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Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract
Brain Research, 460 (1988) 297- 313
297
Elsevier BRE 13928
Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract R.F. Johnson 1'*, R.Y. Moore 2'3 and L.P. M o r i n 1'4 Departments of 1Psychiatry and Behavioral Science, 2Neurology, 3Neurobiology and Behavior and 4psychology, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A,) (Accepted 5 April 1988)
Key words: Circadian rhythm; Entrainment; Retinohypothalamic tract; Neuronal plasticity; Hamster; Geniculohypothalamic tract
The suprachiasmatic nuclei receive photic input information directly through a retinohypothalamic tract (RHT) and indirectly through a projection from the intergeniculate leaflet of the lateral geniculate complex, the geniculohypothalamic tract (GHT). Prior work has established that the RHT is sufficient for entrainment, but has not shown whether it is necessary because it has not been possible to transect that pathway. The present study addresses this problem by employing knife cuts to sever the RHT in male hamsters. Three knife cut procedures were used and one of these succeeded in separating the SCN from the optic chiasm in 8 animals with limited damage to the chiasm and the SCN. The effectiveness of the RHT lesion was confirmed by cholera toxin-HRP histochemistry which demonstrated that the knife cuts eliminate the normal retinal innervation of the SCN while sparing projections to thalamic and tectal visual centers. In a light-dark cycle, the lesioned animals exhibit free-running rhythms indicating that the RHT is necessary for entrainment. A surprising observation is the presence of extensive axonal sprouting of retinal fibers in brains of animals with RHT lesions. The newly-formed axons grow extensively into the SCN, anterior hypothalamus and basal forebrain, but form anomalous axohal plexuses which have no evident function.
INTRODUCTION
that neither the P O T or G H T is necessary for entrainment 4'6'9'11'24 (Johnson et al., submitted). These
The suprachiasmatic nuclei (SCN) function as a clock controlling circadian rhythms in m a m m a l s 15"32.
studies also d e m o n s t r a t e that the R H T is sufficient to maintain entrainment in the absence of all other visual pathways in animals that otherwise a p p e a r functionally blind. It has not been possible, however, to demonstrate that the R H T is necessary for entrainment as no m e t h o d has been available to transect the R H T as it enters the SCN. In the present experiments, we have addressed this p r o b l e m by producing knife cuts at the b o r d e r of the SCN with the optic chiasm. The anatomical d a t a indicate that these cuts destroy the R H T , leaving the chiasm and P O T largely intact and preserving a functional SCN. The behavioral data indicate that the animals maintain visually guided behavior, but circadian entrainment to the l i g h t - d a r k cycle is lost. F u r t h e r , this occurs with a striking growth of retinal axons into the ante-
The SCN are innervated by two visual pathways, a retinohypothalamic tract ( R H T ) and a geniculohypothalamic tract ( G H T ) . E n t r a i n m e n t , the setting of the phase of the clock, could require either of these pathways or, m o r e likely, they could function together in that process. The R H T is a direct projection from the retina to the SCN 16 and to other parts of the hypothalamus ~2"23"29. The G H T is a secondary visual projection that arises from the intergeniculate leaflet ( I G L ) of the lateral geniculate nuclei ( L G N ) 2'3'5'21, a zone that receives direct visual input via the primary optic tracts ( P O T ) 8"z2. Destruction of the P O T of the L G N , without d a m a g e to the R H T , has only minor effects on circadian rhythm e n t r a i n m e n t indicating
* Present address: Department of Psychology, University of Iowa, Iowa City, IA, U.S.A. Correspondence: L.P. Morin, Department of Psychiatry, Health Science Center, SUNY, Stony Brook, NY 11784, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
298 rior hypothalamus in an extensive, and apparently non-functional, anomalous axonal plexus. MATERIALS AND METHODS Male golden hamsters (Charles River) were housed individually in translucent cages under a L D 14:10 p h o t o p e r i o d in a t e m p e r a t u r e - c o n t r o l l e d room. F o o d and water were continuously available. Each cage contained a 17-cm d i a m e t e r running wheel with each revolution of the wheel activating a microswitch, in the initial part of the studies, the microswitch was connected to an Esterline-Angus event recorder and the records were p r e p a r e d as standard actograms. Later, all data were collected by a computer which recorded the n u m b e r of revolutions in 1min bins and later plotted the data in actogram format. Several surgical procedures were tested before fully successful R H T cuts were achieved. To e m p h a size the successful methods, the studies are p r e s e n t e d in reverse order. The experiments in which cuts were not complete (Expts. 2 and 3) have been retained because they provide significant control data concerning the ability of the circadian rhythm system to withstand damage to the SCN.
Experiment 1 Each hamster (n = 17) was o p e r a t e d upon while entrained to LD 14:10. The knife consisted of a stainless-steel tube ground away to form a semicircle (2.0 mm diameter) beveled to a sharp edge. F o r the cut, the knife was fitted into a larger guide cannula to allow vertical m o v e m e n t and lowered at a coordinate
(1.4 mm rostral to bregma; bregma and lambda level) just rostral to the SCN. As the guide cannula was lowered, the chiasm was detected by the vertical slippage of the knife. D o w n w a r d pressure was then combined with a slight back and forth rotation of the knife. This procedure created a hemi-island (Fig. 1A) which included the caudomedial chiasm and the bilateral SCN which maintained all caudal and dorsocaudal connections. Three of these animals died shortly after surgery. A group of control hamsters receiving no surgical intervention was also used in this study. A f t e r surgery, the wheel-running was monitored for 1-2 months while the animals r e m a i n e d on LD 14:10.
Experiment 2 Each hamster (n = 15) received a bilateral rostral cut combined with a cut ventral to the SCN. The knife consisted of a tungsten wire (130 ~ m diameter) soldered into a stainless-steel shaft (320/~m o.d.) for rigidity. The wire p r o t r u d e d 1.5 m m p e r p e n d i c u l a r to the axis of the shaft. The shaft was fitted into a guide cannula which allowed vertical movement. The knife was stereotaxically lowered with the shaft in the midline, the wire cutting in the coronal plane at the same coordinate as in Expt. 1 ;'just rostral to the SCN (Fig. 1B). W h e n the knife hit the optic chiasm (indicated by upward slippage of the shaft in the cannula), the knife was rotated caudally 180 ° between the chiasm and the SCN (Fig. 1C). The knife was then withdrawn contralateral to the side of insertion. This created a bilateral rostral cut c o m b i n e d with a 180 ° cut below the SCN. The cut was m a d e while the hamsters were entrained to L D 14:10. Five experimental
C Fig. 1. Schematic illustration of the methods for making RHT cuts. A: the heavy, semicircular line that roughly follows the contours of the caudal chiasm and medial optic tracts indicates the ideal location of the core cut in Expt. 1. B: the unilateral bar across the optic chiasm represents the initial coronal placement of the L-knife in Expts. 2 and 3. The knife was then rotated to undercut the SCN as indicated by the diagonally filled semicircle. The knife was raised ipsilateral (Expt. 3) or contralateral (Expt. 2) to the side of initial placement. C: a parasaggital view of an ideal SCN undercut made with the L-knife. AC, anterior commissure.
299 animals died shortly after surgery. Seven hamsters in which only the skull was opened and the dura punctured and 5 hamsters which received coronal cuts rostral to the SCN (but no undercuts) were used as controis. Postsurgically, the rhythms were allowed to stabilize in the LD cycle after which the hamsters were placed in constant darkness for at least 8 weeks.
Experiment 3 Hamsters (n = 15) were allowed to generate freerunning circadian rhythms in dim constant light (about 0.2 pW/cm 2 corresponding to about 1.2 lux as measured at midcage level with a J16 Tektronix digital photometer) until the period became stable. Each hamster then received a unilateral coronal plane cut rostral to the SCN combined with a cut ventral to the SCN. The procedure was identical to that in Expt. 2 (Fig. 1B) except that the knife blade was withdrawn ipsilateral to the side of insertion. A group of control hamsters (n = 18) received 0.6 saline injections into the lateral geniculate region. The postsurgical freerunning rhythms were allowed to stabilize, after which the hamsters were switched back to the original LD 14:10 cycle and reentrained. (Mean light intensity was about 10.2 pW/cm 2 corresponding to about 29.3 lux.)
Histology Each experimental hamster received bilateral intraocular injections of 2 pl of 0.1% horseradish peroxidase conjugated to cholera toxin B-subunit, (CTHRP; List Biological) and the brains were prepared for H R P histochemistry 14. The visual projections in each knife cut animalwere evaluated with CT-HRP histochemistry. The R H T projection was evaluated by comparison with control brains as were the retinal projections to the LGN. The brains of several normal hamsters which had received CT-HRP with the same parameters as above 29 were used for determination of changes in the R H T associated with the cuts. In order to evaluate the effects of R H T damage versus general hypothalamic damage, brains of the 5 hamsters which had received coronal knife cuts rostral to the SCN (sparing the chiasm) were also examined with CT-HRP). No histology was performed on the unoperated control group (Expt. 1) or the sham control group from Expt. 2, in which only the skull and dura were opened.
