THE JOURNAL OF COMPARATIVE NEUROLOGY 302:715-728 (1990)

Ontological Study of Calbindin-D,,,-Like and Pawalbumin-likeImmunoreactivities inRat SpinalCordandDorsalRmtGanglia JIAN-HUA ZHANG, YASLJHIR.0MORITA, TAKASHI HIRONAKA, P.C. EMSON, AND MASAYA TOHYAMA Department of Anatomy 11, Osaka University Medical School, Kita-ku, Osaka 530 (J.-H.Z., M.T.), Department of Anatomy, Kagoshima University Medical School, Kagoshima 890 (Y.M.),Iatron Laboratories Inc., Funabashi, Chiba 274 (T.H.),Japan, and MRC Group, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, United Kingdom (P.C.E.)

ABSTRACT The calcium ion plays an important role in some critical developmental events in the nervous system, such as neurulation and neurite elongation. Therefore, as the intracellular calcium-binding proteins calbindin-D,,, (CaB) and parvalbumin (PV) may be expressed in these developmental events. Accordingly, the ontological expression of CaB and PV was examined immunocytochemically in the spinal cord and dorsal root ganglia (DRG) of the rat, in order to evaluate the relationship between CaB and PV expression, and other important developmental events. During the ontogenesis of the spinal cord, the CaB-like immunoreactivity was mainly observed in the cell somata. The immunoreactive cells in the ventral horn of the cervical and thoracic, lumbar, and sacral segments first appeared at embryonic day (El-12, E-13, and E-14, respectively. However, these cells were not detected in the intermediate gray matter of the same segments at E-14, E-15, and E-16, respectively, and in the dorsal horn at E-14-E-15, E-16, and E-17, respectively. The peak of immunoreactive cells, both as to number and intensity, occurred in the perinatal period. However, from postnatal day (PI-14 on, the number and intensity of the positive cells decreased, the adult levels being reached at P-35. The PV-like immunoreactivity was mainly detected in the fibers and punctata during the ontogenesis of the spinal cord. The immunoreactive fibers first appeared on the surface of the dorsal horn in the cervical and thoracic segments at E-14, then entered the dorsal horn at E-15, and reached the intermediate gray matter and ventral horn at E-16. The first appearance of these fibers in the same areas of the lumbar and sacral segments occurred 1 day later than in the cervical and thoracic segments. During the perinatal period, the maximum content of PV-like immunoreactive fibers, together with many punctata, was seen in the gray matter. However, between P-14 and P-17, most of them lost immunoreactivity rapidly, with the exception of the medial region of the intermediate gray matter, where the PV-immunoreactive punctata remained up to the adult stage. In DRG neurons, both CaB and PV was expressed, but in different neurons. Neurons labeled with anti-CaB and anti-PV sera were first detected a t E-16 and E-14, respectively. These neurons were large or medium-sized in the prenatal period. Small neurons were also labeled with these sera after birth. Positive neurons of different sizes in DRG increased in number in the postnatal period. Up to P-35, the expression of CaB and PV in DRG reached the adult levels. The appearance of and developmental changes in CaB-like and PV-like immunoreactivities in the spinal cord and DRG may be maintained during the neurogenesis of the immunoreactive regions, suggesting the possible developmental roles of CaB and PV. Key words: calcium-bindingprotein, differential expression, development

Accepted August 31, 1990

o 1990 WILEY-LISS, INC.

ZHANG ET AL.

716 Many investigations have indicated that Cat+is involved in many biological functions, such as cell division, transmission of nerve signals, release of neuroactive substances, muscle contraction, etc. The intracellular Ca++receptors involve a number of structurally related calcium-binding proteins that can transduce the calcium signals into a variety of physiologicalresponses (Kretsinger, '80; Rasmussen, '80; Campbell, '83; England, '86; Heizmann and Berchtold, '87). For example, the calcium-binding proteins calbindin-D,,, (CaB), a vitamin-D-dependent calciumbinding protein, and parvalbumin (PV), a water-soluble calcium-binding protein, were first isolated from the small intestine of chicks (Wasserman and Taylor, '661, and skeletal muscle of frogs (Deuticke, '341, respectively. More recently, these proteins were also found in many other tissues, including the nervous systems of several species (Celio and Heizmann, '81; Jande et al., '81; Gerday, '82; Berchtold et al., '84; Garcia-Segura et al., '84; Heizmann, '84; Endo et al., '86; Parmentier et al., '87; Philippe and Droz, '88; Sequier et al., '88; Seto-Ohshima et al., '89). In the nervous system of adult animals, CaB and PV are generally expressed in different anatomical regions (Celioet al., '86; Jones and Hendry, '89; Sloviter, '89; Stichel et al., '87). Even in the regions where both CaB and PV are abundant, they are sometimes expressed in different neuronal subpopulations (Gerfen et al., '85; Jones and Hendry, '89). For example, in the cerebellum, Purkinje cells contain both calcium-binding proteins, but basket cells and stellate cells are only labeled with the anti-PV antiserum, i.e., not with the anti-CaB antiserum (Schneeberger et al., '85). Although the cellular localization of CaB and PV has been examined in nervous tissues, little is known about their functions (Heizmann, '84; Heizmann and Celio, '87; Rogers, '89a). Since these proteins bind Cat a role in Cat+ translocation and/or the Ca' buffering system or neuronal transmission in the adult stage is probable (Heizmann, '84; England, '86; McBurney and Neering, '87; Pfyffer et al., '87). On the other hand, many physiological investigations have shown that Ca++may be a key factor in some critical developmental events in the nervous system, such as neural fold elevation (Moore and Stanisstreet, '861, neurulation (Smedley and Stanisstreet, '85), and neurite elongation (Anglister et al., '82; Kater et al., '88; Mattson et a]., '88a, b). All these findings suggest that CaB and PV may be involved in some developmental events during the ontogen+,

