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Migrating luteinizing hormone-releasing hormone (LHRH) neurons and processes are associated with a substrate that expresses S100
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 88 (1995) 148-157
Research report
Migrating luteinizing hormone-releasing hormone (LHRH) neurons and processes are associated with a substrate that expresses SIO0 Diana M. Cummings, Peter C. Brunjes
*
Neuroscience Program and Department of Psychology, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA Accepted 16 May 1995
Abstract
Luteinizing hormone-releasing hormone (LHRH) containing neurons arise in the region of the medial olfactory placode and migrate into the developing olfactory bulbs and basal forebrain along branches of the terminal and vomeronasal nerves. The neurons ultimately come to reside in olfactory and septo-preoptic areas and project extensively to several brain regions, including the preoptic area and median eminence. The present study examined the expression of a glial-associated guidance molecule, S100, as a possible substrate for this migration. Monodelphis domestica (the Brazilian grey, short-tailed opossum) was studied since this species gives birth to very immature young, allowing access to early periods of mammalian forebrain development. Immunoreactivity for both S100 and LHRH-containing neurons and fibers was observed to be closely associated along the entire LHRH migratory route from the vomeronasal organ to the septo-preoptic area as early as the day of birth (PO). By P10, S100-immunoreactivity was also seen in areas containing LHRH-immunoreactive fibers such as the preoptic area and median eminence. We suggest that S100, a protein with neurotrophic properties in vitro, acts as a guidance molecule for migrating LHRH-immunoreactive neurons and elongating processes. Keywords: S100; Glia; Ensheathing cell; LHRH neuron; Olfactory bulb; Development; Cell migration; Immunofluorescence
1. Introduction
Luteinizing hormone-releasing hormone (LHRH) containing neurons and processes play a critical role in the functioning of the hypogonadal axis and in many behaviors associated with vertebrate reproduction. The LHRHcontaining neurons thought to be most actively involved in regulating reproductive behaviors are widely distributed throughout the olfactory system and basal forebrain (see [35] for review). In adult mammals, for example, they are found in regions such as the terminal nerves and ganglia [43,45], main and accessory olfactory bulbs, diagonal band, septum, preoptic area, anterior hypothalamus, and piriform cortex [22,23]. LHRH-containing fibers project extensively throughout the brain and terminate in far-reaching areas, including the median eminence and hypophyseal stalk. LHRH-containing neurons arise in the region of the medial olfactory placode and migrate along branches of
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the terminal and vomeronasal nerves to destinations in the basal forebrain [46,58,60]. This migratory pattern has since been demonstrated in a variety of species including primates [40], chicks [1,32,37], and newts [33], and may also exist in humans [6,47]. Migration of LHRH-containing neurons seems critical for development of the gonadotropin system, since incomplete or disrupted migration of LHRH-containing cells appears to result in the human hypogonadal disorder known as Kalimann's syndrome [42]. Patients with this disorder also suffer from anosmia [24]. Since LHRH-containing cells and processes travel such great distances to reach their targets, possible mechanisms of guidance are currently under investigation. For example, neural cell adhesion molecule (NCAM) is present in the cells and axons of the terminal and vomeronasal nerves which form the substrate for migrating LHRH-containing neurons [21,34,36,41]. Further support of NCAM's role as a guidance molecule has emerged from evidence that perturbation of the NCAM-positive substrate disrupts the migration of LHRH-immunoreactive (ir) cells from the medial olfactory pit of embryonic mice [48]. Another putative guidance factor, peripherin, is a neuron-specific
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intermediate filament [27]. Peripherin is found in olfactory receptor neurons [20] and also appears to be expressed along the LHRH migratory route in embryonic mice [59]. S100 is another central nervous system (CNS) protein which is increasingly becoming recognized as a neurotrophic factor. S100 is at least 10,000-fold more concentrated in the brain than in other tissues [30] and exists in three dimeric forms comprised of alpha and beta subunits: S100aot, S100otfl, and S100/3fl (for reviews see [11,25]). Research in vivo indicates that during development S100 proteins are localized to regions in which they may serve as molecular guides, e.g., chick motor and sensory neurons [5], astrocytes of kitten visual cortex [16,31], and radial glia of the midline raphe [57]. In addition, this protein promotes survival of motor neurons in the chick hindlimb [5]. Recently, S100-immunoreactive cells were found in the vomeronasal organ (VNO) and along the nasal septum of human fetuses and appear localized to regions of migrating LHRH-containing cells [6]. The present study examines the expression of S100 immunoreactivity in relation to migrating LHRH-containing neurons throughout the brain in a developing marsupial, Monodelphis domestica. Monodelphis domestica, the Brazilian grey, short-tailed opossum, is an appropriate species for studying the early maturation of the mammalian brain since offspring are born in a very immature state after only 14 days of gestation. The migration of LHRH-containing neurons has been well characterized in postnatal Monodelphis [44]. Migrating LHRH-containing neurons appear to follow a distinct path from the olfactory-vomeronasal region to the brain. While this migratory route is consistent across many species, Monodelphis is unique in that the terminal nerve is very thick and easily delineated in its projection from the ganglia to the medial septum and preoptic area [44]. Furthermore, unlike rats and mice, LHRH-containing cells appear to migrate into the brain well into the postnatal period in Monodelphis [44]; thus, this species is ideal for studying possible guidance cues. We conducted double-labelling experiments in early postnatal Monodelphis to investigate the localization of immunoreactivity for S100 in relation to LHRH-immunoreactive (ir) neurons and processes. In addition, we investigated the distribution of immunoreactivity for another glial-associated protein, glial fibrillary acidic protein (GFAP), in the brains of developing Monodelphis. Our studies indicate that S100 is present in terminal nerve fibers of developing Monodelphis and that these fibers co-localize with LHRH-containing neurons. This co-localization was observed in the nasal epithelium, olfactory bulb and forebrain, and was also seen in more caudal regions known to contain LHRH-ir fibers, such as the preoptic area and median eminence. 2. Materials and methods
Adult Monodelphisdomestica were purchased from the Southwest Foundation for Biomedical Research (San Anto-
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nio, TX) and maintained for breeding according to the guidelines of Fadem and colleagues [18]. Females were checked daily for litters with the day of birth considered postnatal Day 0 (P0).
2.1. lmmunohistochemistry P0, P5, P10, P20, and adult (n's = 4-5 at each age) animals were given an overdose of barbiturate anesthetics and perfused transcardially with ice-cold 0.1 M phosphate buffer with 0.9% saline (PBS, pH 7.4) followed by 4% paraformaldehyde (pH 7.4). Brains were removed, postfixed in 4% paraformaldehyde with 10% sucrose for 1-4 h, and incubated in 0.1 M PBS with 30% sucrose at 4°C. Due to their small size, P0 and P5 brains were left in the skull, and the whole head was processed for immunohistochemistry. Sagittal sections (10-12 /xm thick) were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and placed under vacuum for 6-8 h at 4°C. Slides were then rinsed 4 times in PBS and incubated in 3% H202 for 15 min at 4°C to block endogenous peroxidases. Afterwards, non-specific binding sites were saturated by immersing sections in 10% normal goat serum (Vector) for 1 hour at 4°C. Sections were then transferred to polyclonal rabbit anti-S100 antibody (S100, DAKO, 1:2000) overnight at 4°C. In additional animals, alternate sections were taken at P5, P10 and P20 (n's---3) and incubated in polyclonal rabbit anti-GFAP antibody (DAKO, 1:5000). To further reduce nonspecific binding and increase penetration, all primary and secondary antisera and the ABC solutions were diluted in PBS containing 0.25% lambda carrageenan and 0.35% Triton X-100. After incubation in primary antisera, slides were washed 4 times in PBS and placed in solutions containing biotinylated secondary antibodies (DAKO, 1:100) for 1 h at room temperature, rinsed in PBS, and transferred to avidin-biotin solution (ABC; Vectastain Elite, Vector) for 90 min. After rinsing slides 3 times in PBS and once in Tris-buffered saline, sections were treated with diaminobenzidine (DAB) in the presence of H202 for 10-15 min until peroxidase labeling was fully visualized. Slides were then rinsed 3 times in phosphate buffer (PB), dehydrated, cleared and coverslipped with DPX (BDH Ltd., Poole UK).
