THE JOURNAL OF COMPARATIVE NEUROLOGY 291~147-161 (1990)

Maturation of the Corpus Callosum of the Rat: II. Influence of Thyroid Homones on the Number and Maturation of Axons CLAUDE GRAVEL, RACHEL SASSEVILLE, AND RICHARD HAWKES Department of Biochemistry and Laboratory of Neurobiology, Faculty of Medicine, Lava1 University, Quebec, Canada

ABSTRACT Quantitative electron microscopy has been used to study the number of callosal axons in the corpus callosum of normal and hypothyroid rats during postnatal development. At birth, the normal corpus callosum contains 4.4 x lo6 axons. This number increases to 11.4 x lo6by 5 days of age (P5) and then, in contrast to cats and primates, remains constant until at least P60, the oldest age examined. The number of axons in the corpus callosum of hypothyroid animals is not significantly different from the values observed in normal rats a t all ages studied, although the callosal axons of hypothyroid rats remain structurally immature. As extensive elimination of callosal axons has been shown to occur in normal rats past P5, we conclude that new callosal processes grow through the corpus callosum past this age that compensate numerically for the loss. Moreover, as the number of callosally projecting neurons seems to be higher in hypothyroid rats than in normal controls, it seems that the constant axon number derives from more parent neurons, and thus that there are more axon collaterals per callosal neuron in a normal animal than in a hypothyroid one. Taken together, these data indicate that although hypothyroidism does not alter the total number of callosally projecting axons, it interferes with the normal processes that define or sculpt the projection fields, thereby leading to a numerically normal projection with abnormal topography. Key words: hypothyroidism, electron microscopy, axon elimination, synaptogenesis

The elimination of immature exuberant or aberrant projections by means of selective cell death or collateral elimination is a common theme in nervous system development (Cowan e t al., '84). For example, during the development of the rat central nervous system a reduction in the number of' neurons that can be retrogradely labeled has been described for many projections, including commissural afferents between both somatosensory (Wise and Jones, '76; Ivy et al., '79; Ivy and Killackey, '81) and visual (Lund et al., '84; Olavarria and Van Sluyters, '85) cortices, the corticospinal (Stanfield et al., '82; Leong, '83; Bates and Killackey, '84; Stanfield and O'Leary, '85; Schreyer and Jones, '88a,b), and the coeruleospinal projections (Chen and Stanfield, '87), and various efferent projections from the retina (for references, see O'Leary et al., '86). In the accompanying article (Gravel and Hawkes, 'go), it was demonstrated that callosally projecting neurons are more widely distributed in the parietal and occipital cortices of hypothyroid rats than in normal animals. Such a difference is also found between neonates and adult animals (Wise and Jones, '76; Ivy et al., 0 1990 WILEY-LISS, INC.

'79; Ivy and Killackey, '81; Lund et al., '84; Olavarria and Van Sluyters, '85). During normal development, the restriction of callosally projecting domains has been shown to be due to axon collateral elimination (rat parietal cortexO'Leary e t al., '81; Ivy and Killackey, '82; cat visual cortexInnocenti, '81), so it seems plausible that neonatal hypothyroidism results in an incomplete maturation of callosal projections by means of a blockade in normal callosal axon elimination. Quantitative electron microscopy has often been used to demonstrate axon elimination directly. In the cortex, about two-thirds of the axons present in the immature corpus callosum are eliminated during development both in cats (Koppel and Innocenti, '83; Berbel and Innocenti, '88) and rhesus monkeys (LaMantia and Rakic, '84),

Accepted August 9,1989. Address reprint requests to Dr. R.B. Hawkes, Department of Anatomy, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.

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148 B

Po

P25

8

H Im m

Fig. 1. Schematic representations of the corpus callosum of a newborn (PO) and a 25-day-old rat (PP5) as seen in Epon-embedded midsagittal sections. The surrounding tissue have not been included. Rostra1 is

to the left. Polygons A, B, and C indicate the regions selected for electron microscopic analysis.

and likewise the number of axons in the developing corticospinal tract is twice that in the adult (hamsters--Reh and Kalil, '82; rats-Schreyer and Jones, '88). To substantiate the hypothesis that the abnormal callosal projections of hypothyroid rats are the consequence of an incomplete maturation of the projection that includes a blockade of callosal axon elimination, an electron microscopic study was made of the postnatal callosal development in normal and hypothyroid rats. I n this study we ask the following questions: (1)is there a reduction in the number of callosal axons during the postnatal development of the rat and (2) what are the effects of hypothyroidism on the number and maturation of callosal axons during postnatal development?

0.25 and 1 mm', were cut from one of 3 sample regions: the genu (block A), the midportion (block B), or the splenium (block C) (Fig. 1).From these Epon blocks, semithin (I pm) and ultrathin (about 70 nm) sections were cut on a microtome. The semithin sections were mounted on glass slides and stained with methylene blue. Thin sections from the Epon-embedded material were mounted on grids, counterstained with uranyl acetate and lead citrate, and 5 fields were photographed at 1 0 , 2 6 0 ~for each Epon block by using a Phillips 300 electron microscope. The magnification was calibrated regularly by means of a cross grating. The photographic fields were spaced evenly across the entire dorsoventral thickness of the corpus callosum. In the genu and splenium, the region where the callosum is the thickest was sampled, and in the midportion of the callosum, the fields were taken from the most central part of the section. All pictures were printed at the same magnification and all axonal profiles within the field were counted. Profiles were considered as myelinated when at least one turn of myelin was present. The compression factor of the thin sections was corrected by comparing photomontages of the thin sections to photomicrographs of the blocks from which they came: averaged together the mean surface compression was 8.6 % . To estimate the number of axons present in the corpus callosum, a mean axonal density was calculated by pooling the data from the 3 sampling regions. After correction for compression, a number of axons per callosum was obtained by using the callosal cross-sectional area measured from the Epon wafer. No correction for the surface occupied by blood vessels or glia was applied as these were not excluded from the photographic fields.

