Circadian activity rhythms in rats with midbrain raphe lesions

240

Brain Re.sear~ h, 3~4 (198~) 240--24t~ Elseviet

BRE 12047

Circadian Activity Rhythms in Rats with Midbrain Raphe Lesions J O E L D . LEVINE, A L A N M . ROSENWASSER, J A C K A . Y A N O V S K l a n d N O R M A N T ADLER

Department o( t~ychology, Universi O' of Pennsyh'ania, Philadelphia, PA 19104 ( U. S. A. , (Accepted 4 March 19861

Key words: Circadian rhythm

Activity

Midbrain

Raphe nucleus

Serotonin

Rat

The dorsal and median mesencephalic raphe nuclei provide a robust projecnon to the hypothalamic suprachiasmatic nucleus, the site of a putative neuronal circadian pacemaker. Although it has been suggested that the raphe may play a rote in the circadian timing system, this role has not yet been specified. In the present report, we examined the circadian activity patterns of rats with large midbrain lesions aimed at the median and dorsal raphe nuclei under conditions of light-dark entrainment, and while free-run ning m constant light and constant darkness. The results indicate that midbrain raphe lesions ma~ interfere with the expression of free-running circadian activity rhythms.

INTRODUCTION

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is critical for the normal display of a wide variety of circadian rhythms in birds and mammals and may indeed be the anatomical locus of a neuronal circadian 'master oscillator' or 'pacemaker '14"16"18'1924. In addition, it has been suggested that an anatomically distributed neuroendocrine network including the putative SCN pacemaker, the retina, pineal gland, ventral lateral geniculate nucleus (vLGN), and raphe nuclei, might comprise the core of the vertebrate circadian timing system sAs. However, the precise role played by these other structures in the generation and entrainment of circadian rhythms has not yet been defined. The dorsal and median mesencephalic raphe nuclei (also designated as cell groups B7 and B8 ~) provide robust serotonergic inputs to the SCN t'25. In addition to these direct projections, the raphe could also influence the SCN indirectly via projections to the vLGN ~'3, which itself projects to the SCN 2~. Retinal fibers are known to reach the S C N Is, the vLGN 9, and possibly the raphe nuclei 7. Furthermore, the SCN and raphe nuclei may be reciprocally intercon-

nected, since projecnons from the SCN to the midbrain raphe have also been described 4. These anatomical considerations seem to suggest a role for the raphe nuclei and their forebrain projections m the control or expression of circadian rhythmicity. The raphe nuclei and serotonergic neurotransmission have been implicated in the regulation of several circadian rhythmic neuroendocrine systems. For example, both raphe lesions and the serotonin synthesis inhibitor, para-chlorophenylalanme (PCPAI, can disrupt or abolish free-running and light-entrained circadian rhythms m adrenocorticotrophic hormone IACTH) and adrenal corticosteroid secretion t°12 1322.23. However. it is not clear whether these effects are due to disruption of the mechamsms underlying the circadian timing of hormone secretion, or to interference with the control of hormone secretion ~tself. The expression of circadian rhythms in behavioral activtty may be completely suppressed, albeit temporarily, following treatment with PCPA m. The time course of this effect approximates that of PCPA-mduced serotonin depletion and recovery. Furthermore. the activity rhythms of PCPA-treated animals re-emerge at a phase displaced by several hours from

Correspondence: J. Levine, Department of Psychology, University of Pennsylvania, 3815 Walnut Street. Philadelphia. PA 191tj4, U.S.A. 0{106-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division )

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Fig. 1. Activity record of a representative animal from the first surgical group (see text). Successive days are represented from top to bottom along the vertical axis and time of day is represented along the horizontal axis. The record is double-plotted in the standard circadian format to facilitate the inspection of rhythmic patterns. The black bar at the top of the right side of the record shows the time of the dark period of the light-dark cycle in effect for the initial segment of the record. LL, the first full day of constant light: D D , thc first day of constant darkness.

