0306-4522/92$5.00+ 0.00 Pergamon Press plc 0 1991IBRO

NeuroscienceVol. 46, No. I, pp. 71-82, 1992 Printed in Great Britain

THE OM SERIES OF TERMINAL FIELD-SPECIFIC MONOCLONAL ANTIBODIES DEMONSTRATE REINNERVATION OF THE ADULT RAT DENTATE GYRUS BY EMBRYONIC ENTORHINAL TRANSPLANTS P. L. WOODHAMS,* H. KAWANO~ and G.

RAISMAN

Norman and Sadie Lee Research Centre, National Institute for Medical Research, The Ridgeway, London NW7 IAA, U.K. Abstract-Monoclonal antibodies OM-1 to OM-4 and IM-I [Woodhams et al. (1991) Neuroscience 46, 57491 have complementary immunostaining patterns in the molecular (dendritic) layer of the adult rat dentate gyrus, with OM-1 to OM-4 selectively recognizing the outer (distal) two-thirds (i.e. the entorhinal afferent zone), and IM-1 the inner (proximal) one-third (i.e. the hippocampal commissural/associational zone). Immunoblotting suggests that OM-I recognizes a single glycoprotein antigen of mol. wt around 93,000, and OM-2, OM-3, and OM-4 all recognize a second glycoprotein antigen of mol. wt around 36,000. At four weeks after removal of the ipsilateral entorhinal cortex the background OM immunostaining of the entorhinal afferent zone is abolished and replaced by a network of densely stained granules, which we interpret as degenerating entorhinal afferent axons. At the same time, the proximal, IM immunoreactive zone expands by about 10pm in width (while the distal deafferented zone shrinks by about 80 pm). Attempts were made to restore the OM immunoreactivity of the distal zone by grafting either small pieces or cell suspensions of embryonic day 18 entorhinal cortex directly into the dentate molecular layer of entorhinally deafferented adult hosts. About half (14/26) of the animals with successfully positioned grafts showed restoration of OM-2 to OM-4 immunostaining throughout the entire width of the outer two-thirds (entorhinal afferent zone) of the dentate molecular layer. Strikingly, however, in adjacent serial sections the restoration of OM-1 immunoreactivity was restricted to the “middle” molecular layer, i.e. the most proximal part of the distal (entorhinal) two-thirds of the dentate molecular layer. In no case did the OM-I immunoreactivity extend to the outer margin of the molecular layer. This did not appear to be associated with incompleteness of the removal of the host entorhinal projection, since it occurred in grafted cases where the hippocampus had been completely isolated from the entorhinal area. The simplest explanation of the observed pattern of OM loss and restitution is that the epitopes are located on the entorhinodentate axons, but it is not clear whether the antigens recognized by OM-1 and OM-2 to OM-4 are expressed in different parts of the same group of axons, or in different subsets of entorhinodentate axons. Nor is it clear why the pattern of OM-1 is only restored to the “middle” molecular layer, while that of OM-2 to OM-4 is restored to the entire outer two-thirds. It is possible that the membrane-associated glycoproteins detected by the OM monoclonals are part of an intrinsic tissue signalling system used to direct and restrict the distribution of entorhinal projection fibres to the appropriate parts of the distal dentate molecular layer during normal ontogeny and during reinnervation of the adult by entorhinal transplants.

In the previous paper2’ we have described a number of monoclonal antibodies which recognize membrane-associated antigens that distinguish between the two major terminal fields of the rat dentate gyrus-the inner commissural/associational field, comprising the proximal one-third of the granule cell dendrites, and the outer, entorhinal field comprising the distal two-thirds. Monoclonals characteristic of the latter region fell into two groups, OM-1 and OM-2_OM-4. They appear to recognize at least two different glycoprotein antigens that differ in their molecular weights (93,000 and 36,000, respectively) and degree of enrichment in lentil-lectin-binding gly

coprotein preparations. The present paper uses lesions and transplantation to examine the extent to which the adult pattern of OM staining depends upon an intact entorhinal projection, and can be restored by transplantation of embryonic entorhinal tissue. EXPERIMENTAL PROCEDURES Animals and surgical procedures

