to 6-16-72
5-16-72 to 6-7-72
10-17-72 to 11-6-72
11-28-73 to 1-16-74
*Vita-Lite fluorescents served as the light sources, approximating the solar spectrum more closely than conventional lamps. Previous research [31] has demonstrated successful entrainment of deep-body temperature with this lamp. For light segments in the entrainment tests (Phases I to IV) Rats 8E and 2G received 15 #W/cm 2 illumination, and Rats 3E and 5E received 390 /~W/cm2. When the animals were tested under constant light (Phase VI), illumination was set at 15 tzW/cm~ in all cases. Use of the dimmer illumination was in response to reports of retinal damage due to constant exposure to bright light (of. [20] ). All light was absent during Phase V (constant dark) and the dark segments of LD cycles. ~'On the first day of Phases II, III and IV, the duration of the dark segment was extended to accomplish the 180° light-dark phase shift. Thus, the 3-day transition pattern between Phases I to II and III to IV was DLLD-DDDL-LDDL; for Phases II to III, LDDL-LDDD-DLLD. 2G's record differs from the others also in that the contrast in proportions between light and dark segments was not so great. Some of the data suggest a relative on-off activity pattern (e.g., Rat 5E, Phase II; Rat 3E, Phase III), though response proportions never fell to zero for an entire 3 hr block. However, the general profile was a gradual transition in response proportions from active to quiet periods and vice versa. To examine the transient behavioral adjustment during LD phase shifts, and free-running drift patterns in constant light and dark conditions, hourly response counts were divided into ranked quartiles in Figs. 2 and 3. The quartile plots indicate at a glance how tightly the periods of high and low responding are correlated with the light-dark schedule, the spread of the oscillation pattern (dense vs. loose packing of symbols) in each condition, and the rapidity and orderliness of response phase shifts when the light-dark cycle is inverted or eliminated. Note that the quartile data are not sensitive to transient or long-term changes in overall daily response output, since the quartile criteria are adjusted each day. In the light-dark conditions, Rat 3E showed responding
in the upper 2 quartiles almost exclusively in the dark segment, once the steady-state was attained after each lightdark cycle inversion. The onset of high activity was quite abrupt (cf. Fig. 1), and there was occasional spilling of high response rates into the first hour of light, especially after 1 - 2 2 - 7 2 . Symbols for the lower 2 quartiles are scattered fairly evenly throughout the light segments, and an occasional high-rate symbol also appears during quiet periods, reflecting transient response accelerations. The transition data at the start of each light-dark inversion are of special interest: the oscillation period abruptly increased beyond 24 hr, and the region of highest density symbols shifted to later hours over successive days, mapping the animal's adjustment to the new entrainment schedule. Note that the behavioral rhythmicity did not disappear during phase adjustment; it appeared similar to the stabilized entrainment rhythm except for its period. During the transition, which required about a week for establishment of the new entrained pattern, considerable responding occurred in the new light segment, which corresponded in clock time to the old dark segment. Thus, while the animal converged on its final steady-state nocturnal activity pattern, light did not
784
TERMAN AND TERMAN RAT 5 E
SEPTAL
RAT 3E SEPTAL 1-3-72
)-[
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L
D
2L D
2-3-72
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RAT BE SEPTAL 5-2-72
Z 0 fOE 0 0 n.o. 03 Z 0 tl. 03 bJ
6-1
5-18-72
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6AM
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12N
6PM
12M
6AM
12N
6PM
12M
2 - D A Y SPAN (HOURS) RAT 5E HYPOTHALAMIC Z
1/18 - 1/24/72
2/9-2/15/72
2/28-3/5/72
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~- A M -"-*- PM ~
R A T 2G H Y P O T H A L A M I C Z k-(3g 0 0 Q_