MATERIALS AND METHODS Experimental hypothyroidism was produced by n-propyl2-thiouracil treatment, as described in the accompanying study (Gravel and Hawkes, '90). Quantitative electron microscopy was carried out on normal and hypothyroid animals aged PO, P5, P10, P25, and P60. At least 3 animals were used in each age group for both normal and hypothyroid litters. Animals were anesthetized with sodium pentobarbital and perfused via the ascending aorta with 50 to 300 ml (depending on body size) of a fixative containing 1%paraformaldehyde, 1.25 % glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed from the skull and immersed overnight in the same fixative a t 4°C. The following day they were transferred to PBS (PB t 0.9% NaC1) and 50-pm-thick sagittal sections of the brains were obtained with a Vibratome. Four to 6 of the most medial sections from each animal were selected, half were mounted on slides and stained with neutral red, and the other half were dehydrated, osmicated in the presence of 3 @*potassium ferrocyanide, infiltrated with Epon, and flat-embedded between Teflon-coated glass coverslips (Romanovicz and Hanker, '77). The most medial Eponembedded section from each animal was selected and photographed, and the cross-sectional area of the corpus callosum measured from the prints by using a graphics tablet. Once the callosal cross-sectional area was determined, blocks containing different regions of the corpus callosum were cut out with a razor blade and mounted on blank Epon stubs by using cyanoacrylate glue. The blocks, measuring between

Fig. 2. Electron micrographs of the genu (A), the midportion (B), and the splenium (C) of the cross-sectioned corpus callosum of a P60 normal rat. Each picture represents about 5% of the surface sampled in each region for axonal counts. A mix of unmyelinated and myelinated axons (respectively labeled u.a. and ma.) is present in all 3 regions. Myelinated axons are usually larger and contain many neurofilaments, microtubules, and some mitochondria, but a population of small myelinated axons also exists (arrowheads in B and C). Most axons are small, unmyelinated, contain mostly microtubules and mitochondria, and are found in increasing densities from region A to C. In P60 animals, glial processes consist mainly of astrocytic profiles (as.) and are pale with irregular shapes (examples indicated in A and C). Scale bar: I pm.

Figure 2

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Fig. 3. Electron micrographs taken in region C (splenium) of normal (A) and an hypothyroid (B) rat at PO. At this age, no differences are seen between the callosa of normal and hypothyroid animals. All axons are unmyelinated and are interspersed with large growth cones and presumptive astroglial profiles. Growth cones (g.c.) have a medium gray cytoplasm, smooth endoplasmic reticulum, and microtubules and vesi-

cles of varying sizes. Immature astroglial profiles (as.) have a light gray cytoplasm and contain mainly round vacuolar profiles. However, the nature of many large profiles is ambiguous (some are indicated by asterisks in A): they have been excluded from the axonal counts. Scale bar: 1 rm.

Statistical analysis was by a 2-way analysis of variance together with a posteriori Duncan tests as appropriate.

sequence is not strictly respected in the figures; to ease comparison of ultrastructural characteristics between normal and hypothyroid developing animals, we have combined electron micrographs of normal and hypothyroid samples in some photomontages. So, in Figures 3,4,5, and 7, A is from a normal and B from a hypothyroid animal.

RESULTS In the following description, we first deal with the results obtained in young adult and developing normal animals and then pass to the corresponding hypothyroid groups. We will close this section with the results on the late corrective effect of thyroxine. However, it has to be noted that this

Normal animals: P60 Electron microscopic observation of cross sections of the corpus callosum of a P60 (Fig. 2) animal reveals the presence

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Fig. 4. Electron micrographs taken in region C of a normal (A) and an hypothyroid (B) rat at P5. No rnyelinated a x n n s are seen. The large majority of axons are small, and surrounded hy immature astroglial

profiles (as.),and growth cones (g.c.). Scale bar: 1 Mm.

of a mixed population of myelinated and unmyelinated axons of widely differing diameters from less than 0.1 to more than 1pm. For example, in region C,the mean diameter averages 0.344 % 0.229 ,urn: N = 363. The myelinated axons (m.a.) are usually larger and contain many microtubules and neurofilaments, together with occasional mitochondria. The diameters are quite heterogeneous and many of them have diameters less than those of the larger unmyelinated fibers. Two such small myelinated axons are indicated by arrowheads in Figure 2B,C. The unmyelinated fibers (u.a.) are more homogeneous in diameter (mostly between 0.1 and 0.3 wm) and their cytoplasm shows many

microtubules, and a few neurofilaments and mitochondria. Intermingled with these fibers are glial profiles, mostly astrocytic processes (as.), identified by their tortuous shape, pale cytoplasm, and the frequent presence of numerous glycogen granules. The mean cross-sectional area of the Epon-embedded callosum at this age is about 2.6 mmz (see Table 2). If we define as a callosal axon, all regularly shaped profiles that contain a t least one or more microtubules, and classify as myelinated axons those that are surrounded by a t least one turn of myelin, then the estimated number of myelinated and unmyelinated axons are 2.3 and 9.7 x lo6,respectively,

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TABLE 1. Densitv of Axons (x lo6)durine Postnatal DeveloDmentof the Cornus Cdosum in Normal and Hwothvroid Flats" ~

