Postnatal development of neuron number and connections in the suprachiasmatic nucleus of the hamster

Postnatal development of neuron number and connections in the suprachiasmatic nucleus of the hamster

Developmental Brain Research 110 Ž1998. 203–213 Research report Postnatal development of neuron number and connections in the suprachiasmatic nucleu...

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Developmental Brain Research 110 Ž1998. 203–213

Research report

Postnatal development of neuron number and connections in the suprachiasmatic nucleus of the hamster Celia Muller, Fernando Torrealba ¨

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Departamento de Ciencias Fisiologicas, Facultad de Ciencias Biologicas, Pontificia UniÕersidad Catolica de Chile, Alameda 340, Casilla 114-D, ´ ´ ´ Santiago, Chile Accepted 14 July 1998

Abstract We had previously found a ca. 30% cell death during the prenatal ontogeny of the suprachiasmatic nucleus ŽSCN. of lambs. The period of neuron death was preceded by the establishment of the retinohypothalamic connections, a major input to this nucleus that allows the entrainment to light of the circadian rhythms generated by the SCN. The present study determined the temporal relationship between the period of ontogenetic neuron death and the establishment of the principal afferent and efferent connections of the SCN in hamsters. We found that during the first 3 postnatal days the SCN volume increases mainly by the addition of cells. After a peak 6140 neurons on each side during the third postnatal day, the SCN underwent an acute decrease of about 40% in neuron number, which led to the final adult complement of neurons, estimated in 3400 neurons per nucleus. The connections of the SCN were studied by placing DiI crystals either into the optic nerve, or into the SCN of brains fixed at different ages. We found, in agreement with previous studies, that retinal axons can be detected after the fifth postnatal day, that is, after the large decrease in neuron number. As for the SCN efferents, they began to invade the appropriate targets during the second postnatal day, followed by a large increase in the density of these efferent projection in the subsequent days. In conclusion, the massive neuronal death in the SCN was preceded by the formation of efferent connections, and followed by the formation of the retinohypothalamic tract. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Circadian; Neuronal death; Retinohypothalamic; Stereological; Suprachiasmatic

1. Introduction The mammalian suprachiasmatic nucleus ŽSCN. is an intrinsic circadian pacemaker that is necessary to produce a daily oscillation in a number of behavioral and physiological variables w14x. Because the principal function of the SCN is quite straightforward, that is to provide a circadian signal to other brain regions, including output nuclei, the study of the structure–function relationships underlying its role seems an addressable issue. In this context, the analysis of the developmental steps that lead to a mature SCN should provide insights on those structure–function relationships. For instance, it has been shown in rats that the fetal SCN presents circadian oscillations of metabolic activity w24x and, within a few days after becoming postmitotic, neurons in the SCN region of the hypothalamus bind

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melatonin w32x. Both events are quite independent of the development of synapses within the SCN, since synaptogenesis is mainly a postnatal event w30x in altricial rodents. Also, circadian outputs like the rhythm of locomotor activity, measured two weeks after birth, can be entrained by maternal signals or by melatonin injections given prenatally w8x. Taken together, these studies suggest that cellular mechanisms very close to the clock itself, like the rhythmic metabolic activity and the ability of the SCN neurons to be entrained, are already functioning in prenatal life, and are not dependent on synaptic activity. In altricial rodents, the development of the major entrainment input in postnatal life, the retinohypothalamic pathway, and the development of the SCN synapses, are both postnatal phenomena w30x. Other important ontogenetic events, like the formation of SCN efferents, the onset of circadian activity of behavioral and physiological outputs, and the possible existence of a period of ontogenetic cell death, have not been studied with the same detail. We had demonstrated a substantial neuron death in the SCN of

0165-3806r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 0 8 - 4

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C. Muller, F. Torrealbar DeÕelopmental Brain Research 110 (1998) 203–213 ¨

fetal lambs, as indicated by a 30% decrease in neuron number w31x. We showed that this major adjustment in the number of SCN neurons follows the arrival of retinal axons to the SCN w31x, suggesting a role for these afferents in regulating neuron number. However, neuron death during late development is more frequently explained by a competition for postsynaptic space, that is, with the development of efferent connections w21,33x. The temporal relationship between the period of cell death in the SCN and the period of afferent and efferent formation should provide a clue on which of these mechanisms—postsynaptic vs. presynaptic—weighs more in the adjustment of neuron number in the mammalian SCN. We decided to investigate in hamsters, a rodent with a short intrauterine life, the

temporal relationship between the retinal innervation and the formation of efferent projections of the SCN with the period of neuronal death in this nucleus. While previous studies on the development of the retinohypothalamic pathway in hamsters w30x have shown that retinal axons can be detected in the SCN from the fourth day after birth, no comparable studies have been conducted on the timing of SCN efferent connections development. The developmental timing of the SCN efferent connections should also shed light on another unsettled issue, which is the nature of the circadian signal from the pacemaker nucleus to the target structures in the brain. These targets in turn control a wealth of behavioral and physiological variables that show a circadian rhythm w14x. In