+

Abbreviations CaB CaB-LI cc df DH dr

DREZ DRG E eP

IG IML IMM If

LSN P PV PV-LI vc vf

VH

calbindin-D,,, calbindin-D,,,-like irnmunoreactive central canal dorsal funiculus dorsal horn dorsal root dorsal root entrance zone dorsal root ganglion embryonic day ependyrna intermediate gray matter intermediolateral nucleus intermediornedial nucleus lateral funiculus lateral spinal nucleus postnatal day parvalburnin parvalbumin-like immunoreactive ventral cornrnissure ventral funiculus ventral horn

esis of nervous tissues. To examine this possibility, the expression of both calcium-binding proteins during ontogenesis should be investigated. Since the ontogenesis of the spinal cord and dorsal root ganglia (DRG) has been thoroughly studied (Lawson et al., '74; Raju et al., '81; Smith, '83; Smith and Hollyday, '83; Altman and Bayer, '84; Harris and McCaig, '84; Hulsebosch et al., '86; Davis et al., '891, it is easy to correlate the expression of CaB and PV with the ontogenic events in nervous tissues using spinal cord as a model. Accordingly, we investigated the developmental changes in the CaB-like and PV-like immunoreactivities in the spinal cord and DRG of the rat.

MAlI3FUALSANDlMETHoDS Animals and tissue preparation Wistar rats of various prenatal and postnatal ages were used in this study, as summarized in Table 1. The day of sperm positivity, determined by checking vaginal smears, was designated as embryonic day 1 (E-1) and the date of birth as postnatal day 1(P-1). Litters of fetuses were dissected out from the uteri of two or three pregnant rats at each prenatal stage after E-10 under sodium pentobarbital (50 mgiml) anesthesia (1.0 ml/kg body weight, i.p.). The crown-rump length (CRL)was determined in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Fetuses younger than E-14 were immersed in 4% paraformaldehyde in 0.1 M PBS or Zamboni's fixative (Zamboni and De Martino, '67) overnight at 4°C. Fetuses older than E-15 and postnatal rats were fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M PBS or Zamboni's fixative; the spinal cord and the DRG were postfixed in the same fresh fixative overnight at 4°C. Next, tissue blocks were rinsed in 20%sucrose (w/v)in 0.1 M PBS at 4°C overnight. After being frozen in powdered dry ice, 10-15 pm sections were cut with a cryostat in the frontal, sagittal, or horizontal plane. Every section of the embryonic tissues or every fourth section of the tissues from the postnatal rats were thaw-mounted on slides, which had been coated with chrome-alum gelatin.

Antibodiesandimmun-hemistry The antigenic materials CaB and PV were purified by HPLC from the small intestines of chicks and muscles of TABLE 1. Numbers of the Experimental Animals Embryonic (E) and postnatal (PIdays E-10 E-11 E-12 E-13 E-14 E-15 E-16 E-17 E-19 E-22 P-1 P-3 P-5 P-7 P-10 P-14 P-17 P-21 P-35 P-56

'CRL. fetalctow-rump length.

CRL (m)'

1-10 1&12 12-14 1415 15-17 22-26 3745

No. of rats used in this study 2 2 3 3 3 3 2 3 3 3 3 2 3 3 2

4 3 3 3 5

CA"-BINDING PROTEINS IN RAT SPINAL CORD mice, respectively (Jones and Hendry, '89). Antibodies against CaB and PV were generated in rabbits and sheep, respectively (Jande et al., '81; Ichimiya et al., '89; Jones and Hendry, '89). As found previously, the anti-PV antibody can be used to detect PV in the rat (Jones and Hendry, '891, while the anti-CaB antibody can recognize calbindin-D,,, and calretinin in the rat (Pochet et al., '85; Rogers, '87) and other species (Parmentier et al., '87; Rogers, '89b). All immunocytochemical procedures were carried out at 15-2OoC, if not stated otherwise. The slide-mounted sections were air-dried for 1hour, rinsed in ice-cold 0.1 M PBS, and then incubated with 1% normal goat serum for rabbit anti-CaB or 1%normal rabbit serum for sheep anti-PV for 1 hour. The sections were in turn incubated with anti-CaB or anti-PV serum (diluted 1:1,000) containing 0.3% Triton X-100 and 0.02% sodium azide. After overnight to 48 hours' incubation, the sections were treated according to the protocol for immunofluorescence histochemistry (Coons, '58) or the avidin-biotin complex method (Hsu et al., '81; Vectastain ABC Kit, Vector Laboratories, Inc.). For immunostaining controls, sections were incubated with antisera preadsorbed with antigens or without primary antibodies. These incubations resulted in no immunoreactivity in the tissue sections. In order to examine the coexistence of both calciumbinding proteins in single cells, double immunofluorescence staining was performed at different prenatal and postnatal stages (Table 2). Tissue sections were incubated simultaneously with rabbit anti-CaB and sheep anti-PV sera without pretreatment with normal sera. The secondary antibodies were donkey and swine IgG, which were directed to each primary serum-IgG, and conjugated with Texas red and fluorescein isothiocyanate, respectively. The fact that there was no cross-reactivity between the second antisera or between the primary antisera and nonspecific second antisera was confirmed by blocking and adsorption tests.

