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Retinohypothalamic tract development in the hamster and rat
Decelopmental Brain Research, 76 (1993) 171-181 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
171
B R E S D 51714
Research Report
Retinohypothalamic tract development in the hamster and rat Joan C. Speh a,, and Robert Y. Moore b Departments o f ,.h Psychiatry. ~' Neurology, h Behauioral Neuroseience and " the Center for Neuroscience, Biomedical Science Tower W 1656, Unicersity of Pittsburgh, Pittsburgh, PA 15261, USA (Accepted 29 June 1993)
Key words." Suprachiasmatic nucleus; Retinohypothalamic tract: Lateral hypothalamus; Retrochiasmatic area; Circadian rhythm; Synaptogenesis: Synapsin
The development of the retinohypothalamic tract ( R H T ) in the albino rat and golden hamster was studied using anterograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP). The R H T has three components in the adult: (1) a dense projection to the ventrolateral subdivision of the suprachiasmatic nucleus (SCN) with some fibers extending into the dorsomedial SCN; (2) a projection to adjacent areas, the anterior hypothalamic area ( A H A ) and retrochiasmatic area (RCA) and in the hamster, into the preoptic area (POA); (3) a projection to the lateral hypothalamic area (LHA).In the rat, the projection to the SCN and adjacent areas first appears as scattered varicosities at the ventral border of the SCN at postnatal day 1 (P1) and gradually increases until the adult pattern is achieved at approximately PI0. The projections to the A H A and R C A are seen first at P 2 - P 3 and gradually increase to the adult appearance by P15. Both the projection to the SCN and adjacent areas and to the LHA, initially are more extensive than in the adult. Many of the axons extend well beyond the zone of the adult pattern but these anomalous fibers are eliminated by P6-P10. The L H A projection first appears at embryonic day 21-22 (E 21-22) and gradually increases in density from P1-P6. In the hamster the projections to the SCN, A H A and L H A appear first on P4 and gradually increase in density to reach the adult pattern by P15. The projections to the R C A and P O A are present by P6 and reach the adult pattern by PI5. None of the R H T projections in the hamster has the initial extended growth followed by pruning back that characterizes R H T development in the rat. Thus, the development of the R H T in both the rat and the hamster is complex with components of the projection appearing at different times with differing patterns of development that indicate specialized interactions of the developing axons with their target neurons. Synaptogenesis in the hamster hypothalamus was analyzed using an antiserum to synapsin I. Few synapses are present at El6, the last day of gestation, in the LHA, SCN and AHA. From P1-P3, synaptogenesis proceeds rapidly and the adult pattern is achieved in all three areas by P4.
INTRODUCTION The retinohypothalamic tract ( R H T ) is known to be an entrainment pathway in the circadian system of mammals ~3"~6'25. This projection was first shown in the rat using the autoradiographic tracing technique z8"l° and has been described in many other mammalian species 6'24"25. The development of the R H T in the rat and hamster has been studied using several techniques 2's'9'21'37. These studies found the R H T projection to the suprachiasmatic nucleus (SCN) of the rat to appear first on postnatal day 3 - 4 (P3-4) and that of the hamster on P6. Subsequent studies in the adult have shown the R H T to be a more complex projection than was initially described with components innervating the lateral hypothalamic area (LHA), anterior hypothalamic area (AHA) and retrochiasmatic area
* Corresponding author. Fax: (1) (412) 648-8376.
(RCA) 13'15"19'3t'33. The most recent studies of the R H T in the adult 13'19 have used a very sensitive anterograde tracer, H R P conjugated to the /3-subunit of cholera toxin 39. This has shown a more extensive and complex pattern of retinal projections to the hypothalamus than was appreciated previously and these observations have prompted the present study which analyzes the development of the R H T in the rat and hamster using the C T - H R P method. In addition, synaptogenesis was analyzed in the hamster anterior hypothalamus using an antiserum to synapsin I to complement a prior study in the rat 26. MATERIALS AND METHODS Rat Timed pregnant female Sprague-Dawley rats (Taconic) used in this study were housed individually with free access to food and water and kept on a diurnal light schedule (lights on 07.00 h-19.00 h). The time of birth was carefully noted and the 24-h period directly after birth designated as postnatal day 1 (P1). Animals from the following postnatal timepoints were used in this study: embryonic
172 day 21/22 (E21/22), P1, P2, P4, P6, P8, PIll and PI5. Mean crown rump lengths of embryonic animals were measured at sacrifice and used as the basis for determining the age of the fetus according to the timetable established by Seiger and Olsen 3~. For E21 injections, the timed pregnant rats were deeply anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and a small incision was made in the abdomen to expose one horn of the uterus. A fiber optic light source was used to illuminate the uterus, allowing the fetus' eye to be located. The injection was made through the uterine wall into the vitreal chamber of the eye. Hypothermia was the anesthesia used for the postnatal timepoints. Each animal received a unilateral injection of cholera toxin conjugated to horseradish peroxidase (CT-HRP, cholera toxin /3-subunit, List Biologicals) diluted in saline with 2% dimethylsulfoxide as vehicle into the vitreal chamber of the eye using a Hamilton microsyringe. Concentrations and volumes of CT-HRP varied with the animals age (Table I). All injections were performed during the middle of the day and the survival period was 24 h.
subdivision of t h e n u cl eu s with s o m e fibers e x t e n d i n g into the d o r s o m e d i a l p o r t i o n o f the nucleus. T h e seco n d c o m p o n e n t o f the R H T is largely an cxtension of the p r o j e c t i o n into the S C N to t h e p r e o p t i c - a n t e r i o r hypothalamic
a r e a ( A H A ) and r e t r o c h i a s m a t i c area
( R C A ) . T h e p r o j e c t i o n to the A H A is first o b s e r v e d in the h a m s t e r at the level o f the o r g a n u m vasculosum l a m i n a t e r m i n a l i s as s c a t t e r e d axons which leave the dorsal b o r d e r o f t h e optic n e r v e with p r o j e c t i o n s into the p r e o p t i c area. This c o n t i n u e s as a loose plexus of t e r m i n a l s that a p p e a r s to be an extension o f the S C N plexus into t h e A l I A in b o t h species which c o n t i n u e s t h e l e n g t h of the SCN. it ex t en d s f u r t h e r into the subparaventricular
zone
and
adjacent
AHA
in the
Hamster
h a m s t e r than in the rat. In b o t h species t h e r e is a large
Timed pregnant golden hamsters (Mesocricetus auratus) were obtained from Charles River and housed in indiviual cages on a diurnal light schedule (lights on 06.00 h-20.00 h) and given free access to food. The time of birth was noted and the first 24-h period designated as PI. Animals from the following timepoints were used in this study: P3, P4, P6, P8, P10, P15. Injection parameters were the same as for the rat experiments as noted above. Volumes and concentrations varied with the age of the animal (Table I). At the end of the survival period, the animals were anesthetized either with ketamine-xylazine or hypothermia. Each animal was perfused transcardially with saline followed by 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. The brains were removed and stored at 4 C in the same fixative for up to 18 h. The brains were cryoprotected in a sucrose series (10-30%) in 0.1 M sodium phosphate buffer. Serial sections were then cut on a freezing microtome in the coronal plane at 40 ~m. Sections were immediately reacted to visualize the horseradish peroxidase (HRP) with tetramethylbenzidine 22. After reaction the sections were mounted on gelatin coated slides, dehydrated in graded series of acetone and coverslipped. Synaptogenesis in the hamster was analyzed using an antiseum to synapsin 17. Two to four animals were sacrificed at El6, P1, P2, P3, P4, P6, P8 and P15. Animals were transcardially perfused or immersion fixed with Bouin's Solution, embedded in paraffin as previously described 26 and 8-10 /zm serial coronal sections through the hypothalamus were collected. Every fourth section through the SCN was stained for synapsin I and the adjacent section was stained with Cresyl violet. The immunohistochemical material was prepared using the peroxidase-anti-peroxidase complex (PAP) method of Sternberger 3s.