Fig. 1. Photomicrographs of Nissl-stained transverse sections through the suprachiasmatic nucleus ŽSCN., at a comparable intermediate level, of postnatal and adult hamsters to show the developmental changes in cytoarchitectonics. Note the high cell density of the SCN, relative to the surrounding hypothalamic regions, at postnatal day 0 ŽP0, A.; P2 ŽB.; P5 ŽC. and adult ŽAd, D.. ox s optic chiasm. Bars s 100 mm.

C. Muller, F. Torrealbar DeÕelopmental Brain Research 110 (1998) 203–213 ¨

principle, the SCN might control the daily change of those variables via humoral andror neural signals. One model assumes that the rhythmic activity of suprachiasmatic neurons influences the activity of other CNS regions via its efferent neural connections to many diencephalic regions in the vicinity of the SCN. Support for a central role of SCN efferents came from studies using knife-cut isolation of the SCN, that caused loss of circadian rhythmicity w19,20x. Transplant studies have provided conflicting evidence as to the role of these efferents w27,29x. While some authors have consistently found a causal relationship between restoration of SCN efferent connections and recovery of circadian outputs, w29x others have not confirmed such a close association w16x. In fact, recent evidence using transplants w27x supports the notion that a diffusible signal from the SCN might control circadian locomotor rhythms. The present study on the development of SCN efferents should contribute to establish the physiological role of these efferent connections in inducing circadian rhythmicity to output nuclei, by relating the formation of these connections to the development Žunknown so far. of these circadian outputs.

2. Material and methods Newborn and adult golden hamsters Ž Mesocricetus auratus) kept in a 12r12-h lightrdarkness cycle were used. The first 24 h after birth were considered as postnatal day zero ŽP0..

3. Preparation of material for counting SCN neurons All animals were killed at CT 04, except when noted. Adult hamsters were deeply anesthetized with sodium thiopental Ž50 mgrkg, i.p.., while newborn hamsters were anesthetized with ether and hypothermia. The animals were perfused through the left ventricle with 2% paraformaldehyde, 2.5% glutaraldehyde and 0.02% picric acid in 0.1 M cacodylate buffer, pH 7.6. The brains were removed and left for 5 days in the same fixative. Blocks containing most of the brain Žnewborns. or just the diencephalon Žadults. were dehydrated in an ascending series of ethanols up to 95%, and embedded in a water soluble methacrylate resin ŽJB-4, Polysciences.. We used rectangular silicone molds and small pins to keep the blocks in the appropriate orientation while the resin polymerized. Serial 2-mm coronal sections were cut with a Huxley ultramicrotome and long edge knives prepared from glass slides w26x. Four adjacent sections were collected, then 7 or 15 sections discarded, and the cycle repeated so as to obtain a series of sections containing the whole rostrocaudal extension of the SCN. The sections were collected on tap water with some drops of ammonia solution; mounted

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on gelatin-coated slides; stained for 2 days in cresyl violet; dehydrated, cleared and covered with Entellan ŽE. Merck, Darmstadt, FGR.. 3.1. Stereological methods To calculate the numerical density of SCN neurons at different ages, we used the physical disector method on the cresyl violet-stained sections w12x. Pairs of adjacent sections, the reference and the look-up sections, served to measure the number of neurons per unit of volume. In the reference section we counted the neurons within a square grid of 100 mm each side, projected through a camera

Table 1 Neuron density Ž D ., volume Ž V . and number of neurons Ž N . of the suprachiasmatic nucleus of newborn and adult hamsters Age Ždays.

D Žper 10 3 mm3 .

V Žmm3 =10y3 .