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FIG. 2. Ranked quartile plot of daily response output for Rat 3E, for 2 LI) 12:12 phase shifts ( 1 - 2 - 7 2 to 2 - 1 6 - 7 2 ) , and for steadystate samples in constant light ( 6 - 1 - 7 2 to 6 - t 6 - 7 2 ) and constant dark ( 6 - 1 7 - 7 2 to 7 - 2 - 7 2 ) . Each day's hourly response counts are divided into ranked quartiles, with symbol size proportional to activity level: f'dled squares, most active 6 hr; rectangles, second highest 6 hr; horizontal lines, third highest 6 hr; empty spaces, lowest 6 hr. The abscissa spans 2 days, facilitating representation of non-24 hr circadian oscillations, where the region of maximal symbol density is seen to drift across days during entrainment phase shifts and constant light conditions. Thus, with successive days on the ordinate, each day's data are presented twice: once at the right of the abscissa, and then one line below at the left. Vertical lines at 6 a.m. and 6 p.m. denote the time of light-dark transitions. Data for 3 days are omitted in this record, resulting in 24 hr blank spaces. On 1 - 8 - 7 2 , brain stimulation intensity was raised in order to increase baseline response output, and the entrained rhythm was briefly disrupted. On 1 - 1 4 - 7 2 , the electrode lead disengaged from the skull cap, resulting temporarily in extinction. (Note that the rhythm immediately recaptured its previous phase on the following day, when stimulation was resumed.) On 2 - 6 - 7 2 , failure of the recording apparatus obliterated the record; the animal was unaffected. The blank space between the final entrainment data and the constant light condition indicates a hiatus of several months between data samples.
>-
~AM--~--PM ~
FIG. 1. Daily response proportions (3 hr blocks) for successive 180 ° LI) 12:12 phase shifts. Data are averaged over 7 day steady-state samples at the end of each entrainment phase. Filled bars denote dark segments within the LI) 12:12 cycle; open bars denote light segments.
suppress responding directly. Under c o n s t a n t light (Fig. 2, starting 6 - 1 - 7 2 ) the oscillation pattern was maintained in a steady-state w i t h a p e r i o d i c i t y greater than 2 4 hr. However, the d e n s i t y o f s y m b o l packing in the quartile plot appears m u c h l o o s e r than under the preceding e n t r a i n m e n t c o n d i t i o n s , indicating less discrete peaking in the daily behavior pattern. As
SELF-STIMULATION AND LIGHT-DARK CYCLES during the transition period at the start of an inverted lightdark condition, the region of highest density symbols shifts to the right over successive days of constant light, indicating that peak responding occurred later every day. If each behavioral oscillation is viewed as one subjective day for the animal under constant conditions, it can be seen that approximately 12 objective hours were lost in 3 weeks of running under constant light. On 6 - 1 7 - 7 2 , Rat 3E was transferred suddenly to constant dark. The quartile plot shows that the region of highest density symbols immediately shifts to the right, and remains at approximately the same position as on the final day of constant light (8 p.m. to 8 a.m.). The loose density of symbols continues to indicate greater spread in the behavioral oscillation than under the former entrainment conditions, though the periods for both entrainment and constant dark approximated 24 hr. Thus, constant light had the effect of slowing the circadian oscillation (in accordance with Aschoff's rule for nocturnal animals [21]), with constant dark maintaining a period indistinguishable from 24 hr. Figure 3 shows a corresponding set of data in quartile format for Rat 2G. The response patterns differed in detail from those of Rat 3E (Fig. 2) in that the region of the highest quartile responding was concentrated around the time of the dark-to-light transition and was not scattered evenly thoughout the dark segment. A gradient of increasing symbol density from early to late hours of dark is apparent, describing a daily gradual rate oscillation pattern, which contrasts to Rat 3E's relative on-off pattern (see also the one-week histogram summaries in Fig. 1). Note that, despite the peak in light, a greater total response output occurred in the dark segment, which would produce a standard nocturnal light:dark activity ratio (cf. [2,33]). Rat 2G showed a transient lengthening of the oscillation p e r i o d at the start of an inverted light-dark cycle ( 8 - 1 0 - 7 3 ) , enabling the behavioral phase shift to be completed in about one week, similar to Rat 3E (Fig. 2). For Rat 2G the constant dark condition preceded constant light, and