z o n e of i n n e r v a t i o n c a u d a l to the S C N into the R C A that
continues
to t h e
tuberal
hypothalamus.
These
p r o j e c t i o n s ar e m o r e d e n s e on the c o n t r a l a t e r a l side in b o t h the h a m s t e r and the rat. T h e third c o m p o n e n t o f the R H T is a p r o j e c t i o n to the l at er al h y p o t h a l a m i c a r e a ( L H A ) . It is c o m p r i s e d of a loosely s c a t t e r e d c o l l e c t i o n o f varicose axons that e x t e n d f r o m the lateral optic chiasm in t h e rostral h y p o t h a l a m u s , b ut m o r e caudally the L H A p r o j e c t i o n is a m o d e r a t e l y d e n s e a c c u m u l a t i o n o f t e r m i n a l s just dorsal to t h e s u p r a o p t i c n u cl eu s (SON). T h e L H A p r o j e c t i o n is substantially l a r g e r on the c o n t r a l a t e r a l side t h a n t h e ipsilateral side in b o t h t h e rat and h a m s t e r .
Development o f the rat retinohypothalamic tract T h e p r o j e c t i o n to t h e L H A is t h e first of t h e t h r e e RHT
components
to d e v e l o p in the rat.
Scattered
axons are p r e s e n t o v e r the lateral o p t i c tract at E 2 1 - 2 2 (Fig. 1). T h e C T - H R P l a b e l e d fibers are p r e s e n t cont r a l a t e r a l to the injected eye and are c o n f i n e d to the
TABLE I RESULTS The
development of the R H T
lntraocular injection parameters for demonstrating retinohypothalamic tract development will be d e s c r i b e d
s e p a r a t e l y for t h e rat a n d t h e h a m s t e r . A b r i e f summ a r y o f t h e n o r m a l a d u lt p a t t e r n o f th e rat a n d h a m ster will p r e c e d e t h e d e s c r i p t i o n s o f t h e R H T d e v e l o p -
For each pre- (E21/22) and postnatal age (P1-P15), the quantity and volume of CT-HRP injected, diluted in saline with 2% DMSO, is listed. The post-injection survival time at each age was 24 h.
Age
me nt. R H T p r o j e c t i o n s can be d i v i d e d into t h r e e com-
Number of animals Rat
Hamster
Quantity injected (~g)
2 6 6 3 6 3 3 4 3
2 4 4 4 3 3
0.5 0.5 0.5 0.5 1.0 1.5 2.0 3.0 4.0
Volume injected (tzl) 0.5 05 0.5 0.5 1.0 1.5 2.0 3.0 4.0
p o n e n t s . T h e largest o f t h e s e is the p r o j e c t i o n to the SCN. This is m o r e d e n s e to t h e c o n t r a l a t e r a l S C N in the rat w h e r e a s in t h e h a m s t e r t h e ipsilateral and c o n t r a l a t e r a l p r o j e c t i o n s a r e a l m o s t equal. A t rostral S C N levels t h e R H T p r o j e c t i o n f o r m s a d e n s e b a n d o f t e r m i n a l s a l o n g t h e dorsal e d g e o f t h e o p ti c chiasm. A s t he S C N e x p a n d s dorsally, retinal fibers p r o d u c e
a
d e n s e plexus o f t e r m i n a l s that fills th e v e n t r o l a t e r a l
E21/22 P1 P2 P3 P4 P6 P8 P10 PI5
173
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Fig. 1. Rat retinohypothalamic development. Schematic drawings of rat hypothalamus in the coronal plane through the rostral-caudal extent of the SCN. Four timepoints of unilateral CT-HRP eye injections are represented: A, E21/22; B, P1; C, P4; D, P15. Each timepoint is illustrated at three rostral through caudal levels. 3V, third ventricle; FX, fornix; OC, optic chiasm; SCN, suprachiasmatic nucleus: SON. supraoptic nucleus•
174 area immediately dorsal to the optic chiasm and tract. No ipsilateral label is evident. At PI, the number and distribution of labeled fibers in the L H A has increased greatly. Labeled fibers are present from the level of the most rostral SCN to the R C A and extend through the L H A into the adjacent A H A and perifornical region. Both the number and distribution of labeled fibers is greater than in the adult. In addition, there are labeled fibers extending in long radial arrays through the L H A (Fig. 2) that differ in apperance from any fibers in the
adult. The LHA projection continues to increase in density to P4 but thereafter appears to be pruned back to form the adult pattern by P10. Projections to the SCN appear first on P1 as scattered fibers in the SCN, particularly in the ventral and ventrolateral parts of the nucleus. The projection increases in density and extent through P4 (Fig. 1). At this point, as in the LHA, apparently anomalous projections extend through the SCN over a wide area of the AHA. These also are pruned back by P10 when the
Fig. 2. Rat retinohypothalamicdevelopment. Darkfield photomicrographsof unilateral CT-HRP eye injections. A, SCN at P1; B, SCN at P4; C, SCN at PI5; D, LH at P1; E, LH at P4: F, RCA at P4. OC, optic chiasm; 3V, third ventricle. Marker bar = 100 #m
175
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Fig. 3. Hamster retinohypothalmic development• Schematic drawings of hamster hypothalamus in the coronal plane through the rostral-caudal extent of the SCN. Four timepoints of unilateral CT-HRP eye injections are represented: A, P4; B, P6; C, P 8; D, P15. Each timepoint is illustrated at three rostral through caudal levels• 3V, third ventricle; FX, fornix; OC, optic chiasm; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus•
l 7~ p r o j e c t i o n to the SCN reaches its a d u l t a p p e a r a n c e .