N

CE N )

P0-a L P0-a R P0-b L P0-b R P0-c L P0-c R P1-a L P1-a R P1-b L P1-b R P1-c L P1-c R P2-a L P2-a R P2-b L P2-b R P2-c L P2-c R P3-a L P3-a R P3-b L P3-b R P4-a L P4-a R P4-b L P4-b R P5-a L P5-a R P5-b L P5-b R P6a L P6a R P6b L P6b R A-1 L A-1 R A-2 L A-2 R A-3 L A-3 R

1.34 1.43 0.85 0.84 0.92 0.93 1.07 0.87 0.89 0.91 0.82 0.8 0.93 0.9 0.76 0.99 0.62 0.66 0.745 0.676 0.86 0.72 0.969 0.894 1.24 0.98 0.739 0.708 0.82 0.77 0.6 0.534 0.603 0.526 0.293 0.329 0.312 0.362 0.29 0.285

4.257 3.826 4.452 3.747 4.81 5.399 5.789 5.982 5.84 5.349 6.864 5.496 7.58 7.038 10.3 8.347 5.93 5.41 5.543 4.921 4.44 3.95 5.822 4.966 3.308 3.346 3.269 3.464 4.67 3.52 7.254 6.381 6.05 5.88 12.3 13.7 9.73 10.95 9.87 7.78

5708 5490 3776 3159 4430 5007 6178 5218 5208 4875 5630 4412 7066 6367 7841 8298 3706 3557 4133 3330 3863 2881 5643 4444 4092 3287 2417 2452 3829 2694 4359 3409 3647 3093 3610 4511 3039 3964 2867 2219

0.059 0.087 0.082 0.05 0.045 0.057 0.065 0.088 0.05 0.045 0.035 0.042 0.08 0.1 0.065 0.089 0.058 0.057 0.044 0.091 0.17 0.15 0.052 0.087 0.096 0.093 0.061 0.068 0.1 0.1 0.041 0.042 0.05 0.056 0.045 0.066 0.049 0.051 0.074 0.12

)Coefficient of error of the estimate of the number of neurons of each SCN.

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lucida over the center of the SCN. All neurons with a sharp nucleus and contained within the grid, and that were not in contact with the superior or the right edges of the grid, were traced on paper. This drawing was superimposed on the adjacent section Žthe look-up section., and the neurons that were present only in the reference section Žtops. were counted. The number of tops Ž Q-. per disector divided by the disector volume Ž VDis s 100)100)2 mm. gave the numerical density of a given disector. The average numerical density of one SCN Ž NÕ . was calculated as in the equation w12x: NÕ s

ÝQ y ÝVDis

The SCN volume was estimated following Cavalieri’s method w12,25x. We traced the area of the SCN from the Nissl-stained 2-mm sections spaced every 10 or 18 sections. The area of each tracing was calculated with the help of a graphic tablet and the SigmaScan measuring system. The SCN volume Ž VRef . was calculated as the average SCN area times the SCN length. The total number of SCN neurons Žon one side. was calculated as the product of NÕ by VRef . The coefficient of error of the systematic observations was estimated w12x by using the equation: CE s

unit of area in each section times the volume of the SCN in that section. 3.2. Tracing of SCN afferents and efferents Newborn hamsters were perfused as above, but with 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.6. The SCN of P0 to P3 hamsters were injected with 1, 1X-dioctadecyl-3,3,3X ,3X-tetramethylindocarbocyanine perchlorate ŽDiI. ŽMolecular Probes, Eugene, OR.. Under a disecting microscope, one small crystal of DiI was pushed, with a very fine pin, into the SCN through the optic chiasm just lateral to the midline. To study the development of the retinohypothalamic pathway we either placed one DiI crystal into to optic nerve 2–3 mm from the chiasm or inserted a thin Ž0.05 mm. and long Ž2 mm. glass pipette covered with dried DiI into the optic disc w1x. The brains were gently brushed to remove loose DiI crystal and left in fixative for 4 to 10 weeks at room temperature. The brains were embedded in 3% agar and cut in the transverse plane with a Vibratome, to obtain 100-mm sections which were mounted on slides using 50% glycerol in 0.1 M phosphate buffer, coverslipped and studied under an epifluorescence microscope.

(Ž 3 A y 4 B q C . r12 Ýx

where A s Ý x i ) x i ; B s Ý x i ) x iq1; C s Ý x i ) x iq2 To calculate the coefficient of error of the SCN volume ŽCE Õ ., x i was the SCN area measured in each section; to calculate the coefficient of error of the total number of neurons, x i was the product of the number of neurons per

4. Results 4.1. Cytoarchitecture of the postnatal SCN deÕelopment The SCN could be readily recognized in semi-thin sections ŽFig. 1. by its relatively higher cell density, by a

Fig. 2. Two adjacent sections, 2 mm in thickness, through the SCN of a P2 hamster, to illustrate the disector method used Žfor more details see Section 2.. The counting frames were aligned using landmarks like blood vessels Žbv. and the ventricle Ž3V.. Only the neuronal profiles which appear in the reference section Žthree of them indicated by 6 in B., but not in the adjacent ŽA, look-up. section were counted. Neurons touching the thick borders of the frame, or present in both sections Žasterisks., and darker cells Žglia or epithelial cells, thick arrow. were excluded. Bars s 50 mm.