2.2. Double-labeling At each developmental age examined (e.g., P0, P5, P10, and P20) sections from 4 to 5 pups were processed for double-labeling experiments. Slides were incubated in primary antisera containing polyclonal anti-S100 (S100, DAKO, 1:2000) and monoclonal anti-LHRH (LRH13, generously provided by Dr. Wakabayashi, Gunma University, Maebashi, Japan, 1:2000) for 48 h at 4°C. LRH13 has been demonstrated to have a high specificity and crossreactivity for LHRH in several species [38]. Slides were rinsed 5 times in PBS, placed into biotinylated goat anti-
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Fig. 1. $100 (dark DAB reaction product) and LHRH (fluorescent labeling appears bright white) immunoreactivity in the vomeronasal and olfactory systems of developing Monodelphis. Note: S100-ir was also expressed in the craniofacial cartilage. A: P5: coronal section through the nose and brain viewed under UV light reveals LHRH-ir cells (small arrows) extending from the vomeronasal organ (VNO; larger arrow) to the terminal nerve ganglion (g). A dense cluster of S100-ir cells (arrowhead) lies dorsal to the VNO and in close apposition to a group of LHRH-ir cells. Smaller groups of S100-ir cells are found along the nasal septum (s), often associated with LHRH-ir cells. B: P10~ sagittal section (rostral to left) through the nose visualized under UV light shows an LHRH-ir cell body (arrowhead) and numerous LHRH-ir fibers (arrows) along S100-ir olfactory and vomeronasal fascicles entering the olfactory nerve layer (onl). ob, olfactory bulb; oe, olfactory epithelium. Scale bars = 100 p~m.
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rabbit secondary for 1 h at room temperature, and processed for ABC-DAB visualization of S100 immunoreactivity as described above. Sections were then rinsed 4 times in PBS and incubated in aminomethylcoumarin acetate (AMCA)-conjugated goat anti-mouse IgG (Jackson,
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1:100) for 2 h at room temperature. Afterwards, slides were rinsed 4 times in PB and coverslipped with glycerol gelatin (Sigma). Both the DAB reaction product which labeled S100-ir and the fluorescence associated with LHRH-ir could be viewed simultaneously with a fluores-
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Fig. 2. S100-ir is co-localized with LHRH-ir cells and processes along the central projection of the terminal nerve. In A - D arrowheads point to the S100-ir centrally projecting branches of the terminal nerve and small arrows point to LHRH-ir cell bodies and processes. In all photomicrographs rostral is at left. A: P0: sagittal section (brightfield) reveals S100-ir cells in the terminal nerve ganglion (g) and along its centrally projecting branches. B: fluorescence photomicrograph of the same section as in A shows LHRH-ir cells and their processes within the terminal nerve ganglion and along its centrally projecting branches. C: P5: fluorescence photomicrograph of a sagittal section of the olfactory bulb and forebrain shows S100-ir in the olfactory nerve layer and along the central branches of the terminal nerve. LHRH-ir cells and processes are positioned along the terminal nerve branches which turn ventrally in the region of the third ventricle (11 lv). D: P10: fluorescence photomicrograph of a sagittal section demonstrates LHRH-ir cells along scattered S 100-ir terminal nerve branches. E: Higher magnification of bracketed region in D shows LHRH-ir processes (large arrows) closely associated with S100-ir terminal nerve branches, oa, olfactory bulb anlage. Scale bars = 100 # m in A - D , 50 /.Lm in E.
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cence microscope using the ultraviolet filter set. In photomicrographs (e.g. Figs. 1 and 2, 4) the DAB reaction product appeared as a dark precipitate while the fluorescent labeling appeared bright white.
2.3. Control experiments
Fig. 3. By P20 S100-ir can be seen in astrocyte-like cells. This brightfield photomicrograph shows a central projection of the terminal nerve (arrow) which projects toward the third ventricle (lily). Dark S100-ir is seen along this nerve projection and in neighboring cells (arrowheads) which resemble astrocytes. Scale bar = 100 p,m.
Control experiments in which the S100 or LRH13 antiserum were omitted revealed no positive staining. In addition preabsorption control experiments were conducted to further verify the S100 immunoreactivity and to determine which of the different forms of the S100 protein exists along the terminal nerve in Monodelphis. In these experiments the S100 antiserum was preabsorbed with an excess of one of the three S100 proteins, e.g., S100aa, S100a/3, or S100/3/3 (Sigma), at either 10 or 20 /.~g per ml of antiserum. After 24 h of preabsorption, the antiserum was used as described above for single-labelling.
Fig. 4. S100-ir is associated with LHRH-ir fibers in the preoptic area and median eminence. A: P5: coronal section (brightfield) shows S100-ir along the terminal nerve branches (arrowheads) in the POA. B: fluorescence photomicrograph of the same section as in A reveals LHRH-ir fibers (arrows) cascading ventrally toward the base of the brain in association with the S100-ir terminal nerve branches. C: P10: sagittal section (brightfield) reveals Sl00-ir along the ventral surface of the median eminence (arrowhead). Light S100-ir is also visible in the hypophyseal stalk (s) and in the posterior hypothalamic area (ph). D: fluorescence photomicrograph of the same section as in C. Many LHRH-ir fibers (arrowhead) are found along the S100-ir of the median eminence, and a few fibers (arrow) were seen along the hypophyseal stalk near S100-ir. Scale bars = 100 /.~m.