Cell countingand charting The identification of segmental levels in early ontogenesis was accomplished in three different planes (coronal, sagittal, and horizontal) by examining the general features of each segment and determining the numbers of DRG. In general, we divided the spinal cord into four different segments, namely, cervical, thoracic, lumbar, and sacral, in a rostra1 to caudal order. To detect the CaB-like and PV-like immunoreactivities at every stage of ontogenesis, all the sections stained with antisera against CaB and PV, respectively, were observed under a light microscope. One frontal section from each segment was selected for several developmental stages and traced with the aid of a camera lucida (Figs. 1-4). After tracing, the positive cells in this section and two adjacent

TABLE 2. Numbers of ExDerimentalAnimals for Double Staining ~~

Embryomc (El and postnatal (PI days E-15 E-17 E-21 P-1 P-7 P 10 P-14 P-21 P-35 P-56

~

No ofrats used in t h study 3

2 4 3 2 3 3 3 4

5

717 sections were counted a t high magnification. Then, the total number of positive cells in these three sections was summarized, as indicated by large dots in the diagrams, one dot representing three to five immunoreactive cells. The immunoreactive fibers and punctata in these sections are represented by dashes and small dots in the diagrams, respectively. The numbers of dashes and small dots in the diagrams roughly represent the relative contents of immunoreactive fibers and punctata in the sections, respectively.

Morphology of and nomenclature for the spinal cord In the present study we employed classical terms of division for the spinal cord (e.g., dorsal horn, intermediate gray matter, and ventral horn), with some additional reference to Rexed's ('52) classification in the case of the dorsal horn (Altman and Bayer, '84).

RESULTS Appearance of CaB-like immunoreactivity in the spinal cord Appearance of CaB-like immunoreactivity in the spinal gray matter Prenatal period Ventral horn (VH). In VH, CaB-like immunoreactive (CaB-LI) cells first appeared at E-12 in the cervical and thoracic segments (Figs. l,E-l2A, B and 5). Cells in the lumbar segments began to be CaB-LI at E-13 and those in the sacral segments at E-14 (Fig. l,E-l4D). Accordingly, CaB-like immunoreactivity in VH appeared to be expressed, with different time courses, in a rostrocaudal direction. These CaB-LI cells were small (about 9-15 pm in diameter) and irregular, having several short processes. Therefore, at this stage it was very difficult to judge whether or not they belonged to neurons or glial cells. In addition, nerve fibers showing CaB-like immunoreactivity were detected in the ventral roots in different segments at the same time as the appearance of the positive cells in VH in the same segments (Fig. 5). The CaB-LI cells in the cervical and lumbar segments could be divided into two groups at E-14, i.e., lateral and medial groups (Fig. l,E-l4A, C). At the same time, the cells became large (about 12-18 pm in diameter) and produced several fine beaded processes. Some of these processes penetrated into the ventral root and reached skeletal muscular structures at this stage, indicating that these CaB-LI cells are motoneurons. At E-15, the motoneurons showed strong CaB-like immunoreactivity (Fig. 6A). In addition, some smaller cells (about 8-12 pm in diameter) were also stained with the anti-CaB antiserum at this stage (Fig. 6A) and Cab-LI end-plate-like structures appeared in the muscular structures. During the period of E-16 to E-22, CaB-LI motoneurons and smaller cells gradually increased in both number and intensity (Figs. 2 and 6C). They also became large (about 20-25 km and 10-18 in diameter at E-17, respectively) and produced more beaded processes. 'Therefore, the typical multipolar form of motoneurons appeared at E-19, with some beaded processes reaching the intermediate gray matter and the ventral funiculus. The smaller CaB-LI cells were spindle-shaped or triangular, with several thin beaded processes (Fig. 6C). From E-16 on, some CaB-LI beaded fibers, which did not connect with the neuronal somata, were seen in VH (Fig. 6C).

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E-12

E-15

E-14

.. A

B

C

D

8

D

Fig. 1. Camera lucida charting of CaB-like and PV-like immunoreactivities (on the left and right half of each scheme, respectively) at selected levels of the spinal cord at E-12, E-14, and E-15. A. Cervical enlargement. B: Thoracic segment. C: Lumbosacral enlargement. D: Caudal sacral segment. Bar = 200 pm.