In inbred adult female PVG rats (180-200 g) under Hypnorm/Hypnovel (Janssen) anaesthesia the left occipital and entorhinal cortices and white matter at the caudal pole of the hemisphere were aspirated until the underlying temporal pole of the hippocampus was visible.25 Following postoperative survival times of five days, four to six weeks, and 12 weeks (six rats in each group), animals were perfused with phosphate-buffered saline and their brains were removed, fixed in acid alcohol, and processed for polyester wax sections as described previously.*2 The part of each forebrain containing the hippocampus was divided into a

*To whom correspondence should be addressed. TPresent address: Department of Anatomy, School of Medicine, University of Tokushima, Tokushima 770, Japan. 71

dorsal portion. which was sectioned coronally from the septal pole for immunohistochemistry of terminal fields rn the dentate gyrus. and a ventral portion which was sectioned horizontal!y to verify the extent of the lesions by thionin staining. In a second series of 27 animals with unilateral entochi~l lesions carried out 7--14 days previously, grafts of i- 2 mmlong fragments of entorhinai cortex dissected from rats aged embryonic day IX (El& taking EO as the day on which vagina1 plugs were recorded) were inserted on the side of the deafferentation into the lateral part of the suprapyramidal blade of the anterior dentate gyrus. The grafts were inserted using a glass can&a (0.8 mm internal diameter) mounted on a steel rod, whose tip was placed 3.0 mm posterior to the bregma, 2.2mm lateral to the midIine,and 5.5mm deep (with the incisor bar 5 mm above the interaural line). After a survival of up to six weeks the animals were killed for histological analysis. A further 18 animals with unilateral entorhinal lesions received microtransplants of suspensions of El 8 entorhinal corttcal cells, injected into the hippocampus of the deafferented side through a glass micropipette.~.” The cefls were prepared by trypsin digestion (10 mgjml for 20 min) of both entorhinal cortices from each animal in a single litter (usually IO-12 pups), and kept on ice at a concentration of 3--S x IO6 cells per ml with 50 ngjml DNAse present to reduce clumping. Immediately prior to injection, 300 ~1 of this cell suspension was spun to a pellet and resuspended in a volume of 50~1, the final injection of 0.5 ,&I thus containing about IO-17 x I@’ cells. The stereotaxic coordinates were 3.0 mm posterior to bregma, 1.5 mm lateral. and 3.0 mm ventral (the incisor bar in this instance being held 5 mm below the interaural line).

Coronal polyester wax sections of the anterior hippocampus from the lesioned and grafted rats were immunostained with monoclonal antibodies OM-1, OM-2, OM-3, OM-4, or IM-1, as described in the accompanying paper.” Antibody RT97 against a phosphorylated epitope on the 2 IO.000 mol. wt neurofilament polypeptide (Anderton et a/., I : 5000) was also used to identify axons in the grafts, and a monoclonal antibody against glial fibriltary acidic protein (Amers~dm product code N358, 1: 1000) to assess the degree of any gliosis. Controls were incubated with I: 1000 normal mouse serum m place of the primary antibody.

The widths of the inner and outer dendritic fields of the molecular layer were measured in five rats at 12 weeks after entorhinal aspiration. Fairs of matched coronal sections through the horizontally orientated rostra) part of the dentate gyrus (taken at the ievel shown in Fig. 2) were stained with IM-I. and the whole molecular layer on both sides was drawn with a camera lucida at a magnification of x 80. The widths of the stained inner zone and of the whole

layer were measured on each section across the radial axis of the dentate gyrus at three equally spaced points along the sup~pyrami~l blade. Values on the lesioned side were compared with similar (control) measurements made on the contralateral, intact side of the same animals. RESULTS

