The olfactory bulb of an embryo (BALB/c strain mouse) was transplanted into the neocortex or hippo- campal formation in a young female adult host (AKR strain mouse) and matured for at least 1 month. Projection fibers from the transplant were demonstrated immunohistochemically using the allelic form of the Thy-1 system between both mice. Fibers from the olfactory bulb transplanted into the frontal cortex showed non-specific elongation in two directions where normally there are no targets of the olfactory bulb fibers: one was toward the cortical surface among radially oriented host fiber systems and another on the corpus callosum running rostrocaudally together with the host's long association bundle. Transplants placed in the hippocampal dentate and hilar regions emit fibers mainly towards the dentate molecular layer and these fibers spread medially and laterally within the molecular layer. The results suggest that projection neurons (mitral and tufted cells) in the transplanted olfactory bulb have the potential ability of non-target-directing axon elongation into the host nerve tissue which usually prevents the penetration of newly growing fibers.
Previous reports 3,4 have demonstrated target-directing long-distance fiber elongations from olfactory bulb (OB) transplants. However, there is still a possibility that all these projections are initially non-target-directing. The projection fibers running aimlessly or running along mechanical cues receive signals from a nearby target on the way, and finally enter there to terminate. Without these considerations, such a long-distance intracerebral fiber elongation (2-5 mm) in the young adult host brain (without host target deafferentation) cannot be explained. This study was thus carried out to determine whether transplanted OB axon elongation occurs towards the host nerve structure where there is no target at all. Successful observation of a non-target-directing axon elongation from the transplanted OB could be obtained immunohistochemically by using the mouse
Correspondence." M. Fujii, 1st Department of Anatomy, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan.
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Thy-1 system (the mouse Thy-l.1/Thy-l.2 allelic form has been known to be a valuable marking tool ~0).
BALB/c OB primordia (about 16 days of the embryonic term) was stereotaxically inserted in the deep frontal cortex or in the hippocampal formation of the septal side in the female AKR strain host (around 5 weeks old, about 20 g in body weight; 21 animals with the developed transplant were offered for immunostaining and the others with the rejected transplant were eliminated).
Thirty-one to 97 days after the operation, the host animals were sacrificed with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (for 5-10 min) under deep Nembutal or ether anesthesia. The brains were cut in a sagittal plane by vibratome (Microcut, Dousaka) at 40-~m thickness. For good sectioning, some brains were embedded with agarose of low gelling temperature.
After lipid extraction and further fixation with chilled 95% ethanol for 10 rain, every 3rd section was reacted immunohistochemically with the monoclonal antibody against Thy-l.2 (1:100, ICN ImmunoBiologicals) overnight in the refrigerator, followed by the avidin-biotin complex (ABC) method (Vector's Kit) and silver gold enhancement 5 All these procedures were carried out on free floating sections. After enhancement, the sections were mounted on a gelatin coated slide, air-dried, lightly counterstained with Cresylviolet and cover-slipped with M.G.K.-S (Matsunami). Transplanted OB projection fibers, then, became visible as silver impregnated fibers. Some sections of adjoining series were also mounted and stained with Cresylviolet alone for cytoarchitectural reference of the transplant.
Normal BALB/c brain sections for the positive control were usually processed together with the experimental ones to check the ABC procedure and silver enhancement. To eliminate non-specific staining, negative control sections without the primary antibody were similarly reacted. No reaction could be obtained in the negative control sections.
OB primordia which developed in these brains regions fused well with the host brain tissue. This could be detected from the surrounding host tissue by its characteristic granular cell and large projection cell aggregations 3,4
When a transplant was placed deep in the frontal cortex (frontal 1.5-2.0 mm from the bregma and lateral 1.0 mm from the midline) numerous non-target-directing axons from the transplant went straight in two directions, one to the cortical surface and the other on and along the corpus callosum where there are long association fibers mainly composed of fibers running rostrocaudally between the frontal and occipital cortices.