the results were similar to those of Rat 3E with the opposite test order. The daily distribution of responses increased under constant conditions, with a wider spread in the high-activity period and less consistent time of peak responding. Under constant dark, the oscillation period appeared close to 24 hr. When constant light was introduced on 1 1 - 2 8 - 7 3 , the oscillation period immediately lengthened: within approximately 3 weeks the highactivity period shifted by 12 hr, closely matching the drift rate for Rat 3E under constant light (Fig. 2). The finding of reliable daily oscillations of operant response output suggests the presence of a continuous periodic process whose parameters (phase, period, amplitude) may be specified by Fourier analysis. Under LD 12:12 entrainment conditions, the data illustrated above suggest a 24 hr behavioral period, which would be represented by a high-amplitude 24 hr fundamental in Fourier analysis (for computational procedures, see [7, 8]). In Fig. 4, the fit of this fundamental waveform to a 7-day averaged steady-state data sample for Rat 8E (Phase I)is illustrated. The close agreement between data and the fundamental show that most of the variance in this sample can be accounted for by a simple 24 hr sinusoid. Indeed, examination of amplitudes of the 2nd, 3rd, and 4th harmonics revealed no component that would significantly improve the Fourier data fit beyond that shown in Fig. 4. (Occasional high-amplitude 2nd and 3rd harmonics appeared in
785
RAT 2G H Y P O T H A L A M I C 7-?_5-73
B-II-73
1-13-7:5
1-29-75
hAM
IZN 6PM IZM 6AM 12N 2 - D A Y SPAN (HOURS)
6PM
12M
FIG. 3. Ranked quartile plot of daily response output for Rat 2G, for one LD 12:12 phase shift (7-24-73 to 9-7-73), and for steady-state samples in constant dark (11-12-73 to 11-27-73) and constant light (11-28-73 to 12-15-73). The graphical procedure is explained in Fig. 2.
+•o-
300u~ t~ o~ z O ~ o3 200t~ tv
RAT BE SEPTAL
;",,
',, ,,o
,,
;'
•
D'"
X+"a"a'
,0o-
. . . . .
+
. . . .
TIME
N. . . . .
in
61,M
. . . .
M
HOURS
FIG. 4. Waveform resynthesis for a 7 day steady-state sample under LD 12:12 (Phase I). Triangles represent averaged hourly response totals. Dots represent amplitude of the 24 hr Fourier fundamental.
786 other data samples, but were not consistently related to any procedure. One effect of such harmonic components is to shift the Fourier waveform into closer agreement with the behavioral data. However, such harmonics do not affect determinations of the circadian period.) Notable deviations from the fundamental sinusoid appear in Fig. 4 as transient response accelerations in the second hour of dark (7 p.m.), the last hour of dark (5 a.m.) and the first hour of light (6 a.m.). Such accelerations are typical of the steady-state entrainment data across animals, and may reflect secondary effects of the light-dark transitions that are distinct from the circadian process per se. The phase angle of the peak of the Fourier fundamental ($) provides a convenient statistical estimator of the moment of peak activity in the 24 hr day. Under LD 12:12 entrainment conditions ~b does not change systematically over days. During LD phase shifts, and under constant illumination conditions, the daily changes in ~b index the rate of the behavioral drift, allowing precise specification of periods deviating from 24.00 hr. Figure 5 shows ~b plotted over successive days across a sequence of light-dark schedules, for Rat 2G. The time of day and corresponding phase angles are replicated on the ordinate to enable following behavioral phase transitions across the daily boundary. U n d e r steady-state entrainment conditions, the peak occurred toward the end of each day's 12 hr dark segment, with an occasional day's peak early in light (cf. Fig. 3). Some variability in the position of the peak over successive days is evident, although the steady-state data show no consistent drift in phase angle over days, indexing a precisely timed 24 hr behavioral cycle. When the light-dark cycle was inverted (starting at 8 - 1 0 - 7 3 and 9 - 8 - 7 3 ) , the peak of the rhythm began to shift to a later hour over days, as suggested earlier in the response quartile plots (Figs. 2 and 3). The slope of these transient data specifies the drift rate exhibited by the animal as it adjusts its behavioral cycle to the new illumination condition. For both phase transitions