From examination of a one in 20 series of thioninstained horizontal sections through the lesioned areas, at least six lesioned animals at each survival time (a total of 18) had almost total removal of the entorhinal cortex (compare Fig. la and b). These

lesions sometimes also involved part of the subicuhim, but cases where there was damage to deeper parts of the ventral hippocampus itself(especially any involving the dentate gyrus) were rejected. In order to avoid severing large vessels associated with the posterior communicating artery (B in Fig. la), a small portion of the retrohippocampal fields was often left (asterisk in Fig. la), but projection fibres from these fragments would have been completely disconnected from the dentate gyrus. Identical histological results in the dentate gyrus were also obtained with a number of additional animals (not included in this series) that had more dorsal lesions of the angular bundle and the perforant path. These lesions would have destroyed the entorhinodentate projection fibres c>nroute, although the ventral entorhinal cortex itsell remained undamaged in these cases. Blocks of the anterior h~ppocampus were selected from successfully lesioned animals, sectioned coronally and immunostained with the panel of OM and IM antibodies. In each section the pattern of staining of the denervated dentate gyrus ipsilateral to the entorhinal lesion was compared with that of the intact contralateral dentate gyrus (as an internal control).

Outer two-thirds of‘ the molecular luyer qfirr lesions Five days after ablation of the entorhinal cortex, a band of OM-1 immunostaining was still apparent in the outer molecular layer of the lesioned dentate gyrus, but no longer as sharply defined as on the intact side (Fig. 2a-d). At higher magnification (Fig. 2d), it was apparent that the immunoreaction product in both the outer dentate molecular layer and the adjacent stratum lacunosum-moleculare of the hippocampus was more granular than it was on the intact side. Whilst a few similar granules were visible in control sections incubated with normal mouse serum, they were very sparsely scattered with only weak reaction product. The specifically immunopositive granules probably represent the debris of fragmented degenerating entorhinal axons or terminals. At longer postoperative times the pattern of OM-I immunostaining in the outer dentate molecular layer was markedly different from that seen at five days. By four to six weeks the outer entorhinal zone had become much paler than the inner zone (Fig. 2e, f), and at I2 weeks (Fig. 2g, h) it was almost completely devoid of OM-I immunoreactivity apart from degeneration granules, which although fewer in number when compared with shorter survival times (Fig. 2f, h) still remained strongly immunoreactive. Almost no such granules were seen in control incubations with normal mouse serum (Fig. Id), and none were visible when antibody incubations or the diamino~nzidine reaction were omitted, indicating that their appearance was not due to non-specific argyrophilia. The OM-1 -positive degeneration granules were often orientated in bead-like rows running parallel to the hippocampal fissure and orthogonal to

a

L AI

Fig. 1. (a), (b) Thionm-stained horizontal sections of entorhmal cortex and hippocampus on the operated side (a) and the intact side (b) six weeks after unilateral entorhinal aspiration. Asterisk marks a remaining fragment of retrohippocampal tissue. B, blood vessels. (c), (d) Details of the outer molecular layer 12 weeks after an ipsilateral entorhinal lesion, (c) stained with OM-1, and (d) control incubation with normal mouse serum. Large arrows indicate the line of the hippocampai fissure (HF), small arrows in c an immunostained degenerating axon. Scale bars = 500 # m (a, b); 20 # m (c, d). Abbreviations used in figures G HF LM MCA ME TP

granule cells hippocampal fissure stratum lacunosum-moleculare of hippocampus commissural/associational field of dentate molecular layer entorhinal field of dentate molecular layer transplant 73

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P.L. WOODHAMS et al.

Fig. 2. OM-1 staining of the deafferented dentate gyrus, on the intact side (a, b); at five days following an ipsilateral entorhinal lesion (c, d); at four weeks (e, f); and at 12 weeks (g, h). Arrowheads delineate the full thickness of the molecular layer. Scale bars = 500 # m (a, c, e, g); 100 p m (b, d, f, h).