that of control animals. A n o t h e r recent report showed that both 5,6-dihydroxytryptamine (5,6D H T ) . a serotonergic neurotoxin, and fluoxetine, a serotonin reuptake blocker, can phase-shift the freerunning activity rhythm in sparrows 5. These results suggest a role for serotonin in the underlying circadian system but do not directly implicate the raphe. Previous studies have examined the effects of raphe lesions on behavioral activity rhythms. In general, raphe lesions have been r e p o r t e d to reduce the amplitude or clarity of entrained and free-running circadian activity rhythms :11"2°'-'3. While this effect

often results from a selective increase in daytime activity, this observation may d e p e n d on the exact locus of the lesion and on the time course of the experiment 11'2°. Despite the reduction in rhythm amplitude, the animals in these lesion studies all showed clearly detectable circadian activity rhythms. However, none of these studies presented long-term observations under both constant light and constant darkness in individual animals. The purpose of the present study was to investigate the role of the raphe nuclei in the circadian timing system by obtaining more extensive and longer-term

242

observations on the entrained and free-running circadian activity rhythms of rats with m i d b r a i n raphe lesions.

dark cycle (LD 14:10), with lights on at 18.00 h. The

MATERIALS AND METHODS

daily n u m b e r of wheel revolutions was used to select animals with m o d e r a t e to high levels of running activity for further experimentation, Surgery was performed when animals weighed 260-300 g.

Subjects

Surgery and histology

Prior to surgery, adult female S p r a g u e - D a w l e y rats (Charles River) were m a i n t a i n e d in running wheel cages in a large colony room u n d e r a l i g h t -

Two separate groups of animals were studied, with different surgical protocols for the lesions m each group• The second group of animals was p r e p a r e d

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and tested after the completion of observations on the first group. Group 1: under ketamine hydrochloride (150 mg/kg i.p.) plus acepromazine (2.4 mg/kg) anesthesia, animals were placed in a stereotaxic instrument with the head level between lambda and bregma. An electrode (0-0-0 stainless-steel insect pin insulated with Formvar except for 1.0 m m at the tip) was low-

ered through a small midline trephine (6.1 mm caudal to bregma) to a point 7.8 m m ventral to the skull surface. The electrode entered the brain at an angle of 10 ° rostral from perpendicular to the level skull surface. A constant-current electrolytic lesion maker was used to pass 1.0 m A anodal current for 45 s. Group 2: based on the behavioral and histological results obtained from the animals in Group 1 (see be-

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low), we r e p e a t e d the e x p e r i m e n t using different stereotaxic coordinates and current p a r a m e t e r s for the lesions. These changes involved lowering the electrode at a 15 ° angle through a trephine drilled 6.7 mm caudal to bregma, and to a point 9.2 mm ventral to the skull surface; these changes were intended to produce a lesion which was c e n t e r e d at a relatively more dorsocaudal position than those in G r o u p 1. In

addition, a 2.0 m A current was passed through the electrode for 60 s, in an a t t e m p t to p r o d u c e lesions which were larger than those in G r o u p 1. Following surgery, all animals w e r e r e t u r n e d to the colony r o o m and carefully observed. Mortality was high following b o t h series of lesions, and we occasionally provided orogastric t u b e feeding f o r animals which b e c a m e aphagic and adipsic in the imme-

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diate postoperative period. Two to three weeks after surgery, 5 recovered animals were placed in running wheels in a separate experimental room for behavioral observations as described below. There was no additional mortality during the collection of behavioral data. Following the completion of behavioral testing, the animals were deeply anesthetized with Nembutal

and perfused intracardially with 10,0% formalin and saline. The brains were removed, frozen in liquid freon, embedded with Lipshaw mounting medium and cut into 20-/~m coronal sections on a cryostat at -18 °C. The sections were then thawed, stained with thionin and examined microscopically to determine the extent of the lesions.

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Fig. 6. Schematic representation of the lesions in both surgical groups. The extent of each lesion is shown on five coronal levels adapted from the atlas of Paxinos and Watson~7; the numbers above each column indicate the distance of the plane caudal to bregma in ram. A: identification of selected structures in the area of the lesions. B: extent of lesions in the first surgical group. C: extent of lesions in the second surgical group. CLi, caudal linear raphe nucleus: IP, interpeduncular nuclei: ml. medial lemniscus- mlf. medial longitudinal fasciculus; MR, median raphe nucleus; Pn, pontine nuclei: DR. dorsal raphe nucleus: xscp, decussation of the superior cerebel lar peduncle: ts, tectospinal tract; RtT, reticulotegmental pontine nucleus: LDT, taterodorsal tegmental nucleus VT, ventral tegmental nucleus; py, pyramidal tracts: DT, dorsal tegmental nucleus: RPn. pontine raphe nucleus.