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darker neuropil than the surrounding hypothalamus, and by the tendency of the SCN neurons to aggregate in clusters. At earlier postnatal ages ŽFig. 1A,B. the SCN had even sharper boundaries than at later ages ŽFig. 1C,D., and the cells looked very much like cells in other hypothalamic regions, that is with an oval nucleus, with three to four clumps of heterochromatin, and no visible cytoplasm. In adults, SCN neurons had scant cytoplasm, and were distinctively smaller than the surrounding hypothalamic neurons; the nuclei of SCN neurons had heterochromatin and a nucleolus. We used stereological techniques to measure the volume and the neuronal density of 40 SCN from 20 hamsters of different ages Žsee Table 1.. We then calculated the number of neurons of each nucleus Žsee Section 2.. Fig. 2 illustrates how we applied the physical disector method to measure neuron density. The coefficient of errors of the estimates of either total SCN neuron number ŽTable 1.,

Fig. 4. One apoptotic cell Žarrow. within the dorsal region of the SCN of a P0 hamster. The ventral tip of the third ventricle Ž3v. is indicated. Bar s 50 mm.

Fig. 3. Changes Žmean"S.E.M.. in neuron number Župper panel., neuronal density Žmiddle panel. and volume Žlower panel. of the SCN of postnatal hamsters. ) ssignificantly different vs. P6 and adult Župper panel; see text.; vs. adult Žmiddle panel. and vs. P0 and P5 Žlower panel..

neuron density or SCN volume Žnot in the table. were below 10% for each animal. Table 1 also shows the high consistency of the values for the two SCN of each animal. During the first 3 days of postnatal development ŽP0– P2. the SCN neuron number linearly increased from 4595 Ž"407 S.E.M.. to 6139 Ž"836 S.E.M.., that is from 136% to 182% of the adult Ž3368 " 337. number ŽFig. 3, upper panel.. We detected in the interval from P2 to P5, a drastic reduction of 30% in the number of SCN neurons Ž p - 0.05; one-way ANOVA and Tukey tests., which persisted into adulthood Ž p - 0.05.. During the interval P3–P6 there was a large interindividual variability Žsee Table 1. but, on average, during these 3 days the SCN reached its adult complement of neurons. This large variability between animals of the same age is thought to be intrinsic to developing CNS w33x and related to differences in the timing of neuron arrival or neuron death. In addition, the variability may also reflect the lower number of animals that we measured within this period. In contrast, the neuronal density steadily decreased ŽFig. 1; Fig. 3, middle panel and Table 1. from about 1 neuron per 10 3 mmy3 at birth to about 0.3 neurons per 10 3 mmy3 in the adult ŽP0 vs. adult; p - 0.05, Kruskal–Wallis’ and Dunn’s tests.. The changes in SCN Õolume ŽFig. 3, lower panel. closely followed the changes in neuron number, up to P6, including the large intersubject variability observed between P3 and P6. At P6, when neuron number had stabilized, the volume was about 63% of 10.7)10y3 mm3 , the average adult size for the hamster SCN Ž p - 0.05, adult

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vs. P0 and P5; Kruskal–Wallis’ and Dunn’s tests.. We did not correct the volume estimations for the shrinkage caused by the histological procedures. This shrinkage Žabout 10% in linear dimensions w11x. may also vary with age. To relate the decrease in neuron number with the phenomenon of developmental cell death, we counted apoptotic cells in the SCN ŽFig. 4. and in a control 150 mm square from the neighboring medial hypothalamic region. The results shown in Table 2 indicated the presence of very few apoptotic cells in the SCN from P0 to P6, without consistent quantitative changes, except for the absence of apoptotic cells in the six nuclei analyzed at P2. We killed additional P3 hamsters at CT 16, to see whether the circadian time influenced the number of apoptotic neurons in SCN. We observed no increases in the number of apoptotic cells compared to the animals killed at noon. 4.2. Injections of DiI into the optic nerÕe Injections in hamsters killed at P3–P5 produced no labeling of retinal axons within the SCN or adjacent hypothalamic regions. At P6 a few fluorescent retinal axons were present mainly in the ipsilateral SCN Žnot shown.. At P7, a dense network of retinal axons was seen in the ipsilateral SCN, and fewer labeled axons in the contralateral SCN. ŽFig. 5A,B.. In contrast, in the retrochiasmatic region much more retinal afferents were present within the contralateral side than in the ipsilateral side ŽFig. 6.. Some retinohypothalamic axons ascended medially along the third ventricle, and seemed to terminate within the anterior hypothalamic area. In occasions we observed labeled perikarya in the lateral hypothalamic area just dorsal to the optic tract, and axons from these neurons entering the optic chiasm. The present and previous studies w30x show that the retinal innervation begins after the period of neuron death in the SCN ŽP3–P4.. In contrast, the other thalamic and midbrain targets of retinal axons are innervated much earlier. A P1 hamster injected into the chiasm and the SCN had labeled axons in the dorsal lateral geniculate nucleus ŽFig. 5C.. Table 2 Average number of apoptotic cells in the suprachiasmatic nuclei ŽSCN, both sides., and the medial hypothalamic region of postnatal hamsters Age Ždays.