'Unmyelinated axodmm2

~~

'Myelinated axons/mm2

Region AC

Region Bc

Region Ce

Region A

Region B

6.396 i 0.119 7.444 i 1.166 5.990 i 0.938 3.236 i 1.037 2.033 i 0.414

4.662 i 1.284 9.301 i 1.277 8.372 i 0.796 5.280 i 0.687 3.346 i 1.056

6.067 i 1.345 9.333 t 2.558 9.537 t 0.605 7.521 t 1.160 6.155 -t 2.049

0 0 0

0

0

0 0

0

0.473 i 0.071 0.648 i 0.129

0.427 i 0.039 0.976 i 0.055

7.238 i 1.319 6.635 f 2.157 3.611 i 1.072 6.805 i 1.895 9.269 i 1.531 6.718 f 1.069

5.681 f 0.198 7.774 i 2.109 5.758 i 0.583 9.082 i 2.606 10.613 i 1.346

6.501 t 1.421 9.210 t 3.409 6.585 t 0.841 11.272 t 1.818 18.163 t 1.955

0

0

-

-

Region C

0 0.225 i 0.012 0.848i 0.215 0 0

0

0

0

0

0

0.030 i 0.003 0.092 i 0.011 0.441 i 0.007

0.040 i 0.011 0.156 i 0.040

0.016 f 0.019 0.040 i 0.0%

~

~

-

~~

valwa are mean i standard deviation Numbers for unmyelineted and 'myelinated axom per mm2have been correded for section compreseion. 'Regions A, B, and C correspond to the genu, midportion,and splenium of the corpus eellasum, mpectively. dNumbersin parenthcsia indicate number of animals. In the hypothyroidgroup. P6oc refers to the p u p that received corrective thyroxinetherapy (seetext).

TABLE 2. Number of Axons ( x lb)during Postnatal Development of the Corpus Cdwum in Normal and Hypothyroid Flats" C d d areab mmz Normal animals PO (3)C P5 (3) PI0 (3) P25 (3) P60 (3) Hypothyroid animals PO (3) P5 (3) P10 (3) P25 (3) P60 (3)

0.784 f 0.102 1.309 i 0.080 1.349 i 0.036 2.096 i 0.170 2.563 i 0.276

4.400 i 0.565 11.364 i 1.963 11.081 i 0.578 11.465 i 0.852 9.691 i 2.061

0.801 t 0.104 1.347 i 0.071 1.866 i 0.184 1.217 i 0.161 1.031 i 0.0'74

5.150 i 0.390 10.632 i 3.540 10.151 i 0.295 10.822 i 1.006 11.796 i 1.387

Myelmated axonS/cdwum

Total axom/ callosum

0 0 0

4.400 i 0.565 11.364 * 1.963 11.081 i 0.578 12.252 i 0.767 11.995 t 1.758

0.787 t 0.088 2.304 -t 0.533 0

0

0 0.031 i 0.012 0.088 f 0.014

5.150 i 0.340 10.632 i 3.540 10.151 t 0.295 10.853 r 1.014 11.884 i 1.373

'All values are mean * standard deviation,Statistid analysis reveals that 1. The total number of axona signi6cantIyincrease with age in both normal and hypothyroid animala(P c 0.01). In both, this is solely due to the PO group. 2. There are no significant differencesin the total number of asom betweennormal and hypothyroid animals with age (p > 0.05). 3. Normal a n i d have significantlymore myelinatad axons than the hypothymidanimalsat both P25 and P60 (p < O.wO1). hCallosalareasare for Epon-embeddedmaterial. Numbersper Callosum have been corrected for section compression and are calculated from the axon densities given in Table 1. CNumbersin parenthesin indicatenumber of animals.

to give a total of about 12 x lo6axons in the corpus callosum of a P60 rat, of which nearly 20% are myelinated (see Table 2). There is much evidence, both physiological and morphological, that the corpus callosum is not homogeneous (Tomasch, '54; Grafstein '63; Valentino and Jones, '82; Vogt and Gorman '82; Koppel and Innocenti '83; Looney and Elberger, '86; Berbel and Innocenti, '88). This is born out by our axon counts from 3 different regions of the corpus callosum (that is, the genu, the midcallosal area, and the splenium, labelled regions A, B, and C, respectively). Whereas the density of myelinated axons is the same in regions A, B, and C (around 0.9 x 106/mm2),the density of unmyelinated axons increases from rostral to caudal with 2 x 106/mm2in A compared to more than 6 x 106/mm2in C (Table 1).Part of the difference is due to the larger axon diameters in the more rostral regions (compare Fig. 2A,C).

Normal animals: postnatal development In the rat, callosal axons cross the midline in two waves, one starting at about gestational day 18.5 (E18.5), the other at about PO (Valentino and Jones, '82; Floeter and Jones, '85). In accordance with these results, we find many immature axons in the corpus callosum of a PO animal (Fig. 3A). In cross section, these appear as circular profiles, usually smaller than 0.2 pm in diameter and containing one or a few microtubules. They tend to be arranged in clusters inter-

spersed by large growth cones and numerous large astroglial profiles. Growth cones (g.c.) are identified by their cytoplasm, which displays about the same electron density as the small axons and contains microtubules, smooth endoplasmic reticulum, and numerous pleomorphic vesicles. Large astroglial processes (as.) are usually paler and may contain compressed elements of smooth endoplasmic reticulum and large clear vesicles. (Many large profiles with an electron density that would classify them as growth cones have also the paucity of organelles characteristic of astrocytic profiles (examples are marked by an asterisk in Fig. 3A). Darker astrocytic processes have been described in the corpus callosum of neonatal rats (Valentino and Jones, '82) and interpreted as either growth cones or vesiculated axons (Berbel and Innocenti, '88). They have been excluded from the axon counts reported here, but as such profiles are quite rare, to include them does not change significantly the values for axonal density or any of our conclusions (see Discussion). No myelinated axon profiles were observed in PO animals. The axon density is 3 times higher in region A (6.4 x lo6,Table I) than in the same region of a P60 animal, but the difference is smaller in region B, and the same density (around 6 x lo6) is encountered in region C. The crosssectional area of the Epon-embedded callosum is only around 0.8 mm2a t PO. This gives the total number of axons in a newborn corpus callosum as 4.4 x lo6,only about a third