SCN with some labeled fibers e x t e n d i n g into the A H A
This includes a d e n s e projection to the v e n t r o l a t e r a l
dorsal to the SCN.
SCN, scattered varicose axons into the d o r s o m e d i a l
T h e largest extra-SCN c o m p o n e n t of the R H T p r o -
Fig. 4. Hamster retinohypothalamic development Darkfield photomicrographs of unilateral CT-HRP eye injections. A, SCN at P4; B, LH at P6; C. RCA at P6; D, SCN at P6; E, SCN at PS; F, RCA at P8. OC, optic chiasm; 3V, third ventricle. Marker bars = 200 ~m
177
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I::!:~:~.;~-.~i...:. • • .." v.::..:...'l-V..:.:......:
Fig. 5. Hamster synaptogenesis schematic drawings of hamster hypothalamus indicating the distribution of synapsin I immunoreactive profiles at four developmental timepoints: A, El6; B, P1; C, P3; D, P4. Broken line indicates the borders of the SCN. OC. optic chiasm
178 jection is to the RCA. This is first evident as scattered varicose axons at P2 and increases markedly between P2 and P4. Like the other projections it contains some anomalous appearing axons at P4 which have been removed by P10 (Fig. 1). Throughout this developmental process, projections to the contralateral structures are greater than those to ipsilateral structures.
extending into the RCA. Over the next 4 days, the projection gradually increases in all areas to reach approximately three-fourths the density of the adult projection in all areas (Fig. 4). The density of innervation gradually increases so that, by P15, the projection has reached the adult pattern.
Synaptogenesis in the hamster anterior hypothalamus Deuelopment of the hamster retinohypothalamic tract The development of the hamster R H T differs from that in the rat in three major ways: (1) it develops significantly later with the first projections appearing at P4 and the adult pattern at Pl5; (2) the projection gradually assumes the adult pattern rather than initally extending beyond the area to be innervated and then being pruned back; (3) the projection to the L H A develops concomitantly with those to the SCN, A H A and RCA. The pattern of development of the hamster R H T is shown in Figs. 3 and 4. The first labeled axons to appear in the hypothalamus are evident at P4. They are present in the ventrolateral SCN, A H A and L H A with occasional fibers
In material prepared with the synapsin I antiserum, there are scattered varicosities, presumably synaptic terminals distributed nearly evenly over the entire anterior hypothalamic area at E l 6 (Fig. 5). Labeled terminals are slightly more dense in the immediate periventricular area, a pattern that continues until the adult distribution of labeled terminals is present at P4 (Fig. 6). The timeing of development of synapsin-immunoreactive varicosities is identical over the SCN and adjacent areas. This differs from the pattern in the rat where synaptogenesis occurs first in the L H A and extends medially with the SCN developing more slowly than adjacent A H A 2~'. The development of synapses in the hamster SCN begins along its borders at E16/P1
Fig. 6. Hamster synaptogenesis photomicrographs of synapsin I immunoreactive synaptic profiles in the hamster SCN. A, P I and B, P4. The asterisk indicates the ependymal lining of the third ventricle. Marker bar = 50/xm.
179 TABLE
II
Retinohypothalamic tract decelopment and synaptogenesis in the hypothalamus in rat and hamster The pattern of development
of the components
+
+ +, moderate;
to + + + + : + , s p a r s e ;
B e r n s t e i n 26 a n d a r e p r e s e n t e d
Age
of the retinohypothalamic
+ + +, dense;
tract and synaptogenesis
are graded from
are taken from Moore
and
here for comparison.
RHT det,elopment SCN
in t h e a n t e r i o r h y p o t h a l a m u s
+ + + +, adult pattern. The data for rat synaptogenesis
LHA
S~vnaptogenesis AHA
RCA
POA
SCN
LHA
AlIA +
Hamster." El6
-
+
+
Pl
_
_
_
++
++
++
P3
_
_
_
+++
+++
+++ ++++
P4
+
+
+
-
_
++++
++++
P6
++
++
++
+
+
++++
++++
++++
P8 P15
+++ ++++
+++ ++++
+++
++
++
++++
++++
++++
++++
++++
++++
++++
++++
++++
+ + ++ +++ ++++
++ +++ ++++ ++++++ +++++ ++++
_
_
++
++
_
_
_
+
+++
+++
+
+
-
++
++++
++++
++
++
-
+++
++++
++++
+++
+ ++
++++
++++
++++
++++
+ +++
++++
++++
++++
Rat.
E21/22 PI P2 P4 P6 PI5
and extends rapidly over the entire nucleus at P4. At this time the adult pattern also has appeared in the remaining A H A , L H A and RCA. DISCUSSION The SCN is a circadian pacemaker in the mammalian brain 25. It receives visual input from a direct retinohypothalamic tract 24'2s and that pathway is sufficient to maintain the entrainment of circadian rhythms ~6. In the present study, we have examined the development of retinohypothalamic projections in the rat and hamster using a sensitive anterograde tracing method, the C T - H R P method. In addition, we have examined the development of synapses in the hamster anterior hypothalamus using an antiserum to the synaptic vesicle protein, synapsin 17, to complement a prior study in the rat 26. The major results of the study are as follows. First, projections to the SCN and adjacent anterior hypothalamic areas, in both the rat and hamster, form significantly later than projections to other visual nuclei. R H T projections in the hamster form significantly later than in the rat. Second, the pattern of R H T development in the rat is one of substantial overgrowth with a subsequent pruning back to the adult pattern. In contrast, in the hamster the pattern is one of gradual growth to the adult pattern. Third, synaptogenesis in the hamster SCN and anterior hypothalamus occurs significantly earlier than in the rat (Table II). Recent studies using the C T - H R P method have shown that R H T projections are more widespread than
previously believed ~3'1'~. These projections have three components. The major terminal site is to the SCN with the contralateral projection greater than the ipsilateral one in both rat and hamster. In both species, additional axons extend beyond the SCN into the adjacent preoptic-anterior hypothalamic area and these projections appear to be more widespread in the hamster than in the rat j~. The dense axonal plexus in the SCN extends caudally into the rctrochiasmatic area and continues to the level of the rostral tuberal hypothalamus. In addition, there is a large projection in both species to the contralateral lateral hypothalamic area immediately dorsal to the optic tract ~'j'~ The axons forming the R H T appear to be, at least in large part, collaterals of retinal axons that innervate the thalamus. Pickard 3° has shown that the same ganglion cells innervate the SCN and the intergeniculate leaflet (IGL) of the thalamus and Millhouse 23 has demonstrated that axons entering the SCN are collaterals of optic chiasm axons. These observations are confirmed by recent studies using the pseudorabies virus that demonstrate that a form of the virus, the Bartha strain, is transported by ganglion cells that only innervate the SCN and the 1GL ~. The optic chiasm and optic tracts form several days before birth in both the rat ~'2° and hamster ~. Prior studies have reported the initiation of R H T innervation to the SCN at P 3 - P 4 in the rat 8"z1'37. Our data indicate that the onset of innervation of the SCN, anterior hypothalamus and retrochiasmatic area begins somewhat earlier but this difference probably reflects the greater sensitivity of the C T - H R P method. It is of interest that the projection to