SCN a

Hypothalamusb

No. of animals

P0 P1 P2 P3 P4 P5 P6

3.3 1.7 0 2 2 2 2

2.7 5.3 9.7 5 0 2 2.5

3 3 3 1 1 1 2

a SCN was not significantly different from hypothalamus Ž ps 0.128; one-way ANOVA.. b We counted apoptotic cells within 2 squares of 150 mm each side, placed on the medial hypothalamic region on both sides of the III ventricle, and in the same sections containing the SCN.

Fig. 5. Fluorescent photomicrographs of DiI labeled axons within the SCN ŽA,B. or the thalamus ŽC., after injections of the tracer into the optic nerve of P1 and P7 hamsters. A, SCN of P7 hamsters. Note the heavier labeling of the ipsilateral Žleft. side. The heavily labeled optic chiasm is in the lower part of the picture. B, Higher magnification of optic axons within the ipsilateral SCN. C, Bundles of labeled axons in the optic tract Žright side and lower left half of the picture.. Note also the finer, single axons entering the dorsal lateral geniculate nucleus Župper left part.. Thalamus contralateral to the injected optic nerve. Barss 50 mm.

4.3. Injections of DiI into the SCN These injections ŽFig. 7. readily labeled axons emerging from the SCN and surrounding regions of P1–P3, but not

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Fig. 6. Fluorescent photomicrographs of DiI labeled axons within the retrochiasmatic area after injections of the tracer into the optic nerve of P7 hamsters. ŽA. Contralateral side. ŽB. Ipsilateral side. The junction between the optic chiasm and the optic tract is in the lower part of the pictures. Bars s 50 mm.

P0 hamsters. Starting at P1, these SCN efferents exhibited a very rapid and simultaneous development to most of the brain regions that are known to be innervated by the adult SCN. In contrast to retinohypothalamic afferents, the SCN efferents started innervating their targets before the period of ontogenetic cell death. Since the injections involved the optic chiasm, in addition to the SCN and immediately surrounding regions, and resulted in heavy axonal labeling in the dorsal ŽFig. 5C. and ventral lateral geniculate nuclei and the intergeniculate leaflet, it was not possible to distinguish SCN projections to these thalamic nuclei. However, no labeled neurons were found in the intergeniculate leaflet at P0–P3, suggesting that the geniculohypothalamic pathway had not yet developed. As described above, the retinohypothalamic tract had not invaded the SCN, or other

hypothalamic territories at P3, so that from P0 to P3, the axons present in the hypothalamus and related structures could only be labeled by the SCN injections. We injected DiI crystals into the SCN of seven P0 Žthree of them perfused 6 h after birth, and the other four at 12 h after birth. pups and, even though well stained axons were visible within the optic chiasm, the optic tracts and the lateral geniculate nuclei, no axons or cell bodies were labeled in the known SCN targets within the neighboring hypothalamus. In addition, we placed thin glass micropipettes covered with DiI Žsee Section 2. in the region of the paraventricular nucleus of the hypothalamus Žtwo P0 pups. or into the region just ventral to it Žtwo P0 pups., and obtained no retrogradely labeled cells in the respective SCN. These findings indicate that at P0 the

Fig. 7. Fluorescent photomicrographs of sections through the anteroventral hypothalamus of P0 and P1 hamsters, to show the location and extent of the DiI injections into the SCN and the optic chiasm Žox.. Bars s 200 mm.

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SCN has not yet given off axonal efferents to the surrounding hypothalamus. Efferents from the SCN were first noticed in P1 hamsters, where the distribution of SCN efferents was as widespread as it was sparse. A much denser projection was observed in P2 and P3 hamsters. In three P1 pups a moderate number of axons emerged from the injection site and followed a dorsal direction along the midline; fewer axons took a more oblique course in a dorsal and lateral direction. Many of these axons had terminal and en passant boutons in the paraventricular hypothalamic nucleus ŽPaV., in the anterior hypothalamic area ŽAHA., and in the periventricular region dorsal to SCN. Less frequently, we observed typical growth cones at the tip of the axons. In contrast to this axonal labeling, very few labeled neuronal perikarya were present in P1 hamsters, and always within the ipsilateral PaV or the AHA. In the P2 and P3 hamsters