OM-immunostainmg of entorhinodentate axons

75

Fig. 3. (a), (b) OM-4 staining of the outer molecule layer (ME) in the intact (a) and lesioned (b) dentate gyrus 12 weeks after unilateral entorhinal aspiration. (c), (d) IM-1 staining of the inner molecular layer on the intact (c) and deafferented (d) sides at six weeks postlesion. Arrows at HF indicate the hippocampal fissure: note the shrinkage of the outer molecular layer in b and d. Scale bar = 100 #m. the radial axis of the granule cell dendrites (Fig. lc), providing a strong indication that they represent degenerating axons. Similar OM-l-positive granules were observed in the adjacent entorhinal projection

zone (stratum lacunosum-moleculare) of the hippocampal field C A 1, although here the loss of immunostaining in the surrounding neuropil was variable and often incomplete, suggesting incompleteness of

76

I’.

L. Wool)HAMS (‘I I!/.

the entorhinal deafferentation of this part of the hipp~ampus (see Discussion). Staining with antibodies OM-2. OM-3, and OM-4 (for the lower molecular weight antigen) gave similar results to those seen with OM- 1. i.e. a marked loss of the strongly immunoreactive band in the outer molecular layer (Fig. 3a, b). As with OM-1, the “degeneration” granules in the outer layer retained OM-2. OM-3, and OM-4 immunoreactivity up to the longest survival time examined, 12 weeks (Fig. 3b). At all survival times, the OM-1 to OM-4 immunostaining of the contralateral dentate molecular layer resembled that of the normal intact animal (indicating that loss of any crossed entorhinal proj~tions~,‘~,‘~ is too small to cause a detectable change). OM immunostaining of areas not receiving entorhinal afferents (e.g. hippocampal stratum oriens and stratum radiatum) were unaffected by the lesions, and perisomatic staining in the granule cell and pyramidal cell layers by antibodies OM-2 to OM-4 (see accompanying paper” ) remained strong. Inner one-third

of the molecular layer ufter tesions

Following entorhinal lesions the overall pattern 01 OM-I immunostaining in the dentate molecular layer was altered not only by a loss of OM-I staining from the outer layer, but also by an enhancement of OM-1 immunoreactivity in the inner zone. This was readily apparent in comparison with intact controls at four to six weeks, and the staining remained at 12 weeks (Fig. 2e-h). In contrast, there was no enhancement of expression of the antigen recognized by OM-2. OM-3, and OM-4 in the inner zone of the molecular layer (Fig. 3a, b). There was no marked change in the intensity of IM-1 staining in the inner molecular layer, either at six weeks or at 12 weeks following ipsilateral entorhinal lesions (Fig. 3c, d). However, at 12 weeks the mean width (+S.E.M.) of the inner IM-I-positive zone had increased by 16%. from 72 f 2.2 pm on the control side to 83 f 2.4 pm (significant at P < O.Ot. Student’s r-test). Due to shrinkage of the outer zone. the molecular layer as a whole had decreased in width by 27% on the lesioned side, from 245 rl: 5.0 pm to 179 t_ 4.4 pm. The combination of these two factors caused the proportion of the molecular layer occupied by the IM-1 positive field to increase from 29% to 47% of the total width (compare Fig. 3c and d). Entorhinul tissue transplanted dent&e g,vrus

into the deafikrented

Grafts of excised entorhinal tissue fragments or cell sus~nsions derived from the El8 entorhinal area survived well when transplanted into the adult entorhinally deafferented host dentate gyrus. Both types of transplants formed compact masses, which were larger and less regularly shaped in the cases with solid grafts, and smaller and more rounded with the suspensions. Most of the surviving grafts were SUCcessfully placed in the dorsal (suprdpyramidal) blade