RESULTS The R H T projections in the normal hamster will be reported in detail elsewhere (Johnson et al., submitted). Briefly, however, the terminal plexus in the SCN begins in the ventral portion of the nucleus at its rostral extreme. It expands caudally to fill the nucleus with the ventrolateral portions having the densest label. The R H T projection to the SCN is densest in the intermediate and caudal parts of the nucleus (Fig. 2A) and continues caudally into the retrochiasmatic area to occupy a zone between the ventral limits of the paraventricular nucleus and the dorsal border of the anterior hypothalamic nucleus. In addition to its primary termination in the SCN, and dorsocaudal to it, the R H T of normal animals also projects to the lateral hypothalamic area via fibers arising from the lateral optic tract near the supraoptic nucleus, the anterior hypothalamus via fibers extending beyond the dense plexus in the SCN and to the anterior amygdala and the cortical amygdaloid nucleus via a sparse group of fibers which leaves the very rostral chiasm and travels along the base of the medial preoptic area and basal forebrain.
Experiment 1 In all cases, the semicircular cuts extend through the chiasm at the ventral surface of the brain. Some of these cuts fall behind the chiasm and others are located about midway through the rostrocaudal extent of the chiasm. The majority of hamsters have cuts which destroy the rostral to intermediate portions of the SCN, leaving the caudal portion of the nucleus at least partially intact. The cuts extend along the lateral boundaries of the SCN and into the retrochiasmatic area. In 8 of the animals (XT1,XT9,XT11,XT12,XT14,XT15, XT16,XT17), the cuts appear to have completely severed connections between the SCN and the optic chiasm and optic tract. In 3 animals (XT3,XT4,XT6), the cuts are incomplete, leaving part of the optic tract connected to the SCN. In three other animals (XT5, XT8,XT18), the cuts are placed across the SCN, greatly disrupting the middle to caudal portions of the nuclei. The R H T projection to the SCN, as demonstrated by the CT-HRP material, is absent in two animals with complete cuts (XT12,XT9; Fig. 3B) and appears
;tral Fig. 2. A: heavy (normal) RHT label in the intermediate rostrocaudal level of the SCN in an animal that received a coronal cut rob Iced to the SCN and no undercut. B: reduced RHT label in an animal that received a unilateral rostra1 cut and SCN undercut. C‘: redu and RHTlabel in an animal that received a bilateral rostra1 cut and an SCN undercut. The ventral scarring caused by the knife cut in B latic C is indicated by the arrows and the line of bright artifact between the SCN and the chiasm. OC‘. opttc chiasm; SC‘N. \upr;tchiasn nucleus.
301 as very sparse or anomalous fibers (Fig. 3A) in the remaining animals. In the 3 animals with incomplete cuts, two have robust R H T innervation of the SCN (XT3,XT4) and one (XT6) has sparse terminations in the caudal SCN. In the three hamsters with heavy damage through the caudal SCN, there is very little innervation of the SCN and occasional, anomalous large fibers. Although the R H T projection to the SCN is greatly reduced or anomalous in all animals with semicircular cuts, the terminal field in the L G N is extensive and normal. Fig. 4 shows the retinal innervation of
the L G N for one animal in which no retinal fibers were present in the SCN (XT12). All animals with reductions in the R H T projection to the SCN also show marked reductions in projections of the R H T to other hypothalamic areas. The semicircular cut is associated with anomalous retinal axons that extend far rostrally through the preoptic area to areas not normally receiving retinal input (Fig. 5A). The anomalous fibers are coarse and thick, often appearing as a tangle of large fibers rather than as a pattern of dense terminals. At the level of the SCN, such fibers are clearly present in the nu-
Fig. 3. The RHT in the SCN of animals receiving the semicircular knife cuts. A,B: examples from two animals with complete cuts; an RHT terminal plexus is not identifiable within the SCN, although both animals have robust label in the LGN (Fig. 4). Both animals have free-running rhythms in LD 14:10 after the cuts (e.g., Fig. 7B). OC, optic chiasm; SCN, suprachiasmatic nucleus.
302
Fig. 4. Retinal innervation of the LGN in an animal (XT12) in which the R H T to the SCN is completely severed and no labeled fibers in the SCN are visible (Fig. 3B). The plexus in the L G N in this animal is normal and shows heavy label in the IGL. The empty region within the IGL is a blood vessel. D L G , dorsolateral geniculate; IGL, intergeniculate leaflet; POT, primary optic tract; V L G , ventrolateral geniculate.
cleus, but also extend far outside of the nucleus dorsally along the ventricle (Fig. 3A). in other cases, the fibers appear to orient and concentrate along the line of the cut. This apparent sprouting was also indicated
by a much heavier than normal innervation of the rostral SCN which extended periventricularly beyond the SCN into the anterior hypothalamic area (Fig, 5B). The massive extent of sprouting evident in
Fig. 5. A n o m a l o u s growth of retinal fibers observed after damage to the RHT. Large fibers are olten found far rostral to the S('N near the level of the o r g a n u m vasculosum ol the lamina terminalis (A,C). In other cases, abnormally dense label is prcsent in the very ro,,tral SCN and extends far beyond the SCN laterally and periventricularly (D). At this level, the R H T to the SCN is normally vcr~ faim and restricted to the ventral portion of the nucleus. The chiasm is completely severed coronally immediately caudal to the photomicrograph shown in (D), Some animals also show large anomalous fibers caudal to the cut, in the region of the SCN (BI. but these fibcr~ also extend far beyond the SCN in a periventricular orientation. The arrows in (B) indicate the lateral boundary of the cut. l bc ph~tomicrograph shown in (B) is a magnification and dorsal extension of the photomicrograph shown in Fig. 3A. O(', optic chke, m: ()N. ~t~tic nerve.
303
zl
31)4 some individuals is shown in Fig. b. Photomicrographs of the brain of this animal are also shown in Figs. 3A and 5A,B.
Behavior was recorded from all hamsters for at least 30 days after surgery, although some individuals required recording beyond this time because activity
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Fig. 6. Extent of a n o m a l o u s fibers in animal XT1 which sustained an R H T cut in Expt. 1. The broken lines indicate location of the knife cut. Blackened areas show extremely dense label in which individual fibers could not be identified. A C , anterior commissure; D B , diagonal band of Broca: OC, optic chiasm; ON, optic nerve; OT, optic tract; P O A , preoptic area: PVN, paraventricular nucleus: R C h , retrochiasmatic area; TC, tuber cinereum.
305 patterns were unclear. All of the hamsters showed transient disruptions in activity rhythms after the cuts (Table I). One hamster (XT3; Fig. 7A) rapidly regained rhythmicity and remained normally entrained. Six of the hamsters ( X T 1 7 , X T 1 , X T 1 2 , X T 1 6 , X T 9 , XT15) showed clear free-running rhythms within two weeks of surgery (Figs. 7 B - D and 8B). The free-running rhythms were consistently greater than 24 h,. clearly could be traced to the phase of previously entrained activity and continued for the entire survival period. Three hamsters (XT6,XT4,XT8) showed transient free runs of >24 h within a week of surgery which eventually switched to periods of <24 h and reestablished an entrained phase. One of these animals had a normal phase angle of entrainment while two had unstable activity onsets which were phase-advanced and preceded light offset (Fig. 8A). This pattern of entrainment continued throughout the remaining survival period. Two hamsters (XT11,XT18) showed very disrupted rhythms with no clear or consistent phasing. The remaining two hamsters (XT14, XT5) had two components in their rhythms, one coinciding with, and synchronized to, the dark phase of the cycle and one free-running with a >24 h period (Fig. 8C). In these two cases, the free-running component could be traced to the previous phase of entrainment. In each, the free-running rhythm continued, without obvious phase changes, through the
light portion of the cycle, eventually becoming phased with the component synchronized to the LD cycle. Because none of these animals was ever placed in constant lighting conditions, it is not known whether the rhythmicity which was phase locked to the LD cycle postsurgically represented true entrainment of a circadian rhythm or exogenously driven activity. Experiment 2
In the group receiving the bilateral rostral cuts with SCN undercuts, two of the animals died before the brains were taken for histology. The remaining 8 animals show variable amounts of damage to the SCN (Table II). In one animal (U3) the cut severs the rostral and ventral connections of the SCN, but leaves the SCN intact. In two animals (U9,U11), the cut destroys the rostral extreme of the SCN on both sides, but leaves the middle to caudal portions of the SCN intact. In the other 5 animals ( U 5 , U 7 , U 8 , U12,U14), a large portion of the middle SCN is destroyed (accompanied by an enlarged ventricle) leaving only a small rostral and caudal portion of the SCN intact. In two of these latter animals (U7,U8), the caudal portion of the SCN is present, but it is greatly distorted and appears damaged by the cut. In each of the animals, the SCN appears to be severed from the optic chiasm. The extent of the R H T in the SCN in
TABLE I Rhythmicity of hamsters sustaining semicircular cuts around the rostral SCN (Expt. 1)
Strong, a robust rhythm with stable phase. Moderate, a rhythm with slightly unstable phase and increased activity during the "rest' phase. Weak, a rhythm that is apparent, but very unstable, and which has a high level of activity during the 'rest' phase. Very weak, a rhythm which is barely discernible in the actogram, but which is statistically significant. Animal
SCN innervation
SCN damage
Rhythmicity in LD post-cut
XT1 XT3 XT4 XT5 XT6 XT8 XT9 XT11 XT12 XT14 XT15 XT16 XT17 XT18
anomalous normal normal sparse sparse sparse none anomalous none anomalous anomalous anomalous anomalous sparse/anomalous
rost-mid rost-mid rost-mid mid-caud rost-mid mid-caud rost-mid rost-mid rost-mid rost-mid rost-mid rost-mid rost-mid mid-caud
moderate free-run = 24.18 h strong entrainment transient free-run, weak entrained two weak components, 24.0 and 24.57 h transient free-run, strong ent~'ainment transient free-run, strong entrainment weak free-run = 24.14 h none moderate free-run = 24.10 h two weak components, 24.0 and 24.43 h weak free-run = 24.1 h moderate free-run = 24.14 h strong free-run 24.14 h none
306 the animals is reduced (Fig. 2C), but the primary visual projection appears normal in all animals.