Figure 1 shows a transplant in the neocortex just anterior to the corpus callosum. Numerous axons from the transplant proceeded to the cortical surface among the surface directing apical dendrites of cortical neurons and the afferent and efferent fibers (Fig. 1A, C and D). Near the cortical surface, these axons changed their straight form and some fibers emitted collaterals. After arriving at the cortical surface, the fibers further elon- gated rostrally or caudally, waving in the molecular layer of the cortex. A few fibers again entered the cortical layers obliquely. The fibers ran within fascicles of the host vertical fiber system in restricted regions, although near the cortical surface the fasciculation became obscure.
Some of the fibers of the fasciculation near the insertion point of the guide tube (see small arrowhead in Fig. 1A) left the faseiculation and turned towards the surface lesion due to the insertion. Usually many fibers accumulated at the point where some trophic factors might go out 11. The direction of the cortical vertical fiber system in this area had been different from the guide insertion line (Fig. 1A), so that the axon elongation in the cortical vertical fiber system was only partly affected by the graft insertion.
2 8 7
V
f , t ~'°, '. ,.
Fig. 1. Axon elongation towards the cortical surface in the host radially oriented fiber system from B A L B / c olfactory bulb transplanted into the frontal cortex in an A K R mouse host. Seventy-three days of survival after transplantation. All sagittal sections. (A) Schematic drawing of axon elongation from the transplant. The small arrowhead on the cortical surface shows the insertion point of the guide tube. The larger arrowhead indicates the projection fibers among the host long association fibers. (B) Cytoarchitecture of the transplant. Note that projection neurons are in the anterior part of the transplant. Segregation of the large projection neuron and granular neuron groups is clear. Cresylviolet stain. (C) Photomicrograph of Thy-l .2 positive fibers in the host fiber system vertical to the cortical surface. Approximate location is indicated by an open arrow in A. (D) Higher-power magnification of a part of the fasciculation. Arrowheads in C and D show the same vessel. aca = anterior commissura, anterior part; Acb = accumbens nucleus; AO = anterior olfactory nucleus; cc = corpus callosum; CPu = caudate putamen; DG = dentate glanular layer; fi = fimbria of hippocampus; Fr = frontal cortex; Hi = hippocampus; L V = lateral ventricle; OB = olfactory bulb; Oc = occipital cortex; Tu =
olfactory tubercle. Magnification bar at bottom: A, 1 mm; B, 500/.tm; C, 200/.tm; D, 50 #m.
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Other non-target-directing fibers occurred among the host association fibers running longitudinally between the frontal and occipital cortices (see large arrowhead in Fig. IA). It extended caudally about 2 mm at maximum, emitting a few perpendicular collaterals towards the cortical surface on the way (not shown in Fig. 1A). The dominant projection of OB fibers towards the cortical surface is perhaps due to the anterior location of the large projection neuron aggregation within the transplant (Fig. 1B). Another transplant which also revealed a large neuron area in the caudal part sent massive fibers caudally over a considerable distance on the corpus callosum (about 4 mm with calibration of the mouse brain atlas of Sidman et al. ~4) with numerous perpendicular collaterals. In these cases, another fiber bundle issued from the ventral aspect of the transplant. These fibers may have had opportunities to receive signals from the host targets which are located mostly in the basal part of the forebrain (including host OB). In fact, these fibers were found to enter the OB, olfactory tubercle or piriform cortex.
The other 4 transplants similarly placed in the host frontal cortex show, more or less, the radial (toward the cortical surface) and longitudinal (on the corpus callosum) axon elongation. Among them, transplants placed on the corpus callosum (but not in front of it) showed exclusively non-target-directing fibers within the cerebral cortex (lack of ventrally running fibers).
