Brain Research, 229 (1981) 85-101 85 Elsevier/North-Holland Biomedical Press

T E M P O R A R Y D E S Y N C H R O N I Z A T I O N A M O N G C I R C A D I A N R H Y T H M S

W I T H LATER AL F O R N I X ABLATION*

CHRISTINE T. FISCHETTE, HENRY M. EDINGER and ALLAN SIEGEL

Departments of Physiology and Neurosciences, College of Medicine and Dentistry of New Jersey~ Newark, NJ (U.S.A.)

(Accepted April 23rd, 1981)

Key words: circadian rhythms - - fornix - - adrenal corticosteroids - - locomotor activity - - body temperature - - feeding and drinking

SUMMARY

Lateral or medial fornix suction ablations were performed on adult male rats in order to selectively ablate or leave intact, respectively, fibers which terminate in the region of the suprachiasmatic nucleus and hypophysiotropic area of the hypo- thalamus. Plasma adrenal corticosteroid secretion, locomotor activity, body tem- perature, and food and water intake were recorded at 4 h intervals over a period of 48 h in individual animals 7-10 days postoperatively. Lateral fornix ablation specifically disrupted adrenal cotticosteroid periodicity. A least-squares spectrum analysis of the data indicated that corticosteroid secretion may be under ultradian control after this lesion. All animals, regardless of treatment, exhibited normal circadian locomotor activity patterns. Aberrations in feeding, drinking and body temperature rhythms were occasionally observed. This represents a temporary dissociation between the rhythmic expression of corticosteroid secretion and activity, temperature, feeding and drinking. The evidence presented lends support to the multi-oscillator theory of circadian organization, and suggests that the anteroventral subiculum, via the medial cor- t icohypothalamic tract, is important in the regulation of some, but not all, circadian parameters.

In addition to the observations on the rhythmicity of locomotor activity, the ex- tent to which the animals are active is also significantly different between groups; ie., the hyperactivity of fornix-transected animals previously reported by others was found to be associated with lateral and not medial fornix ablation.

* A preliminary report on this subject was presented at the Society for Neuroscience Meeting, November, 1979. This work was submitted in partial requirement for the degree of Ph.D.

0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press

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INTRODUCTION

Total fornix lesions disrupt the cilcadian rhythmicity of plasma adrenal corticosteroid secretion that is normally synchronized to the light-dark cycle when sampling occurs 1-2 weeks postoperatively 17. However, the rhythm returns three weeks postoperatively 15 which suggests that recovery or re-arrangement of neural elements due to neural plasticity may have occurred. Lateral, but not medial fornix ablation disrupts the adrenal hormone secretory rhythm when animals are sampled 7-10 days after surgery3, 4. The disrupted pattern does not follow the same time course when animals are resampled 2-3 days later 3.

The observed physiological effects are in accord with the known anatomical sub- strate. Meibach and SiegeP 6 have found in the rat that fibers from the dorsal hippo- campal formation travel in the medial part of the fornix, while fibers from the ventral hippocampai formation travel in the lateral part of the fornix and fimbria. The medial corticohypothalamic tract (mcht), whose cell bodies of origin lie in the anteroventral subiculum, travels in the most lateral tip of the fornix and fimbria. These fibers terminate in the region of the suprachiasmatic nuclei (SCN) and the medial basal hypothalamus in. The SCN is a postulated pacemaker of many rhythmic activities, including corticosterone release, locomotor activity, feeding and drinking 18,22,24,~5.

Because corticosterone periodicity is normally determined by the habitual sleep-wake cycle 2°, and since corticosterone fluctuations correlate well with bursts of locomotor activity under constant illumination 7, the circadian rhythm of locomotor activity was monitored in differentially fornix-ablated rats in order to determine whether the disruptive influences that occur in lateral fornix-ablated rats with regard to corticosterone release are reflected in locomotor activity patterns. In addition, the effects of differential fornix ablation were investigated with regard to the hyperactivity exhibited by fornix-transected rats z9 or those sustaining hippocampal lesion 12,26, along with its effect on other known circadian parameters, ie., body temperature, and food and water intake.