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3. Results 3.1. Vomeronasal organ and nasal cavity LHRH-ir cells and processes were found associated with S100-positive cells at all points along their migratory route from the VNO to the olfactory bulb. At the earliest ages examined (P0 and P5), LHRH-ir neurons were observed both within and outside of the VNO (Fig. 1A). These cells were closely associated with S100-ir cells observed both in and around the VNO and in a distinct cluster just dorsal to this structure (arrowhead, Fig. 1A). The primary ganglion of the terminal nerve contained many S100-ir cells as well as LHRH-ir neurons (Fig. 1A). In P10 pups, S100-ir appeared denser along the edges of nerve fascicles. LHRH-ir cell bodies and fibers were in close apposition to the dark S100-ir nerve fibers as they approached the olfactory bulb (Fig. 1B). 3.2. Olfactory bulbs and forebrain At P0, S100-ir cells of the terminal nerve ganglion were seen ventral to the anlage of the olfactory bulbs (Fig. 2A, B). Sagittal sections revealed S100-positive processes extending bilaterally from these ganglia toward the third ventricle along the medial walls of the developing telencephalon. LHRH-ir neurons and processes were seen in the ganglion of the terminal nerve and in close apposition to its centrally-projecting branches (Fig. 2B). LHRH-immunopositive cells were often found in clusters while leading processes were observed running along the S100-ir terminal nerve branches as they arched through the developing telencephalon (Fig. 2B). The olfactory nerve layer (onl) of the olfactory bulb was S100-ir at all ages examined, although developmental increases in staining density were observed. At P0 light S100-ir could be seen around the olfactory bulb anlage (Fig. 1A,B). By later ages, e.g.,
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P5-P20, S100-ir in the onl was very dense (Fig. 1A,B, Fig. 2C,D). At P5 more S100-ir terminal nerve fibers could be seen coursing dorsocaudally over the ventromedial telencephalon (Fig. 2C). Just rostral to the third ventricle many S100-ir fibers turned ventrally into the subfornical organ or extended along the lamina terminalis, rostral to the third ventricle. Many other S100-positive fibers extended radially into the ventral telencephalon. LHRH-ir perikarya and processes were found in the same locations identified at earlier ages, along the S100-positive terminal nerve branches projecting toward the third ventricle (Fig. 2C). LHRH-ir fibers were found along S100-ir terminal nerve branches that either arched ventrally just rostral to the third ventricle or projected radially into the telencephalon. By P10, the S100-positive terminal nerve branches appeared more widespread and LHRH-ir neurons and processes were visible at many rostral-caudal positions along the S100-ir fibers (Fig. 2D). Observations at higher magnification confirmed the consistent juxtaposition between LHRH-ir cell bodies and processes and S100-ir terminal nerve branches. At P20, S100-immunopositive labelling was still present along terminal nerve fibers in the olfactory bulbs and telencephalon. In addition cells which resembled astrocytes were immunoreactive for S100 at this age (Fig. 3). In adult Monodelphis S100-ir was found predominantly in astrocyte-like cells, but was not observed along the terminal nerve. 3.3. Other targets By P5, S100-positive extensions of the terminal nerve could be seen coursing ventro-laterally from the third ventricle and splaying into the preoptic area (POA) in a triangular array (Fig. 4A, B). From P5 to P20 LHRH-ir processes were seen in close apposition to the S100-ir
Fig. 5. S100-ircells of the olfactorybulb and terminal nerveare also immunoreactivetor GFAP.A: P10: sagittal sectionshows dark S100-irin the onl and along the centrallyprojectingterminal nerve(arrowhead).B: adjacentsectionrevealslight GFAP-irin the oni, ob, and centrallyprojectingterminal nerve (arrowheads). Scale bar = 100 /xm.
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terminal nerve branches of the POA, and to extend along the ventral surface of the brain (Fig. 4B). S100-immunoreactivity was also observed along the ventral aspect of the median eminence at P10 (Fig. 4C,D) and P20 (data not shown). Dark S100-ir could be seen along the ventral median eminence. This dense immunoreactive labeling was associated with numerous LHRH-ir processes. In contrast, only light S100-ir was visible in the hypophyseal stalk by P10; and likewise only a few LHRH-ir fibers were observed in the region of S100 labeling. (Fig. 4D). Neither S100-ir nor LHRH-ir fibers were present in the median eminence or hypophyseal stalk at P5. Light S100-ir was also observed in the optic tract along the lateral aspect of the diencephalon at P5, P10, and P20 (data not shown). No LHRH-ir cell bodies or fibers were observed in the eye, otic nerve, or optic chiasm at any age examined. S100-ir was also found along the developing hippocampus and fimbria at P5, P10, and P20. These fibers were found along S100-ir cells which bordered the developing dentate gyrus and fimbria (data not shown).
3.4. GFAP immunoreactivity Staining alternate sections for GFAP and S100 revealed that the terminal nerve is also immunoreactive for GFAP at P5, P10, and P20. At P10, for example, sagittal sections demonstrated dark immunoreactivity for S100 in the olfactory nerve layer and along the centrally projecting branches of the terminal nerve (Fig. 5A). An adjacent section stained for GFAP revealed similar, although less distinct, immunoreactive product in the same locations (Fig. 5B).
3.5. Preabsorption control experiments Preabsorption with SlOOa/3 and S100/3/3 resulted in no immunoreactive product (Fig. 6C,D), whereas preabsorption with S l O 0 a a resulted in positive immunoreactivity that was not discernable from staining observed in adjacent sections which were processed with S100 antiserum that had not been preabsorbed (Fig. 6A,B). Immunoreactivity for S100 in the S100aa-preabsorbed sections was ob-
Fig. 6. Preabsorption control experiments reveal that S100-ir is specific for the glial form of the S100 protein. A: P6: sagittal section that was incubated in primary antiserum preabsorbed with the neuronal form of the S100 protein (S100aa) at 10 /zg per ml of antiserum. B: adjacent section which was processed with S100 primary antiserum that was not preabsorbed. Dark S100-ir appears identical in both sections in the olfactory nerve layer (onl) and along the centrally projecting branches of the terminal nerve (arrows). Scale bar = 200 ~m C: PT: sagittal section that was treated with primary antiserum preabsorbed with one of the glial forms of the S100 protein (S100flfl) at 10 /.~gper ml of antiserum. No positive staining is present in any of the regions which normally exhibit immunoreactivityfor S100. D: adjacent section, exposed to S100 primary antiserum which was not preabsorbed, reveals S100-ir in the onl and along the centrally projecting branches of the terminal nerve (arrowhead). Scale bar = 200 ~m. OB = olfactory bulb.
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served in the same locations as found previously, e.g., the olfactory nerve layer and along the branches of the terminal nerve.