occupies an abnormally restricted area of the nu-
In the animal with the least destruction of the SCN
cleus. The plexus in the SCN also contains numerous
(U3), the R H T in the SCN is very faint and restricted to an abnormally small portion of the nucleus. The
anomalous fibers. In the remaining 5 animals with heavy damage to the middle SCN, the R H T appears
R H T in the SCN of the two animals with damage to
as faint terminals or anomalous tangles of fibers in
XT3
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0 24 0 24 Hr Fig. 7. Actograms of animals receiving semicircular cuts around the SCN on the day indicated by the arrow. In an animal with a cut which failed to completely sever the SCN from the chiasm (A), the rhythm remains entrained to the LD cycle. In other animals in which the cuts were complete (B-D), the rhythms appear to free-run under LD conditions immediately after surgery. In (A), the RHT plexus in the SCN is robust, whereas the RHT plexus of the animals that free-run is absent (B) or drastically reduced (C,D). See text for details. The black and white bar at the top of each actogram indicates the LD 14:10cycle.
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Fig. 8. Actograms from animals that required extended monitoring after the cuts (arrow) because of ambiguous patterns. One animal (A) shows an unstable activity onset phase. Another animal (B) shows a free-running rhythm with a period near 24 h. A third has two disrupted components (C), one of which appears entrained and the other free-running with a long period. See text for details. The LD 14:10 cycle is indicated by the black and white bar at the top of each actogram.
p o r t i o n s of the S C N c a u d a l to the cut. In t h e s e animals,
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In 6 of the 8 a n i m a l s , an a b n o r m a l l y large n u m b e r
abnormally
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coarse and o f t e n e x t e n d p e r i v e n t r i c u l a r l y rostral to
that n o r m a l l y r e c e i v e few, if any, retinal fibers. This
the cut. A b n o r m a l l y c o a r s e fibers also a p p e a r in the
a b n o r m a l p a t t e r n of the R H T is m o s t o f t e n o b s e r v e d
r e t r o c h i a s m a t i c a r e a of two animals ( U 7 , U 1 4 ) . T h e
as an unusually d e n s e plexus rostral to the cut. T h e
sprouting e v i d e n t in this e x p e r i m e n t did not s e e m as h e a v y as in E x p t . 1.
p r o j e c t i o n also differs f r o m n o r m a l dorsally and laterally. T h e fibers e x t e n d into the m e d i a l p r e o p t i c
TABLE II Circadian rhythmicity of hamsters with bilateral coronal cuts rostral to the SCN and an SCN undercut (Expt. 2)
Strength of rhythmicity definitions are as in Table I. Animal
U1 U3 U4 U5 U7 U8 U9 U 11 U 12 U 14
SCN innervation
unknown sparse unknown sparse sparse sparse sparse sparse sparse sparse
SCN damage
unknown none unknown middle mid-caudal all levels rostral rostral middle middle
Rhythmicity
Period of
LD
DD
moderate mode rate moderate weak very weak very weak weak very weak moderate moderate
moderate moderate moderate moderate transient, weak none transient, weak none moderate moderate
free-rbln 24.20 23.89 24.10 24.10 23.62 24.07 24.05 24.10
Phase advance
1-2h n o n e
none Bone
7-8h 1-2 h 2-3h B o n e
308
U
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Fig. 10. Actograms from animals that received unilateral rostral cuts and SCN undercuts. The animals were allowed to flee-run in constant dim light. The cuts were performed on the days indicated by the circles in the actograms. For animals in which the ventral cuts passed through or below the chiasm, missing the SCN (A,B), there is little effect on the rhythm. For animals in which the cuts pass between the SCN and chiasm, damaging the ventral SCN (C,D), the rhythms are disrupted. Under LD conditions (indicated by the black and white bar), all animals show synchronization of wheel-running to the LD cycle.
310 area and are particularly concentrated in a periventricular zone. In 3 animals (U9,UII,U14), dense anomalous fibers are observed far rostral to the SCN where only occasional fibers are observed in normal animals (Fig. 5C). The anomalous fibers are observed as far rostral as the organum vasculosum and some extended dorsally into the septum. In addition to abnormal sprouting rostral to the cut, abnormal patterns of retinal innervation are present in intact SCN caudal to the cuts. This is most often seen as very sparse and restricted innervation of the SCN and adjacent areas (Fig. 2C). In some animals, the residual innervation also deviates from the normal pattern of dense terminals ventrally and laterally diminishing to light innervation dorsomedially and, instead, appears as a tangle of large, varicose fibers in the middle of the nucleus. The exact route by which the residual SCN is innervated with these anomalous fibers is not evident. All hamsters except one eventually showed a 24 h rhythm wit'," activity associated with the dark phase of the LD cycle (Table I). However, in some animals the rhythm was severely disrupted for several days immediately after the cuts (Fig. 9C,D). Further, the activity patterns of these animals, although clearly synchronized to the LD cycle, tended to be poorly organized and showed disruptions in phase of entrainment. When the hamsters were transferred to constant darkness, 6 of them showed sustained free-running rhythms (Fig, 9B,C) and 4 had activity rhythms which eventually disintegrated into arrhythmicity (Fig. 9A,D). Interestingly, 3 of the hamsters which maintained free-running rhythms show some of the greatest losses of SCN tissue (e.g., U12: Fig. 9B). Animals with hypothalamic damage from coronal cuts aimed rostral to the SCN sustain very little, if any, damage to those nuclei. The cuts generally are located between the organum vasculosum lamina terminalis (OVLT) and the rostral extreme of the SCN. The RHT projection to the SCN of these hamsters is robust and normal (Fig. 2A) as are retinal projections to the lateral geniculate region.
Experiment 3 The group receiving unilateral rostral cuts and SCN undercuts can be divided roughly into three subgroups. In the first (n = 6), the cut goes through the base of the brain and enters the ventral side of the
chiasm. In the second (n = 5), the cut passes between the chiasm and the SCN on one side and extends into the optic tracts on the opposite side. In the third subgroup (n = 4), the knife appears to completely sever the SCN from the chiasm (Fig. 2B). In the latter two subgroups, there is variable, slight damage to the ventral extremes of the SCN and unilateral, but not contralateral, damage rostral (100-200 um) to the SCN in all animals. In general, however, the SCN of all animals is remarkably well preserved. The HRP histochemistry shows, in all animals, an RHT terminal plexus in the SCN. Unfortunately, in these brains there was a poor quality reaction resulting in rapid, variable fading of the reaction product which prevented a more detailed analysis of changes which might have occurred in the RHT. All hamsters except one (no. 17) continued to show free-running circadian rhythms after the cut (Fig. 10). The exception became arrythmic. The other animals usually resumed a free-running rhythm within a day or two after the cut and maintained it as long as the animals were in constant dim light. However, the extent of disruption of the free-running rhythm was associated with the placement of the cuts. The subgroup in which the cut separates the SCN from the chiasm most effectively (and also causes the greatest damage to the ventral SCN) showed the greatest rhythm disruption (the arrhythmic hamster is in this group; Fig. 10C,D). The subgroup with incomplete cuts showed variable amounts of disruption, whereas in the subgroup with cuts which entirely miss the SCN, the animals were indistinguishable from control animals according to visual inspection of the actograms (Fig. 10A,B). Upon reintroduction of the LD cycle, all hamsters in this experiment showed a clearly entrained, apparently normal, activity rhythm. DISCUSSION The present results demonstrate that the hamster RHT can be selectively destroyed, leaving the primary visual projections to the thalamus virtually intact. Following effective RHT cuts, the hamster wheel-running rhythm fails to entrain to LD cycles (even after prolonged exposure to LD; unpublished data). In addition, such damage to the RHT produces a dramatic reorganization of visual projections to
311 the hypothalamus with extensive sprouting of retinal axons to form anomalous plexuses in the hypothalamus and extending beyond its borders.