we observed a large increase in the density of SCN efferents, and also a moderate increase in the number of retrogradely labeled neurons in the ipsilateral hypothalamus. In addition to the above mentioned terminal fields found in P1 hamsters, we observed labeled axons with boutons just medial to the ventral hypothalamic nucleus, and few if any boutons in the retrochiasmatic area. Rostral to the SCN there were labeled axons with boutons in the medial preoptic nucleus and the medial preoptic area ŽFig. 8B., in the bed nucleus of the stria terminalis and within the lateral septal area. Dorsally directed axons and boutons were found into the periventricular and paraventricular hypothalamic nuclei ŽFig. 8A.; into the AHA ŽFig. 8C., and very few fibers in the paraventricular nucleus of the thalamus ŽFig. 8D.. DiI injections into the chiasm and SCN of P0 to P2 hamsters labeled radial glial processes arching from the

Fig. 8. DiI labeled efferents from the SCN of P2 hamsters, ipsilateral side. ŽA. Axons in the paraventricular nucleus of the hypothalamus ŽPaV.. ŽB. Labeling in the rostral part of the anterior hypothalamic area ŽAHA. and in the medial preoptic nucleus ŽMPA.. ŽC. Projection to the anterior hypothalamic area ŽAH. just dorsal to the SCN. ŽD. Single axon Žarrowhead. within the paraventricular nucleus of the thalamus ŽPVA.. D3Vs dorsal part of the third ventricle. Bars s 50 mm.

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tract to the third ventricle. At P3, these fluorescent glial processes were scarce and from P4 on they were no longer visible. The labeling of radial processes when the optic tract was stained with DiI had a time course that coincided with the involution of radial glial cells immunoreactive to vimentine in postnatal hamsters w2x. As in the present study, Botchkina and Morin w2x showed the existence of radial glial processes at birth, while at P3 most of the vimentin immunoreactivity had disappeared.

5. Discussion The present study established the temporal relationship between ontogenetic cell death, the formation of efferent pathways and the development of the retinohypothalamic afferent pathway in the SCN of hamsters. All these events take place after birth. We determined that SCN neurons begin to give off efferents during P1, that is, 2 days before the abrupt decrease in neuron number we detected between P2 and P5, and we confirmed a late retinohypothalamic innervation, where the retinal afferents start invading the SCN immediately after this period of cell death. 5.1. Morphometric deÕelopment The stereological techniques rely on a precise delimitation of the structure to be measured. In spite of being one of the most clearly demarcated nuclei of the hypothalamus, the precise limits of the SCN are debatable, and the criteria that may be useful to demarcate the nucleus in adult animals, may be of little use during development. No single immunocytochemical marker defines the boundaries of the SCN at all ages, since the major neurotransmitters are restricted to portions of the SCN w3x, and the antigens present in glial cells do not delineate this nucleus, at least during the first postnatal weeks w2,15,18x. Similarly, the principal SCN afferents w22x are either present only in a single SCN subdivision or, in addition to a terminal field within this nucleus, they also innervate surrounding regions that clearly do not belong into the SCN. For these reasons we decided to rely on widely used cytological criteria to trace the boundaries of the SCN. We applied as operational criteria to trace the SCN, a higher density of small neurons that tended to group in clusters, and immersed in a darker neuropil. The SCN can be easily identified at birth, when it has 36% more cells than the adult nucleus. During the first 3 postnatal days ŽP0–P2., the SCN keeps growing by the addition of postmigratory neurons. Two studies on the SCN neurogenesis in hamsters have shown that SCN neurons are born over a 2 1r2-day period w5,6x, with a peak around day 11.5 postgestation ŽE11.5.. A comparison of the rate of neuron birth with the postnatal time course of neuronal addition to the SCN Žpresent study., indicates that