MATERIALS AND METHODS

Adult male Sprague-Dawley rats were housed in individual cages and allowed to acclimate to the lighting schedule (lights on 06.00 to 20.00 h) for at least 3 weeks prior to experimentation. Food and water were available ad libitum. Ambient temperature was 23 ± 1 °C. Animals were divided into the following groups: lateral or medial for- nix-ablated, and cortical or intact controls. Surgical procedures were performed as previously described 3. In brief, either the medial or lateral portions of the fornix were removed bilaterally by suction ablation after removal of the overlying parietal cortex and corpus callosum. Cortical controls were subjected to the same operative procedure except that the fornix was left intact. Animals were transferred from individual cages to individual plastic tubs a few days prior to or immediately following surgery. The 48 h sampling period began 7-10 days postoperatively. During the recuperative period, animals were adapted to a restrainer at variable intervals. One day prior to the com-

87

mencement of the sampling regimen, tubs were cleaned and placed on individual activity monitors (Lafayette Instruments, Model 86010). The output of the activity monitors was set at 'counts/10 min' and reflected all types of movement including exploration, grooming, etc. At 4 h intervals, 0.05-0.30 ml of blood was obtained by tail venipuncture in less than 2 rain from the time of cage opening and placed into chilled heparinized glass tubes. Plasma was separated and frozen until subsequent radio- immunoassay for adrenal corticosteroids according to the method of Krey et a1.18 with minor modifications 3. Body temperature, as measured by a rectal probe thermistor (Yellow Springs Instrument), and ambient temperature were recorded immediately following tail venipuncture. Food and water intake were also recorded at each 4 h interval. In order to minimize any effect due to sequential sampling, subgroups of animals commenced the sampling regimen at different times and then were sampled throughout a subsequent 48 h period. Only 4 animals were sampled during any 48 h session which always included at least one intact or cortical control animal.

At the conclusion of the experiment rats were deeply anesthesized, perfused transcardially with saline followed by 10 ~ buffered formalin and decapitated. Brains were removed and stored in 10 ~ buffered formalin until subsequent embedding in paraffin. Blocks were cut at 15 #m and stained with cresyl violet for histological veri- fication of neural damage. Lateral fornix-ablated animals included only those in which the most lateral tip of the fornix and fimbria was damaged bilaterally. Medial fornix- ablated animals sustained bilateral damage to the medial fornix, while cortical controls sustained damage only to the overlying parietal cortex and corpus callosum.

Any observed periodic function can always be exactly represented by an infinite series of sines and cosines 2s. For data which consists of samples taken over time periods which are of the order of the period of the function, harmonic analysis provides a systematic procedure for estimating the Fourier coefficients z8. When dealing with biological data which exhibit periodic behavior, it is necessary to separate the signal (the true periodic nature of the response) from the observed data, which contains both the signal and the experimental variability, or noise. Sufficient replicates must be measured to ensure statistical reproducibility of the signal. This is true whether the measurements are carried out over a time period which is long (a longitudinal sample of the time series) or short (a transverse sample of the time series) compared with the period of the function. Simplified procedures, such as the cosinor analysis 6, are available which allow estimates of the contribution of a particular frequency (rhythm) to the observed periodic signal. The cosinor analysis can be used to estimate rhythmic parameters for even one 24 h span with data available at 4 h intervals (the transverse sample) 6. A number of individuals must be sampled, however, to ensure ieproducibility of the effect (hence, N = 14, lateral fornix-ablated; N = 8, medial fornix-ablated; N = 5, cortical control; N = 12 intact rats). The cosinor analysis for transverse samples is particularly useful when there is independent evidence available as to the reproducibility of the effect. In prior experiments we have established the disruption of the adrenal corticosteroid rhythm with lateral fornix ablation 3. Furthermore, animals sampled 2-3 days after the first sampling regimen exhibit a fluctuating pattern of corticosteroid secretion that is different from the first 3.

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Least-squares spectra were computed according to a modification of the method of Halberg et al. 6 Fourier coefficients were estimated recursively by linear least-squares regression at each frequency over a predetermined range of frequencies. Amplitudes computed from these best-fit parameters indicate the relative contribution, or strength, of the cosine component at a particular frequency. Analysis of variance on regression was used to evaluate statistical significance. Only series containing at least 10 points were included in this analysis. Least-squares spectral components were classified according to period (~). Rhythms were called circadian if20 h < z _<_ 28 h, ultradian if ~ < 20 h, and infradian if ~ > 28 h. Two-tailed t-tests were used to compare differences between two means, and a two-way analysis of variance was used to compare treatment vs. time effects.