4. Discussion
The present study demonstrates that S100, a neurotrophic factor commonly found in astroglia and Schwann cells, is expressed by cells of the terminal nerve during early development in Monodelphis. In the periphery, S100-ir was observed in and around the vomeronasal organ, along the olfactory, vomeronasal, and terminal nerves, and in the terminal nerve ganglion. Centrally, S100 was expressed in the olfactory nerve layer of the olfactory bulbs and by the bilateral branches of the terminal nerve coursing through septal and preoptic areas. Double-labelling experiments revealed a consistent juxtaposition between S100-ir and LHRH-immunopositive cell bodies and fibers, suggesting that S100 may be involved in the migration of LHRH-containing neurons from the olfactory placode to the forebrain. S100 may also play a role in regions which do not contain LHRH-ir perikarya but do contain LHRH-ir fibers, e.g., the preoptic area and median eminence. 4.1. $100 may act as a neurotrophic factor Although $100 has often been used as a marker for glial cells, its role as a guidance molecule in the developing central nervous system has only begun to be explored. Recent studies have shown that S100 is present during the formation of several brain areas, including the visual cortex [16,31] and midline raphe [57]. In vitro experiments indicate that the /3 subunit of the S100 protein, found in S100 proteins of astroglia and Schwann cells, stimulates neurite extension in cortical [26], dorsal root ganglia [56] and motor [5] neurons. S100/3 also influences glial cell morphology and proliferation [49,50]. Thus, S100 proteins which contain the /3 subunit are believed to serve as a neurotrophic factor for neurons, while regulating proliferation and perhaps other activities in glia. The preabsorption control experiments conducted in the present study suggest that the S100/3 subunit is present along the terminal nerve in developing Monodelphis. Preabsorption with the two glial-specific proteins, S100a/3 and S100fl/3, did block S100 immunoreactivity while preabsorption with the neuronal-specific form, S100aa, failed to block immunopositive staining. This finding suggests that the glial-specific S100/3 subunit is present along the terminal nerve; thus the S100 protein expressed along this nerve may serve in a neurotrophic capacity for migrating LHRH-containing cells and processes. Previous studies suggest mechanisms by which S100 may affect cells at the intracellular level. $100 is released into the extracellular space [51] and may be taken up into
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target cells by binding to membrane receptors or by passing through the lipid bilayer. Once inside a cell, S100 appears to affect the cytoskeleton [25]. For example, in vivo, S100 promotes the disassembly and inhibits the assembly of brain microtubules [10,12,17]. $100 also appears to influence the intracellular workings of a cell via calcium-dependent second messenger systems. For example, the S100/3 subunit stimulates increases in intracellular calcium in both glial and neuronal cells [4]. Thus, S100 may affect neurons and glia through several pathways. 4.2. Is $100 involved in LHRH-containing cell migration and fiber outgrowth ? The spatial and temporal expression of S100 is consistent with the possibility that the molecule may play a role in guiding the migration of LHRH-containing neurons. First, Sl00-ir was consistently found along the terminal nerve in close apposition to LHRH-containing cell bodies and in areas containing outgrowing and/or terminating LHRH-ir fibers. As far as we know, S100 is the only protein that demonstrates such continuous association with LHRH-containing fibers. Second, the timing of S100 expression during development supports our hypothesis that this molecule serves in a guidance capacity. At P0 and P5, S100-ir was limited to nerve fascicles in the nasal cavity, the olfactory nerve layer, and the terminal nerve; and LHRH-ir cells and processes were not seen beyond the caudal extension of this nerve. By P10, when LHRH-ir fibers were first observed in the median eminence and hypophyseal stalk, S100-ir was found in these regions. In adulthood, when LHRH migration is complete, S100-ir could no longer be found along the terminal nerve and was only observed in cells that resembled astroglia. Thus, the expression of S100-ir in Monodelphis coincides both temporally and spatially with LHRH-containing cell migration and fiber elongation. 4.3. SlO0-positive cells along the terminal nerve may be ensheathing cells Immunohistochemical studies indicate that the $100 protein is expressed by ensheathing cells of the olfactory epithelium [52,61]. These cells surround and insulate bundles of olfactory and vomeronasal nerves and form the glial limitans at their entrance to the olfactory bulbs [13,39]. Ensheathing cells exhibit characteristics of both astroglia and Schwann cells [3]. For example, the cells contain GFAP [2] and exhibit morphological features found in astroglia [3,14,29]. Unlike astrocytes, however, ensheathing cells do not originate in the neural tube; instead they are derived from precursor cells in the olfactory placode [7,8,13,19]. Controversy exists regarding how far ensheathing cells extend into the CNS. Some investigators report having observed the cells deep within the glomerular layer of the
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olfactory bulbs [29,54]. In fact Valverde and colleagues [55] suggest that ensheathing cells play a vital role in delineating olfactory bulb glomeruli, much like the role of antennal lobe glia in the olfactory lobe of Manduca sexta (see [53] for review). In contrast, others propose that ensheathing cells do not enter the CNS [15,39]. The cells observed in the present study expressed the glial form of the S100 protein and were also GFAP-posirive, and therefore may have been ensheathing ceils. At all developmental timepoints examined, S100-ir cells were observed in the olfactory nerve layer of the olfactory bulbs. The fact that the labeling did not extend into deeper bulb layers supports the suggestion that ensheathing cells are prohibited from entering the CNS region of the olfactory bulbs, findings consistent with those reported elsewhere [28]. Our observations of S100-ir cells along the terminal nerve, however, indicate that an S100-ir glial cell type exists along both its peripheral and central projections during development. Thus, if the S100-ir cells of the terminal nerve are in fact ensheathing cells, they may extend far into other CNS regions. Analysis at the electron microscopic level is warranted to further determine the S100-ir cell type associated with the terminal nerve. 4.4. Conclusion
In recent years, an increasing number of proteins have been shown to exert neurotropic effects in the nervous system. Studies have also indicated that neuronal migration and axon guidande may be influenced by multiple factors (see [9] for review). Often neurotropic factors are expressed only during critical periods of development. The S100 protein appears to be expressed by glial cells of the terminal nerve during the time that LHRH-containing neurons migrate from the vomeronasal organ into the developing brain and extend their processes. The consistent and close apposition between S100-ir and migrating LHRHcontaining neurons and their processes suggests a role for this glial-associated molecule during LHRH migration along the terminal nerve. Acknowledgements This work was supported by grants from the National Institutes of Health (DC-00338) and National Sciences Foundation (BNS-8919751). We would like to thank Dr. Katsumi Wakabayashi for the generous contribution of the LRH13 antibody. Thanks to Drs. Christine Byrd, Tammy Dellovade, and Dagmar Malun for their helpful comments on the manuscripts. References [1] Akutsu, S., Takada, M., Ohki-Hamazaki, H., Murakami, S. and Arai, Y., Origin of luteinizing hormone-releasing hormone (LHRH) neu-
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Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) in the brain and nervus terminalis of the adult and early neonatal gray short-tailed opossum (Monodelphis domestica), J. Comp. Neurol,, 276 (1988) 44-60. [45] Schwanzel-Fukuda, M., Morrell, J.l. and Pfaff, D.W., Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat, J. Comp. Neurol., 238 (1985) 348-364. [46] Schwanzel-Fukuda, M. and Pfaff, D.W., Origin of luteinizing hormone-releasing hormone neurons, Nature, 338 (1989) 161-164. [47] Schwanzel-Fukuda, M., Pfaff, D.W., Bouloux, P.M.G., Hardelin, J.-P. and Petit, C,, Migration of LHRH neurons in early human embryos: Association with neural cell adhesion molecules, Soc. Neurosci. Abstr., 20 (1994) 1491 (614.9). [48] Schwanzel-Fukuda, M., Reinhard, G.R., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W., Antibody to neural cell adhesion molecule can disrupt the migration of luteinizing hormone-releasing hormone neurons into the mouse brain, J. Comp. NeuroL, 342 (1994) 174-185. [49] Selinfreund, H., Barger, S.W., Pledger, W.J. and Van Eldik, UJ., Neurotrophic protein S100fl stimulates glial cells proliferation, Proc. Natl. Acad. Sci., 88 (1991) 3554-3558. [50] Selinfreund, R.H., Barger, S.W., Welsh, M.J. and Van Eldik, L.J., Antisense inhibition of glial S100/3 production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation, J. Cell Biol., 111 (1990)2021-2028. [51] Shashoua, V.E., Hesse, G.W. and Moore, B.W., Proteins of the brain extracellular fluid: evidence for release of S-100 protein, J. Neurochem., 42 (1984) 1536-1541. [52] Takahashi, S., Iwanaga, T., Takahashi, Y., Nakano, Y. and Fujita, T., Neuron-specific enolase, neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses. An immunohistochemical study, Cell, Tissue Res., 238 (1984) 231-234. [53] Tolbert, L.P. and Oland, L.A., Glial cells form boundaries for developing insect olfactory glomeruli, Exp. Neurol., 109 (1990) 19-28. [54] Valverde, F. and Lopez-Mascaraque, L., Neuroglial arrangements in the olfactory glomeruli of the hedgehog, J. Comp. NeuroL, 307 (1991) 658-674. [55] Valverde, F., Santacana, M. and Heredia, M., Formation of an olfactory glomerulus: morphological aspects of development and organization, Neuroscience, 49 (1992) 255-275. [56] Van Eldik, L.J., Christie-Pope, B., Bolin, L.M., Shooter, E.M. and Whetsell, W.O., Neurotrophic activity of S-100/3 in cultures of dorsal root ganglia from embryonic chick and fetal rat, Brain Res., 542 (1991) 280-285. [57] Van Hartesveldt, C., Moore, B. and Hartman, B.K., Transient midline raphe glial structure in the developing rat, J. Comp. NeuroL, 253 (1986) 175-184. [58] Wray, S., Grant, P. and Gainer, H., Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode, Proc. Natl. Acad. Sci., 86 (1989) 8132-8136. [59] Wray, S., Key, S., Qualls, R. and Fueshko, S.M, A subset of peripherin positive olfactory axons delineates the luteinizing hormone releasing hormone neuronal migratory pathway in developing mouse, Dev. Biol., 166 (1994) 349-354. [60] Wray, S., Nieburgs, A. and Elkabes, S., Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev. Brain Res., 46 (1989) 309-318. [61] Yamagishi, M., Hasegawa, S., Takahashi, S., Nakano, T. and lwanaga, T., Immunohistochemical analysis of the olfactory mucosa by use of antibodies to brain proteins and cytokeratin, Ann. Otol, Rhinol. Laryngol., 98 (1989) 384-388.