of the rostra1 third of the host dentate gyrus, largely occupying the molecular layer, but occasionally extending down through the granule cell layer. Both solid and suspension grafts contained large, well developed neurons of pyramidal type, embedded in a dense and uniformly OM-positive neuropil. Whilst a number of cases with both kinds of graft showed similar results after OM immunostaining (see below), we found that graft placement was less accurate and consistent for the solid grafts, which often exhibited significant degrees of gliosis (examined by staining for glial fibrillary acidic protein) and necrosis. The cell suspension method was much more reproducible, and produced negligible gliosis at the graft-host interface. Non-specific silver impregnation of blood capillaries by the silver enhancement technique indicated that the suspension grafts were well vascularized (e.g. Fig. 6a, c, d). The intervai of seven to IO days between making lhe lesions and transplanting the cells followed a protocol which was successful for reinnervation of the mouse dentate gyrus using solid EIX entorhinal grafts stained for Thy 1,l.2s Gibbs and Cotman suggested that such a delay before transplantation may be beneficial. due to the accumulation of trophic factors. Forty-five animals received grafts, 27 of them solid and I8 cell suspensions. The histological results from five solid and one suspension graft were uninterpretable due to damage and necrosis, and a further nine solid grafts which were found to have been placed far from the dentate molecular layer were examined in thionin-stained sections only. Sections from the remaining 30 cases were examined with each of the OM antibodies, and the results are summarized in Table I. The four cases in which the suspension grafts were not in contact with the host dentate molecular layer (Table I) showed persistent loss of OM immunoreactivity. comparable to the lesion results described earlier. Grafts suc~ssfully placed in contact with the host dentate molecular layer (n = 26) fell into two groups: I1 cases in which the pattern of OM-I staining was the same as in entorhinal lesions with no grafts (i.e. where restoration of the normal staining pattern had not occurred), and 15 cases where there was restoration of OM immunoreactivity. Of these, Table I. Results of immunostaining the entorhinally dean‘erented outer molecular layer of the dentate gyrus in 30 animals, six weeks after grafting embryonic entorhinal cortex

No. of cases

Total

Solid

susp.

Graft in place

OM-I

OM-:! to OM-4

14

5

9

+

+

i-

I II

x

I* 3

+ +

4

4

30 *Case EC?-96 (see text).

-. _

-t

-

OM-immunostaining

of entorhinodentate

Fig. 4. Camera lucida tracing of a coronal section showing the extent of the entorhinal lesion (hatched area) in case EG-8 1. Scale bar = 1 mm.

14 had a band of OM-1 immunoreactivity in the middle third of the molecular layer, and all 15 showed restoration of the normal pattern of OM-2 to OM-4 ~mmunostaining throughout the full thickness of the outer molecular layer. Case EG-81 exemplifies the differences in pattern of restoration of OM- 1 and OM-2 to OM-4 immunostaining. It is chosen for illustration because in this case the lesion was large, resulting in destruction of the whole of the caudal pole of the cortex and the ventrocaudal hippocampus, except for a very small isolated hippocampal fragment at its most ventral aspect (Fig. 4). Thus in this animal entorhinal deafferentation of the surviving rostra1 fragment of dentate gyrus containing the graft was unequivocally complete. OM-I staining of this case showed a band of immunoreactivity in the middle one-third of the dentate molecular layer (Fig. 5a, c), densest towards and sharply delineated from the adjacent inner commissural/associational field, but merging diffusely into the unstained outermost one-third of the dendritic field (the area to which the lateral entorhinal cortex normally projects’*). In no case did the restoration of OM-1 immunostaining extend throughout the full thickness of the outer molecular layer, but in all 14 grafted cases which showed a band of OM-I immunoreactivity restricted to the middle third of the molecular layer, staining for the OM-2, OM-3, and OM-4 immunoreactive antigen was fufiy restored (i.e. to its normal pattern in intact animals) across the entire width of the outer two-thirds of the molecular layer (Fig. Sb, d; compare with Fig. 3a). Both kinds of projection extended from the graft along the entire mediolateral extent of the dentate gyrus (Fig. Sa, b), not only in the suprapyramidal blade close to the graft but also extending evenly round the crest and through the