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Fig. 5. Electron micrographs taken in region C of a normal (A) and an hypothyroid (B) rat at PIO. No myelinated axons are present in either group. Note in the hypothyroid animal that many large (over 1pm in diameter) immature astroglial processes are still present (some of them are labelled). In the normal animal, immature astroglial profiles are smaller (the largest one of the field is labelled), but some unmyelinated axons (ma.) have started to increase in diameter. Scale bar: 1p m .

of the value found at P60 (Table 2). Even when only the unmyelinated axons are compared, the number at PO is less than half that at P60 (Table 2). Thus the data indicate that nearly two-thirds of the callosal axons present in a young adult rat crossed the midline postnatally. The ultrastructure of the corpus callosum at P5 (Fig. 4A) is similar to that described for the PO animal. Again, numerous small unmyelinated axons containing a few microtubules are packed between large astroglial processes and growth cones. The mean cross-sectional area of the callosum has increased to 1.3 mm2 (Table 2) and the axon density has

also increased, most notably in region B where it attains an average of 9.3 x 106axons/mm2 (Table 1). This results in a total number of axons per callosum of 11.4 x lo6,a value not significantly different from that obtained in P60 animals (Table 2). Quantitative analysis of PI0 and P25 normal callosa reveals that the total number of callosal axons remains constant at between 11 and 1 2 x lo6(Table 2). At PlO, the mean cross-sectional area of the corpus callosum is very close to that at P5, and 'although the rostral-to-caudal gradient in axon density has started to emerge, the total number of

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Fig. 6. Electron micrographs taken in region C of a normal animal at PI0 to illustrate the maturation of glial elements. In A, a partial view of a mature astroqte (As.). Note the pale appearance of the cytoplasm and the presence of glial filaments in the main process. In B, immature oli-

godendrocytic profiles (01.) showing dark cytoplasm and numerous ribosome rosettes seen here are intermingled with an astrocytic process (As.). Note that some large vesiculated immature astroglial elements can still be found at this age (one is labelled as in B).Scale bars: 1 pm.

axons is not significantly different with a mean 11 x lo6 axons per callosum. Ultrastructurally (Fig. 5),there are still no myelinated profiles, and most unmyelinated axons are less than 0.2 pm in diameter, although a small proportion has started to increase in diameter (examples are labelled in Fig. 5A). The immature astroglial fibers (as.) are generally smaller than in younger animals (compare Fig. 5A to Fig. 3A), and more mature astrocytic elements (labelled As to differentiate them from immature profiles, labelled as.) are present that contain glial filaments and glycogen granules

(Fig. 6A). Immature oligodendrocytic profiles are now seen a t this age that can easily be identified by their dark cytoplasm and the presence of ribosome rosettes (01. in Fig. 6B). At P25, the mean callosal cross-sectional area has increased to 2 mm'. Myelinated axons (m.a.) are now present (Fig. 7A), although they still represent only about 6% of the total population of axons (Table 2). Most myelinated axons are relatively large compared to the unmyelinated ones, but as noted for P60 animals, there is also a population

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Fig. 7. Electron micrographs taken in region C of a normal (A) and a hypothyroid (B) rat at PZ5. At this age, about 3% of the s o n s are myelinated (m.a.) in region C of normal animals (about 6 %when regions A, B, and C are pooled), but this proportion falls to 0.15% (0.3% of total) in hypothyroids. An immature oligodendrocyte profile (01.) is labelled in B, as well as some immature and mature astrocytic profiles (respectively as and As). Scale bar: 1 wm.

of small (around 0.2 fim in diameter) myelinated profiles. The distribution of myelinated profiles is heterogeneous in the P25 corpus callosum with the highest density found in region A and the lowest in region C (Table 1).This is consistent with numerous studies showing a general caudorostral maturation gradient in both the corpus callosum and the cerebral hemispheres (Berry and Rogers, '65; Hicks and D'Amato, '68; densen and Altman, '82; Silver et al., '82; Valentino and Jones, '82; Looney and Elberger, '86; Berbel and Innocenti, '88). The density of unmyelinated axons has dropped a little compared to the density found a t PlO, but

due to the concomitant increase in callosal cross-sectional area the total number of unmyelinated axons remains not significantly changed at 11.5 x lo6 (Table 2). Adding the total number of myelinated axons brings the total number of axons to a little over 1 2 x lo6, the same as was obtained for P60 animals (Table 2). To recapitulate, at birth the corpus callosum of normal rats contains 4.4 x lo6 small unmyelinated axons. Between PO and P5 there is a substantial gain in the cross-sectional area of the corpus callosum, a gain that is paralleled by an increase in the total number of axons to about 11.3 x lo6.

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The cross-sectional area of the corpus callosum continues to increase a t least until P60, a t which time it is almost twice as large as at P5. Despite the increase in cross-sectional area, the total number of callosal axons remains stable between 11 and 12.2 x lo6 from P5 to P60. Myelination starts between P10 and P25 and is slower and/or starts later in the splenium (region C) than in the genu or midcallosum (regions A and B, respectively).