18() the lateral hypothalamus is initiated significantly earlier, before birth in the rat. This could reflect either a difference in the rate of maturation of the lateral hypothalamus innervation or that this area is innervated by a separate set of ganglion cells from those providing the other R H T input. There is insufficient information to choose between these alternatives at this time. A second difference between the observations in this study and those done previously is the marked overgrowth of R H T projections that occurs during development in the rat. In all areas innervated, there is an initial overgrowth followed by a remodelling of the projections to gradually form the adult pattern. That this was not observed in the prior studies almost certainly reflects that they were done using less sensitive methods. The formation of the final pattern of connections probably reflects several events. The formation of R H T projections probably reflects formation of collaterals from optic chiasm and tract axons in response to a signal from the adjacent hypothalamus. The axonal overgrowth may reflect growth of axons that are not intended to innervate hypothalamus as described by Bhide and Frost 1 for other visual centers or it may represent a pruning back of axonal arbors that do not form functional synaptic contacts ~4'3s. For example, it is evident that there is significant cell death in the rat retina and loss of optic nerve axons during early postnatal development ~7'32. This indicates that these ganglion cells did not make functional connections and some of these might be the ones anomalously innervating hypothalamus. It is noteworthy that the pattern of R H T development in the hamster is totally different with no overgrowth of axons but, rather, a steady progression of input to achieve the adult pattern, as in the rat, between P6 and P10. The striking difference in development of the R H T between the rat and hamster is also reflected in the pattern of synaptogenesis. In the rat, synaptogenesis is initiated in the lateral hypothalamus and moves medially 26. The SCN is the last anterior hypothalamic area to show synapse development and it does not achieve an adult pattern until at least P10. In contrast, in this study we find a very rapid synaptogenesis, shown by synapsin I immunoreactivity, in the early postnatal period throughout the anterior hypothalamus in the hamster. The significance of these differences is unclear but they probably are the result of the very different evolutionary history of these two, rather disparate, rodent species. The R H T develops with three components, a projection to the lateral hypothalamic area and one principally to the SCN with additional projections extending
from it to the adjacent anterior hypothalamic area and retrochiasmatic area. In both the hamster and rat, the projection to the lateral hypothalamus develops before that to the SCN and adjacent areas suggesting that it may arise from different ganglion cells. There are two significant differences between the patterns of development in the rat and hamster. All R H T projections in the rat develop with an original overgrowth followed by a pruning back to the adult pattern whereas R H T development in the hamster occurs by a pattern of gradual increase in innervation to the adult pattern. Synaptogenesis in the hamster SCN and anterior hypothalamus occurs much earlier than the rat. This work was supported by NIH Grant, NS16304. We are grateful to Nadine Suhan and Doreen Hock for their assistance with this study. Acknowledgements.
REFERENCES 1 Bhide, P.G. and Frost, D.O., Stages of growth of hamster retinofugal axons: implications for developing axonal pathways with multiple targets, J. Neurosci., 11 (1991) 485-504. 2 Bunt, S.M., Lund, R.D. and Land, P.W., Prenatal development of the optic projection in albino and hooded rats, Dec. Brain Res., 6 (1983) 149-168. 3 Campbell, G. and Frost, D.O., Target-controlled differentiation of axon terminals and synaptic organization, Proc. Natl. Acad. Sci. USA, 84 (1987) 6929-6933. 4 Card, J.P. and Moore, R.Y., The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience, 13 (1985) 415-431. 5 Card, J.P., Whealy, M.E., Robbins, A.K., Moore, R.Y. and Enquist, L.W., Two alpha herpes virus strains are transported differentially in the rodent visual system, Neuron, 6 (1991) 957-969. 6 Cassone, V.M., Speh, J.C., Card, J.P. and Moore, KY., Comparative anatomyof the mammalian suprachiasmatic nucleus, J. Biol. Rhythms, 3 (1988) 71-91. 7 DeCamilli, P., Cameron, R. and Greengard, P., Synapsin I (Protein I), A nerve terminal specific phosphoprotein: its specific association with synaptic vesicles demonstrated by immunocytochemisty in agarose-embedded synaptosomes, J. Cell Biol., 96 (1983) 1355-1373. 8 Felong, M., Development of the retinohypothalamicprojection in the rat, Anat. Rec., 184 (1976) 400-401. 9 Frost, D.O., So, K.-F. and Schneider, G.E., Postnatal development of retinal projections in Syrian hamster: a study using autoradiographic and anterograde degeneration techniques, Neuroscience, 4 (1979) 1649-1677. 10 Hendrickson, A.E., Wagoner, N. and Cowan, W.N., An autoradiographic and electron microscopicstudy of retino-hypothalamic connections, Z. Zellforsch. Mikrosk. Anat., 135 (1972) 1-26. 11 Horsburgh, G.M. and Sefton, A.J., The early development of the optic nerve and chiasm in the embryonic rat, J. Comp. Neurol., 243 (1986) 5477-560. 12 Inouye, S.I.T. and Kawamura, H., Characteristics of a circadian pacemaker in the supracbiasmatic nucleus, J. Comp. Physiol., 146 (1982) t53-166, 13 Johnson, R.F., Moore, R.Y. and Morin, L.P., Retinohypothalamic projections in the rat and hamster demonstrated using cholera toxin, Brain Res., 462 (1988) 301-312. 14 Kalil, R.E., Dubin, M.W., Scott, G. and Stark, L.A., Elimination of action potentials blocks the structural development of the retinogeniculate synapses, Nature, 323 (1986) 156-158. 15 Kita, H. and Oomura, Y., An anterograde HRP study of retinal
181
16
17
18
19
20
21
22
23 24 25
26
27
projections to the hypothalamus in the rat, Brain Res. Bull., 8 (1982) 249-253. Klein, D.C. and Moore, R.Y., Pineal N-acetyltransferase and hydroxyl-indole-o-methyltranferase: control by the retino hypothalamic tract and the suprachiasmatic nucleus, Brain Res., 174 (1979) 245-262. Lain, K., Sefton, J. and Bennett, M.R., Loss of axons from the optic nerve of the rat during early postnatal development, De~'. Brain Res, 3 (1982) 487-491. Langdon, R.B. and Frost, D.O., Transient retinal axon collaterals to visual and somatosensory thalamus in neonatal hamsters, J. Comp. Neurol., 310 (1991) 200-214. Levine, J.D., Weiss, M.L., Rosenwasser, A.M, and Miselis, R.R., Retinohypothalamie tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin, J. Comp. Neurol., 306 (1991) 344-360. Lund, R.D. and Bunt, A.H., Prenatal development of central optic pathways in albino rats, J. Comp. Neurol., 165 (1976) 247-264. Mason, A.M., Sparrow, N. and Lincoln, D.W., Structural features of the retinohypothalamic projection in the rat during development, Brain Res., 132 (1977) 141-148. Mesulam, M.M., Tetramethylbenzidine for horseradish peroxidase neurochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. Millhouse, O.E., Optic chiasm collaterals afferent to the suprachiasmatic nucleus, Brain Res., 137 (1977) 351-355. Moore, R.Y., Retinohypothalamic projection in mammals: a comparative study, Brain Res., 49 (1973) 403-409. Moore, R.Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. Moore, R.Y. and Bernstein, M.E., Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin-I immunoreactivity, J. Neurosci., 9 (1989) 2151-2162. Moore, R.Y. and Card, J.P., Visual pathways and entrainment of circadian rhythms, Ann. NYAcad. Sci., 453 (1985) 123-133.