These results indicate that host parallel-running (radially or longitudinally) fibers offer some cue for transplant OB projection fiber elongation. There is a possibility that some immaturity of cortical myelination in the 5-week-old host 8 supports the axon elongation because mature oligodendroglia prevents the penetration of newly growing fibers into the host tissue 13
A recent study 16 on the behavior of transplanted neocortical astrocytes into the neonate cerebral cortex demonstrates that astrocytes from the transplant move along the radially oriented host fiber system towards the cortical surface, as found for the trans- planted OB projection fibers in this study. There is a possibility that the transplanted astrocyte guides the late-coming axon elongation. The coincidence of the timing of the astrocyte immigration and elongation of the OB projection fibers will be examined in a future study.
When a transplant was placed more medially in the neocortex close to the midline where the dominant nerve tissue is the gray matter of the medial cortical surface, numerous axons entered the border between the indusium griseum (IG) and the neocor- tex. Posteriorly, the axons between the IG and neocortex gradually decreased in number and disappeared within the border, whereas rostrally the fibers ran similarly to the rostral end of the IG and diverged into the frontal cortex, without elongating along the continuous gray of the tenia tecta. A few of them arrived at the cortical surface.
Similar running fibers at the border between IG and neocortex were observed in the other two transplants which extended into the tenia tecta just adjacent to the IG or located close to the medial cortical surface. Unlike human IG, which locates on the corpus callosum independently, mouse IG is embedded in the neocortex. The surface-re- lated cue v may still operate for the OB axon elongation.
Figure 2 shows transplant fiber projections in the host hippocampal formation. When a transplant was placed in the dentate granular layer and hippocampal hilar part (posterior 2.0 mm from the bregma and 1.0 mm lateral from the midline) numerous fibers entered the molecular layer of the dentate gyrus (Fig. 2B). In spite of the anterior position of the large projection neuron area (Fig. 2A), numerous fibers issued from the posterior aspect of the transplant (Fig. 2B) and accumulated in the dentate molecular layer. Some fibers were also found to run towards the hippocampus proper (Fig. 2B), but these fibers
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Fig. 2. Axon elongation from transplants (BALB/c) placed in and on the host hippocampal formation in A K R mouse hosts. All sagittal sections. (A) Cytoarchitecture of the transplant. Large neuron area is upper right in the transplant facing the hilar part (hi) and CA 3. For approximate location in the brain, see Fig. 1A. Cresylviolet section. (B) Photomicrograph of Thy-l.2 positive fiber outgrowth from the transplant in the sections adjacent to A. The bulk of the fibers enter the dentate molecular layer; at this level, however, some fibers spread into the hippocampal hilar part and CA 3. (C) Photomicrograph of the dentate gyrus just lateral to the transplant. Numerous Thy-l.2 positive fibers accumulate in the molecular layer (DM) and on the inner border of the granular layer (DG). Note that the fibers in the hilar part and the continuous hippocampal pyramidal layer (CA3) appear to move gradually towards the dentate gyrus. (D) Photomicrographs through the same dentate gyrus just medial to the transplant. Massive Thy-l.2 positive fibers accumulate in similar regions as in C but their number is much greater. A few fibers in the hilar part are moving towards the dentate gyrus. (E) Photomicrograph of the most medial part of the molecular layer (DM) of the same dentate gyrus. Massive fibers accumulate at the medial end. Note that a few fibers issue from the medial end and enter the hippocampus as if they are partly forced out. (A-E) A mouse which survived for 77 days after transplantation. (F) A transplant placed in contact with the dorsal surface of the hippocampus (black area in the upper left of the photomicro- graph) emits diffuse fibers into the hippocampus. Ninety-two days survival after transplantation. DM = dentate molecular layer; hi = hippocampal hilar part. For other abbreviations, see Fig. 1. Magnifications are all the
same. Bar at the bottom: 200 #m.
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gradually moved back posteriorly to enter the dentate molecular layer, directly or through the inner surface of the dentate granular layer (Figs. 2C and D). Except for these fibers, only a few fibers were scattered within the granular layer and in the hippocampus proper (Fig. 2C and D).