ELSEVIER
Developmental Brain Research 88 (1995) 148-157
Research report
Migrating luteinizing hormone-releasing hormone (LHRH) neurons and processes are associated with a substrate that expresses SIO0 Diana M. Cummings, Peter C. Brunjes
*
Neuroscience Program and Department of Psychology, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA Accepted 16 May 1995
Abstract
Luteinizing hormone-releasing hormone (LHRH) containing neurons arise in the region of the medial olfactory placode and migrate into the developing olfactory bulbs and basal forebrain along branches of the terminal and vomeronasal nerves. The neurons ultimately come to reside in olfactory and septo-preoptic areas and project extensively to several brain regions, including the preoptic area and median eminence. The present study examined the expression of a glial-associated guidance molecule, S100, as a possible substrate for this migration. Monodelphis domestica (the Brazilian grey, short-tailed opossum) was studied since this species gives birth to very immature young, allowing access to early periods of mammalian forebrain development. Immunoreactivity for both S100 and LHRH-containing neurons and fibers was observed to be closely associated along the entire LHRH migratory route from the vomeronasal organ to the septo-preoptic area as early as the day of birth (PO). By P10, S100-immunoreactivity was also seen in areas containing LHRH-immunoreactive fibers such as the preoptic area and median eminence. We suggest that S100, a protein with neurotrophic properties in vitro, acts as a guidance molecule for migrating LHRH-immunoreactive neurons and elongating processes. Keywords: S100; Glia; Ensheathing cell; LHRH neuron; Olfactory bulb; Development; Cell migration; Immunofluorescence
1. Introduction
Luteinizing hormone-releasing hormone (LHRH) containing neurons and processes play a critical role in the functioning of the hypogonadal axis and in many behaviors associated with vertebrate reproduction. The LHRHcontaining neurons thought to be most actively involved in regulating reproductive behaviors are widely distributed throughout the olfactory system and basal forebrain (see [35] for review). In adult mammals, for example, they are found in regions such as the terminal nerves and ganglia [43,45], main and accessory olfactory bulbs, diagonal band, septum, preoptic area, anterior hypothalamus, and piriform cortex [22,23]. LHRH-containing fibers project extensively throughout the brain and terminate in far-reaching areas, including the median eminence and hypophyseal stalk. LHRH-containing neurons arise in the region of the medial olfactory placode and migrate along branches of
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the terminal and vomeronasal nerves to destinations in the basal forebrain [46,58,60]. This migratory pattern has since been demonstrated in a variety of species including primates [40], chicks [1,32,37], and newts [33], and may also exist in humans [6,47]. Migration of LHRH-containing neurons seems critical for development of the gonadotropin system, since incomplete or disrupted migration of LHRH-containing cells appears to result in the human hypogonadal disorder known as Kalimann's syndrome [42]. Patients with this disorder also suffer from anosmia [24]. Since LHRH-containing cells and processes travel such great distances to reach their targets, possible mechanisms of guidance are currently under investigation. For example, neural cell adhesion molecule (NCAM) is present in the cells and axons of the terminal and vomeronasal nerves which form the substrate for migrating LHRH-containing neurons [21,34,36,41]. Further support of NCAM's role as a guidance molecule has emerged from evidence that perturbation of the NCAM-positive substrate disrupts the migration of LHRH-immunoreactive (ir) cells from the medial olfactory pit of embryonic mice [48]. Another putative guidance factor, peripherin, is a neuron-specific
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intermediate filament [27]. Peripherin is found in olfactory receptor neurons [20] and also appears to be expressed along the LHRH migratory route in embryonic mice [59]. S100 is another central nervous system (CNS) protein which is increasingly becoming recognized as a neurotrophic factor. S100 is at least 10,000-fold more concentrated in the brain than in other tissues [30] and exists in three dimeric forms comprised of alpha and beta subunits: S100aot, S100otfl, and S100/3fl (for reviews see [11,25]). Research in vivo indicates that during development S100 proteins are localized to regions in which they may serve as molecular guides, e.g., chick motor and sensory neurons [5], astrocytes of kitten visual cortex [16,31], and radial glia of the midline raphe [57]. In addition, this protein promotes survival of motor neurons in the chick hindlimb [5]. Recently, S100-immunoreactive cells were found in the vomeronasal organ (VNO) and along the nasal septum of human fetuses and appear localized to regions of migrating LHRH-containing cells [6]. The present study examines the expression of S100 immunoreactivity in relation to migrating LHRH-containing neurons throughout the brain in a developing marsupial, Monodelphis domestica. Monodelphis domestica, the Brazilian grey, short-tailed opossum, is an appropriate species for studying the early maturation of the mammalian brain since offspring are born in a very immature state after only 14 days of gestation. The migration of LHRH-containing neurons has been well characterized in postnatal Monodelphis [44]. Migrating LHRH-containing neurons appear to follow a distinct path from the olfactory-vomeronasal region to the brain. While this migratory route is consistent across many species, Monodelphis is unique in that the terminal nerve is very thick and easily delineated in its projection from the ganglia to the medial septum and preoptic area [44]. Furthermore, unlike rats and mice, LHRH-containing cells appear to migrate into the brain well into the postnatal period in Monodelphis [44]; thus, this species is ideal for studying possible guidance cues. We conducted double-labelling experiments in early postnatal Monodelphis to investigate the localization of immunoreactivity for S100 in relation to LHRH-immunoreactive (ir) neurons and processes. In addition, we investigated the distribution of immunoreactivity for another glial-associated protein, glial fibrillary acidic protein (GFAP), in the brains of developing Monodelphis. Our studies indicate that S100 is present in terminal nerve fibers of developing Monodelphis and that these fibers co-localize with LHRH-containing neurons. This co-localization was observed in the nasal epithelium, olfactory bulb and forebrain, and was also seen in more caudal regions known to contain LHRH-ir fibers, such as the preoptic area and median eminence. 2. Materials and methods
Adult Monodelphisdomestica were purchased from the Southwest Foundation for Biomedical Research (San Anto-
149
nio, TX) and maintained for breeding according to the guidelines of Fadem and colleagues [18]. Females were checked daily for litters with the day of birth considered postnatal Day 0 (P0).