28 Moore, R.Y. and Lenn, N.J., A retinohypothalamic projection in the rat, J. Cornp. Neurol., 146 (1972) 1-14. 29 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. 30 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. 31 Pickard, G.E. and Silverman, A.J., Direct retinal projections to the hypothalamus, piriform cortex and accessory nuclei in the golden hamster demonstrated by a sensitive anterograde horseradish peroxidase technique, J. Comp. Neurol., 196 (1081) 155-172. 32 Potts, R.A., Drehr, B. and Bennett, M.R., The loss of ganglion cells in the developing retina of the rat, Det'. Brain Res., 3 (1982) 481-486. 33 Riley, J.N., Card, J.P. and Moore, R.Y., A retinal projection to the lateral hypothalamus in the rat, Cell Tiss. Res., 214 (1981) 257-269. 34 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rec., 59 (1979) 449-526. 35 Schmidt, J.T., Formation of retinotopic connections: Selective stabilization by an activity-dependent mechanism, Cell. Mol. Neurobiol., 5 (1985) 65-84. 36 Seiger, A. and Olsen, L., Late prenatal ontogeny of the central monamine neurons in the rat: fluorescence histochemicat observations, Z. Anat. Entwickl.-Gesch., 140 (1973) 281-318. 37 Stanfield, B. and Cowan, W.M., Evidence for change in the retinohypothalamic projection in the rat following early removal of one eye, Brain Res., 104 (1976) 129-136. 38 Sternberger, L.A., The unlabeled antibody peroxidase-anti-peroxidase (PAP) method, In L.A. Sternberger (Ed.), lmmunocytochemistry, Wiley, New York, 1986, pp. 104-169. 39 Trojanowski, J.Q., Gonatas, J.O. and Gonatas, N.K., Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers, Brain Res., 223 (1981) 381-385.
171
B R E S D 51714
Research Report
Retinohypothalamic tract development in the hamster and rat Joan C. Speh a,, and Robert Y. Moore b Departments o f ,.h Psychiatry. ~' Neurology, h Behauioral Neuroseience and " the Center for Neuroscience, Biomedical Science Tower W 1656, Unicersity of Pittsburgh, Pittsburgh, PA 15261, USA (Accepted 29 June 1993)
Key words." Suprachiasmatic nucleus; Retinohypothalamic tract: Lateral hypothalamus; Retrochiasmatic area; Circadian rhythm; Synaptogenesis: Synapsin
The development of the retinohypothalamic tract ( R H T ) in the albino rat and golden hamster was studied using anterograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP). The R H T has three components in the adult: (1) a dense projection to the ventrolateral subdivision of the suprachiasmatic nucleus (SCN) with some fibers extending into the dorsomedial SCN; (2) a projection to adjacent areas, the anterior hypothalamic area ( A H A ) and retrochiasmatic area (RCA) and in the hamster, into the preoptic area (POA); (3) a projection to the lateral hypothalamic area (LHA).In the rat, the projection to the SCN and adjacent areas first appears as scattered varicosities at the ventral border of the SCN at postnatal day 1 (P1) and gradually increases until the adult pattern is achieved at approximately PI0. The projections to the A H A and R C A are seen first at P 2 - P 3 and gradually increase to the adult appearance by P15. Both the projection to the SCN and adjacent areas and to the LHA, initially are more extensive than in the adult. Many of the axons extend well beyond the zone of the adult pattern but these anomalous fibers are eliminated by P6-P10. The L H A projection first appears at embryonic day 21-22 (E 21-22) and gradually increases in density from P1-P6. In the hamster the projections to the SCN, A H A and L H A appear first on P4 and gradually increase in density to reach the adult pattern by P15. The projections to the R C A and P O A are present by P6 and reach the adult pattern by PI5. None of the R H T projections in the hamster has the initial extended growth followed by pruning back that characterizes R H T development in the rat. Thus, the development of the R H T in both the rat and the hamster is complex with components of the projection appearing at different times with differing patterns of development that indicate specialized interactions of the developing axons with their target neurons. Synaptogenesis in the hamster hypothalamus was analyzed using an antiserum to synapsin I. Few synapses are present at El6, the last day of gestation, in the LHA, SCN and AHA. From P1-P3, synaptogenesis proceeds rapidly and the adult pattern is achieved in all three areas by P4.
INTRODUCTION The retinohypothalamic tract ( R H T ) is known to be an entrainment pathway in the circadian system of mammals ~3"~6'25. This projection was first shown in the rat using the autoradiographic tracing technique z8"l° and has been described in many other mammalian species 6'24"25. The development of the R H T in the rat and hamster has been studied using several techniques 2's'9'21'37. These studies found the R H T projection to the suprachiasmatic nucleus (SCN) of the rat to appear first on postnatal day 3 - 4 (P3-4) and that of the hamster on P6. Subsequent studies in the adult have shown the R H T to be a more complex projection than was initially described with components innervating the lateral hypothalamic area (LHA), anterior hypothalamic area (AHA) and retrochiasmatic area
* Corresponding author. Fax: (1) (412) 648-8376.