illustrated, the analysis shows that the 180 ° shift in behavior required 9 - 1 0 days following the light-dark inversion. A summary of the number of days required for the 180 ° behavioral transition across all subjects in Phases I to IV is shown in Table 2. The two rats receiving bright light for daytime segments (3E and 5E) required from 3 to 7 days, while the two rats receiving dim light (8E and 2G) required 7 to 11 days, suggesting that greater light intensity controls more rapid behavioral phase adjustment to a new light-dark cycle. The time of the peak of the Fourier fundamental is shown for all animals across entrainment conditions, in Fig. 6. With the exception of Rats 5E and 8E under LDDL, two independent determinations (Phases I and III, Phases II and IV) of the peak were made for each condition. In all cases, the fundamental peak occurred in the middle-to-late period of the dark segment, with only two determinations out of 14 before 6 hr of dark had elapsed (Rat 5E, DLLD, 10.76 PM; and LDDL, 11.46 AM). Repeated determinations for a given condition showed close agreement, with a maximum discrepancy of 1.43 hr (Rat 5E, DLLD). Individual animals showed peaks at different times relative to dark onset. From earliest to latest, the ordering was Rat 5E, Rat 8E, Rat 3E, Rat 2G. These results do not suggest that the locus of electrode placement or illumination intensity during light segments determines the time of peak of the fundamental, since the ordering of animals is mixed with respect to these variables.
TERMAN AND TERMAN @
HR
RAT
2G
HYPOTHALAMIC
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DAYS FIG. 5. Phase angle ($) and corresponding clock time of daily peak in the Fourier fundamental, over three LD 12:12 phase shifts for Rat 2G. Rapid drifting in the daily peak coincides with the start of each 180°LD 12:12 phase shift. Under steady-state entrainment conditions the peaks tend to fall in the late portion of the dark segment. TABLE 2 NUMBER OF DAYS REQUIRED FOR 180° LD RE-ENTRAINMENT 15 uW/cm: Phase Transition I -
Rat 8E
390 uW/cm2
Rat 2G
Rat 3E
Rat 5E
II
7
9
5
7
II - I I I
9
10
6
5
-
11
3
-
III -
IV
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PHASE II • PHASE IV
LDDL
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8E
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8E
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FIG. 6. Time of day of peak of the Fourier fundamental, for steadystate entrainment samples across DLLD and LDDL conditions (Phases I to IV for Rats 3E and 2G; Phases I to III for Rats 5E and 8E).
SELF-STIMULATION AND LIGHT-DARK CYCLES
787 TABLE 3
PERIOD (r IN HR) AND 95 PERCENT CONFIDENCE INTERVAL UNDER LIGHT-ENTRAINED AND FREE-RUNNING CONDITIONS Septal
Hypothalamic
Phase
Rat 3E
Rat 8E
Rat 5E
Rat 2G
I (DLLD)
23.99 -+ 0.26
24.08 -+ 0.11
24.01 -+ 0.07
24.04 -+ 0.08
III (DLLD)
23.98 -+ 0.41
24.06 -+ 0.13
24.07 -+ 0.30
24.00 -+ 0.10
II (LDDL)
23.98 -+ 0.13
23.97 -+ 0.15
24.04 -+ 0.21
24.09 -+ 0.12
IV (LDDL)
24.07 -+ 0.22
V (DDDD)
23.94 -+ 0.12
23.13 -+ 0.10"
24.01 +- 0.05
23.96 -+ 0.03 t
VI (LLLL)
24.82 -+ 0.11~
24.90 -+ 0.17~
24.58 -+ 0.10~t
24.48 -+ 0.13~
24.05 -+ 0.12
*p<0.02 tp<0.01 ~tp<0.002 Quantitative estimates of the period (r) of the behavioral rhythm under free-running or entrainment conditions can be obtained by computing the slope of the least-squares fit line though successive daily ¢ values in a given condition. Successful entrainment to an LD 12:12 schedule is indicated if a 2-tailed regression test [ 11 ] on the data points around the least-squares f i t does not support the hypothesis that the slope differs from zero, where ~- = 24.00 hr. Table 3 summarizes r's for all four animals under the entrainment regimes (DLLD and LDDL) as well as constant dark and constant light. Data samples for ~- determinations were based on steady-state performance in each condition, omitting transient data at the start of each LD phase shift. An obtained r not significantly different from 24 hr may reflect successful entrainment to the light-dark cycle (as was found in all cases for Phases I - I V ) , or an absence of drift of the free-running rhythm under constant conditions (as was found for Rats 3E and 5E in Phase V, constant dark). The 24 hr periodicity of all entrainment data following LD phase shifts shows that the relatively low intensity light (15 tzW/cm2) used for Rats 8E and 2G was able to entrain the behavior rhythm as well as the higher intensity light (390 uW/cm 2) used for Rats 3E and 5E. Significant drifting from 24 hr periodicity was found for Rats 8E and 2G under constant dark, and for all animals under constant light. The range of obtained r's all exceeded 24 hr under constant light (24.48 to 24.90 hr, mean = 24.70 hr) and, in agreement with Aschoff's generalization for nocturnal animals [21], r's for constant light were always greater than 7% for constant dark. DISCUSSION In our earlier study [29], brain self-stimulation rates were shown to oscillate with circadian periodicity in con-