RESULTS

Animals in the intact, cortical control, or medial fornix-ablated groups demon- strated clear circadian patterns of adrenal corticosteroid secretion, locomotor activity, body temperature, and food and water intake that were synchronized to the light-dark cycle. Lateral fornix-ablated rats, however, exhibited fluctuations of adrenal corti- costeroid secretion that were not synchronized to the light--dark cycle nor to locomotor, body temperature, feeding or drinking rhythms. Fig. 1 represents patterns of adrenal corticosteroid secretion superimposed on locomotor activity in addition to the data obtained on food and water intake and body temperature in order to observe phase interrelationships in individual animals. The intact, cortical control, and medial fornix-ablated groups (Fig. IA) display prominent circadian periodicities of all parameters with peaks in the dark periods and troughs in the light periods. Typical aberrations observed in the lateral fornix-ablated group are displayed in Fig. 1B and C. All of the corticosteroid patterns were abnormal; L-13 (Fig. 1B) in particular displayed a rhythm that was strikingly independent of the locomotor activity rhythm.

In an attempt to further define the fluctuating pattern of adrenal corticosteroid secretion characteristic of lateral fornix-ablated rats, a least-squares spectrum analysis was performed on the data. In this analysis the height of the amplitude of each component indicates the relative dominance of that rhythm component over other components. Fig. 2A and B demonstrates the least-squares spectra of plasma adrenal corticosteroid secretion and locomotor activity for representative cases. In some of the lateral fornix-ablated rats clear dissociations between corticosteroid and activity periodicities were obtained, in contrast to intact, cortical control or medial fornix- ablated rats which demonstrate circadian patterns in both steroid secretion and activity.

Total activity differences between treatments are presented in Table 1. Because of varying levels of sensitivity in the activity monitors, differentially fornix-ablated animals were compared only to the controls that were placed on the same activity platform. In 3 out of 4 cases, lateral fornix-ablated animals displayed a significantly higher number of counts than controls. In contrast the number of counts in the medial fornix-ablated group approximated the controls, except for one case.

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TABLE I

Total activity differences between treatments

Results are expressed as per cent of control.

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* Control animals include intact and cortical control rats since no significant difference was found between them. N, number of animals; NS, non-significant; P, P vs control.

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95

The group pattern of rectal body temperatures for the 48 h sampling session is depicted in Fig. 3. In all t~eatment groups the highest values which occurred in the dark phase of the cycle were significantly different (P < 0.0005) from the lowest values which occurred in the light phase of the cycle. Although valleys of the lateral and medial fornix-ablated rats appear to be higher than intact or cortical control groups, this is not truly the case. Aberrations of temperature rhythms in specific individuals obscured the true rhythm; for example, in the lateral fornix-ablated group two animals exhibited a 180 ° phase change, and three animals displayed multipeak patterns during the observed 48 h (see Fig. 1C for examples). Therefore, a mean value for both the peak and trough of each group was derived by averaging the highest or lowest point, respectively, of each animal's cycle (Table II). Although valleys are not signifi- cantly different between any of the groups, the peaks in the lateral and medial fornix- ablated groups are significantly higher than in intact animals (P < 0.005, P < 0.02, respectively), but not cortical controls.

Group food and water intake patterns over the 48 h sampling period are depicted in Fig. 4 and 5. Peaks of food and water intake occur in the dark phase of the cycle, while troughs occur during the light phase in all groups. A few individual animals deviated from the normal pattern. One cortical control displayed a peak of food intake immediately prior to the lights-off period, and one lateral fornix-ablated animal exhibited a relatively unvarying pattern of food intake concomitant with circadian drinking patterns (Fig. IB, L-13). Multipeak patterns of food and water intake were observed in two lateral fornix-ablated rats (Fig. 1 C; L-38, L-33). A least- squares spectral analysis of the data indicated that all of the animals had dominating circadian components with regard to food intake except L-13 (aperiodic) and L-33 (dominant ultradian component). The total amount of food consumed over the 48 h period did not vary significantly (P > 0.05) among the 4 treatment groups, nor did the percentage of food consumed in the dark phase of the cycle (Table III). Cortical controls, medial or lateral fornix-ablated rats do not significantly vary from each other with regard to total amount of water consumed over the 48 h period (P > 0.05), although cortical controls and lateral fornix-ablated animals drank more than intact animals. The percentage of water consumed in the dark was lower in the lateral fornix- ablated group than in intact, cortical control, or medial fornix-ablated groups (Table IV). The two lateral fornix-ablated animals that exhibited the least amount of drinking in the dark (L-33, 42.07 ~/o; L-28, 52.18 ~) were the only animals within the group that did not display dominant circadian components in the least-squares spectral analysis. Instead, both of these animals had dominant rhythms in the ultradian range. Nearly all of the intact, cortical control or medial fornix-ablated animals, however, were dominated by circadian components.