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the increase of neuron number in the SCN is a much slower process. In a 2-day interval, from P0 to P2, the SCN increased from 75% to its maximum number of neurons Ž6139, or 100%.. In 2 days, all SCN precursors have become postmitotic. This temporal difference indicates that some, but necessarily not all, SCN precursors have a delayed or a slower migration from the subventricular zone to their final destination. The capacity of SCN neurons to be entrained is present prenatally, before the arrival of retinal afferents, and before the formation of SCN efferents. In hamsters, Davis and Gorski w7x demonstrated that the ablation of the maternal SCN at 7 days of gestation disrupted the normal synchrony within the litter, measured at weaning, that is, at 2 weeks after birth. In contrast, little effect of a maternal SCN lesion performed at 14 days of gestation was observed at weaning. Further evidence for prenatal phase setting of the circadian rhythm in hamsters comes from transplant studies where the SCN grafts from E13–E14 fetal hamsters w29x determine the period and phase of the induced circadian rhythms of the host. These results were interpreted as indicating that the phase of the circadian rhythms of the pups was set between E7 and E14. In addition, melatonin-binding sites are densely present in the region of the SCN at E14, i.e., 2 days before birth w32x. Our present results show that during the first 3 postnatal days the last 25% of the SCN neurons are arriving to the nucleus. The extrapolation to the fetal hamster of the postnatal time course of the SCN morphogenesis makes it likely that at E14 Ži.e., 2 days before birth. not much of the SCN neurons were already forming the nucleus. Taking our findings and those of Davis and Gorski w7x together, it seems clear that the capacity of the SCN neurons to be entrained by maternal signals is present before most of the SCN neurons have migrated to their final destination dorsal to the chiasm. In other words, neurons that are just postmitotic, and are not connected to other brain regions, do already show a central property of the circadian oscillator, that is the capacity of being entrained. 5.2. Neuron death Between P2 and P5 there was a large, about 40% decrease in neuron number, and a larger intersubject variability in the number of neurons. The values fluctuated around the final adult neuron number. We expected to find the largest number of apoptotic cells in the sections corresponding to animals having the steepest decrease in neuron number, that is from P3 to P5. We found no such a correspondence. Although the number of SCN neurons hastily decreased within a short period of time after reaching the maximal number at P2, we observed similar numbers of apoptotic cells from P0 to P6. Cell death occurred before and after this short period when the actual number of SCN neurons fell to about 60% of its highest value. The low number of apoptotic cells is partly

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explained by the rapid removal of the dying cells w21,28x. However, it was surprising that no increase in the number of apoptotic cells occurred between P2 and P3, the period of acute decrease in the number of neurons. The change in the number of neurons during this developmental stage depends on the balance between newly arrived neurons and dying neurons. It is possible that during SCN growth, arriving neurons outnumber dying neurons, and that the fall in neuron number is caused by a short period of time when the arrival of neurons decreased below the number of dying neurons w10x. This might explain a steady number of apoptotic cells in the face of a large decrease in neuron number. Perhaps we are grossly underestimating the number of cells that undergo ontogenetic cell death when we just count cell numbers w10x. The presence of apoptotic cells at P0 and P1, before the formation of SCN extrinsic connections, raises the question of whether other factors—like a successful entrainment by maternal signals or the formation of intrinsic connections—may also be playing a role in the process of ontogenetic neuron death. 5.3. Ontogenetic cell death and the formation of connections It is commonly accepted that the massive and widespread neuron death occurring during late development of the nervous system is a mechanism to finely adjust the number of neurons of a given structure, and to remove cells that made inappropriate connections. One model involves competition for postsynaptic space and trophic substances. In this model, ontogenetic cell death should occur during the development of the efferent projections. Less frequently invoked as a mechanism for regulating neuron number is the establishment of adequate inputs. In a previous work on fetal lamb SCN development w31x we had shown that the retinal axons start invading the ipsilateral SCN while neuroblasts were still migrating to this nucleus, as assessed by increases in neuron number. These findings indicated that the retinal innervation, was established before the period of ontogenetic neuron death in the fetal lamb, in contrast to the present findings in hamsters. If the development of efferents is also followed by cell death in the lamb SCN, then we would predict that these connections should be present before E84, that is before the large decrease in SCN neuron number in this species. 5.4. DeÕelopment of SCN connections Our results on the development of the retinohypothalamic tract using DiI agree with more detailed studies using cholera toxin as an anterograde tracer w15,30x. We found a bilateral retinal projection at P6 and P7, and no retinal axons in the SCN at P3. We also found that the major thalamic targets are innervated by retinofugal axons at birth. These findings confirm that in hamsters, as in other mammals, the retinal projections to thalamic and mesencephalic targets are present much earlier than the retinohy-