stant light. The present data further define the dynamics of the circadian system by successful entrainment of the freerunning oscillator to strict 24 hr periodicity under imposed light-dark cycles. Successive LD phase shifts allowed an analysis of the transient adjustment of the baseline oscillation to new environmental conditions. Comparison of freerunning rates under constant light and constant dark demonstrated the sensitivity of the circadian system to the presence and absence of ambient illumination. In general, these results indicate that the brain self-stimulation rhythm follows rules of circadian organization similar to other popularly measured motor activity and physiological variables in the rodent (cf. [9] ). The 12 hr on-off entrainment procedures in the present experiment revealed successful control over the free-running rhythm by light intensities ranging from 15 uW/cm 2 (fairly dim to the human eye) to 390 ~W/cm 2 (extremely bright). Steady-state entrainment patterns were similar under both bright and dim illumination levels, although the 180 ° phase transitions appeared to show more rapid reentrainment under bright light. The behavioral phase adjustment to a 12 hr LD shift was not immediate: the oscillation periods increased above 24 hr for as long as 11 days (cf. Table 2), until peak activity stabilized in the new dark segment. Thus, the function of light and dark is not simply to turn the behavior off and on, but to guide the phase of a baseline rhythm which would persist and free-drift under constant illumination conditions. The 180 ° light inversion paradigm may require the longest duration of any possible rhythmic re-entrainment, since the former peak of the fundamental circadian process must be replaced by its most extreme opposite, the trough of the sinusoid. Several procedures may be applied to expedite the rate of entrainment to new LD schedules: (a) Table 2 suggests that bright illumination during light segments is optimal; (b) graded dawn-dusk
788
TERMAN AND TERMAN
light transitions [ 13] may provide stronger control by the LD cycle than sudden on-off light transitions; and (c) a succession of LD phase shifts less than 12 hr [ 16] may be used to "fade in" a full 12 hr shift with maximum speed. The operant response output showed graded probabilities throughout the day. Some responding usually occurred during quiet periods, and the data suggested a gradual acceleration in hourly output (although not necessarily momentary rate) preceding the most active period, followed by a gradual deceleration. Such a pattern contrasts with the often-observed sudden transition patterns in behavioral activity studies (e.g., [3, 21, 24] ) which suggest a circadian square wave oscillator. In those studies, the time of activity onset is a satisfactory index for determining the periodicity of oscillation across days. However, our operant measure as well as many circadian physiological functions (e.g., [10]) show gradual transitions, suggestive of a sine wave oscillator. In these cases, one cannot specify a discrete moment of activity onset to index the oscillation period and phase. Instead, estimation of the time of peak activity in the cycle may be substituted as the anchor point for such calculations. An intriguing result of the constant dark condition (Phase V) was that all of the animals showed free-running rhythms very close to 24.00 hr (Table 3). The behavioral oscillation period (7") for Rats 3E and 5E did not differ significantly from the r's obtained under the LD entrainment regimes, although the time of the peak of the Fourier fundamental was not anchored to any programmed external synchronizer. Such results suggest that the function of constant light (which always produces r's greater than 24.00 