(RCA) 13'15"19'3t'33. The most recent studies of the R H T in the adult 13'19 have used a very sensitive anterograde tracer, H R P conjugated to the /3-subunit of cholera toxin 39. This has shown a more extensive and complex pattern of retinal projections to the hypothalamus than was appreciated previously and these observations have prompted the present study which analyzes the development of the R H T in the rat and hamster using the C T - H R P method. In addition, synaptogenesis was analyzed in the hamster anterior hypothalamus using an antiserum to synapsin I to complement a prior study in the rat 26. MATERIALS AND METHODS Rat Timed pregnant female Sprague-Dawley rats (Taconic) used in this study were housed individually with free access to food and water and kept on a diurnal light schedule (lights on 07.00 h-19.00 h). The time of birth was carefully noted and the 24-h period directly after birth designated as postnatal day 1 (P1). Animals from the following postnatal timepoints were used in this study: embryonic
172 day 21/22 (E21/22), P1, P2, P4, P6, P8, PIll and PI5. Mean crown rump lengths of embryonic animals were measured at sacrifice and used as the basis for determining the age of the fetus according to the timetable established by Seiger and Olsen 3~. For E21 injections, the timed pregnant rats were deeply anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and a small incision was made in the abdomen to expose one horn of the uterus. A fiber optic light source was used to illuminate the uterus, allowing the fetus' eye to be located. The injection was made through the uterine wall into the vitreal chamber of the eye. Hypothermia was the anesthesia used for the postnatal timepoints. Each animal received a unilateral injection of cholera toxin conjugated to horseradish peroxidase (CT-HRP, cholera toxin /3-subunit, List Biologicals) diluted in saline with 2% dimethylsulfoxide as vehicle into the vitreal chamber of the eye using a Hamilton microsyringe. Concentrations and volumes of CT-HRP varied with the animals age (Table I). All injections were performed during the middle of the day and the survival period was 24 h.
subdivision of t h e n u cl eu s with s o m e fibers e x t e n d i n g into the d o r s o m e d i a l p o r t i o n o f the nucleus. T h e seco n d c o m p o n e n t o f the R H T is largely an cxtension of the p r o j e c t i o n into the S C N to t h e p r e o p t i c - a n t e r i o r hypothalamic
a r e a ( A H A ) and r e t r o c h i a s m a t i c area
( R C A ) . T h e p r o j e c t i o n to the A H A is first o b s e r v e d in the h a m s t e r at the level o f the o r g a n u m vasculosum l a m i n a t e r m i n a l i s as s c a t t e r e d axons which leave the dorsal b o r d e r o f t h e optic n e r v e with p r o j e c t i o n s into the p r e o p t i c area. This c o n t i n u e s as a loose plexus of t e r m i n a l s that a p p e a r s to be an extension o f the S C N plexus into t h e A l I A in b o t h species which c o n t i n u e s t h e l e n g t h of the SCN. it ex t en d s f u r t h e r into the subparaventricular
zone
and
adjacent
AHA
in the
Hamster
h a m s t e r than in the rat. In b o t h species t h e r e is a large
Timed pregnant golden hamsters (Mesocricetus auratus) were obtained from Charles River and housed in indiviual cages on a diurnal light schedule (lights on 06.00 h-20.00 h) and given free access to food. The time of birth was noted and the first 24-h period designated as PI. Animals from the following timepoints were used in this study: P3, P4, P6, P8, P10, P15. Injection parameters were the same as for the rat experiments as noted above. Volumes and concentrations varied with the age of the animal (Table I). At the end of the survival period, the animals were anesthetized either with ketamine-xylazine or hypothermia. Each animal was perfused transcardially with saline followed by 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. The brains were removed and stored at 4 C in the same fixative for up to 18 h. The brains were cryoprotected in a sucrose series (10-30%) in 0.1 M sodium phosphate buffer. Serial sections were then cut on a freezing microtome in the coronal plane at 40 ~m. Sections were immediately reacted to visualize the horseradish peroxidase (HRP) with tetramethylbenzidine 22. After reaction the sections were mounted on gelatin coated slides, dehydrated in graded series of acetone and coverslipped. Synaptogenesis in the hamster was analyzed using an antiseum to synapsin 17. Two to four animals were sacrificed at El6, P1, P2, P3, P4, P6, P8 and P15. Animals were transcardially perfused or immersion fixed with Bouin's Solution, embedded in paraffin as previously described 26 and 8-10 /zm serial coronal sections through the hypothalamus were collected. Every fourth section through the SCN was stained for synapsin I and the adjacent section was stained with Cresyl violet. The immunohistochemical material was prepared using the peroxidase-anti-peroxidase complex (PAP) method of Sternberger 3s.
z o n e of i n n e r v a t i o n c a u d a l to the S C N into the R C A that
continues
to t h e
tuberal
hypothalamus.
These
p r o j e c t i o n s ar e m o r e d e n s e on the c o n t r a l a t e r a l side in b o t h the h a m s t e r and the rat. T h e third c o m p o n e n t o f the R H T is a p r o j e c t i o n to the l at er al h y p o t h a l a m i c a r e a ( L H A ) . It is c o m p r i s e d of a loosely s c a t t e r e d c o l l e c t i o n o f varicose axons that e x t e n d f r o m the lateral optic chiasm in t h e rostral h y p o t h a l a m u s , b ut m o r e caudally the L H A p r o j e c t i o n is a m o d e r a t e l y d e n s e a c c u m u l a t i o n o f t e r m i n a l s just dorsal to t h e s u p r a o p t i c n u cl eu s (SON). T h e L H A p r o j e c t i o n is substantially l a r g e r on the c o n t r a l a t e r a l side t h a n t h e ipsilateral side in b o t h t h e rat and h a m s t e r .
Development o f the rat retinohypothalamic tract T h e p r o j e c t i o n to t h e L H A is t h e first of t h e t h r e e RHT
components
to d e v e l o p in the rat.
Scattered
axons are p r e s e n t o v e r the lateral o p t i c tract at E 2 1 - 2 2 (Fig. 1). T h e C T - H R P l a b e l e d fibers are p r e s e n t cont r a l a t e r a l to the injected eye and are c o n f i n e d to the
TABLE I RESULTS The
development of the R H T
lntraocular injection parameters for demonstrating retinohypothalamic tract development will be d e s c r i b e d
s e p a r a t e l y for t h e rat a n d t h e h a m s t e r . A b r i e f summ a r y o f t h e n o r m a l a d u lt p a t t e r n o f th e rat a n d h a m ster will p r e c e d e t h e d e s c r i p t i o n s o f t h e R H T d e v e l o p -
For each pre- (E21/22) and postnatal age (P1-P15), the quantity and volume of CT-HRP injected, diluted in saline with 2% DMSO, is listed. The post-injection survival time at each age was 24 h.