In the molecular layer of the dentate gyrus, the fibers tended to elongate mediolater- ally. Fiber outgrowth from the transplant appeared to be more prosperous towards the medial direction than laterally (Figs. 2C and D). At the medial end of the dentate molecular layer numerous fibers were still massive there. At this level some fibers again entered the hippocampal area as if they had been partly forced out from their position, perhaps because of a standstill at the medial end (Fig. 2E).
The other 3 transplants which were placed in the same region also showed a preference for the dentate molecular layer even if large projection neurons accumulated in the anterior surface region of the transplant and made contact with the hippocampus proper.
When transplants (2 cases) were placed just dorsal to the hippocampus proper (contact with the hippocampus), projection fibers from the OB transplants diffusely entered the hippocampus (Fig. 2F), showing no special preference for the dentate molecular layer. These findings may partly be due to the small number of projection fibers from the transplanted OB which did not contain distinct large neuron aggregations.
These results suggest that the transplanted OB projection fibers have a preference for the dentate molecular layer which consists of dendrites of dentate granular neurons and the afferent fibers. The reason for such a preference is not clear at present. Does it indicate that there is a common trophic factor arising from a small granular type of neuron? This is probable because collaterals of normal OB projection fibers terminate within the OB exclusively in the granular layer 9. The hippocampal tissues appear to have some acceptability of the OB projection fibers as well (Figs. 2E and F). Perhaps it may be partly due to the relative lack of myelinated fibers in the host hippocampal formation 2.
In some of the neocortical groups of OB transplants, projection fibers issuing from the ventral aspect arrived at the host olfactory tubercle, piriform cortex or even OB even if they were not dominant projections. The possibility that the non-specifically elongated fibers are a group of long axon collaterals a2 of these target-directing fibers cannot be completely discounted. However, the OB axon elongation into the dentate molecular layer is excluded from this possibility.
From the results of the OB transplantations into neocortical and hippocampal regions, it is suggested that transplanted embryonic OB has original abilities to elongate its projection axons for a considerable distance, even if there is no nearby target at all. Under unnatural conditions the transplanted neurons may reveal their potential proper- ties which are normally masked in a developing brain. In adult CNS, long-distance axon elongation from regenerating neurons and transplanted tissues occurred only with some special conditions such as guidance with peripheral nerve accompaniment 1, host target deafferentation 15 or surface cue 7. However, it is evident from the present results that transplanted embryonic OB neurons can elongate their axons for a considerable distance even if there is no such guide mechanism.
Recent experimental study on the primary olfactory nerve suggests that terminal glomeruli of the nerve are formed with and also without targets in OB 6. The fact that target signals appeared to be unnecessary for glomerular formation suggests an intrinsic non-target-directing capacity of the olfactory nerve.
It is of interest that the secondary olfactory neurons, OB projection mitral and tufted cells also reveal similar intrinsic characteristics: axon elongation as contrasted with the glomerular formation occurred in a non-specific form, which suggests the restricted role
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of the sur rounding host extracellular matrix. The p h e n o m e n a may express the except ional characteristics of the OB neurons in the m a m m a l i a n CNS which usual ly refuses to accept
newly growing fibers. The non-specif ic characteristics are perhaps essential also to the no rma l OB developing system, al though it is revealed only in a strange env i ronmen t with heterotopic t ransplanta t ion . The t ransplanted OB axonal behavior, however, appears to be affected by regional cues of the host brain. That may determine the route of axon elongation, i ndependen t of the presence of the target and independen t of the normal olfactory project ion route. The long-distance fiber e longat ion from the t ransp lan ted OB 3,4 can be par t ly explained by these findings. At tempts to analyze the other determin- ing factors of the project ions are now in progress.
ACKNOWLEDGEMENTS
The author expresses her grat i tude to Misako Nakamich i for making excellent prepara- t ions of the mouse bra in and thanks Mar tha Alexander for her k indness in correcting the
English in this manuscr ipt .
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