Entrainment The lesion which most effectively reduces or destroys the R H T and allows free-running rhythms under LD conditions is a semicircular isolation of the SCN from all rostral, ventral and lateral connections. The other two types of lesion, a unilateral or bilateral knife cut rostral to the SCN combined with an SCN undercut are less effective, always leaving at least a portion of the R H T terminal plexus in the SCN intact. The undercuts also produce a qualitatively different effect on the rhythms. Although they acutely disrupt the locomotor rhythm, there is little indication that entrainment is lost. The undercuts may leave fibers intact which arise from the chiasm rostral or lateral to the SCN. In the case of the effective semicircular cuts, fibers arising rostral or lateral to the SCN would be destroyed as would fibers which remain in the chiasm to enter the SCN from a ventral direction. The loss of direct retinal innervation of the SCN is probably the primary contributor to the loss of entrainment consequent to the R H T cut. Animals displaying free-running rhythms had the greatest reduction of normal R H T projections. Although these animals also sustained damage to the rostral SCN, it is unlikely that loss of entrainment is specifically related to this damage. Animals with similar destruction of the rostral SCN (Expt. 2 and ref. 10) remain entrained to the LD cycle. Perhaps equally important for the loss of entrainment may be the loss of G H T innervation of the SCN. Several studies have indicated that the G H T is not necessary for entrainment 4'6"9'11'24 (Johnson et al., submitted). But the G H T may be a sufficient entrainment pathway and the present cuts may have destroyed both the R H T and GHT. The exact route by which the G H T reaches the SCN is not clear, but recent evidence suggests that the NPY portion of the G H T arrives at the SCN both via the optic tracts and via the zona incerta 27. The present semicircular cuts would have destroyed the optic tract portion of the G H T , but probably spared the zona incerta portion if it enters the SCN from a caudolateral direction. Destruction of the optic tracts by parasaggital cuts ira-
mediately lateral to the SCN leaves a robust plexus of NPY terminals of geniculate origin in the ventrolateral SCN (n = 3; Johnson, unpub.), whereas destruction of the LGN (and thereby the entire G H T ) eliminates all NPY innervation of the SCN of geniculate origin (Johnson et al., submitted). We were not able to perform NPY immunohistochemistry on the material prepared for CT-HRP and could not assess the integrity of the GHT. Therefore, we cannot answer the question of whether the G H T is sufficient for entrainment.
Rhythmicity With regard to SCN function, several conclusions concerning cut placement and subsequent changes in rhythmicity can be made: (1) hamsters are able to sustain major loss of SCN tissue and still show relatively normal behavioral rhythms (Expt. 2); (2) SCN efferents rostral and lateral to the nucleus are not required for the expression of wheel-running circadian rhythmicity (Expt. 1); and (3) damage to the ventral region of the SCN has only slightly disruptive effects on the wheel-running rhythms (Expt. 3). The minor effect of damage to the ventral portion of the nucleus on circadian rhythmicity is in agreement with work on rats I. The persistence of free-running rhythms after partial isolation of the SCN also supports earlier observations from r a t s 19"2°'33 that the rostral and lateral efferents of the SCN 34 are not required for the expression of circadian rhythmicity. In particular, the pathway from the dorsomedial SCN to the LGN (which takes a lateral route above the optic tracts 34) appears not to be required for rhythm generation or expression. This observation is in agreement with resuits from rats 4 and hamsters 6'9'24 showing that the LGN is not required for entrainment. The ability to destroy large portions of the SCN and yet maintain circadian rhythmicity(18.26.33; Expt. 2 and unpublished work) indicates that either the rhythm generating capacity of the SCN is very diffuse or redundant or that a small, but as yet undefined portion, is required. Sprouting The sprouting identified in these studies supports several conclusions: (1) in experimentally altered and normal animals, CT-HRP is a very sensitive tracer of normal and anomalous visual projections;
312 (2) there is r e m a r k a b l e anomalous growth after damage to the RHT; and (3) as indicated by the existence of anomalous fibers in the SCN of hamsters showing free-running rhythms under L D conditions, presence of retinal efferents does not always indicate functional connections. It is likely that the sprouting is derived from axons of the R H T , but we cannot exclude the possibility that the axons giving rise to the anomalous innervation are from other c o m p o n e n t s of the optic chiasm injured by the lesions. Sprouting of retinal efferents after retinal fiber damage may also occur in the lateral hypothalamus ~3. The neuronal plasticity of the R H T suggests the possibility that recovery of function of residual tissue may occasionally follow successful lesions or knife cuts. A f t e r apparently successful SCN lesions or R H T damage, hamsters often show residual rhythmicity which entrains to an LD cycle -'~'. The possibility exists that such entrainment represents recovery of R H T function. The discovery of sprouting in the present work is particularly intriguing because it occurs in adult animals. Plasticity after damage to the R H T has been shown in the neonatal rat 3°. Plasticity of the h a m s t e r visual system has been d o c u m e n t e d , both at the anatomical and behavioral levels, after the damage in very young animals, but not if the damage occurs in adults -'~. The sprouting seen in the h y p o t h a l a m u s may have been paralleled by sprouting of axon collaterals which project to the geniculate l-s'2z. H o w e v e r ,
REFERENCES l Brown, M.H. and Nunez, A.A., Hypothalamic circuits and circadian rhythms: effects of knife cuts vary with their placement within the suprachiasmatic area, Brain Res. Bull., 16 (1986) 705-711. 2 Card, J.P. and Moore, R.Y., Ventral lateral geniculate efferents to rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity, J. Comp. Neurol., 206 (1982) 390-396. 3 Card, J.P.. Meade, R.P. and Moore, R.Y., The origin, organization and projections of visual afferents to the rat hypothalamus: implications for the control of circadian rhythmicity, Soc. Neurosci. Abstr., 13 (1986) 211. 4 Dark, J.G. and Asdourian, D., Entrainment of the rat's activity rhythm by cyclic light following lateral geniculate lesions, Physiol. Behav., 15 (1975) 295-301. 5 Harrington, M.E., Nance, D.W. and Rusak, B., Neuropeptide Y immunoreactivity in the geniculo-suprachiasmatic tract, Brain Res. Bull., 15 (1985) 465-472. 6 Harrington, M.E. and Rusak, B., Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms, J. Biol. Rhythms, I (1987)309-325.
the possibility that s(mlc sprouting occurrcd in the geniculate complex cannot be excluded ~ls small changes in the innervation would not be appreciated because of the intense C ] - H R P label that is transported by the intact PO~I to the L G N region. It is of interest that evidence for extensive sprouting of R H T fibers was not found after neonatal SCN lesions in the rat 17. In summary, the data from this study d e m o n s t r a t e that lesions of the R H T in the hamster result in loss of circadian rhythm entrainment. Because the lesions also ablated significant parts of the SCN, the data support previous studies which have found that only about 25% of the SCN is required to maintain circadian rhythm generation r2s'3~, The extensive sprouting induced by the lesions in this study is further evidence that the adult visual system is capable of responding to injury and also emphasizes that such responses are not necessarily functionally significant. ACKNOWLEDGEMENTS This work was s u p p o r t e d by N I H Grants NS22168 and AG05773. The manuscript was p r e p a r e d while one of the authors ( R . Y . M . ) was a Fellow-inResidence, The Neurosciences Institute, Neuroscience Research Program, New York, NY. We thank D. Caselles for assistance in preparing the manuscript.
7 Hendrickson, A.E., Wagoner, N. and Cowan, W.M., An autoradiographic and electron microscopic study of retinohypothalamic connections, Z. Zellforsch.. 135 (1972) 1-26. 8 Hickey, T.L. and Spear, P.D., Retinogeniculate projections in hooded and albino rats: an autoradiographic study. Exp. Brain Res., 24 (1976) 523-529. 9 Johnson, R.F., Morin, L.P. and Moore, R.Y., Circadian effects of lesions of the lateral geniculate nucleus and cuts of the primary optic tracts in hamsters, Soc. Neurosci. Abstr., 12 (1986) 48.9. 10 Johnson, R.F., Moore. R.Y. and Morin, L.P., Suprachiasmatic knife cuts disrupt circadian wheelrunning in hamsters, Soc. Neurosci. Abstr., 13 (1987) 120. 11 Klein, D.C. and Moore, R.Y., Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase:control by the retinohypothalamic tract and suprachiasmatic nucleus, Brain Research, 174 (1979) 245-262. 12 Levine, J.D., Weiss, M.L., Gosin, D., Rosenwasser, A.M. and Miselis, R.R., Re-examination of the retino-hypothalamic projections of the rat using HRP conjugated to cholera toxin (CT-HRP), Soc. Neurosci. Abstr., 12 (1986) 148. 13 Mai, J.K., Distribution of retinal axon within the lateral hypothalamic area. Exp. Brain Res., 34 (1979) 373-377.