Intermediate gray matter (IG). The CaB-like immunoreactivity appeared in IG 2 days later than in VH. Therefore, the first stained cells appeared at E-14 in the cervical and thoracic segments (Fig. l,E-l4A, B), followed by the appearance of CaB-LI cells in the lumbar and sacral segments at E-15 and E-16, respectively (Figs. 1,E-15and 2,E-16).After E-16, the cells in IG increased in both number and intensity (Fig. 2), especially in the thoracic segments, where positive cells accumulated to form the intermediolateral nucleus (IML) (Figs. 2,E-l6B and 6C). Some cells in IML extended laterally, with their fine processes, into the lateral funiculus. In addition, some small cells (about 7-10 Frn in diameter) appeared in the area of the intermediomediate nucleus (IMM) at E-17 (Fig. 6 0 , and some fibers running from IML to IMM were seen at this stage.

Dorsal horn (OH). The appearance of CaB-like immunoreactivity in DH was latest in the spinal cord compared to that in VH and IG. Some faintly stained cells (about 6-8 Fm in diameter) first appeared in the deep layers of the cervical and thoracic segments at E-14 (Fig. l,E-l4A, B) and extended into superficial layers at E-15 (Figs. l,E-l5A, B and 6A). CaB-LI cells appeared in the lumbar and sacral segments at E-15 (Fig. l,E-l5C) and E-16 (Fig. 2,E-l6D), respectively. Mature features of the structures of DH appeared at E-16 (Fig. 2,E-16).At this time, CaB-LI cells in DH were located in both the deep layers (layers IV-VI) and superficial layers (layers 1-111). The cells in the deep layers showed strong CaB-like immunoreactivity at E-17 (Fig. 6C), most of them being spindle-shaped or triangular. Their fine beaded processes extended dorsally and ventrally into

CA"-BINDING PROTEINS IN RAT SPINAL CORD

E-19

E-16

719

E-2 2

df

Fig. 2. Camera lucida charting of CaB-like and PV-like immunoreactivities (on the left and right half of each scheme, respectively) at selected levels of the spinal cord at E-16, E-19, and E-22. A: Cervical enlargement. B: Thoracic segment. C : Lumbosacral enlargement. D: Caudal sacral segment. Bar = 200 km.

neighboring areas. The cells in the superficial layers increased in number and some of their processes extended into layer IV at E-19 (Fig. 2,E-19). In addition, some CaB-LI punctata were also found in DH after E-19, especially in the superficial layers (Fig. 2,E-19,E-22). Postnatal period VH. After birth, the CaB-like immunoreactivity in VH was at the same level as that seen at E-22 (Fig. 3,P-3).At this time, many large motoneurons and some smaller cells (about 20-25 pm in diameter) were immunoreactive for CaB. This level was maintained during the first 2 weeks following birth (Fig. 3), although the size of the CaB-LI cells continued to increase in this period. In addition, during this period, the CaB-LI processes lost their beaded form, becoming smooth. During the period of P-15 to P-35, the number of CaB-LI cells in VH decreased gradually, especially the motoneurons. Therefore at P-35 and P-56, only a few medium-sized

(about 30-35 pm in diameter) and small (about 15-18 pm in diameter) neurons were found in VH. ZG. Most of the cells in IG became CaB-LI after birth (Fig. 3). In the thoracic segments, many CaB-LI neurons accumulated in the IML and IMM. During the first 2 postnatal weeks, a high level of expression of CaB was maintained in IG (Fig. 3). From P-15 on, the number of CaB-LI cells in IG gradually decreased. At P-35 and P-56, only a few CaB-LI cells were found in IG. DH. In contrast to VH and IG, the CaB-like immunoreactivity in DH continued to increase during the first week after birth, especially in the superficial layers (Fig. 3,P-3, P-7). Therefore, at P-7, many CaB-LI cells and punctata were found in all layers. At this stage, cells in the superficial layers were about 11-14 pm in diameter, while those in deeper layers were 18-22 pm in diameter. Occasionally, a few large multipolar neurons were also found in the deep layers of DH after P-3.

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P-3

P-7

P-14

Fig. 3. Camera lucida charting of CaB-like and PV-like immunoreactivities (on the left and right half of each scheme, respectively) at selected levels of the spinal cord at P-3, P-7, and P-14. A: Cervical enlargement. B: Thoracic segment. C : Lumbosacral enlargement. D: Caudal sacral segment. Bar = 200 km.

During the period of P-15 to P-35, the CaB-LI neurons in the deep layers of DH decreased gradually, while those in the superficial layers remained at the same level at P-35 (Fig. 7A) and a later stage (P-56). Appearance of CaB-like immunoreactivity in the spinal white matter. CaB-like immunoreactivity in the spinal white matter was first observed in the ventral funiculus at E-14 (Fig. 1,E-14) and then in the lateral funiculus at E-15 (Fig. l,E-15). These immunoreactive structures were identified as punctata (Figs. 6A, C). CaBlike immunoreactivity in the funiculi gradually increased, a plateau being reached around birth (Figs. 2 and 3). This plateau level of CaB-LI fibers was maintained during early postnatal life (Fig. 3 ) .After P-17, the number of immunoreactive punctata decreased, while those in the dorsal part of the lateral funiculus remained (Figs. 4 and 7A). Only the latter area contained CaB-LI punctata and a cross section of fibers after P-35. In the posterior funiculus, CaB-LI fibers appeared at E-16 in all segments (Fig. 2 ) , but they decreased in number