Hypothyroid animals In the accompanying study (Gravel and Hawkes, '90) it was shown that experimental congenital hypothyroidism results in an abnormally wide tangential distribution of callosally projecting cells, accompanied by the reduced myelination of callosal axons. Added to the previously demonstrated blockade in the expression of the NF-H neurofilament antigen in the corpus callosum (Plioplys et al., '86), these results suggest a protracted immaturity in the callosal afrerents (Gravel and Hawkes, '90). To check if this immaturity is reflected ultrastructurally and can modify the number of callosal axons found during development, we have applied the same quantitative electron microscopic methods used for normal animals to a series of hypothyroid animals of corresponding postnatal ages. Both qualitative and quantitative electron microscopic analyses of sagittal sections taken from the corpus callosum of PO and P5 hypothyroid animals failed to detect any significant differences from their normal counterparts. At PO, the hypothyroid corpus callosum has a mean cross-sectional area of 0.8 mm2 and contains about 5 x lo6 axons (Table 2). As in normal animals, these axons are small and unmyelinated, contain few microtubules, and are surrounded by large immature glial profiles and growth cones (Pig. 3B). At P5 the mean cross-sectional area has increased to 1.3 mm2 and the total number of axons has reached 10.6 x lo6,values similar to those found in normal controls a t P5 (Table 2). Again, the typical electron microscopic image shows small, unmyelinated axons surrounded by growth cones and large, immature astroglial elements (Figure 4B). By P10 the cross-sectional area of the hypothyroid corpus callosum has reached nearly 1.9 mm2, 38% more than the normal PI0 corpus callosum (Table 2). However, this difference is not due to a higher number of axons in the hypothyroid corpus callosum: there are about 10.2 x lo6 axons in the entire callosum compared to about 11.1x 106in a normal P10 (Table 2). As in normal animals, there is a rostrocaudal gradient in axon density in the P10 hypothyroid corpus callosum with region A containing about 3.6 x lo6 axons/mm2 and region C 6.6 x 106/mm2and all axons are unmyelinated (Fig. 5B). The difference in callosal areas may tentatively be explained by the observation that the large, pale vesiculated profiles identified previously as astroglial processes appear less common and smaller in the normal P10 callosum than in the hypothyroid one (compare Fig. 5A,B), and that a t least in the cerebellum, astrocytes have been shown to be more numerous in hypothyroid animals (Nicholson and Altman, '72; Clos and Legrand, '73). Although no quantitative study was made, it appears to us that in normal animals the size and number of the large astroglial profiles decreases between P5 and P10. The larger size of those profiles in the P10 hypothyroid corpus callosum might be another sign of the blockade in maturation provoked by a shortage in thyroid hormones. This hypothesis is consistent with the observation that mature astrocytic profiles containing glial filaments and glycogen granules ap-

pear less common in hypothyroid animals, and immature oligodendrocytes, common at P10 in normal animals, are almost absent,. Finally, it is interesting to note that undifferenciated glial elements are still visible at P50 in the subependymal germinal zone near the lateral ventricles of hypothyroid animals, but are gone by P20 in normal animals (Bass and Young, '73). In contrast to the data obtained in normal animals, the cross-sectional area of the corpus callosum is smaller at P25 than at PlO in hypothyroid animals, averaging only 1.2 mm2 (Table 2). This represents a 35% decrease from the value obtained a t P10, and only 58% of the value obtained for normal P25 animals. But again, the change in cross-sectional surface area is not due to a change in the number of axons: the axon density is higher in all three regions of the corpus callosum at P25 resulting in a total of about 10.9 x lo6axons in the entire callosum, a value not significantly different from that at PlO. In the hypothyroid corpus callosum at P25, the ultrastructural appearance is dominated by the presence of densely packed, small (0.2 ym or less) unmyelinated s o n s containing almost exclusively microtubules and a few mitochondria, interspersed by glial profiles (Fig. 7B). Larger, myelinated axons are very rare and account for only 0.3% of the total population of axons: this is less than 5% of the number found in normal P25 animals. Most of these rare myelinated axons are found in regions A and B, confirming that myelination begins later or proceeds more slowly in posterior regions of the corpus callosum in hypothyroid a n mals. Some astrocytic processes contain glycogen granules and so have apparently matured (As.), but most astroglial profiles appear immature (as.), although of a smaller diameter than found a t P10 (compare Figs. 7B and 5B). This difference in diameter may at least in part explain the reduction in cross-sectional area observed between P10 and P25. The corpus callosum of P60 hypothyroid rats shows a further decrease in cross-sectional area when compared to the callosum of hypothyroid animals at P25 (Table 2). The mean cross-sectional area has passed from 1.2 to about 0.9 mm', compared to nearly 2.6 mm2 in normal P60 animals. However, the axon density is about 3 times higher in all 3 regions of' the hypothyroid corpus callosum compared to their normal counterparts, resulting in a total average number of callosal axons of 11.9 x lo6, not significantly different from the normal P60 animals. The rostrocaudal gradient in density of unmyelinated axons is still present, with region A displaying around 9.3 x lo6 axons/mm2 and region C over 18 x 106(Table 1). Myelination has progressed somewhat between P25 and P60 in hypothyroid animals, but at P60 (Fig. 8) both the density and total number of myelinated axons remains well under normal value with only 0.7% of axons possessing one turn or more of myelin compared to near 20% in normal controls (a P60 control is shown in Fig. 2). These rare myelinated axons are usually bigger than the unmyelinated axons and contain the usual microtubules, neurofilaments, and mitochondria. Again, the posterior region has the lowest density in myelinated axons of the 3 examined. Unmyelinated axons show no increase in diameter and their appearance is identical to that found for the vast majority of axons from PO onward (mean diameter in region C averages 0.191 0.128 ym: N = 589).