313 14 Mesulam, M., Tetramethyl benzidine for horseradish peroxidase neurochemistry: a non-carcinogenic blue reactionproduct with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 15 Millhouse. O.E., Optic chiasm collaterals afferent to the suprachiasmatic nucleus, Brain Research, 137 (1977) 351-355. 16 Moore, R.Y. and Card, J.P., Visual pathways and the entrainment of circadian rhythms, Ann. N.Y. Acad. Sci., 453 (1985) 123-133. 17 Mosko, S.S. and Moore, R.Y., Retinohypothalamic tract development: alteration by suprachiasmatic lesions in the neonatal rat, Brain Research, 164 (1979) 1-15. 18 Mosko, S.S. and Moore, R.Y., Neonatal suprachiasmatic nucleus lesions effects on the development of circadian rhythms in the rat, Brain Research, 164 (1979) 17-38. 19 Nishio, T., Shiosaka, S., Nakagawa, H., Sakumoto, T. and Satoh, K., Circadian feeding rhythm after hypothalamic knife-cut isolating suprachiasmatic nucleus, Physiol. Behay., 23 (1979) 763-769. 20 Nunez, A.A. and Stephan, F.K., The effects of hypothalamic knife cuts on drinking rhythms and the estrous cycle of the rat, Behav. Biol., 20 (1977) 224-234. 21 Pickard, G.E., The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection, J. Comp. Neurol., 211 (1982) 65-83. 22 Pickard, G.E., Bifurcating axons of retinal ganglion cells terminate in the hypothalamic suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus, Neurosci. Lett., 55 (1985) 211-217. 23 Pickard, G.E. and Silverman, A., Direct retinal projections to hypothalamus, piriform cortex and accessory optic nuclei in the golden hamster as demonstrated by a sensitive anterograde horseradish peroxidase technique, J. Comp. Neurol., 196 (1981) 155-172. 24 Pickard. G.E.. Ralph. M.R. and Menaker, M., The inter-
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32 33
34
geniculate leaflet partially mediates effects of light on circadian rhythms. J. Biol. Rhythms. 2 (1987) 35-56. Raisman, G. and Brown-Grant, K., The 'Suprachiasmatic Syndrome': endocrine and behavioral abnormalities following lesions of the suprachiasmatic nuclei in the female rat, Proc. Roy. Soc. Lond. B, 198 (1977) 297-314. Rusak, B., The role of the suprachiasmatic nuclei in the generation of circadian rhythms in the golden hamster, Mesocricetus auratus, J. Comp. Physiol., 118 (1977) 145-164. Sabatino, F.D., Murnane, J.M., Hoffman, R. and McDonald, J.K., Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster, J. Comp. Neurol,, 257 (1987) 93-104. Schneider, G.E., Early lesions of superior colliculus: factors affecting the formation of abnormal retinal projections, Brain Behav. Evol., 8 (1973) 73-109. Smale, L., Johnson, R.F., Morin, L.P. and Moore, R.Y., The retinohypothalamic tract (RHT) in hamster: normal organization and response to injury, Soc. Neurosci. Abstr., 13 (1987) 120. Stanfield, B. and Cowan, W.M., Evidence for a change in the retinohypothalamic projection in the rat following early removal of one eye, Brain Research, 104 (1976) 129-136. Stetson, M.H. and Watson-Whitmyre, M., Nucleus superchiasmaticus: the biological clock in the hamster?, Science, 191 (1976) 197-199. Turek, F.W., Circadian neural rhythms in mammals, Annu. Rev. Physiol., 47 (1985) 49-64. Van den Pol, A.N. and Powley, T., A fine-grained anatomical analysis of the rat suprachiasmatic nucleus in circadian rhythm of feeding and drinking, Brain Research, 160 (1979) 307-326. Watts, A.G. and Swanson, L.W., Efferent projections of the suprachiasmatic nucleus. II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat, J. Cornp. Neurol., 258 (1987) 204-229.
297
Elsevier BRE 13928
Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract R.F. Johnson 1'*, R.Y. Moore 2'3 and L.P. M o r i n 1'4 Departments of 1Psychiatry and Behavioral Science, 2Neurology, 3Neurobiology and Behavior and 4psychology, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A,) (Accepted 5 April 1988)
Key words: Circadian rhythm; Entrainment; Retinohypothalamic tract; Neuronal plasticity; Hamster; Geniculohypothalamic tract
The suprachiasmatic nuclei receive photic input information directly through a retinohypothalamic tract (RHT) and indirectly through a projection from the intergeniculate leaflet of the lateral geniculate complex, the geniculohypothalamic tract (GHT). Prior work has established that the RHT is sufficient for entrainment, but has not shown whether it is necessary because it has not been possible to transect that pathway. The present study addresses this problem by employing knife cuts to sever the RHT in male hamsters. Three knife cut procedures were used and one of these succeeded in separating the SCN from the optic chiasm in 8 animals with limited damage to the chiasm and the SCN. The effectiveness of the RHT lesion was confirmed by cholera toxin-HRP histochemistry which demonstrated that the knife cuts eliminate the normal retinal innervation of the SCN while sparing projections to thalamic and tectal visual centers. In a light-dark cycle, the lesioned animals exhibit free-running rhythms indicating that the RHT is necessary for entrainment. A surprising observation is the presence of extensive axonal sprouting of retinal fibers in brains of animals with RHT lesions. The newly-formed axons grow extensively into the SCN, anterior hypothalamus and basal forebrain, but form anomalous axohal plexuses which have no evident function.
INTRODUCTION
that neither the P O T or G H T is necessary for entrainment 4'6'9'11'24 (Johnson et al., submitted). These
The suprachiasmatic nuclei (SCN) function as a clock controlling circadian rhythms in m a m m a l s 15"32.
studies also d e m o n s t r a t e that the R H T is sufficient to maintain entrainment in the absence of all other visual pathways in animals that otherwise a p p e a r functionally blind. It has not been possible, however, to demonstrate that the R H T is necessary for entrainment as no m e t h o d has been available to transect the R H T as it enters the SCN. In the present experiments, we have addressed this p r o b l e m by producing knife cuts at the b o r d e r of the SCN with the optic chiasm. The anatomical d a t a indicate that these cuts destroy the R H T , leaving the chiasm and P O T largely intact and preserving a functional SCN. The behavioral data indicate that the animals maintain visually guided behavior, but circadian entrainment to the l i g h t - d a r k cycle is lost. F u r t h e r , this occurs with a striking growth of retinal axons into the ante-
The SCN are innervated by two visual pathways, a retinohypothalamic tract ( R H T ) and a geniculohypothalamic tract ( G H T ) . E n t r a i n m e n t , the setting of the phase of the clock, could require either of these pathways or, m o r e likely, they could function together in that process. The R H T is a direct projection from the retina to the SCN 16 and to other parts of the hypothalamus ~2"23"29. The G H T is a secondary visual projection that arises from the intergeniculate leaflet ( I G L ) of the lateral geniculate nuclei ( L G N ) 2'3'5'21, a zone that receives direct visual input via the primary optic tracts ( P O T ) 8"z2. Destruction of the P O T of the L G N , without d a m a g e to the R H T , has only minor effects on circadian rhythm e n t r a i n m e n t indicating
* Present address: Department of Psychology, University of Iowa, Iowa City, IA, U.S.A. Correspondence: L.P. Morin, Department of Psychiatry, Health Science Center, SUNY, Stony Brook, NY 11784, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
298 rior hypothalamus in an extensive, and apparently non-functional, anomalous axonal plexus. MATERIALS AND METHODS Male golden hamsters (Charles River) were housed individually in translucent cages under a L D 14:10 p h o t o p e r i o d in a t e m p e r a t u r e - c o n t r o l l e d room. F o o d and water were continuously available. Each cage contained a 17-cm d i a m e t e r running wheel with each revolution of the wheel activating a microswitch, in the initial part of the studies, the microswitch was connected to an Esterline-Angus event recorder and the records were p r e p a r e d as standard actograms. Later, all data were collected by a computer which recorded the n u m b e r of revolutions in 1min bins and later plotted the data in actogram format. Several surgical procedures were tested before fully successful R H T cuts were achieved. To e m p h a size the successful methods, the studies are p r e s e n t e d in reverse order. The experiments in which cuts were not complete (Expts. 2 and 3) have been retained because they provide significant control data concerning the ability of the circadian rhythm system to withstand damage to the SCN.
Experiment 1 Each hamster (n = 17) was o p e r a t e d upon while entrained to LD 14:10. The knife consisted of a stainless-steel tube ground away to form a semicircle (2.0 mm diameter) beveled to a sharp edge. F o r the cut, the knife was fitted into a larger guide cannula to allow vertical m o v e m e n t and lowered at a coordinate
(1.4 mm rostral to bregma; bregma and lambda level) just rostral to the SCN. As the guide cannula was lowered, the chiasm was detected by the vertical slippage of the knife. D o w n w a r d pressure was then combined with a slight back and forth rotation of the knife. This procedure created a hemi-island (Fig. 1A) which included the caudomedial chiasm and the bilateral SCN which maintained all caudal and dorsocaudal connections. Three of these animals died shortly after surgery. A group of control hamsters receiving no surgical intervention was also used in this study. A f t e r surgery, the wheel-running was monitored for 1-2 months while the animals r e m a i n e d on LD 14:10.