Histological verification of the lesion was performed on all animals. Photomi- crographs of neural damage with this type of surgical procedure have been presented in a previous study 3.

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TABLE IT

Body temperature rhythms

Comparison of peaks and troughs in individual animals obtained by averaging the highest or lowest points in each individual cycle regardless of time of occurrence (°C).

Treatment N Trough Peak (2 ± S.E.) (2 :k S.E.)

Lateral fornix-ablated 14 38.19 ± 0.12 39.38 ± 0.06* Medial fornix-ablated 8 38.16 :t- 0.12 39.33 ± 0.08** Cortical control 5 37.62 ± 0.16 39.06 ± 0.16 Intact 4 37.71 ± 0.24 39.00 ± 0.09

* P < 0.005 vs intact; ** P < 0.02 vs. intact.

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97

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TABLE III

Total amount of food consumed over 48 h (g), followed by the percentage of food consumed in the dark phase of the cycle N = number of animals. (~) -t- S.E.)

Treatment Total amount of % in dark phase food consumed (9)

Lateral fornix-ablated (14) 36.71 d: 2.14 65.38 -4- 3.10 Medial fornix-ablated (8) 40.86 d: 1.81 66.92 ± 2.49 Cortical control (5) 44.92 :k: 3.23 67.71 -4- 5.69 Intact (12) 35.82 -4- 2.67 66.69 i 2.29

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TABLE IV

Total amount o f water consumed over 48 h (ml), followed by the percentage o f water consumed in the dark phase o f the cycle

N number of animals. (2 ± S.E.).

Treatment Total water °/o in dark phase consumed (ml)

Lateral fornix-ablated (14) 57.00 ± 3.23** 65.34 ± 2.40*** Medial fornix-ablated (8) 53.41 ~ 6.54 71.87 -k 3.94 Cortical controls (5) 56.04 ~ 2.87* 72.53 :k 2.65 Intact (12) 45.52 ~_ 2.97 78.72 ~ 2.52

* vs intact, P ~.. 0.025; ** P -~. 0.02; *** P < 0.001.

DISCUSSION

Medial fornix-ablated, cortical control and intact rats all display clear circadian patterns of adrenal corticosteroid secretion, locomotor activity, body temperature,

and food and water intake that are synchronized with the light-dark cycle. In contrast, lateral fornix-ablated rats exhibit fluctuations of corticosteroid secretion that are not synchronized with the light-dark cycle, nor with locomotor activity, temperature,

feeding or drinking rhythms. It has been suggested that the disruption of adrenal hormone rhythmicity with lateral fornix ablation may be due to severance of the mcht,

whose cell bodies of origin lie in the anteroventral subiculum of the hippocampal for-

mation z. This fiber tract travels in the most lateral tip of the fornix and fimbria, and terminates in the region of the suprachiasmatic nuclei and medial basal hypothalamus (ref. 16)*. Although it is clear that other fibers travelling in the lateral fornix and

terminating in the septum, hippocampal formation and mammillary bodies are also damaged with this type of surgical procedure, septal lesion, for example, has no effect

on corticosteroid periodicity ~3. Corticosteroid secretion patterns were not aligned with locomotor activity

rhythms in the lateral fornix-ablated group indicating a dissociation between the rhythmic expression of these parameters. Although both these rhythmic functions have been proposed to be governed by a common oscillator 7,8,11, free-running loco-

motor activity rhythms have been observed in conjunction with non-circadian

secretory episodes of corticosterone following catecholamine depletion in animals

housed under constant illumination 1°. It had been proposed by these investigators that the transmission pathways from the common oscillator may be affected differently by various manipulations 10, although the possibility that separate oscillators may govern

each function cannot be ruled out.

* It has been recently demonstrated that following horseradish peroxidase injection in or around the suprachiasmatic nucleus, labeled neurons are found in the anteroventral subiculum (G. Pickard, personal communication). In addition, electrical stimulation of the anteroventral subiculum results in metabolic activation of the suprachiasmatic nucleus region as indicated by the radioactive 2-deoxy- glucose method ('R. Watson, personal communication).

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It has also been suggested that body temperature and locomotor activity rhythms are governed by a common oscillator, since 'bursts' of fluctuations in temperature correlate closely with 'bursts' of activity when animals are exposed to prolonged continuous light 7-9. However, humans kept in isolated underground bunkers devoid of time cues have body temperature rhythms and activity rhythms that are vastly different with respect to period length 1.