Late corrective effects of thyroxine The blockade in the expression of the mabN210 epitope observed in the basket cell axons of the cerebellum and the

MATURATION OF THE CORPUS CALLOSUM

Fig. 8. Electron micrographs taken in region A (A),region B (B), and region C (C) of a P60 hypothyroid rat. Note the low density of my-

157

elinated profiles (some labelled m.a.) compared to a normal P60 animal (shown in Fig. 2). Scale bar: 1 pm.

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axons of the corpus callosum of animals kept on a PTU diet from intrauterine life can be reversed by daily thyroxine administration started a t birth (Plioplys et al., '86; Gravel et al., '87b). However, thyroxine therapy started later is less effective in correcting abnormalities found in the cerebellum of congenital hypothyroids, and past the first postnatal month, results in little if any correction (Legrand, '83; Marc e t al.. '86; Gravel and Hawkes, '87b). In the accompanying work (Gravel and Hawkes, 'go), we have shown that in contrast to what was observed for the cerebellum, a thyroxine therapy started a t P30 on congenital hypothyroid animals effectively restores mabN21O immunoreactivity in callosal axons to apparently normal levels. In the present study, we have checked whether this late corrective effect also applies to the ultrastructural characteristics of the hypothyroid corpus callosum. Thin sections were collected from Epon blocks of tissue taken from the genu of the corpus callosum of P60 hypothyroid animals having received 1pg T4/100 g body weight/day from P30 to P60 but otherwise kept on the propylthiouracil diet Quantitative results are given in Table 1 . In the genu of a normal P60 animal, the density of unmyelinated axons averages 2 x lo6 axons/mm2 and the density of myelinated axons is over 0.8 x 106/mm2(i.e. 42% myelinated). In contrast. unmyelinated axons reach a density of more than 9 x 10' profiles/mm2 in the genu of the hypothyroid corpus callosum at P60, but the density of myelinated axons is only 0.09 x 106/mm2(1% myelinated). A daily thyroxine treatment given from P30 to hypothyroid animals induces significant changes in the distribution with the density of unmyelinated axons falling to 6.7 x 10' profiles/mm2, and the density of myelinated axons increasing to 0.4 x 106/mm2 (P60c: 7 % myelinated). Thus the corpus callosum of hypothyroid animals is still responsive to corrective thyroxine therapy after 1 postnatal month; however, the correction of the myelination deficit is only partial and it remains to be tested whether a longer thyroxine treatment or a higher daily dosage would be more effective.

DISCUSSION Methodological considerations We have used quantitative electron microscopy to estimate the numbers of axons in the corpus callosum of normal and hypothyroid rats during postnatal development. The methodology relies on sampling the axonal density from 3 different regions of the corpus callosum, and subsequently extrapolating the total axon number per callosum by using area measurements of the total cross-sectioned callosal surface. The 3 regions chosen for density sampling were not randomly selected, and each sample were given equal weight in the calculations. Our results show that the axonal density can vary between these regions. As there is no a priori reason to believe that these 3 regions are each representative of a third of the corpus callosum, this may have introduced a bias that could lead to either an over- or underestimate of the total number of callosal axons. Nonetheless, we decided to adopt this protocol for 2 reasons. First, we were interested to see if hypothyroidism equally affected callosal fibers from different cortical regions. As it has been shown that axons from restricted regions of the cortex pass through restricted regions of the corpus callosum in both neonatal and adult rats (Olavarria and Van Sluyters, '861, this method permits us to follow the development of callosal axons from 3 different rostrocaudal locations in the cortex. Second, the first

report of an absolute decline in the number of callosal axons during development (Koppel and Innocenti, '83) used a similar sampling technique, and so comparisons with our data are more straightforward. Our criteria for identifying the various constituents of the developing corpus callosum of the rat under the electron microscope have been based on the works of Seggie and Berry ('72) and Valentino and Jones ('82). This has led us to consider most of the large vesiculated profiles that are especially prominent between PO and P10 as immature astroglial processes, on the basis of their electron lucent cytoplasm, their lack of microtubules, and the presence of large clear vesicles and of cisternae of smooth endoplasmic reticulum (by contrast mature astrocytic processes display few vesicular bodies, some glycogen granules, and glial filaments). However, in the developing cat corpus callosum many profiles that we would classify as immature astroglial processes using the above criteria were described as vesiculated axons, on the basis that some very similar profiles were seen containing microtubules when observed longitudinally or sagittally (Berbel and Innocenti, '88). So it remains possible that a fraction of the profiles identified in the present study as astroglial processes are in fact axons or growth cones. We have nonetheless retained the presence of microtubules as a condition sine qua non for classifying a profile in the axon category; the presumptive astroglial processes are numerous at PO and almost gone by P10, but we estimate that at P5, when the maximal number of axons is attained, they represent less than 10% of the total number of profiles observed and thus that their inclusion in axonal counts would not bring significant changes in any of our estimates. In the developing corpus callosum of the cat, the proportion of vesiculated axons reach a maximum of 8.6% (Berbel and Innocenti, '88).