Age
me nt. R H T p r o j e c t i o n s can be d i v i d e d into t h r e e com-
Number of animals Rat
Hamster
Quantity injected (~g)
2 6 6 3 6 3 3 4 3
2 4 4 4 3 3
0.5 0.5 0.5 0.5 1.0 1.5 2.0 3.0 4.0
Volume injected (tzl) 0.5 05 0.5 0.5 1.0 1.5 2.0 3.0 4.0
p o n e n t s . T h e largest o f t h e s e is the p r o j e c t i o n to the SCN. This is m o r e d e n s e to t h e c o n t r a l a t e r a l S C N in the rat w h e r e a s in t h e h a m s t e r t h e ipsilateral and c o n t r a l a t e r a l p r o j e c t i o n s a r e a l m o s t equal. A t rostral S C N levels t h e R H T p r o j e c t i o n f o r m s a d e n s e b a n d o f t e r m i n a l s a l o n g t h e dorsal e d g e o f t h e o p ti c chiasm. A s t he S C N e x p a n d s dorsally, retinal fibers p r o d u c e
a
d e n s e plexus o f t e r m i n a l s that fills th e v e n t r o l a t e r a l
E21/22 P1 P2 P3 P4 P6 P8 P10 PI5
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Fig. 1. Rat retinohypothalamic development. Schematic drawings of rat hypothalamus in the coronal plane through the rostral-caudal extent of the SCN. Four timepoints of unilateral CT-HRP eye injections are represented: A, E21/22; B, P1; C, P4; D, P15. Each timepoint is illustrated at three rostral through caudal levels. 3V, third ventricle; FX, fornix; OC, optic chiasm; SCN, suprachiasmatic nucleus: SON. supraoptic nucleus•
174 area immediately dorsal to the optic chiasm and tract. No ipsilateral label is evident. At PI, the number and distribution of labeled fibers in the L H A has increased greatly. Labeled fibers are present from the level of the most rostral SCN to the R C A and extend through the L H A into the adjacent A H A and perifornical region. Both the number and distribution of labeled fibers is greater than in the adult. In addition, there are labeled fibers extending in long radial arrays through the L H A (Fig. 2) that differ in apperance from any fibers in the
adult. The LHA projection continues to increase in density to P4 but thereafter appears to be pruned back to form the adult pattern by P10. Projections to the SCN appear first on P1 as scattered fibers in the SCN, particularly in the ventral and ventrolateral parts of the nucleus. The projection increases in density and extent through P4 (Fig. 1). At this point, as in the LHA, apparently anomalous projections extend through the SCN over a wide area of the AHA. These also are pruned back by P10 when the
Fig. 2. Rat retinohypothalamicdevelopment. Darkfield photomicrographsof unilateral CT-HRP eye injections. A, SCN at P1; B, SCN at P4; C, SCN at PI5; D, LH at P1; E, LH at P4: F, RCA at P4. OC, optic chiasm; 3V, third ventricle. Marker bar = 100 #m
175
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Fig. 3. Hamster retinohypothalmic development• Schematic drawings of hamster hypothalamus in the coronal plane through the rostral-caudal extent of the SCN. Four timepoints of unilateral CT-HRP eye injections are represented: A, P4; B, P6; C, P 8; D, P15. Each timepoint is illustrated at three rostral through caudal levels• 3V, third ventricle; FX, fornix; OC, optic chiasm; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus•
l 7~ p r o j e c t i o n to the SCN reaches its a d u l t a p p e a r a n c e .
SCN with some labeled fibers e x t e n d i n g into the A H A
This includes a d e n s e projection to the v e n t r o l a t e r a l
dorsal to the SCN.
SCN, scattered varicose axons into the d o r s o m e d i a l
T h e largest extra-SCN c o m p o n e n t of the R H T p r o -
Fig. 4. Hamster retinohypothalamic development Darkfield photomicrographs of unilateral CT-HRP eye injections. A, SCN at P4; B, LH at P6; C. RCA at P6; D, SCN at P6; E, SCN at PS; F, RCA at P8. OC, optic chiasm; 3V, third ventricle. Marker bars = 200 ~m
177
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'. •
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• ~-:iii~//:!.}!i: ....k--i~:;"..-i:
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Fig. 5. Hamster synaptogenesis schematic drawings of hamster hypothalamus indicating the distribution of synapsin I immunoreactive profiles at four developmental timepoints: A, El6; B, P1; C, P3; D, P4. Broken line indicates the borders of the SCN. OC. optic chiasm
178 jection is to the RCA. This is first evident as scattered varicose axons at P2 and increases markedly between P2 and P4. Like the other projections it contains some anomalous appearing axons at P4 which have been removed by P10 (Fig. 1). Throughout this developmental process, projections to the contralateral structures are greater than those to ipsilateral structures.
extending into the RCA. Over the next 4 days, the projection gradually increases in all areas to reach approximately three-fourths the density of the adult projection in all areas (Fig. 4). The density of innervation gradually increases so that, by P15, the projection has reached the adult pattern.
Synaptogenesis in the hamster anterior hypothalamus Deuelopment of the hamster retinohypothalamic tract The development of the hamster R H T differs from that in the rat in three major ways: (1) it develops significantly later with the first projections appearing at P4 and the adult pattern at Pl5; (2) the projection gradually assumes the adult pattern rather than initally extending beyond the area to be innervated and then being pruned back; (3) the projection to the L H A develops concomitantly with those to the SCN, A H A and RCA. The pattern of development of the hamster R H T is shown in Figs. 3 and 4. The first labeled axons to appear in the hypothalamus are evident at P4. They are present in the ventrolateral SCN, A H A and L H A with occasional fibers
In material prepared with the synapsin I antiserum, there are scattered varicosities, presumably synaptic terminals distributed nearly evenly over the entire anterior hypothalamic area at E l 6 (Fig. 5). Labeled terminals are slightly more dense in the immediate periventricular area, a pattern that continues until the adult distribution of labeled terminals is present at P4 (Fig. 6). The timeing of development of synapsin-immunoreactive varicosities is identical over the SCN and adjacent areas. This differs from the pattern in the rat where synaptogenesis occurs first in the L H A and extends medially with the SCN developing more slowly than adjacent A H A 2~'. The development of synapses in the hamster SCN begins along its borders at E16/P1
Fig. 6. Hamster synaptogenesis photomicrographs of synapsin I immunoreactive synaptic profiles in the hamster SCN. A, P I and B, P4. The asterisk indicates the ependymal lining of the third ventricle. Marker bar = 50/xm.