during the late embryonic stages (E-19 to E-221, reaching a plateau after birth (Fig. 3). After P-14, they began to decrease in number again and were undetectable at P-35 (Fig. 4,P-35). Appearance of CaB-like immunoreactiuity in DRG. In DRG, a few large (about 12-15 pm in diameter) and some smaller (about 8-10 pm in diameter) CaB-LI neuronal somata appeared at E-16. Thereafter the number and intensity of these immunoreactive neurons increased. The central and peripheral processes of these neurons showed immunoreactivity at E-17 (Fig. 8A). The CaB-LI central processes ran along the dorsal root to the dorsal funiculus, while the peripheral processes extended peripherally to innervate peripheral organs. After birth, a large number of DRG neurons expressed CaB (Fig. 8C); large (about 20-25 pm in diameter) as well as medium-sized (about 15-18 pm in diameter) and small (about 10-15 pm in diameter) neurons were immunoreactive. All the CaB-LI neurons increased in number during the first 2 weeks following birth (Fig. 8E). Thus, three

CA"-BINDING PROTEINS IN RAT SPINAL CORD

P--17

72 1

P-35

P-21

A

B

Fig. 4. Camera lucida charting of CaB-like and PV-like immunoreactivities (on the left and right half of each scheme, respectively) at selected levels of the spinal cord at P-17, P-21, and P-35. A Cervical enlargement. B: Thoracic segment. C: Lumbosacral enlargement. D: Caudal sacral segment. Bar = 500 Fm. -

~

kinds of CaB-LI neurons were seen in DRG in adult animals: large (about 45-50 pm in diameter), mediumsized (about 30-35 pm in diameter), and small (about 18-22 pm in diameter), although most of them were small neurons. Appearance of CaB-like immunoreactivity in the ependymal layer. In addition to neuronal elements, CaB-like immunoreactivity was found in the ependymal cells lining the central canal of the spinal cord. Cells in the roof plate expressed CaB at E-12, and increased in number and density up to E-15 (Fig. 6A). At E-15, CaB-LI cells also appeared in the ventral part of the wall of the central canal, whose apical processes extended into the basal plate. From E-16 on, most ependymal cells and the dorsal median septum were stained (Fig. 6C). Around the perinatal period, CaB-like immunoreactivity in median septum disappeared and that in the ependymal reached the level Seen in adult rats, in which only a few ePendYmal cells were the CaB-LI.

~

~

Fig. 5. photomicrograph showing the first appearance of CaB-LI motoneurons in a cervical segment of the spinal cord at E-12, The neurons are irregular in form and send immunoreactive axons into the ventral root (solid arrow). Bar = 100 wm.

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Figure 6

CA"-BINDING PROTEINS IN RAT SPINAL CORD

723

Fig. 7. Photomicrographs showing CaB-like (A) and PV-like (B) immunoreactivities in the dorsal horn of the thoracic spinal cord at P-21. A: Small CaB-LI neurons and fibrous structures can be seen in the superficial layers. A large neuron in the lateral area of lamina V and

neurons in the lateral spinal nucleus (LSN) are also positive. B: Small PV-LI neurons and axon terminals can be seen in layers I1 and 111. Bar in B = 100 pm; A is the same magnification.

Appearance ofPV-likeimmunoreactivity in the spinal cord

prenatal period, the PV-LI fibers and punctata increased in number and intensity, and extended into all parts of IG (Fig. 2,E-22). No PV-LI cells were detected in IG during the prenatal period. DH. Immunoreactive fibers arising in DRG were not observed to enter the dorsal root entrance zone (DREZ) in the cervical and thoracic segments until E-14 (Fig. l,E-l4A, B). At E-15, PV-LI fibers were seen in DREZ of all segments of the spinal cord, and those in the cervical and thoracic segments extended into the superficial part of DH (Figs. 1,E-15and 6B). At E-16, DREZ shifted medially to form the dorsal funiculus, which received more PV-LI fibers from DRG (Fig. 2,E-16). At the same time, these fibers entered the spinal gray matter from the medial aspects of DH. in all segments and some of them reached IG and VH in the cervical and thoracic segments. During the later prenatal period, PV-LI fibers showed a steep increase in number and most of them terminated in IG and VH in all segments (Fig. 2,E-19, E-22). In addition, a few PV-LI punctata were also found during the later prenatal period. No PV-LI cells were found in DH during the prenatal period. Postnatal period VH. During the early postnatal period (P-1 to P-71, many PV-LI fibers and punctata exhibiting strong immunoreactivity were seen in VH (Fig. 3). Some punctata were found around motoneurons. During the second week after birth, the numbers and intensities of PV-LI fibers and punctata decreased slightly. Such decreases in the upper segments preceded those in the lower segments.