The number of callosal axons during postnatal development The value obtained for the total number of callosal axons in a P60 rat is around 12 x lo6.This number may not be the final adult value as the corpus callosum is still not fully mature: for example, myelination continues up to 8 months postnatally in the mouse (Sturrock, '80) and up to 10 months in the rat (Seggie and Berry, '72). In the genu of our normal P60 animals, myelinated axons represent 29% of the population, compared to 53.6% in the genu of adult rats (Seggie and Berry, '72). Moreover, quantitative analyses have shown a decline in the number of callosal axons extending up to P150 in the cat (Berbel and Innocenti, '88) and up to the 6th month in the rhesus monkey (LaMantia and Rakic, '84). Nonetheless, we believe that this value represents a better estimate of the total number of callosal axons in the rat than the previous figures of 1.1and 2 x lo6 axons obtained by light microscopic techniques (Doty and Negrao, '73). The most surprising result of this study was that the number of axons in the corpus callosum of the rat increases from PO to P5 and then remains virtually the same at least until P60. Numerous investigations have reported a decrease in the number of callosal neurons that can be retrogradely labelled from the opposite hemisphere in the parietal and occipital cortices of the rat a t least through the second postnatal week (Ivy and Killackey, '81, '82; O'Leary et al., '81; Olavarria and Van Sluyters, '85). In the rat parietal cortex, double labelling studies have shown that the main mechanism involves elimination of callosal processes

MATURATION OF THE CORPIJS CALLOSUM

(O'Leary e t al., '81; Ivy and Killackey, '82), a mechanism previously identified in the visual cortex of the cat (Innocenti, '81). Should the elimination of exuberant callosal collaterals being restricted to regions destined to become "weakly callosal" in the adult (roughly midareas 17 and 18a in the occipital cortex and the barrelfield area in the parietal cortex), the disappearance of callosal axons would easily remain undetected in this quantitative study as these regions represent only a small fraction of the total cortical surface. But it appears that the reduction in the number of callosally projecting neurons during development also affects regions that retain extensive callosal projections in the adult animal (Ivy and Killackey, '82; Koppel and Innocenti, '83; Innocenti, '86). Moreover, in the cat the elimination of exuberant callosal collaterals during development is not restricted to parts of homotopic callosal connections: some grossly heterotopic juvenile callosal projections are also eliminated (Innocenti and Clarke, '84a,b), and there is a suggestion that the same phenomenon may exist in the rat (Gravel and Hawkes, '90; see below). Thus it appears that the reduction in the number of callosally projecting neurons is widespread and should involve extensive elimination of callosal axons. T o our knowledge, quantitative electron microscopic studies of the number of callosal axons present during development of the corpus callosum have been reported previously for only 2 species. In the cat, the number of axons increases during prenatal development, reaches a peak of about 79 x lo6axons at birth and decreases subsequently to 23 x 106 in adulthood (Koppel and Innocenti, '83; Berbel and Innocenti, '88). Similarly, in the rhesus monkey, a similar profile of prenatal increase and postnatal decrease in the number of callosal axons was identified that also results in a 70% loss of axons compared to the maximal number attained (LaMantia and Rakic, '84). We do not know why the development of the rat corpus callosum should be so different. One possibility is that the mature complement of callosal axons is attained by competing processes of axon outgrowth and elimination. In some species (such as the cat), these phases may occur sequentially, to yield extensive numerical elimination, whereas in others (such as the rat), they are concomitant and gross axon number remains constant. This view treats the developmental profile of callosal axon numbers as a secondary consequence of opposing processes: alternatively, the fact that total axon number is tightly controlled from P5 onwards, despite radically different developmental timetables in normal and hypothyroid cortices, may reflect an alternative level of regulation. For example, one straightforward interpretation is that 12 x lo6 axons is the maximum number of callosal inputs that the cortical target can sustain. Because much axon elimination in the cat occurs during the first few postnatal months (Berbel and Innocenti, '88), a period during which much of the exuberant callosal projections are being eliminated (Innocenti and Caminiti, '80; Innocenti and Clarke, '84a,b), a causal link was postulated between the postnatal restriction of callosally projecting domains and the postnatal decrease in the number of callosal axons. This relationship is less evident in the primate, as the axon elimination appears to occur only after the restriction of callosal efferents (LaMantia and Rakic, '84). Although we do not. question that the postnatal sharpening of callosal connections involves selective axon elimination, the results obtained in the present study show that this sharpening need not be reflected in an absolute decrease in the number

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of callosal axons. From PO to P4-P5 in the rat, the somata of callosally projecting cells of both visual and somatosensory cortices are distributed in 2 continuous tangential laminae occupying layers Va and Vc-VIa, and there is no evidence yet of a restriction in the number of callosally projecting cells (Ivy and Killackey, '81; Olavarria and Van Sluyters, '85). During this same period the number of callosal axons more than doubles, passing from 4.4 to 11.4 x lo6.It is possible that a higher value is reached at some point between PO and P5 and then that some axon elimination escaped detection: in any case, this would not be involved in the restriction of callosally projecting domains. The topographical restriction of the callosal projections starts at about P5 and continues until the adult pattern is attained by about P15 (somatosensory cortex-Ivy and Killackey, '81; visual cortex-Olavarria and Van Sluyters, '85). In the somatosensory cortex the restriction of the projection is reflected in a decrease in the number of cells in layers Va and Vc-VIa of parietal cortex (notably the barrelfield area) that can be retrogradely labelled from the opposite hemisphere, together with an increase in the number of callosally projecting cells in layer I11 of regions destined to remain callosally connected in the adult (Ivy and Killackey, '81). In the visual cortex, the decrease in the number of callosal cells affects particularly layer Va of midareas 17 and 18a in the occipital cortex, and an increased number of cells can be retrogradely labelled from the opposite hemisphere in layers 11-111of area 18b, lateral area 17-medial area 18a and lateral area 18a (Olavarria and Van Sluyters, '85). Thus in the rat the sharpening of callosal connections involves a combination of selective decreases and increases in the number of callosally projecting cells. This may be different in the cat where, with minor exceptions (Innocenti and Clarke, '84b), the radial distribution of callosal cells is similar in neonates and adults (Innocenti, '86), and the sharpening of callosally projecting domains seems restricted to a decrease in the number of cells that can be retrogradely labelled from the contralateral hemisphere in some specific cortical locations (Innocenti et al., '77; Innocenti and Caminiti, '80; Innocenti and Clarke, '84a,b).