hr) is to slow a circadian clock which would match the solar period in the dark. A reliable deviation from strict 24 hr rhythmicity is determined by a statistical test of slope difference between the linear fit to the fundamental phase angle (~b) across days, and zero slope. With greater day-today variability in ~b, it is increasingly difficult to demonstrate a significant, albeit minimal, free-running drift away from 24.00 hr in constant dark. Different experimental procedures may help to reduce such variability in future experiments. For example, Zimmerman and Terman [32] successfully reduced the variability of 4) by lowering the temporal density of reinforcing brain stimulation below that available under continuous reinforcement, using a differential-reinforcement-of-low-ratesprocedure [ 28]. The function of light in controlling behavioral circadian oscillation is a problem for further experimental investigation. We have demonstrated the effectiveness of the LD cycle as synchronizer of the 24 hr operant rhythm, but how does it work? In addition to its entrainment function, light may serve to suppress responding (perhaps with a delay after onset) below an otherwise higher baseline, or dark may serve to facilitate responding above an otherwise lower baseline. To investigate these possibilities, comparisons of
the shapes of the circadian waveform (including amplitude characteristics, phase, presence of harmonics, local rates of response acceleration and deceleration) are necessary under LD, constant light and constant dark conditions. Such an analysis proved unfeasible in the present experiment, where baseline shifts in overall response output over the many months of testing would confound the comparisons. Procedures are necessary that will allow behavior sampling within shorter time periods, where rates are stable and adjustments in reinforcer magnitude are not required to maintain the baseline. Light onset may have different (non-sensory) physiological effects than light offset, as has been shown for pineal activity [30]. Such effects may be reflected in the fine grain of operant activity, either as local rate transients or as prolonged influences on the circadian waveform. Figure 1 suggests that response acceleration in dark often was not symmetrical with the deceleration in light. Procedures employing different ratios of light-to-dark time, brief light probes during dark segments (and vice versa), and different light intensities may help to clarify the function of light in determining parameters of the circadian waveform. The demonstration of successful entrainment of operant behavior to the LD 12:12 cycle is but a first step toward such an analysis. Measurement of concurrent physiological rhythms such as deep body temperature [ 12,31] and noradrenergic activity [ 15 ] may reveal light-sensitive functions that affect behavior rates on the oscillating baseline. It may be instructive to monitor behavior patterns concurrent with the operant measure, such as ingestion, grooming and sleeping [2, 26, 33], since they may show circadian periodicities that correlate or interact with brain self-stimulation. Taken together, these measures form a group of highly probable units in an animal's repertory, yet each is incompatible with the others. When the animal does not self-stimulate, there is the opportunity to engage in the other behaviors, and one might expect to find specific classes that show peak probability when operant output is lowest. Available evidence suggests that ingestive activity follows a circadian rhythm quite closely phased with brain self-stimulation [2,29], and informal observations suggest that bouts of grooming and sleeping occur throughout the 24 hr day. Direct tests are required to determine if one rhythmic behavioral class entrains - or obscures - others. It is possible that all effortful behaviors (i.e., excluding sleep) oscillate together, as subsets of a general activity rhythm (cf. [2,22]). The reinforcement contingency may be viewed as selecting a subset of activity which will predominate in a given environment and in a given rhythmic state (cf. [25]). As such, we need not presently posit that some aspect of reinforcement per se oscillates with circadian rhythmicity (e.g., the detection threshold for brain stimulation [ 5] ), though this may also be the case.
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