179 TABLE
II
Retinohypothalamic tract decelopment and synaptogenesis in the hypothalamus in rat and hamster The pattern of development
of the components
+
+ +, moderate;
to + + + + : + , s p a r s e ;
B e r n s t e i n 26 a n d a r e p r e s e n t e d
Age
of the retinohypothalamic
+ + +, dense;
tract and synaptogenesis
are graded from
are taken from Moore
and
here for comparison.
RHT det,elopment SCN
in t h e a n t e r i o r h y p o t h a l a m u s
+ + + +, adult pattern. The data for rat synaptogenesis
LHA
S~vnaptogenesis AHA
RCA
POA
SCN
LHA
AlIA +
Hamster." El6
-
+
+
Pl
_
_
_
++
++
++
P3
_
_
_
+++
+++
+++ ++++
P4
+
+
+
-
_
++++
++++
P6
++
++
++
+
+
++++
++++
++++
P8 P15
+++ ++++
+++ ++++
+++
++
++
++++
++++
++++
++++
++++
++++
++++
++++
++++
+ + ++ +++ ++++
++ +++ ++++ ++++++ +++++ ++++
_
_
++
++
_
_
_
+
+++
+++
+
+
-
++
++++
++++
++
++
-
+++
++++
++++
+++
+ ++
++++
++++
++++
++++
+ +++
++++
++++
++++
Rat.
E21/22 PI P2 P4 P6 PI5
and extends rapidly over the entire nucleus at P4. At this time the adult pattern also has appeared in the remaining A H A , L H A and RCA. DISCUSSION The SCN is a circadian pacemaker in the mammalian brain 25. It receives visual input from a direct retinohypothalamic tract 24'2s and that pathway is sufficient to maintain the entrainment of circadian rhythms ~6. In the present study, we have examined the development of retinohypothalamic projections in the rat and hamster using a sensitive anterograde tracing method, the C T - H R P method. In addition, we have examined the development of synapses in the hamster anterior hypothalamus using an antiserum to the synaptic vesicle protein, synapsin 17, to complement a prior study in the rat 26. The major results of the study are as follows. First, projections to the SCN and adjacent anterior hypothalamic areas, in both the rat and hamster, form significantly later than projections to other visual nuclei. R H T projections in the hamster form significantly later than in the rat. Second, the pattern of R H T development in the rat is one of substantial overgrowth with a subsequent pruning back to the adult pattern. In contrast, in the hamster the pattern is one of gradual growth to the adult pattern. Third, synaptogenesis in the hamster SCN and anterior hypothalamus occurs significantly earlier than in the rat (Table II). Recent studies using the C T - H R P method have shown that R H T projections are more widespread than
previously believed ~3'1'~. These projections have three components. The major terminal site is to the SCN with the contralateral projection greater than the ipsilateral one in both rat and hamster. In both species, additional axons extend beyond the SCN into the adjacent preoptic-anterior hypothalamic area and these projections appear to be more widespread in the hamster than in the rat j~. The dense axonal plexus in the SCN extends caudally into the rctrochiasmatic area and continues to the level of the rostral tuberal hypothalamus. In addition, there is a large projection in both species to the contralateral lateral hypothalamic area immediately dorsal to the optic tract ~'j'~ The axons forming the R H T appear to be, at least in large part, collaterals of retinal axons that innervate the thalamus. Pickard 3° has shown that the same ganglion cells innervate the SCN and the intergeniculate leaflet (IGL) of the thalamus and Millhouse 23 has demonstrated that axons entering the SCN are collaterals of optic chiasm axons. These observations are confirmed by recent studies using the pseudorabies virus that demonstrate that a form of the virus, the Bartha strain, is transported by ganglion cells that only innervate the SCN and the 1GL ~. The optic chiasm and optic tracts form several days before birth in both the rat ~'2° and hamster ~. Prior studies have reported the initiation of R H T innervation to the SCN at P 3 - P 4 in the rat 8"z1'37. Our data indicate that the onset of innervation of the SCN, anterior hypothalamus and retrochiasmatic area begins somewhat earlier but this difference probably reflects the greater sensitivity of the C T - H R P method. It is of interest that the projection to
18() the lateral hypothalamus is initiated significantly earlier, before birth in the rat. This could reflect either a difference in the rate of maturation of the lateral hypothalamus innervation or that this area is innervated by a separate set of ganglion cells from those providing the other R H T input. There is insufficient information to choose between these alternatives at this time. A second difference between the observations in this study and those done previously is the marked overgrowth of R H T projections that occurs during development in the rat. In all areas innervated, there is an initial overgrowth followed by a remodelling of the projections to gradually form the adult pattern. That this was not observed in the prior studies almost certainly reflects that they were done using less sensitive methods. The formation of the final pattern of connections probably reflects several events. The formation of R H T projections probably reflects formation of collaterals from optic chiasm and tract axons in response to a signal from the adjacent hypothalamus. The axonal overgrowth may reflect growth of axons that are not intended to innervate hypothalamus as described by Bhide and Frost 1 for other visual centers or it may represent a pruning back of axonal arbors that do not form functional synaptic contacts ~4'3s. For example, it is evident that there is significant cell death in the rat retina and loss of optic nerve axons during early postnatal development ~7'32. This indicates that these ganglion cells did not make functional connections and some of these might be the ones anomalously innervating hypothalamus. It is noteworthy that the pattern of R H T development in the hamster is totally different with no overgrowth of axons but, rather, a steady progression of input to achieve the adult pattern, as in the rat, between P6 and P10. The striking difference in development of the R H T between the rat and hamster is also reflected in the pattern of synaptogenesis. In the rat, synaptogenesis is initiated in the lateral hypothalamus and moves medially 26. The SCN is the last anterior hypothalamic area to show synapse development and it does not achieve an adult pattern until at least P10. In contrast, in this study we find a very rapid synaptogenesis, shown by synapsin I immunoreactivity, in the early postnatal period throughout the anterior hypothalamus in the hamster. The significance of these differences is unclear but they probably are the result of the very different evolutionary history of these two, rather disparate, rodent species. The R H T develops with three components, a projection to the lateral hypothalamic area and one principally to the SCN with additional projections extending
from it to the adjacent anterior hypothalamic area and retrochiasmatic area. In both the hamster and rat, the projection to the lateral hypothalamus develops before that to the SCN and adjacent areas suggesting that it may arise from different ganglion cells. There are two significant differences between the patterns of development in the rat and hamster. All R H T projections in the rat develop with an original overgrowth followed by a pruning back to the adult pattern whereas R H T development in the hamster occurs by a pattern of gradual increase in innervation to the adult pattern. Synaptogenesis in the hamster SCN and anterior hypothalamus occurs much earlier than the rat. This work was supported by NIH Grant, NS16304. We are grateful to Nadine Suhan and Doreen Hock for their assistance with this study. Acknowledgements.
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