Developmental mechanisms leading to the restriction of callosally projecting domains in the rat Although a higher number of callosal neurons are present in hypothyroid animals at P25 and Y35, the callosal connections of hypothyroid rats share many similarities with those of normal animals. For example, the radial distribution of callosally projecting cells is normal, as are the radial and tangential distributions of callosal terminals. However, the hypothyroid corpus callosum displays many features of an immature axon tract: almost all axons are small and unmyelinated, glial maturation is delayed, and there is an almost complete ahsence of an epitope of the late-appearing high molecular weight subunit of neurofilaments (NF-H: Plioplys et al., '86; Gravel and Hawkes, '90). Electrophysiologically, the transcallosal response is also immature, displaying long latency, long wave duration, high threshold, and small amplitude (Hatotani and Timiras, '67). Coupled with the fact that callosal neurons are more widely distributed in the parietal and occipital cortices of both hypothyroid and immature (up to P5) normal animals, these data suggest that the elimination of callosal axons that occurs from P5 in normal animals is blocked in hypothyroid animals. As a result, the callosal connections of hypothyroid rats would

160 remain immature, being neither able to retract axons issuing from inappropriate cortical locations nor to stabilize preferentially those coming from appropriate cortical zones. The blockade in the elimination of axons from inappropriate cortical territories would explain the wider tangential distribution of callosally projecting neurons in the hypothyroid cortex, the higher number of those cells, as well as the suspected supernumerary auditory-to-visual callosal projection. An abnormal developmental progression in hypothyroid animals might also explain the small size and incomplete cytoskeleton of callosal axons, and the paucity of myelinated profiles. On this point, i t is interesting that both NF-H and myelinated axons appear after the period of axon elimination in the optic nerve of the rat (Pachter and Liem, '84; Crespo et al., '85) and rabbit (Willard and Simon, '83; Robinson et al., '87). In the corpus callosum of the cat, NF-H levels and the number of myelinated axons rise slowly from the end of the third postnatal week, when axon elimination is well underway (Looney and Elberger, '86; Figlewicz et al., '88). The failure to stabilize callosal axons normally in hypothyroid rats would also explain the immature electrophysiological characteristics of their callosal connections and contribute to the deficit in the number of synapses found in the hypothyroid cerebral cortex (Cragg, '70) and in the number of dendritic spines observed on pyramidal cells of visual and auditory cortices (Ruiz-Marcos e t al., '79, '83, '85). In normal development, the number of callosally projecting somata is reduced substantially during the second postnatal week (O'Leary et al., '81; Ivy and Killackey, '82). Likewise, in the accompanying study (Gravel and Hawkes, '90, this issue), it was demonstrated that there are more callosally projecting cells in the parietal and occipital cortices of P25 or P35 congenital hypothyroid rats than in normal controls. These supernumerary cells were particularly evident in midareas 17 and 18a of the visual cortex and the barrelfield area of the somatosensory cortex, but there was a suggestion that more callosal cells are present throughout the occipital and parietal cortices. Moreover, the data suggested that an auditory-to-visual callosal projection is present in those animals, but is absent from normal animals of this age. This widespread elimination of immature projections is not reflected in the total axon numbers, which remain constant. Therefore, it must be assumed that neurons destined to retain their callosal projections in normal adults emit new callosally projecting collaterals after P5, in numbers sufficient to match the loss due to elimination. The most interesting quantitative result from hypothyroid animals is that these processes have become uncoupled: the callosal axon count is normal, whereas the number of callosally projecting neurons is abnormally high. Thyroid hormones are not necessary to regulate total axon number, but rather seem important to the selection process through which inappropriate projections are eliminated. Both in normal and hypothyroid rats each hemisphere support between 5.5 and 6 x lo6callosal inputs, independent of the number of cells from which these processes originate. In the presence of normal levels of thyroid hormones, the postnatal sculpting of the callosal projection results in the highly selective elimination of some collaterals, to create the mature acallosal domains, together with the sprouting of new collaterals to maintain a constant density of innervation. In hypothyroidism, it seems that whereas the axon recycling continues, the topographical information has been suppressed, so that the density of

C. GRAVEL ET AL. innervation remains normal but the source of that innervation is less well defined, more callosal cells each send on average fewer collaterals, and topographical specificity is lost. This is reminiscent of another projection in which topographical specificity is achieved through selective elimination-the rat retinocollicular projection (O'Leary e t al., '86). In that case, elimination occurs through retinal ganglion cell death, but there is an interesting parallel. If electrical activity in the projection is eliminated by the application of tetrodotoxin, axon elimination is quantitatively normal but positional information is lost. The same may be the case in the hypothyroid corpus callosum: the lack of thyroid hormones may interfere with the signals-electrophysiological or molecular-that normally provide detailed positional information, so that whereas the size of the projection is normal, the topography is not.

ACKNOWLEDGlMENTS We thank Jamel Rafrafi for technical aid and Suzanne Bilodeau for secretarial assistance. This work was supported by grants from MRC (Canada), the FRSQ, and the FCAR (R.B.H.) and by a studentship from the FRSQ (C.G.).

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Maturation of the corpus callosum of the rat: II. Influence of thyroid hormones on the number and maturation of axons.

Quantitative electron microscopy has been used to study the number of callosal axons in the corpus callosum of normal and hypothyroid rats during post...
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