Appearance of PV-like irnrnunoreactivity i n the spinal gray matter. PV-like immunoreactivity in the spinal cord was clearly distinguishable from CaB-like immunoreactivity throughout ontogenesis in the rat. Prenatal period VH. Several PV-like immunoreactive (PV-LI)cells (about 14-16 km in diameter) were first observed in the lateral motoneuron group of VH in the cervical and thoracic segments at E-14 (Fig. l,E-l4A, B). However, these cells soon began to loose their immunoreactivity, i.e., from E-16 (Figs. 1,E-l5Aand 2,E-l6A),which completely disappeared after E-17 and in the following prenatal period (Fig. 2,E-19, E-22). On the other hand, a few PV-LI fibers and punctata appeared in the cervical and thoracic segments at E-16 (Fig. 2,E-l6A, B), followed by their appearance in the lumbar and sacral segments at E-17. These fibers arose from the dorsal root and entered VH through the medial aspects of DH and IG. They were mainly located in the medial and lateral areas of VH. In the later prenatal period (Fig. 21, the PV-LI fibers and punctata both increased in number rapidly. Just before birth (E-22), many PV-LI fibers and punctata were seen in VH in each segment, with the most dense localization in the medial and lateral areas. ZG. At E-16, a few PV-LI fibers and punctata were observed in all the segments except for the sacral segments (Fig. 2,E-l6A, B, C), in which PV-LI fibers were first observed at E-19 (Fig. 2,E-19). At first, PV-LI fibers and punctata were located in the middle part of IG and the areas adjacent to the central canal. However, during the later

Fig. 6. Photomicrographs showing CaB-like (A and C) and PV-like immunoreactivities (B and D) at the level of the cervical spinal cord at E-15 (A and B) and at the level of the thoracic spinal cord at E-17 (C and D). A: CaB-LI motoneurons in the ventral horn extend their processes into the ventral funiculus. Cells in the roof plate (rp)are deeply stained. B: PV-LI primary afferent fibers can be seen in the dorsal root (dr) and dorsal root entrance zone of the spinal cord. Several PV-LI fibers enter the dorsal part of the dorsal horn (arrows). The ventrolateral group of motoneurons in the ventral horn (VH) also shows PV-like immunoreactivity at this stage. C: In addition to an increased number and density of

CaB-LI neurons in the gray matter, numerous cross-sectioned fibers in the white matter show CaB-like immunoreactivity in the ventral and lateral funiculi. In addition, several immunoreactive fibers cross the ventral commissure (vc) to the contralateral side. Ependymal cells are also stained immunohistochemicallyat this step. D: A number of PV-LI fibers can be seen in the dorsal funiculus. Some PV-LI fibers run ventrally to the inner part of the intermediate gray matter (IG) and others run to the ventral horn (VH). Bar in D = 100 km; A-C are the same magnification.

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ZHAN'G ET AL.

Fig. 8. Photomicrographs showing CaB-LI (A, C, and E) and PV-LI (B, D, and F) neurons in the dorsal root ganglia of the rat at different ages. A and B: E-17. C and D: P-1.E and F: P-14. Large- and medium-sized neurons in the DRG at each stage express CaB and PV,

which remain until adulthood. Small neurons become positive after P-1, especially CaB-LI neurons. Bar in F = 100 km; A-E are the same magnification.

After the second postnatal week, steep decreases in PV-LI fibers and punctata were seen in all levels of the spinal cord. At P-17, PV-LI fibers and punctata disappeared in VH in the cervical segments (Fig. 4,P-17), followed by their disappearance from the other spinal segments at P-21 (Fig. 4,P-21). Therefore, at P-35 and P-56, no PV-LI fibers or punctata could be detected in VH of the spinal cord.

After P-10, a few small (about 15-20 km in diameter) and medium-sized (about 25-35 km in diameter) PV-LI neurons appeared in VH in the lumbar and sacral segments (Figs. 3,P-14 and 4). These neurons could still be observed at P-35 (Fig. 4,P-35) and P-56. IG. After birth, more PV-LI fibers and punctata were found in IG than in the prenatal period. These fibers and