Experiment 2 Each hamster (n = 15) received a bilateral rostral cut combined with a cut ventral to the SCN. The knife consisted of a tungsten wire (130 ~ m diameter) soldered into a stainless-steel shaft (320/~m o.d.) for rigidity. The wire p r o t r u d e d 1.5 m m p e r p e n d i c u l a r to the axis of the shaft. The shaft was fitted into a guide cannula which allowed vertical movement. The knife was stereotaxically lowered with the shaft in the midline, the wire cutting in the coronal plane at the same coordinate as in Expt. 1 ;'just rostral to the SCN (Fig. 1B). W h e n the knife hit the optic chiasm (indicated by upward slippage of the shaft in the cannula), the knife was rotated caudally 180 ° between the chiasm and the SCN (Fig. 1C). The knife was then withdrawn contralateral to the side of insertion. This created a bilateral rostral cut c o m b i n e d with a 180 ° cut below the SCN. The cut was m a d e while the hamsters were entrained to L D 14:10. Five experimental
C Fig. 1. Schematic illustration of the methods for making RHT cuts. A: the heavy, semicircular line that roughly follows the contours of the caudal chiasm and medial optic tracts indicates the ideal location of the core cut in Expt. 1. B: the unilateral bar across the optic chiasm represents the initial coronal placement of the L-knife in Expts. 2 and 3. The knife was then rotated to undercut the SCN as indicated by the diagonally filled semicircle. The knife was raised ipsilateral (Expt. 3) or contralateral (Expt. 2) to the side of initial placement. C: a parasaggital view of an ideal SCN undercut made with the L-knife. AC, anterior commissure.
299 animals died shortly after surgery. Seven hamsters in which only the skull was opened and the dura punctured and 5 hamsters which received coronal cuts rostral to the SCN (but no undercuts) were used as controis. Postsurgically, the rhythms were allowed to stabilize in the LD cycle after which the hamsters were placed in constant darkness for at least 8 weeks.
Experiment 3 Hamsters (n = 15) were allowed to generate freerunning circadian rhythms in dim constant light (about 0.2 pW/cm 2 corresponding to about 1.2 lux as measured at midcage level with a J16 Tektronix digital photometer) until the period became stable. Each hamster then received a unilateral coronal plane cut rostral to the SCN combined with a cut ventral to the SCN. The procedure was identical to that in Expt. 2 (Fig. 1B) except that the knife blade was withdrawn ipsilateral to the side of insertion. A group of control hamsters (n = 18) received 0.6 saline injections into the lateral geniculate region. The postsurgical freerunning rhythms were allowed to stabilize, after which the hamsters were switched back to the original LD 14:10 cycle and reentrained. (Mean light intensity was about 10.2 pW/cm 2 corresponding to about 29.3 lux.)
Histology Each experimental hamster received bilateral intraocular injections of 2 pl of 0.1% horseradish peroxidase conjugated to cholera toxin B-subunit, (CTHRP; List Biological) and the brains were prepared for H R P histochemistry 14. The visual projections in each knife cut animalwere evaluated with CT-HRP histochemistry. The R H T projection was evaluated by comparison with control brains as were the retinal projections to the LGN. The brains of several normal hamsters which had received CT-HRP with the same parameters as above 29 were used for determination of changes in the R H T associated with the cuts. In order to evaluate the effects of R H T damage versus general hypothalamic damage, brains of the 5 hamsters which had received coronal knife cuts rostral to the SCN (sparing the chiasm) were also examined with CT-HRP). No histology was performed on the unoperated control group (Expt. 1) or the sham control group from Expt. 2, in which only the skull and dura were opened.
RESULTS The R H T projections in the normal hamster will be reported in detail elsewhere (Johnson et al., submitted). Briefly, however, the terminal plexus in the SCN begins in the ventral portion of the nucleus at its rostral extreme. It expands caudally to fill the nucleus with the ventrolateral portions having the densest label. The R H T projection to the SCN is densest in the intermediate and caudal parts of the nucleus (Fig. 2A) and continues caudally into the retrochiasmatic area to occupy a zone between the ventral limits of the paraventricular nucleus and the dorsal border of the anterior hypothalamic nucleus. In addition to its primary termination in the SCN, and dorsocaudal to it, the R H T of normal animals also projects to the lateral hypothalamic area via fibers arising from the lateral optic tract near the supraoptic nucleus, the anterior hypothalamus via fibers extending beyond the dense plexus in the SCN and to the anterior amygdala and the cortical amygdaloid nucleus via a sparse group of fibers which leaves the very rostral chiasm and travels along the base of the medial preoptic area and basal forebrain.
Experiment 1 In all cases, the semicircular cuts extend through the chiasm at the ventral surface of the brain. Some of these cuts fall behind the chiasm and others are located about midway through the rostrocaudal extent of the chiasm. The majority of hamsters have cuts which destroy the rostral to intermediate portions of the SCN, leaving the caudal portion of the nucleus at least partially intact. The cuts extend along the lateral boundaries of the SCN and into the retrochiasmatic area. In 8 of the animals (XT1,XT9,XT11,XT12,XT14,XT15, XT16,XT17), the cuts appear to have completely severed connections between the SCN and the optic chiasm and optic tract. In 3 animals (XT3,XT4,XT6), the cuts are incomplete, leaving part of the optic tract connected to the SCN. In three other animals (XT5, XT8,XT18), the cuts are placed across the SCN, greatly disrupting the middle to caudal portions of the nuclei. The R H T projection to the SCN, as demonstrated by the CT-HRP material, is absent in two animals with complete cuts (XT12,XT9; Fig. 3B) and appears
;tral Fig. 2. A: heavy (normal) RHT label in the intermediate rostrocaudal level of the SCN in an animal that received a coronal cut rob Iced to the SCN and no undercut. B: reduced RHT label in an animal that received a unilateral rostra1 cut and SCN undercut. C‘: redu and RHTlabel in an animal that received a bilateral rostra1 cut and an SCN undercut. The ventral scarring caused by the knife cut in B latic C is indicated by the arrows and the line of bright artifact between the SCN and the chiasm. OC‘. opttc chiasm; SC‘N. \upr;tchiasn nucleus.
301 as very sparse or anomalous fibers (Fig. 3A) in the remaining animals. In the 3 animals with incomplete cuts, two have robust R H T innervation of the SCN (XT3,XT4) and one (XT6) has sparse terminations in the caudal SCN. In the three hamsters with heavy damage through the caudal SCN, there is very little innervation of the SCN and occasional, anomalous large fibers. Although the R H T projection to the SCN is greatly reduced or anomalous in all animals with semicircular cuts, the terminal field in the L G N is extensive and normal. Fig. 4 shows the retinal innervation of
the L G N for one animal in which no retinal fibers were present in the SCN (XT12). All animals with reductions in the R H T projection to the SCN also show marked reductions in projections of the R H T to other hypothalamic areas. The semicircular cut is associated with anomalous retinal axons that extend far rostrally through the preoptic area to areas not normally receiving retinal input (Fig. 5A). The anomalous fibers are coarse and thick, often appearing as a tangle of large fibers rather than as a pattern of dense terminals. At the level of the SCN, such fibers are clearly present in the nu-
Fig. 3. The RHT in the SCN of animals receiving the semicircular knife cuts. A,B: examples from two animals with complete cuts; an RHT terminal plexus is not identifiable within the SCN, although both animals have robust label in the LGN (Fig. 4). Both animals have free-running rhythms in LD 14:10 after the cuts (e.g., Fig. 7B). OC, optic chiasm; SCN, suprachiasmatic nucleus.
302
Fig. 4. Retinal innervation of the LGN in an animal (XT12) in which the R H T to the SCN is completely severed and no labeled fibers in the SCN are visible (Fig. 3B). The plexus in the L G N in this animal is normal and shows heavy label in the IGL. The empty region within the IGL is a blood vessel. D L G , dorsolateral geniculate; IGL, intergeniculate leaflet; POT, primary optic tract; V L G , ventrolateral geniculate.
cleus, but also extend far outside of the nucleus dorsally along the ventricle (Fig. 3A). in other cases, the fibers appear to orient and concentrate along the line of the cut. This apparent sprouting was also indicated
by a much heavier than normal innervation of the rostral SCN which extended periventricularly beyond the SCN into the anterior hypothalamic area (Fig, 5B). The massive extent of sprouting evident in
Fig. 5. A n o m a l o u s growth of retinal fibers observed after damage to the RHT. Large fibers are olten found far rostral to the S('N near the level of the o r g a n u m vasculosum ol the lamina terminalis (A,C). In other cases, abnormally dense label is prcsent in the very ro,,tral SCN and extends far beyond the SCN laterally and periventricularly (D). At this level, the R H T to the SCN is normally vcr~ faim and restricted to the ventral portion of the nucleus. The chiasm is completely severed coronally immediately caudal to the photomicrograph shown in (D), Some animals also show large anomalous fibers caudal to the cut, in the region of the SCN (BI. but these fibcr~ also extend far beyond the SCN in a periventricular orientation. The arrows in (B) indicate the lateral boundary of the cut. l bc ph~tomicrograph shown in (B) is a magnification and dorsal extension of the photomicrograph shown in Fig. 3A. O(', optic chke, m: ()N. ~t~tic nerve.