2.1. lmmunohistochemistry P0, P5, P10, P20, and adult (n's = 4-5 at each age) animals were given an overdose of barbiturate anesthetics and perfused transcardially with ice-cold 0.1 M phosphate buffer with 0.9% saline (PBS, pH 7.4) followed by 4% paraformaldehyde (pH 7.4). Brains were removed, postfixed in 4% paraformaldehyde with 10% sucrose for 1-4 h, and incubated in 0.1 M PBS with 30% sucrose at 4°C. Due to their small size, P0 and P5 brains were left in the skull, and the whole head was processed for immunohistochemistry. Sagittal sections (10-12 /xm thick) were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and placed under vacuum for 6-8 h at 4°C. Slides were then rinsed 4 times in PBS and incubated in 3% H202 for 15 min at 4°C to block endogenous peroxidases. Afterwards, non-specific binding sites were saturated by immersing sections in 10% normal goat serum (Vector) for 1 hour at 4°C. Sections were then transferred to polyclonal rabbit anti-S100 antibody (S100, DAKO, 1:2000) overnight at 4°C. In additional animals, alternate sections were taken at P5, P10 and P20 (n's---3) and incubated in polyclonal rabbit anti-GFAP antibody (DAKO, 1:5000). To further reduce nonspecific binding and increase penetration, all primary and secondary antisera and the ABC solutions were diluted in PBS containing 0.25% lambda carrageenan and 0.35% Triton X-100. After incubation in primary antisera, slides were washed 4 times in PBS and placed in solutions containing biotinylated secondary antibodies (DAKO, 1:100) for 1 h at room temperature, rinsed in PBS, and transferred to avidin-biotin solution (ABC; Vectastain Elite, Vector) for 90 min. After rinsing slides 3 times in PBS and once in Tris-buffered saline, sections were treated with diaminobenzidine (DAB) in the presence of H202 for 10-15 min until peroxidase labeling was fully visualized. Slides were then rinsed 3 times in phosphate buffer (PB), dehydrated, cleared and coverslipped with DPX (BDH Ltd., Poole UK).
2.2. Double-labeling At each developmental age examined (e.g., P0, P5, P10, and P20) sections from 4 to 5 pups were processed for double-labeling experiments. Slides were incubated in primary antisera containing polyclonal anti-S100 (S100, DAKO, 1:2000) and monoclonal anti-LHRH (LRH13, generously provided by Dr. Wakabayashi, Gunma University, Maebashi, Japan, 1:2000) for 48 h at 4°C. LRH13 has been demonstrated to have a high specificity and crossreactivity for LHRH in several species [38]. Slides were rinsed 5 times in PBS, placed into biotinylated goat anti-
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Fig. 1. $100 (dark DAB reaction product) and LHRH (fluorescent labeling appears bright white) immunoreactivity in the vomeronasal and olfactory systems of developing Monodelphis. Note: S100-ir was also expressed in the craniofacial cartilage. A: P5: coronal section through the nose and brain viewed under UV light reveals LHRH-ir cells (small arrows) extending from the vomeronasal organ (VNO; larger arrow) to the terminal nerve ganglion (g). A dense cluster of S100-ir cells (arrowhead) lies dorsal to the VNO and in close apposition to a group of LHRH-ir cells. Smaller groups of S100-ir cells are found along the nasal septum (s), often associated with LHRH-ir cells. B: P10~ sagittal section (rostral to left) through the nose visualized under UV light shows an LHRH-ir cell body (arrowhead) and numerous LHRH-ir fibers (arrows) along S100-ir olfactory and vomeronasal fascicles entering the olfactory nerve layer (onl). ob, olfactory bulb; oe, olfactory epithelium. Scale bars = 100 p~m.
D.M. Cummings, P.C. Brunjes /Developmental Brain Research 88 (1995) 148-157
rabbit secondary for 1 h at room temperature, and processed for ABC-DAB visualization of S100 immunoreactivity as described above. Sections were then rinsed 4 times in PBS and incubated in aminomethylcoumarin acetate (AMCA)-conjugated goat anti-mouse IgG (Jackson,
151
1:100) for 2 h at room temperature. Afterwards, slides were rinsed 4 times in PB and coverslipped with glycerol gelatin (Sigma). Both the DAB reaction product which labeled S100-ir and the fluorescence associated with LHRH-ir could be viewed simultaneously with a fluores-
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Fig. 2. S100-ir is co-localized with LHRH-ir cells and processes along the central projection of the terminal nerve. In A - D arrowheads point to the S100-ir centrally projecting branches of the terminal nerve and small arrows point to LHRH-ir cell bodies and processes. In all photomicrographs rostral is at left. A: P0: sagittal section (brightfield) reveals S100-ir cells in the terminal nerve ganglion (g) and along its centrally projecting branches. B: fluorescence photomicrograph of the same section as in A shows LHRH-ir cells and their processes within the terminal nerve ganglion and along its centrally projecting branches. C: P5: fluorescence photomicrograph of a sagittal section of the olfactory bulb and forebrain shows S100-ir in the olfactory nerve layer and along the central branches of the terminal nerve. LHRH-ir cells and processes are positioned along the terminal nerve branches which turn ventrally in the region of the third ventricle (11 lv). D: P10: fluorescence photomicrograph of a sagittal section demonstrates LHRH-ir cells along scattered S 100-ir terminal nerve branches. E: Higher magnification of bracketed region in D shows LHRH-ir processes (large arrows) closely associated with S100-ir terminal nerve branches, oa, olfactory bulb anlage. Scale bars = 100 # m in A - D , 50 /.Lm in E.
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D.M. Cummings, P.C. Brunjes/ Developmental Brain Research 88 (1995) 148-157
cence microscope using the ultraviolet filter set. In photomicrographs (e.g. Figs. 1 and 2, 4) the DAB reaction product appeared as a dark precipitate while the fluorescent labeling appeared bright white.
2.3. Control experiments
Fig. 3. By P20 S100-ir can be seen in astrocyte-like cells. This brightfield photomicrograph shows a central projection of the terminal nerve (arrow) which projects toward the third ventricle (lily). Dark S100-ir is seen along this nerve projection and in neighboring cells (arrowheads) which resemble astrocytes. Scale bar = 100 p,m.
Control experiments in which the S100 or LRH13 antiserum were omitted revealed no positive staining. In addition preabsorption control experiments were conducted to further verify the S100 immunoreactivity and to determine which of the different forms of the S100 protein exists along the terminal nerve in Monodelphis. In these experiments the S100 antiserum was preabsorbed with an excess of one of the three S100 proteins, e.g., S100aa, S100a/3, or S100/3/3 (Sigma), at either 10 or 20 /.~g per ml of antiserum. After 24 h of preabsorption, the antiserum was used as described above for single-labelling.