pothalamic projection w9x. Some of the retinal afferents to SCN are collaterals of axons innervating the intergeniculate leaflet of the thalamus w23x. Since the SCN is innervated almost a week later, it remains to be determined whether these collaterals sprout after their arrival to the leaflet, or if they represent a different axonal population to that invading the leaflet earlier. Also, we confirmed that the ipsilateral SCN is invaded a bit earlier than the contralateral nucleus w4,31x. We showed that the SCN of hamsters innervates its targets starting from the second postnatal day. The adult SCN provides efferent projections to medial preoptic nucleus, the paraventricular nucleus of the thalamus, the parvicellular division of the hypothalamic paraventricular nucleus, the subparaventricular zone, the dorsomedial nucleus of the hypothalamus, the ventral lateral septum, the intergeniculate leaflet, the bed nucleus of the stria terminalis, and the olivary pretectal nucleus w13,17x. Of these terminal fields, we were able to trace projections to most of them, in the new born animals. Exceptions were the projections to the olivary pretectal nucleus, to the contralateral SCN w13x, and to the retrochiasmatic area. However other authors have not found the last two projections w17x. These negative results may reflect an inability for certain tracers to label all types of connections to a given area w17x. Alternatively, it is possible that these connections had not yet developed, since we made no SCN injections in hamsters older than P3. It is remarkable, though, that SCN projections arise from a nucleus that is still growing by the addition of neurons. The present findings suggest that the SCN of newborn hamsters might be in a position to influence the activity of other regions of the brain by way of its neural connections. The precise role of SCN neural connections in setting circadian outputs is a matter of debate. Transplant studies have provided clues about the role of SCN efferent connections in the restoration of overt circadian outputs. Recent studies w29x have shown that the recovery of a circadian rhythm of locomotor activity by the host requires the previous existence of connections from the grafted SCN to diencephalic structures of the host brain. In contrast, previous studies w16x argued that the neural connections from the graft were not essential to restore circadian rhythmicity of behavior in the host animal, since the recovery was observed in the absence of neural connections. In addition, recent evidence from the same group w27x supports the idea that a diffusible signal from the grafted SCN controls circadian locomotor rhythms in the host. The possibility still remains that neural connections are important, or even essential in the intact animal to control the many circadian rhythms. To support the role of SCN efferents in setting the many circadian outputs, it is important to establish the temporal relationship between the onset of circadian outputs and the formation of those axonal connections. At present this information is not available, because the onset of circadian rhythms of behavioral or physiological vari-

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ables during prenatal or early postnatal life has not been established in rodents. 6. Conclusions During the first 3 postnatal days, the last 25% of neuroblasts are added to the SCN. This is followed by an acute decrease of 40% of the maximal number of neuron, to reach the final adult complement of neurons in 1–2 days. This period of neuron death is preceded by a rapid and widespread postnatal development of the SCN efferents. In contrast, retinal afferents invade the SCN after the major decrease in neuron number. Our results support a central role of competition for postsynaptic space as determinant in the process of ontogenetic cell death in the hamster SCN. Acknowledgements We thank Drs. M. A. Carrasco and M. Seron-Ferre ´ ´ for comments and help with the readability of this paper. This research was financed by Fondecyt grant 1940652. References w1x P.G. Bhide, D.O. Frost, Stages of growth of hamster retinofugal axons: implications for developing axonal pathways with multiple targets, J. Neurosci. 11 Ž1991. 485–504. w2x G.I. Botchkina, L.P. Morin, Ontogeny of radial glia, astrocytes and vasoactive intestinal peptide immunoreactive neurons in hamster suprachiasmatic nucleus, Dev. Brain Res. 86 Ž1995. 48–56. w3x J.P. Card, R.Y. Moore, The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience 13 Ž1984. 415–431. w4x L.A. Cavalcante, C.E. Rocha-Miranda, Development of retinohypothalamic and accessory optic projections in the opossum, Brain Res. 144 Ž1978. 378–382. w5x W.J. Crossland, C.J. Uchwat, Neurogenesis in the central visual pathways of the golden hamster, Dev. Brain Res. 5 Ž1982. 99–103. w6x F.C. Davis, R. Boada, J. LeDeaux, Neurogenesis of the hamster suprachiasmatic nucleus, Brain Res. 519 Ž1990. 192–199. w7x F.C. Davis, R.A. Gorski, Development of hamster circadian rhythms: role of the maternal suprachiasmatic nucleus, J. Comp. Physiol. A 162 Ž1988. 601–610. w8x F.C. Davis, J. Mannion, Entrainment of hamster pup circadian rhythms by pre-natal melatonin injections, Am. J. Physiol. 255 Ž1988. R439–R448. w9x D.O. Frost, K.-F. So, G.E. Schneider, Postnatal development of retinal projections in syrian hamsters: a study using autoradiographic and anterograde degeneration techniques, Neuroscience 4 Ž1979. 1649–1677. w10x L. Galli-Resta, M. Ensini, An intrinsic time limit between genesis and death of individual neurons in the developing retinal ganglion cell layer, J. Neurosci. 16 Ž1996. 2318–2324. w11x A.M. Glauert, Fixation, dehydration and embedding of biological specimens, in: A.M. Glauert ŽEd.., Practical Methods in Electron Microscopy, Elsevier, Amsterdam, 1975, 207 pp.

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