Circadian food and water intake patterns were evident in all groups suggesting a synchronization of neural circuits which control locomotor activity, feeding, drinking and, in the majority of cases, body temperature. A few isolated examples in the lateral fornix-ablated group, however, indicate that these rhythms need not necessarily be synchronized, and can, in fact, be dissociated from one another. This is evidenced by a relatively unvarying feeding pattern along with clear circadian drinking, locomotor and temperature rhythms (Fig. 1B, L-13); a temperature rhythm that is 180 ° out of phase with locomotor activity, feeding and drinking (Fig. 1C, L-15); a multipeak feeding and drinking pattern that is independent of a multipeak temperature rhythm in combination with a circadian locomotor activity rhythm (Fig. 1 C, L-38); and a case where one cycle 'split' into two distinct peaks in feeding, drinking and body temperature (Fig. 1C, L-33). Fuller et al. 5 have demonstrated occasional splitting of the colonic temperature rhythm under constant illumination. Transient splitting of the body core temperature rhythm also occurs in chair-acclimatized monkeys exposed to light- dark shiftslL

Although the precise mechanism for the temporary desynchronization of these rhythms from each other, and in some cases, from the light-dark zeitgeber is unknown, it is hypothesized that removal of the mcht upsets the neuronal balance of activity within an integration area, such as the SCN. Although corticosteroid periodicity is the most vulnerable to lateral fornix ablation, other rhythmic parameters may also be temporarily affected.

There are interesting parallels between the effects of constant light and lateral fornix ablation on body temperature and plasma corticosterone levels. The 'splitting' phenomenon with regard to temperature is occasionally seen in animals exposed to light-dark alterationsS, 19 and in animals subjected to lateral fornix ablation. Exposure to constant illumination disrupts the circadian rhythmicity of corticosterone 2 resulting in the appearance of ultradian componentsT, 9. Mean plasma corticosteroid concen- tration over a 48 h period is higher in animals studied under constant light than under light-dark conditions 14. Similarly, lateral fornix ablation results in fluctuations of plasma corticosteroid secretion in animals sampled 7-10 days postoperatively 3,4 that suggest the presence of ultradian rhythms; however, multiple uncoupled circadian oscillations are also a viable possibility. Data collection from a period longer than 48 h is necessary to define the system completely. In addition, mean plasma corticosteroid concentrations of lateral fornix-ablated animals are significantly higher than medial fornix-ablated, cortical control or intact animals 3. The interpretation that has been given to the effects of constant light upon circadian rhythmicity is that there is a loss of internal circadian organization 21. It is tempting to speculate that lateral fornix ablation affects corticosterone release by a similar mechanism, although it is clear that

100

a discussion of true endogenous circadian mechanisms requires that the study be performed under constant conditions. In contrast, the present study must of necessity be interpreted in terms of synchronization to the light-dark cycle, since the possibility remains that the observed effects of lateral fornix ablation may be due to a defect in visual processing. However, it is difficult to explain why a visual defect of this kind would not also affect locomotor activity, body temperature, feeding or drinking rhythms.

The hyperactivity of fornix-transected rats 29 is confirmed in this study along with the retention of circadian rhythmicity of locomotion 27. It appears that hyperac- tivity is due to severance of lateral fornix fibers only, since medial fornix-ablated rats were rarely significantly different from controls in terms of total activity.

In summary, despite a strong entraining cue (light-dark cycle), the rhythms ob- served still dissociated from each other and displayed differences not only in phase but also in period. The adrenal corticosteroid rhythm was the most vulnerable to lateral fornix ablation and followed what appeared to be ultradian frequencies despite the presence of circadian patterns of locomotor activity, body temperature, feeding and drinking. These findings suggest that the circadian timing system is a multi-oscillator system composed of more than one rhythm generating area, or at least more than one transmission pathway; these systems or pathways are normally linked, but may become uncoupled temporarily by lateral fornix ablation. It is proposed that the anteroventral subiculum via the mcht is an important modulator of SCN function, and that different neural circuits exist for the regulation of different rhythms.

ACKNOWLEDGEMENTS

This research has been supported by NIH Research Grant NS 07941- 1 ININCDS.

The authors are indebted to Dr. Walt Tapp who wrote the computer program for the spectral analysis and Dr. Phil A. Femano. Work areas were generously provided by Drs. H. Feder (biochemistry), L. Michaelson (computer graphics) and B. Natelson (computer analysis). Technical instruction was provided by Christian Reboulleau (biochemistry), H. Hauser and D. Krieger (blood collection). Technical assistance was supplied by Ray Basri. All are gratefully acknowledged.

REFERENCES

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