77

axons

entire length of the infrapyramidal blade (Fig. 5c, d). In only one instance (EG-96; Table 1) was there restoration of staining for the OM-2 to OM-4 antigens without any restoration of staining for the OM- 1 antigen in the outer molecular layer. In contrast to these 15 reinnervating grafts (typified by case EG-81), 11 animals with grafts successfully located in the entorhinally deafferented outer molecular layer failed to show even partial restoration of any type of OM immunostaining in the outer molecular layer. The loss of host OM immunostaining, and the histology of the lesions, provided good evidence that the host target field had been successfully deafferented of its entorhinal input. In these cases the grafts had developed an extensive OM immunopositive neuropil between iind around the grafted cell bodies, which stained for OM-1 with an intensity similar to that of the intact outer molecular layer on the contralateral side (Fig. 6a, b). This positive neuropil did not, however, appear to integrate with that of the deafferented host molecular layer (Fig. 6~). Neurofilament immunostaining showed numerous axons within the neuropil of the grafts and around their margins, but they tended to course circumferentially around the bolus of transplanted neurons rather than run out into the host molecular layer neuropil (Fig. 6d). In a number of grafted animals (two with restoration of OM-1 staining to the middle molecular layer and four without), occasional small fascicles of OM-l-positive axons (not illustrated) could be distinguished close to the grafts in areas of the host molecular layer which were otherwise ON- 1-negative apart from degeneration granules. These fascicles were occasionally seen to run out of the graft beside a blood capillary, although they were more often close to but not in contact with the graft neuropil.

~ISCU~ION

Entorhinal deaferentation The loss of OM staining in the outer terminal field of the dentate gyrus after lesions to the entorhinal cortex can be interpreted in one of two ways. The results could be due to degeneration of OM-positive entorhinodentate axons, or they could be due to loss of antigen from the granule cell dendrites (or even other, non-entorhinal axons) on which OM expression is transneuronally dependent upon the presence of intact afferent entorhinal innervation. On the grounds of economy, we favour the former explanation, although the presence of OM-1 antigen on entorhinal axons does not preclude expression on the granule cell dendrites or other axons as well. OM-immunoreactive epitopes are present on a wide variety of non-entorhinal structures throughout the hippocampus and other parts of the brain, and our ultrastructural data on the dentate molecular layer did not distinguish between axons and dendrites.“’

78

P. L. WOODHAMS et al.

O0, t..4 ~

.~8~ o. ~ . s

tt'~

OM-immunostaining of entorhinodentate axons

79

Fig. 6. Cell suspension graft, case EG-89. (a) At low magnification strong OM-1 staining of the bolus of transplanted cells (TP) contrasts with the OM-l-negative deafferented outer molecular layer (ME). Arrowheads delineate the full width of the molecular layer. (b) OM-1 staining on the intact side. (c) Detail of a, showing the graft-host interface. (d) Serial section to c, stained for neurofilaments. Scale bars = 100/~m. The present study showed trails of granules traversing the molecular layer at right angles to the axis of the dentate granule cell dendrites, suggesting fragments of axons. It is noteworthy that for many weeks after the lesion they retain a degree of OM immuno-

reactivity, indicating that the epitopes are stable in the tissue under conditions of axonal degeneration. We observed a modest (10#m) increase in the width of the inner IM-1-positive field of the molecular layer of the entorhinally deafferented dentate

80

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WOODHAMS e/ al.

gyms at 12 weeks, and this was accompanied by a nearly 80pm shrinkage of the outer field. The net result was that as a fraction of the entire layer the proportion occupied by the inner field rose from about one-third in controls to one-half in the entorhinally deafferented dentate gyrus. These figures are in good agreement with the published magnitude of lesion-induced sprouting of commissural and associational fibres into the deafferented outer entorhinal field.2*5.9,”Whatever the cause of the increased OM-1 immunoreactivity of the inner molecular layer, it is certainly not due to entorhinal axons since no entorhinal input to the inner molecular layer has ever been described in the many articles on this subject. Moreover, this OM- 1-immunoreactive component of the inner molecular layer in the entorhinally denervated dentate gyrus is incapable of invading the outer molecular layer. The OM immunoreactivity of the hippocampal stratum lacunosum-moleculare is not as pronounced as that of the outer dentate molecular layer,2’ and lesion-induced changes in the hippocampus were less marked than in the dentate gyrus. Although degeneration granules were seen, the background level of OM immunoreactivity was not greatly reduced on the lesioned side when compared with the intact. To some extent, this remaining OM staining could be due to an incomplete removal of the surgically less accessible rostrolateral strip of the entorhinal area which projects to this part of the hippocampus. It should also be recalled that the entorhinal projections to the hippocampus arise at least in part from different cell groups to those innervating the dentate gyrus, and it is possible that the entorhinodentate subset (arising from the superficial cells of layer II of the entorhinal area”) are especially rich in OM- 1-immunoreactive epitopes.