Fig. 4. S100-ir is associated with LHRH-ir fibers in the preoptic area and median eminence. A: P5: coronal section (brightfield) shows S100-ir along the terminal nerve branches (arrowheads) in the POA. B: fluorescence photomicrograph of the same section as in A reveals LHRH-ir fibers (arrows) cascading ventrally toward the base of the brain in association with the S100-ir terminal nerve branches. C: P10: sagittal section (brightfield) reveals Sl00-ir along the ventral surface of the median eminence (arrowhead). Light S100-ir is also visible in the hypophyseal stalk (s) and in the posterior hypothalamic area (ph). D: fluorescence photomicrograph of the same section as in C. Many LHRH-ir fibers (arrowhead) are found along the S100-ir of the median eminence, and a few fibers (arrow) were seen along the hypophyseal stalk near S100-ir. Scale bars = 100 /.~m.
D.M. Cummings,P.C. Brunjes/DevelopmentalBrain Research 88 (1995) 148-157
3. Results 3.1. Vomeronasal organ and nasal cavity LHRH-ir cells and processes were found associated with S100-positive cells at all points along their migratory route from the VNO to the olfactory bulb. At the earliest ages examined (P0 and P5), LHRH-ir neurons were observed both within and outside of the VNO (Fig. 1A). These cells were closely associated with S100-ir cells observed both in and around the VNO and in a distinct cluster just dorsal to this structure (arrowhead, Fig. 1A). The primary ganglion of the terminal nerve contained many S100-ir cells as well as LHRH-ir neurons (Fig. 1A). In P10 pups, S100-ir appeared denser along the edges of nerve fascicles. LHRH-ir cell bodies and fibers were in close apposition to the dark S100-ir nerve fibers as they approached the olfactory bulb (Fig. 1B). 3.2. Olfactory bulbs and forebrain At P0, S100-ir cells of the terminal nerve ganglion were seen ventral to the anlage of the olfactory bulbs (Fig. 2A, B). Sagittal sections revealed S100-positive processes extending bilaterally from these ganglia toward the third ventricle along the medial walls of the developing telencephalon. LHRH-ir neurons and processes were seen in the ganglion of the terminal nerve and in close apposition to its centrally-projecting branches (Fig. 2B). LHRH-immunopositive cells were often found in clusters while leading processes were observed running along the S100-ir terminal nerve branches as they arched through the developing telencephalon (Fig. 2B). The olfactory nerve layer (onl) of the olfactory bulb was S100-ir at all ages examined, although developmental increases in staining density were observed. At P0 light S100-ir could be seen around the olfactory bulb anlage (Fig. 1A,B). By later ages, e.g.,
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P5-P20, S100-ir in the onl was very dense (Fig. 1A,B, Fig. 2C,D). At P5 more S100-ir terminal nerve fibers could be seen coursing dorsocaudally over the ventromedial telencephalon (Fig. 2C). Just rostral to the third ventricle many S100-ir fibers turned ventrally into the subfornical organ or extended along the lamina terminalis, rostral to the third ventricle. Many other S100-positive fibers extended radially into the ventral telencephalon. LHRH-ir perikarya and processes were found in the same locations identified at earlier ages, along the S100-positive terminal nerve branches projecting toward the third ventricle (Fig. 2C). LHRH-ir fibers were found along S100-ir terminal nerve branches that either arched ventrally just rostral to the third ventricle or projected radially into the telencephalon. By P10, the S100-positive terminal nerve branches appeared more widespread and LHRH-ir neurons and processes were visible at many rostral-caudal positions along the S100-ir fibers (Fig. 2D). Observations at higher magnification confirmed the consistent juxtaposition between LHRH-ir cell bodies and processes and S100-ir terminal nerve branches. At P20, S100-immunopositive labelling was still present along terminal nerve fibers in the olfactory bulbs and telencephalon. In addition cells which resembled astrocytes were immunoreactive for S100 at this age (Fig. 3). In adult Monodelphis S100-ir was found predominantly in astrocyte-like cells, but was not observed along the terminal nerve. 3.3. Other targets By P5, S100-positive extensions of the terminal nerve could be seen coursing ventro-laterally from the third ventricle and splaying into the preoptic area (POA) in a triangular array (Fig. 4A, B). From P5 to P20 LHRH-ir processes were seen in close apposition to the S100-ir
Fig. 5. S100-ircells of the olfactorybulb and terminal nerveare also immunoreactivetor GFAP.A: P10: sagittal sectionshows dark S100-irin the onl and along the centrallyprojectingterminal nerve(arrowhead).B: adjacentsectionrevealslight GFAP-irin the oni, ob, and centrallyprojectingterminal nerve (arrowheads). Scale bar = 100 /xm.
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terminal nerve branches of the POA, and to extend along the ventral surface of the brain (Fig. 4B). S100-immunoreactivity was also observed along the ventral aspect of the median eminence at P10 (Fig. 4C,D) and P20 (data not shown). Dark S100-ir could be seen along the ventral median eminence. This dense immunoreactive labeling was associated with numerous LHRH-ir processes. In contrast, only light S100-ir was visible in the hypophyseal stalk by P10; and likewise only a few LHRH-ir fibers were observed in the region of S100 labeling. (Fig. 4D). Neither S100-ir nor LHRH-ir fibers were present in the median eminence or hypophyseal stalk at P5. Light S100-ir was also observed in the optic tract along the lateral aspect of the diencephalon at P5, P10, and P20 (data not shown). No LHRH-ir cell bodies or fibers were observed in the eye, otic nerve, or optic chiasm at any age examined. S100-ir was also found along the developing hippocampus and fimbria at P5, P10, and P20. These fibers were found along S100-ir cells which bordered the developing dentate gyrus and fimbria (data not shown).
3.4. GFAP immunoreactivity Staining alternate sections for GFAP and S100 revealed that the terminal nerve is also immunoreactive for GFAP at P5, P10, and P20. At P10, for example, sagittal sections demonstrated dark immunoreactivity for S100 in the olfactory nerve layer and along the centrally projecting branches of the terminal nerve (Fig. 5A). An adjacent section stained for GFAP revealed similar, although less distinct, immunoreactive product in the same locations (Fig. 5B).
3.5. Preabsorption control experiments Preabsorption with SlOOa/3 and S100/3/3 resulted in no immunoreactive product (Fig. 6C,D), whereas preabsorption with S l O 0 a a resulted in positive immunoreactivity that was not discernable from staining observed in adjacent sections which were processed with S100 antiserum that had not been preabsorbed (Fig. 6A,B). Immunoreactivity for S100 in the S100aa-preabsorbed sections was ob-
Fig. 6. Preabsorption control experiments reveal that S100-ir is specific for the glial form of the S100 protein. A: P6: sagittal section that was incubated in primary antiserum preabsorbed with the neuronal form of the S100 protein (S100aa) at 10 /zg per ml of antiserum. B: adjacent section which was processed with S100 primary antiserum that was not preabsorbed. Dark S100-ir appears identical in both sections in the olfactory nerve layer (onl) and along the centrally projecting branches of the terminal nerve (arrows). Scale bar = 200 ~m C: PT: sagittal section that was treated with primary antiserum preabsorbed with one of the glial forms of the S100 protein (S100flfl) at 10 /.~gper ml of antiserum. No positive staining is present in any of the regions which normally exhibit immunoreactivityfor S100. D: adjacent section, exposed to S100 primary antiserum which was not preabsorbed, reveals S100-ir in the onl and along the centrally projecting branches of the terminal nerve (arrowhead). Scale bar = 200 ~m. OB = olfactory bulb.