CA"-BINDING PROTEINS IN RAT SPINAL CORD

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punctata were distributed throughout IG, the densities of Appearance of PV-like immunoreactivity in the punctata being high in the medial part of IG and the area ependymal layer. In contrast to CaB-like immunoreacadjacent to the central canal (Fig. 3,P-3). This high expres- tivity, the cells lining the central canal did not show any sion level of PV in IG was maintained in the first 2 postnatal PV-like immunoreactivity during the ontogenesis of the spinal cord. weeks (Fig. 3). As in VH, most of the PV-LI fibers and punctata in IG Colacalizationof CaB-likeand PV-like disappeared during the period of P-15 to P-17 (Fig. 4,P-17) immunoreactivitiesin ontogenesis in all segments. Therefore, in the following and adult stages, only a few PV-LI punctata remained in the areas In order to examine colocalization of CaB and PV, tissue adjacent to the central canal (Fig. 4,P-21, P-35). sections were treated simultaneously with rabbit anti-CaB No PV-LI cells were seen in IG during the postnatal and sheep anti-PV sera. Double-stained neurons first apperiod. peared in DRG after birth, then inVH at P-10, and finally in DH. In the first 2 postnatal weeks, many PV-LI fibers DH at P-35. However, it should be noted that these were seen in the dorsal funiculus and the medial part of DH double-stained neurons consisted only of a small population (Fig. 3). A few PV-LI fibers and punctata were also scat- throughout ontogenesis (Fig. 9A-D). tered in the deep layers of DH (Fig. 3). However, between P-15 and P-17, most of the PV-LI fibers in the dorsal AlmicnhsandD_H.in_thc~y_pll~r ,ym~nt~A&pru?medQj~ aT>aaJ.-wN ' 2,FlrT,'foiiofieh DY tn6' a1sappearances"of tnes'Z m e i s in Ontologyof CaB-Likeimmunoreactivityin the lower segments later from P-17. After P-21, no PV-LI relationto developmentof the spinalcord fibers were found in the dorsal funiculus in any segments andDRG (Fig. 4,P-21, P-35). Since some calcium-binding proteins have been detected On the other hand, some PV-LI small neurons (about 10-15 pm in diameter) and punctata were found in the in glial cells (Perlmutter et al., '88; Bronstein et al., '891, it superficial layers (layers 11-111) of DH after P-21 (Figs. is important to determine whether the CaB-LI cells during 4,P-21 and 7B), and they increased in number slightly until ontogenesis are neurons or glial cells. In the present study, CaB-like immunoreactivity was first observed in the cells P-35 (Figs. 4,P-35). Appearance of PV-like immunoreactivity in the located in VH of the spinal cord at E-12. At this stage, it was spinal white matter. PV-LI fibers were observed in very difficult to judge whether these CaB-LI cells were DREZ at E-14 and E-15 or in the dorsal funiculus (do after neurons or glial cells due to their small and irregular size, E-16 until P-17 (Figs. 1-3, and 5B, D). These fibers with few processes. However, at the same stage, immunoreoriginated from DRG through the dorsal root. They in- active fibers were also seen in the ventral root and in creased in number in the prenatal period along with subsequent stages. In addition, these cells seemed to dedevelopment (Fig. 2). After birth, most of the PV-LI fibers velop into motoneurons; therefore, it is likely that they were seen in df during the early postnatal period (Fig. 3). were motoneurons. As to the CaB-LI cells in IG and DH, it However, they began to decrease after P-14 (Fig. 4). At was also very difficult to determine their features in the P-35, PV-LI fibers in the dorsal funiculus were undetect- early embryonic stages. However, the first appearance of the CaB-LI cells in IG and DH occurred somewhat earlier able (Fig. 4,P-35). than the appearance of glial cells in the same areas, which No PV-LI fibers or punctata were found in the ventral or seems to begin only from E-17 (Raju et al., '81; Altman and lateral funiculus during the ontogenesis of the spinal cord. Bayer, '84). In addition, during ontogenesis, CaS-LI cells in Appearance of PV-like immunoreactivity in DRG. IG and DH follow the development pattern of neurons, as Neurons in DRG began to show PV-like immunoreactivity discussed below. It is possible that most of the CaB-LI cells at E-14 in the cervical and thoracic segments. These during ontogenesis are neurons. However, CaB is indeed neurons were bipolar in shape (about 10-12 pm in diame- expressed in some glial cells during ontogenesis, e.g., roof ter) and their central processes were also positive in the plate and ependymal cells. dorsal root and DREZ of the spinal cord at the same time. During the development of the spinal cord, it is well At E-15, many DRG neurons and their processes in all known that neurons are produced in a ventrodorsal direcsegments became PV-LI. Most of them were spindle- tion (Altman and Bayer, '84; Langman and Haden, '70; shaped. From E-17 on, they became unipolar in shape and Nornes and Das, '72, '74): In the cervical spinal segments, they increased in size and number in later developmental motoneurons are produced from E-11 to E-12, relay neustages (Fig. 8B, D, F). The smaller PV-LI neurons (about rons in IG from E-12 to E-14, and interneurons in DH from 16-18 pm in diameter at E-22) appeared in the perinatal E-15 to E-16. The present study, in which CaB immunoreperiod and increased in number quickly during the first 2 activity was examined, also demonstrated a ventrodorsal weeks following birth (Fig. 8B, D, F). After birth, some pattern as to the appearance of immunoreactive cells in all neurons about 10-15 pm in diameter in DRG also became the spinal segments. In addition, the first appearance of PV-LI. These small neurons increased in number gradually CaB-LI cells also followed a rostral to caudal gradient, in the postnatal period (Figs. 8D, F). Therefore, in adult CaB-LI cells in the rostral part of the spinal cord appearing animals, similar to CaB-like immunoreactivity, there were earlier then in the caudal part. Thus the pattern of developalso three kinds of PV-LI neurons in DRG: large (about ment and the appearance of CaB-LI cells correspond to the 46-52 pm in diameter), medium-sized (about 34-38 pm in segmental developmental sequence of the neurons in the diameter), and small (about 20-24 pm in diameter). How- spinal cord. Therefore, the CaB-LI cells appearing in the ever, most of the PV-LI neurons in DRG were large and early embryonic stages seem to be neurons, and it is medium-sized, although some small neurons also expressed possible that once neurons are generated in every segment PV. of the spinal cord, they begin to express CaB.

Fig. 9. Immunofluorescent photomicrographs of the same section of the ventral horn (VH) at P-14(A and B) and dorsal root ganglia (C and D) at P-lafter double staining with antisera to CaB (A and C) and PV (Band D). Arrows indicate the double-stainedneurons. Note that these neurons are few in number. Bar in B = 100 km; A is the same magnification. Bar in D = 100 km; C is the same magnification.