303
zl
31)4 some individuals is shown in Fig. b. Photomicrographs of the brain of this animal are also shown in Figs. 3A and 5A,B.
Behavior was recorded from all hamsters for at least 30 days after surgery, although some individuals required recording beyond this time because activity
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Fig. 6. Extent of a n o m a l o u s fibers in animal XT1 which sustained an R H T cut in Expt. 1. The broken lines indicate location of the knife cut. Blackened areas show extremely dense label in which individual fibers could not be identified. A C , anterior commissure; D B , diagonal band of Broca: OC, optic chiasm; ON, optic nerve; OT, optic tract; P O A , preoptic area: PVN, paraventricular nucleus: R C h , retrochiasmatic area; TC, tuber cinereum.
305 patterns were unclear. All of the hamsters showed transient disruptions in activity rhythms after the cuts (Table I). One hamster (XT3; Fig. 7A) rapidly regained rhythmicity and remained normally entrained. Six of the hamsters ( X T 1 7 , X T 1 , X T 1 2 , X T 1 6 , X T 9 , XT15) showed clear free-running rhythms within two weeks of surgery (Figs. 7 B - D and 8B). The free-running rhythms were consistently greater than 24 h,. clearly could be traced to the phase of previously entrained activity and continued for the entire survival period. Three hamsters (XT6,XT4,XT8) showed transient free runs of >24 h within a week of surgery which eventually switched to periods of <24 h and reestablished an entrained phase. One of these animals had a normal phase angle of entrainment while two had unstable activity onsets which were phase-advanced and preceded light offset (Fig. 8A). This pattern of entrainment continued throughout the remaining survival period. Two hamsters (XT11,XT18) showed very disrupted rhythms with no clear or consistent phasing. The remaining two hamsters (XT14, XT5) had two components in their rhythms, one coinciding with, and synchronized to, the dark phase of the cycle and one free-running with a >24 h period (Fig. 8C). In these two cases, the free-running component could be traced to the previous phase of entrainment. In each, the free-running rhythm continued, without obvious phase changes, through the
light portion of the cycle, eventually becoming phased with the component synchronized to the LD cycle. Because none of these animals was ever placed in constant lighting conditions, it is not known whether the rhythmicity which was phase locked to the LD cycle postsurgically represented true entrainment of a circadian rhythm or exogenously driven activity. Experiment 2
In the group receiving the bilateral rostral cuts with SCN undercuts, two of the animals died before the brains were taken for histology. The remaining 8 animals show variable amounts of damage to the SCN (Table II). In one animal (U3) the cut severs the rostral and ventral connections of the SCN, but leaves the SCN intact. In two animals (U9,U11), the cut destroys the rostral extreme of the SCN on both sides, but leaves the middle to caudal portions of the SCN intact. In the other 5 animals ( U 5 , U 7 , U 8 , U12,U14), a large portion of the middle SCN is destroyed (accompanied by an enlarged ventricle) leaving only a small rostral and caudal portion of the SCN intact. In two of these latter animals (U7,U8), the caudal portion of the SCN is present, but it is greatly distorted and appears damaged by the cut. In each of the animals, the SCN appears to be severed from the optic chiasm. The extent of the R H T in the SCN in
TABLE I Rhythmicity of hamsters sustaining semicircular cuts around the rostral SCN (Expt. 1)
Strong, a robust rhythm with stable phase. Moderate, a rhythm with slightly unstable phase and increased activity during the "rest' phase. Weak, a rhythm that is apparent, but very unstable, and which has a high level of activity during the 'rest' phase. Very weak, a rhythm which is barely discernible in the actogram, but which is statistically significant. Animal
SCN innervation
SCN damage
Rhythmicity in LD post-cut
XT1 XT3 XT4 XT5 XT6 XT8 XT9 XT11 XT12 XT14 XT15 XT16 XT17 XT18
anomalous normal normal sparse sparse sparse none anomalous none anomalous anomalous anomalous anomalous sparse/anomalous
rost-mid rost-mid rost-mid mid-caud rost-mid mid-caud rost-mid rost-mid rost-mid rost-mid rost-mid rost-mid rost-mid mid-caud
moderate free-run = 24.18 h strong entrainment transient free-run, weak entrained two weak components, 24.0 and 24.57 h transient free-run, strong ent~'ainment transient free-run, strong entrainment weak free-run = 24.14 h none moderate free-run = 24.10 h two weak components, 24.0 and 24.43 h weak free-run = 24.1 h moderate free-run = 24.14 h strong free-run 24.14 h none
306 the animals is reduced (Fig. 2C), but the primary visual projection appears normal in all animals.
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coarse and o f t e n e x t e n d p e r i v e n t r i c u l a r l y rostral to
that n o r m a l l y r e c e i v e few, if any, retinal fibers. This
the cut. A b n o r m a l l y c o a r s e fibers also a p p e a r in the
a b n o r m a l p a t t e r n of the R H T is m o s t o f t e n o b s e r v e d
r e t r o c h i a s m a t i c a r e a of two animals ( U 7 , U 1 4 ) . T h e
as an unusually d e n s e plexus rostral to the cut. T h e
sprouting e v i d e n t in this e x p e r i m e n t did not s e e m as h e a v y as in E x p t . 1.
p r o j e c t i o n also differs f r o m n o r m a l dorsally and laterally. T h e fibers e x t e n d into the m e d i a l p r e o p t i c
TABLE II Circadian rhythmicity of hamsters with bilateral coronal cuts rostral to the SCN and an SCN undercut (Expt. 2)
Strength of rhythmicity definitions are as in Table I. Animal
U1 U3 U4 U5 U7 U8 U9 U 11 U 12 U 14
SCN innervation
unknown sparse unknown sparse sparse sparse sparse sparse sparse sparse
SCN damage
unknown none unknown middle mid-caudal all levels rostral rostral middle middle
Rhythmicity
Period of
LD
DD
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moderate moderate moderate moderate transient, weak none transient, weak none moderate moderate
free-rbln 24.20 23.89 24.10 24.10 23.62 24.07 24.05 24.10
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Fig. 10. Actograms from animals that received unilateral rostral cuts and SCN undercuts. The animals were allowed to flee-run in constant dim light. The cuts were performed on the days indicated by the circles in the actograms. For animals in which the ventral cuts passed through or below the chiasm, missing the SCN (A,B), there is little effect on the rhythm. For animals in which the cuts pass between the SCN and chiasm, damaging the ventral SCN (C,D), the rhythms are disrupted. Under LD conditions (indicated by the black and white bar), all animals show synchronization of wheel-running to the LD cycle.
310 area and are particularly concentrated in a periventricular zone. In 3 animals (U9,UII,U14), dense anomalous fibers are observed far rostral to the SCN where only occasional fibers are observed in normal animals (Fig. 5C). The anomalous fibers are observed as far rostral as the organum vasculosum and some extended dorsally into the septum. In addition to abnormal sprouting rostral to the cut, abnormal patterns of retinal innervation are present in intact SCN caudal to the cuts. This is most often seen as very sparse and restricted innervation of the SCN and adjacent areas (Fig. 2C). In some animals, the residual innervation also deviates from the normal pattern of dense terminals ventrally and laterally diminishing to light innervation dorsomedially and, instead, appears as a tangle of large, varicose fibers in the middle of the nucleus. The exact route by which the residual SCN is innervated with these anomalous fibers is not evident. All hamsters except one eventually showed a 24 h rhythm wit'," activity associated with the dark phase of the LD cycle (Table I). However, in some animals the rhythm was severely disrupted for several days immediately after the cuts (Fig. 9C,D). Further, the activity patterns of these animals, although clearly synchronized to the LD cycle, tended to be poorly organized and showed disruptions in phase of entrainment. When the hamsters were transferred to constant darkness, 6 of them showed sustained free-running rhythms (Fig, 9B,C) and 4 had activity rhythms which eventually disintegrated into arrhythmicity (Fig. 9A,D). Interestingly, 3 of the hamsters which maintained free-running rhythms show some of the greatest losses of SCN tissue (e.g., U12: Fig. 9B). Animals with hypothalamic damage from coronal cuts aimed rostral to the SCN sustain very little, if any, damage to those nuclei. The cuts generally are located between the organum vasculosum lamina terminalis (OVLT) and the rostral extreme of the SCN. The RHT projection to the SCN of these hamsters is robust and normal (Fig. 2A) as are retinal projections to the lateral geniculate region.