Experimental protocols Each rat was m a i n t a i n e d in a standard W a h m a n n running wheel with attached side cage. The wheels were placed within light-shielded cabinets e q u i p p e d with exhaust fans and p r o g r a m m a b l e lighting schedules. Lighting was provided by three 25-W incandescent lamps on each shelf of the cabinets, resulting in about 200 lux illumination at cage level. F o o d (Purina Rat Chow) and water were p r o v i d e d ad lib. E a c h revolution of the running wheels closed a microswitch and was t h e r e b y r e c o r d e d on an Esterline Angus event

r e c o r d e r running at a speed of 18 inches/24 h. These event records were cut into 24-h segments and p a s t e d on boards with each successive day below the previous day to produce standard circadian "actograms'. The animals from each surgical group were first maintained under L D cycles and then under constant light (LL) and constant darkness ( D D ) . G r o u p 1 animals were m a i n t a i n e d under LD 12:12 for 51 days. LL for 40 days, and D D for 1 l days. G r o u p 2 animals received the identical sequence of lighting conditions but were maintained under L D 14:10 for 33 days. LL for 43 days. and D D for 44 days.

247 RESULTS

Activity rhythms The circadian activity record of a representative animal from the first surgical group is shown in Fig. 1. All 5 animals in Group 1 showed clearly detectable circadian activity rhythms which entrained under the LD cycle and which persisted with free-running periodicity throughout the entire duration of LL and DD conditions. Free-running periods were always shorter under DD than under LL, as is typical of the nocturnal rat. In addition, the clarity and cohesiveness of free-running rhythms were generally greater in DD than in LL. In our experience, the activity rhythms displayed by the animals in this group were typical and could not be distinguished from those we have observed previously in intact animals under similar conditions. Animals in the second surgical group showed normal-appearing entrained activity rhythms under LD. In contrast, the free-running activity rhythms displayed by the animals in Group 2 were always less coherent, and in some cases appeared to be severely disrupted, relative to those seen in Group l. Figs. 2-5 present the activity records obtained from 4 of the 5 Group 2 animals: the fifth animal was almost totally inactive and is not depicted. Although these animals all showed free-running rhythms during the first few clays to about two weeks of EL, rhythmicity decayed during continued exposure to this condition. Indeed, by the end of LL observations, two of the animals (Figs. 4 and 5) showed no discernible rhythmicity in the activity records, while two others (Figs. 2 and 3) appeared to show some detectable, but noisy, rhythmicity. Under subsequent DD, these animals displayed activity rhythms which were generally more coherent than those seen under LL. However, even the DD rhythms of two of these animals (Figs. 3 and 5) were clearly less coherent than those seen in Group 1.

Histology Schematic representations of the lesions in both surgical groups are shown in Fig. 6. The areas included in 4 of the 5 lesions in the first group (one brain was damaged and not usable) and by all 5 lesions in the second group are shown at 5 standardized coronal levels of the midbrain and rostral pons.

These planes were adapted from the atlas of Paxinos and Watson 17 and span the rostral-caudal extent of the median raphe and include nearly all of the dorsal raphe nucleus. In Group 1, the most caudal extent of one of the lesions appears in the most rostral plane shown, and therefore only 3 lesions are represented at the next 3 levels; none of the lesions in this group extend into the most caudal of these levels. Although lesions in Group 1 resulted in considerable damage to the median raphe, the more dorsocaudal aspects of this nucleus were always spared. In addition, none of these lesions impinged on the dorsal raphe nucleus at any level. Other structures partially compromised by these lesions include the interpeduncular nuclei, medial lemniscus, decussation of the superior cerebellar peduncle, ventral tegmental area, reticulotegmental area, and the fibers of the tectospinal tract. In Group 2 the lesions tended'to be larger and more dorsocaudally placed than lesions in the first group, as we intended (see Materials and Methods). These lesions produced more complete damage over the entire extent of the median raphe than those in the first group. In all the animals in Group 2, midline damage extended into the rostral aspects of the pontine raphe nucleus. Furthermore, all but one (see below) of these lesions included at least some of the ventral aspects of the dorsal raphe, and the most dorsally placed lesions in this group resulted in fairly substantial damage (perhaps 50%) to this nucleus. However, none of the lesions involved the entire extent of the dorsal raphe. In addition to the structures mentioned above for the Group 1, the lesions in Group 2 also included parts of the ventral and laterodorsal tegmental nuclei and the medial longitudinal fasciculUS.