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CA"-BINDING PROTEINS IN RAT SPINAL CORD

727

Enderlin et al. ('87) reported that CaB-LI neurons could be found at E-14 and E-19 in the basal part of the spinal cord and DRG, respectively, while we found that they appeared at E-12 in VH and at E-16 in DRG. The discrepancy may be due to the different strains of experimental animal or the fixatives used in the studies. In the perinatal period, the neurons in the spinal cord and DRG undergo many maturation processes, such as the generation of dendrites and axons, and the establishment of connections with their targets (Lawson et al., '74; Altman and Bayer, '84). The present study shows that CaB-like immunoreactivity also increases quickly and retains a regional distribution in these tissues, suggesting that CaB is involved in the maturation processes of the neurons in the spinal cord and DRG. From P-14 on, the developmental pattern of CaB-like immunoreactivity differs according to the spinal region. The CaB-LI neurons in VH and IG began to decrease in number gradually, while no significant decrease in CaB-like immunoreactivity was seen in DH or DRG. There are at least two possible reasons for this decrease: cell death (Cowan et al., '84) and a decrease in CaB synthesis in the neurons. The former possibility seems to be unlikely, because cell death in the motoneuron groups of the spinal cord has already stopped before birth (Lance-Jones, '82; Harris and McCaig, '84).

rapidly during the first 2 weeks following birth, even though the number of DRG neurons remains constant during the same period (Hulsebosch et al., '86). The stage at which the disappearance of the PV-LI fibers occurs is highly similar to the stage at which the elimination of fibers in the dorsal root occurs. After the disappearance of PV-LI fibers in VH and IG, some PV-LI terminals appear in layers 11-111 of DH from P-21. Because most of the small and medium-sized DRG neurons terminate in the superficial layers of DH and these terminals appear later than in VH during the ontogenesis of the spinal cord (Davis et al., '891, the PV-LI terminals in layers 11-111may be derived from small and medium-sized neurons generated later during prenatal development of DRG. Since these terminals remain in adult animals, it is assumed that in this fiber system, PV may play an important role in adult animals as well as in neurogenesis.

OntologyofPV-likeimmunoreadivityin relation to development of the spinalcord andDRG PV-LI fibers in the spinal cord first appear in DREZ at E-14. Subsequently, these fibers enter the spinal cord from the medial aspect of DH to reach IG and VH between E-15 and E-17. From E-21 to P-14, they reach the maximum level. These fibers seem to originate from larger and medium-sized DRG neurons, because: (1)immunoreactive fibers can be traced to the dorsal root; (2) PV-LI fibers, and large and medium-sized neurons in DRG appear at the same time; and (3) dorsal rhizotomy at P-7 results in the disappearance of PV-LI fibers in the ipsilateral gray matter at P-14 (unpublished data). It has been reported that a significant level of monosynaptic transmission from primary afferent fibers to motoneurons first appears at E-19 in the lumbar segments of the rat spinal cord and the magnitude of monosynaptic reflex responses gradually increases with age during the prenatal period (Kudo and Yamada, '87). Accordingly, the stages at which PV-LI primary sensory afferent fibers appear and develop in the gray matter coincide with the establishment of the monosynaptic reflex pathway, suggesting that PV plays an important role in this event. The subsequent disappearance of these fibers supports this hypothesis. In addition, the present results also show that PV-LI can be used as an early marker for the development of primary afferent fibers. After P-14, PV-LI fibers disappear in VH and IG. At the same time, however, the PV-LI large and medium-sized neurons in DRG do not show significant decreases. These results suggest that the PV transport in the central processes of the large and medium-sized neurons may decrease after axon maturation/myelination. This is very similar to the developmental expression of PV in Purkinje cells and their axons (Braun et al., '86). Another possibility is the cessation of PV-LI fiber pruning or the selective loss of supernumerary fibers (Cowan et al., '84). It has been shown that the number of fibers in the dorsal root decreases

Dif€erente x p d o n s of CaB andPV It has been shown that CaB and PV are localized in different neuron populations in the brains of adult animals (Gerfen et al., '85; Schneeberger et al., '85; Celio et al., '86; Jones and Hendry, '89; Sloviter, '89). In the present study, we found that they are mainly localized in separated neuron groups in the spinal cord and DRG throughout ontogenesis, suggesting the heterogeneity of spinal neurons from the viewpoint of the ontogeny of calcium-binding proteins present in the neurons. However, our results are in contrast to those reported by Carr et al. ('89). They found that CaB was highly colocalized with PV in DRG neurons in the adult rat. Although it is difficult to explain the discrepancies between the results of Carr et al.'s study and those of others including ours, the different nature of the antisera and the immunostaining techniques used may be two reasons.

ACKNOWLEDGMENTS Dr. Jian-Hua Zhang is a visiting researcher from the Department of Anatomy of the Fourth Military Medical College, Shaan Xi, Xi'an, China, and is supported by the Kanohara Foundation. This work was supported by the Ministry of Education, Science, and Culture of Japan, and the Japan Brain Foundation.

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Ontological study of calbindin-D28k-like and parvalbumin-like immunoreactivities in rat spinal cord and dorsal root ganglia.

The calcium ion plays an important role in some critical developmental events in the nervous system, such as neurulation and neurite elongation. There...
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