D.M. Cummings, P.C. Brunjes /Developmental Brain Research 88 (1995) 148-157
served in the same locations as found previously, e.g., the olfactory nerve layer and along the branches of the terminal nerve.
4. Discussion
The present study demonstrates that S100, a neurotrophic factor commonly found in astroglia and Schwann cells, is expressed by cells of the terminal nerve during early development in Monodelphis. In the periphery, S100-ir was observed in and around the vomeronasal organ, along the olfactory, vomeronasal, and terminal nerves, and in the terminal nerve ganglion. Centrally, S100 was expressed in the olfactory nerve layer of the olfactory bulbs and by the bilateral branches of the terminal nerve coursing through septal and preoptic areas. Double-labelling experiments revealed a consistent juxtaposition between S100-ir and LHRH-immunopositive cell bodies and fibers, suggesting that S100 may be involved in the migration of LHRH-containing neurons from the olfactory placode to the forebrain. S100 may also play a role in regions which do not contain LHRH-ir perikarya but do contain LHRH-ir fibers, e.g., the preoptic area and median eminence. 4.1. $100 may act as a neurotrophic factor Although $100 has often been used as a marker for glial cells, its role as a guidance molecule in the developing central nervous system has only begun to be explored. Recent studies have shown that S100 is present during the formation of several brain areas, including the visual cortex [16,31] and midline raphe [57]. In vitro experiments indicate that the /3 subunit of the S100 protein, found in S100 proteins of astroglia and Schwann cells, stimulates neurite extension in cortical [26], dorsal root ganglia [56] and motor [5] neurons. S100/3 also influences glial cell morphology and proliferation [49,50]. Thus, S100 proteins which contain the /3 subunit are believed to serve as a neurotrophic factor for neurons, while regulating proliferation and perhaps other activities in glia. The preabsorption control experiments conducted in the present study suggest that the S100/3 subunit is present along the terminal nerve in developing Monodelphis. Preabsorption with the two glial-specific proteins, S100a/3 and S100fl/3, did block S100 immunoreactivity while preabsorption with the neuronal-specific form, S100aa, failed to block immunopositive staining. This finding suggests that the glial-specific S100/3 subunit is present along the terminal nerve; thus the S100 protein expressed along this nerve may serve in a neurotrophic capacity for migrating LHRH-containing cells and processes. Previous studies suggest mechanisms by which S100 may affect cells at the intracellular level. $100 is released into the extracellular space [51] and may be taken up into
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target cells by binding to membrane receptors or by passing through the lipid bilayer. Once inside a cell, S100 appears to affect the cytoskeleton [25]. For example, in vivo, S100 promotes the disassembly and inhibits the assembly of brain microtubules [10,12,17]. $100 also appears to influence the intracellular workings of a cell via calcium-dependent second messenger systems. For example, the S100/3 subunit stimulates increases in intracellular calcium in both glial and neuronal cells [4]. Thus, S100 may affect neurons and glia through several pathways. 4.2. Is $100 involved in LHRH-containing cell migration and fiber outgrowth ? The spatial and temporal expression of S100 is consistent with the possibility that the molecule may play a role in guiding the migration of LHRH-containing neurons. First, Sl00-ir was consistently found along the terminal nerve in close apposition to LHRH-containing cell bodies and in areas containing outgrowing and/or terminating LHRH-ir fibers. As far as we know, S100 is the only protein that demonstrates such continuous association with LHRH-containing fibers. Second, the timing of S100 expression during development supports our hypothesis that this molecule serves in a guidance capacity. At P0 and P5, S100-ir was limited to nerve fascicles in the nasal cavity, the olfactory nerve layer, and the terminal nerve; and LHRH-ir cells and processes were not seen beyond the caudal extension of this nerve. By P10, when LHRH-ir fibers were first observed in the median eminence and hypophyseal stalk, S100-ir was found in these regions. In adulthood, when LHRH migration is complete, S100-ir could no longer be found along the terminal nerve and was only observed in cells that resembled astroglia. Thus, the expression of S100-ir in Monodelphis coincides both temporally and spatially with LHRH-containing cell migration and fiber elongation. 4.3. SlO0-positive cells along the terminal nerve may be ensheathing cells Immunohistochemical studies indicate that the $100 protein is expressed by ensheathing cells of the olfactory epithelium [52,61]. These cells surround and insulate bundles of olfactory and vomeronasal nerves and form the glial limitans at their entrance to the olfactory bulbs [13,39]. Ensheathing cells exhibit characteristics of both astroglia and Schwann cells [3]. For example, the cells contain GFAP [2] and exhibit morphological features found in astroglia [3,14,29]. Unlike astrocytes, however, ensheathing cells do not originate in the neural tube; instead they are derived from precursor cells in the olfactory placode [7,8,13,19]. Controversy exists regarding how far ensheathing cells extend into the CNS. Some investigators report having observed the cells deep within the glomerular layer of the
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olfactory bulbs [29,54]. In fact Valverde and colleagues [55] suggest that ensheathing cells play a vital role in delineating olfactory bulb glomeruli, much like the role of antennal lobe glia in the olfactory lobe of Manduca sexta (see [53] for review). In contrast, others propose that ensheathing cells do not enter the CNS [15,39]. The cells observed in the present study expressed the glial form of the S100 protein and were also GFAP-posirive, and therefore may have been ensheathing ceils. At all developmental timepoints examined, S100-ir cells were observed in the olfactory nerve layer of the olfactory bulbs. The fact that the labeling did not extend into deeper bulb layers supports the suggestion that ensheathing cells are prohibited from entering the CNS region of the olfactory bulbs, findings consistent with those reported elsewhere [28]. Our observations of S100-ir cells along the terminal nerve, however, indicate that an S100-ir glial cell type exists along both its peripheral and central projections during development. Thus, if the S100-ir cells of the terminal nerve are in fact ensheathing cells, they may extend far into other CNS regions. Analysis at the electron microscopic level is warranted to further determine the S100-ir cell type associated with the terminal nerve. 4.4. Conclusion
In recent years, an increasing number of proteins have been shown to exert neurotropic effects in the nervous system. Studies have also indicated that neuronal migration and axon guidande may be influenced by multiple factors (see [9] for review). Often neurotropic factors are expressed only during critical periods of development. The S100 protein appears to be expressed by glial cells of the terminal nerve during the time that LHRH-containing neurons migrate from the vomeronasal organ into the developing brain and extend their processes. The consistent and close apposition between S100-ir and migrating LHRHcontaining neurons and their processes suggests a role for this glial-associated molecule during LHRH migration along the terminal nerve. Acknowledgements This work was supported by grants from the National Institutes of Health (DC-00338) and National Sciences Foundation (BNS-8919751). We would like to thank Dr. Katsumi Wakabayashi for the generous contribution of the LRH13 antibody. Thanks to Drs. Christine Byrd, Tammy Dellovade, and Dagmar Malun for their helpful comments on the manuscripts. References [1] Akutsu, S., Takada, M., Ohki-Hamazaki, H., Murakami, S. and Arai, Y., Origin of luteinizing hormone-releasing hormone (LHRH) neu-
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