Experiment 3 The group receiving unilateral rostral cuts and SCN undercuts can be divided roughly into three subgroups. In the first (n = 6), the cut goes through the base of the brain and enters the ventral side of the
chiasm. In the second (n = 5), the cut passes between the chiasm and the SCN on one side and extends into the optic tracts on the opposite side. In the third subgroup (n = 4), the knife appears to completely sever the SCN from the chiasm (Fig. 2B). In the latter two subgroups, there is variable, slight damage to the ventral extremes of the SCN and unilateral, but not contralateral, damage rostral (100-200 um) to the SCN in all animals. In general, however, the SCN of all animals is remarkably well preserved. The HRP histochemistry shows, in all animals, an RHT terminal plexus in the SCN. Unfortunately, in these brains there was a poor quality reaction resulting in rapid, variable fading of the reaction product which prevented a more detailed analysis of changes which might have occurred in the RHT. All hamsters except one (no. 17) continued to show free-running circadian rhythms after the cut (Fig. 10). The exception became arrythmic. The other animals usually resumed a free-running rhythm within a day or two after the cut and maintained it as long as the animals were in constant dim light. However, the extent of disruption of the free-running rhythm was associated with the placement of the cuts. The subgroup in which the cut separates the SCN from the chiasm most effectively (and also causes the greatest damage to the ventral SCN) showed the greatest rhythm disruption (the arrhythmic hamster is in this group; Fig. 10C,D). The subgroup with incomplete cuts showed variable amounts of disruption, whereas in the subgroup with cuts which entirely miss the SCN, the animals were indistinguishable from control animals according to visual inspection of the actograms (Fig. 10A,B). Upon reintroduction of the LD cycle, all hamsters in this experiment showed a clearly entrained, apparently normal, activity rhythm. DISCUSSION The present results demonstrate that the hamster RHT can be selectively destroyed, leaving the primary visual projections to the thalamus virtually intact. Following effective RHT cuts, the hamster wheel-running rhythm fails to entrain to LD cycles (even after prolonged exposure to LD; unpublished data). In addition, such damage to the RHT produces a dramatic reorganization of visual projections to
311 the hypothalamus with extensive sprouting of retinal axons to form anomalous plexuses in the hypothalamus and extending beyond its borders.
Entrainment The lesion which most effectively reduces or destroys the R H T and allows free-running rhythms under LD conditions is a semicircular isolation of the SCN from all rostral, ventral and lateral connections. The other two types of lesion, a unilateral or bilateral knife cut rostral to the SCN combined with an SCN undercut are less effective, always leaving at least a portion of the R H T terminal plexus in the SCN intact. The undercuts also produce a qualitatively different effect on the rhythms. Although they acutely disrupt the locomotor rhythm, there is little indication that entrainment is lost. The undercuts may leave fibers intact which arise from the chiasm rostral or lateral to the SCN. In the case of the effective semicircular cuts, fibers arising rostral or lateral to the SCN would be destroyed as would fibers which remain in the chiasm to enter the SCN from a ventral direction. The loss of direct retinal innervation of the SCN is probably the primary contributor to the loss of entrainment consequent to the R H T cut. Animals displaying free-running rhythms had the greatest reduction of normal R H T projections. Although these animals also sustained damage to the rostral SCN, it is unlikely that loss of entrainment is specifically related to this damage. Animals with similar destruction of the rostral SCN (Expt. 2 and ref. 10) remain entrained to the LD cycle. Perhaps equally important for the loss of entrainment may be the loss of G H T innervation of the SCN. Several studies have indicated that the G H T is not necessary for entrainment 4'6"9'11'24 (Johnson et al., submitted). But the G H T may be a sufficient entrainment pathway and the present cuts may have destroyed both the R H T and GHT. The exact route by which the G H T reaches the SCN is not clear, but recent evidence suggests that the NPY portion of the G H T arrives at the SCN both via the optic tracts and via the zona incerta 27. The present semicircular cuts would have destroyed the optic tract portion of the G H T , but probably spared the zona incerta portion if it enters the SCN from a caudolateral direction. Destruction of the optic tracts by parasaggital cuts ira-
mediately lateral to the SCN leaves a robust plexus of NPY terminals of geniculate origin in the ventrolateral SCN (n = 3; Johnson, unpub.), whereas destruction of the LGN (and thereby the entire G H T ) eliminates all NPY innervation of the SCN of geniculate origin (Johnson et al., submitted). We were not able to perform NPY immunohistochemistry on the material prepared for CT-HRP and could not assess the integrity of the GHT. Therefore, we cannot answer the question of whether the G H T is sufficient for entrainment.
Rhythmicity With regard to SCN function, several conclusions concerning cut placement and subsequent changes in rhythmicity can be made: (1) hamsters are able to sustain major loss of SCN tissue and still show relatively normal behavioral rhythms (Expt. 2); (2) SCN efferents rostral and lateral to the nucleus are not required for the expression of wheel-running circadian rhythmicity (Expt. 1); and (3) damage to the ventral region of the SCN has only slightly disruptive effects on the wheel-running rhythms (Expt. 3). The minor effect of damage to the ventral portion of the nucleus on circadian rhythmicity is in agreement with work on rats I. The persistence of free-running rhythms after partial isolation of the SCN also supports earlier observations from r a t s 19"2°'33 that the rostral and lateral efferents of the SCN 34 are not required for the expression of circadian rhythmicity. In particular, the pathway from the dorsomedial SCN to the LGN (which takes a lateral route above the optic tracts 34) appears not to be required for rhythm generation or expression. This observation is in agreement with resuits from rats 4 and hamsters 6'9'24 showing that the LGN is not required for entrainment. The ability to destroy large portions of the SCN and yet maintain circadian rhythmicity(18.26.33; Expt. 2 and unpublished work) indicates that either the rhythm generating capacity of the SCN is very diffuse or redundant or that a small, but as yet undefined portion, is required. Sprouting The sprouting identified in these studies supports several conclusions: (1) in experimentally altered and normal animals, CT-HRP is a very sensitive tracer of normal and anomalous visual projections;
312 (2) there is r e m a r k a b l e anomalous growth after damage to the RHT; and (3) as indicated by the existence of anomalous fibers in the SCN of hamsters showing free-running rhythms under L D conditions, presence of retinal efferents does not always indicate functional connections. It is likely that the sprouting is derived from axons of the R H T , but we cannot exclude the possibility that the axons giving rise to the anomalous innervation are from other c o m p o n e n t s of the optic chiasm injured by the lesions. Sprouting of retinal efferents after retinal fiber damage may also occur in the lateral hypothalamus ~3. The neuronal plasticity of the R H T suggests the possibility that recovery of function of residual tissue may occasionally follow successful lesions or knife cuts. A f t e r apparently successful SCN lesions or R H T damage, hamsters often show residual rhythmicity which entrains to an LD cycle -'~'. The possibility exists that such entrainment represents recovery of R H T function. The discovery of sprouting in the present work is particularly intriguing because it occurs in adult animals. Plasticity after damage to the R H T has been shown in the neonatal rat 3°. Plasticity of the h a m s t e r visual system has been d o c u m e n t e d , both at the anatomical and behavioral levels, after the damage in very young animals, but not if the damage occurs in adults -'~. The sprouting seen in the h y p o t h a l a m u s may have been paralleled by sprouting of axon collaterals which project to the geniculate l-s'2z. H o w e v e r ,
REFERENCES l Brown, M.H. and Nunez, A.A., Hypothalamic circuits and circadian rhythms: effects of knife cuts vary with their placement within the suprachiasmatic area, Brain Res. Bull., 16 (1986) 705-711. 2 Card, J.P. and Moore, R.Y., Ventral lateral geniculate efferents to rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity, J. Comp. Neurol., 206 (1982) 390-396. 3 Card, J.P.. Meade, R.P. and Moore, R.Y., The origin, organization and projections of visual afferents to the rat hypothalamus: implications for the control of circadian rhythmicity, Soc. Neurosci. Abstr., 13 (1986) 211. 4 Dark, J.G. and Asdourian, D., Entrainment of the rat's activity rhythm by cyclic light following lateral geniculate lesions, Physiol. Behav., 15 (1975) 295-301. 5 Harrington, M.E., Nance, D.W. and Rusak, B., Neuropeptide Y immunoreactivity in the geniculo-suprachiasmatic tract, Brain Res. Bull., 15 (1985) 465-472. 6 Harrington, M.E. and Rusak, B., Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms, J. Biol. Rhythms, I (1987)309-325.
the possibility that s(mlc sprouting occurrcd in the geniculate complex cannot be excluded ~ls small changes in the innervation would not be appreciated because of the intense C ] - H R P label that is transported by the intact PO~I to the L G N region. It is of interest that evidence for extensive sprouting of R H T fibers was not found after neonatal SCN lesions in the rat 17. In summary, the data from this study d e m o n s t r a t e that lesions of the R H T in the hamster result in loss of circadian rhythm entrainment. Because the lesions also ablated significant parts of the SCN, the data support previous studies which have found that only about 25% of the SCN is required to maintain circadian rhythm generation r2s'3~, The extensive sprouting induced by the lesions in this study is further evidence that the adult visual system is capable of responding to injury and also emphasizes that such responses are not necessarily functionally significant. ACKNOWLEDGEMENTS This work was s u p p o r t e d by N I H Grants NS22168 and AG05773. The manuscript was p r e p a r e d while one of the authors ( R . Y . M . ) was a Fellow-inResidence, The Neurosciences Institute, Neuroscience Research Program, New York, NY. We thank D. Caselles for assistance in preparing the manuscript.
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