Relationship between histological and behavioral observations The activity record shown in Fig. 1 is from the animal in the first surgical group which sustained the most dorsally placed lesion, and consequently the greatest extent of damage to the median raphe nucleus. This lesion was clearly consistent with the expression of a robust free-running activity rhythms, under both LL and DD. Similarly, the animal from Group 2 which displayed the most well-organized free-running rhythms (Fig. 2) had the smallest and

248 most ventrally placed lesion in this group. Although this lesion destroyed nearly all of the median raphe, the dorsal raphe was not involved. On the other hand, the animal from Group 2 which displayed the least coherent free-running rhythms (Fig. 5) had the largest lesion, which resulted in extensive damage to both the dorsal and median raphe nuclei. This rat was the only one in the study to display the unusually high daytime activity which has been reported to occur following raphe lesions 2tl .20 In summary, circadian activity rhythms were less coherent in Group 2 than in Group 1 animals. There also appeared to be a trend within Group 2 for larger lesions to result in greater rhythmic disruption. However, we cannot at present separate the possible relative contributions of lesion size and lesion placement to these behavioral observations. DISCUSSION The results of this study indicate that large midbrain lesions which include the raphe nuclei may disrupt the expression of free-running circadian activity rhythms in the rat. Comparison of the behavioral and histological observations from the two groups of animals suggests that these disruptions probably result from either (1) extensive overall damage to the median and dorsal raphe nuclei, or (2) damage to some specific intra-raphe locus in Group 2 but not Group 1 lesions. However, we cannot exclude the involvement of other, non-raphe structures which were also damaged by the lesions, and it is even possible that the larger size of Group 2 lesions, rather than their specific locus, was responsible for the differences noted between the two surgical groups. The least coherent rhythms observed in this study were displayed by Group 2 animals under LL. At least some of these animals also showed apparent disruptions under DD (e.g., Figs. 3 and 5). However, the animals showing the most disrupted rhythmicity under LL did not necessarily show similar disruptions under DD (e.g., Fig. 5). While LL is itself capable of producing rhythmic disruption even in normal animals, the lighting intensity employed in this study did not prevent the expression of long-term free running rhythmicity in the animals in Group 1. Although there may have been an 'interactive' effect between lighting conditions and lesions, it is also possible that the lesions simply reduced the amplitude of free-run-

ning rhythmicity under both EL and DD. The most disrupted free-running rhythms observed in this experiment appear to be less coherent than those seen in previous studies using raphe lesions 2'-~3. possibly as a result of differences in lesion location or size, light intensity, or duration of exposure to free-running conditions. Ft~r example, Block and Zucker 2 reported that lesions which included most of the median raphe nucleus were most effective in reducing the amplitude o! circadian activity rhythms, while damage to the dorsal raphe and other nearby structures seemed less critical for this effect. In contrast, the present results suggest that the most disrupted rhythms are seen following lesions which involve both median and dorsal raphe nuclei. However, it is not yet possible to draw firm conclusions concerning the anatomical basis for the reported effects. The present results are consistent with the hypothesis that the raphe nuclei, and possibly their serotonergic projections to the SCN and vLGN, play a role in an anatomically distributed network underlying the control of circadian activity rhythms. Both pharmacological intervention in serotonergic systems and raphe lesions can alter the expression of circadian activity rhythms. Since the SCN is itself an anatomically heterogeneous structure which may comprise multiple, intra-SCN oscillatory subcomponents ~a, it is possible that serotonergic input from the raphe nuclei serves to facilitate oscillator coupling relationships within the SCN. In addition, it has been reported that the (feline) raphe nuclei receive a direct retinal projection r. This observation, as well as the well-documented raphe projections to the vLGN, suggests that the raphe may play a role in the modulation of SCNmediated circadian rhythms. Further studies are required to implicate directly either serotonergic neurotransmission or raphe projections to the SCN in the mediation of the effects observed m this study.

ACKNOWLEDGEMENTS We thank Dr. Peter Hand for anatomical assistance. We also thank Louise Levine for help with the figures. This study was supported in part by NSF Grant BNS 8217281 to N.T.A. and A.M.R.; J.A;Y. is a Medical Scientist Training Program trainee, NIH Grant 5-T32-GM07170.

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