Transplantation All the grafts consisted of large, pyramidal-like neurons dispersed in a strongly and uniformly OMimmunoreactive neuropil, although, as described in occurrence of the previous paper, 2’ the widespread OM immunoreactivity in many cortical and other areas means that this is not a specific indication that the grafts contain entorhinodentate projection neurons. The solid grafting method presented a number of practical difficulties, such as variation in location of the tissue fragments (the pieces having a tendency to move as the cannula is withdrawn), the possibility that the graft may not have been sufficiently closely apposed to the host neuropil (in part due to bleeding or exudate at the time of operation), and the frequent occurrence of a dense astroghal scar at the graft-host interface. The use of cell suspensions.’ which can be microinjected in a virtually atraumatic manner, largely avoided these technical problems, producing a successful restoration of the normal staining pattern for the OM-2 to OM-4 antigen in IO out of a total

of 18 animals, compared with five out of the 27 receiving solid grafts. We are at present unable to explain why about half of the successfully positioned grafts failed to show reinnervation of the host molecular layer despite their being closely integrated into their appropriate and denervated target field. They were, however. able to elaborate a considerable OM-positive neuropil in the immediate vicinity of the transplanted neuronal perikarya and in some cases appeared to send out narrow fascicles of OM-l-positive axons, indicating that the viability of these grafts was not compromised. Other possible factors which might explain the failure to restore the OM staining patterns in these cases include the size of the graft, and the degree of accuracy with which the grafted entorhinal cortex could be identified and dissected out from the embryonic donors. There was, however, no clear correlation between graft size and success or failure of reinnervation: as a result of keeping the cell suspension injections small and thus as atraumatic as possible, the final number of cells in all the present grafts was quite low. The injection of I@-17 x lo3 donor cells produced after six weeks a bolus of cells and neuropil which was usually about 300 pm across and extended for a maximum of SO&600 pm in the rostrocaudal direction, often along the hippocampal fissure. These grafts were thus smaller than the solid mouse transplants described by Zhou et a1.,25 and the solid rat transplants of Gibbs and co-workers.6,7 We consider that the 15 cases of restoration of OM-2, OM-3 and OM-4 immunoreactivity throughout the full thickness of the dentate molecular layer are a result of reinnervation of the denervated host tissue by entorhinal axons from the transplants. Since it does not occur in the entorhinally deafferented but non-transplanted animals, it seems unlikely that this restoration of OM-2 to OM-4 immunoreactivity could bc due to sprouting by, for example, axons of the crossed projection from the contralateral entorhinal cortex, which would be expected to show a pattern of OM antigen expression similar to that of the ipsilateral entorhinal axons. Our failure to find immunohistochemically detectable OM-2 to OM-4 restoration in non-transplanted animals with ipsilatera1 entorhinal lesions may be due to the fact that the contralateral projection is relatively small and represents only 3-5% of the total entorhinal input to the dentate gyrus;‘3,‘4 possibly any sprouted contralateral entorhinal projection (which may itself be relatively limited3~‘3~‘4~‘6)is of insufficient density to provide detectable levels of antigen. The pattern of OM-2, OM-3 and OM-4 immunostaining restored by entorhinal transplants in the rat exactly resembles the reconstructed mouse entorhinodentate projection described by Zhou cut u/..?~ using Thy-1.1/1.2 allelic marking. While we cannot exclude the possibility that the restoration of OM immunoreactivity could have been due to transncuronal reinduction of host OM antigen by the

OM-immunostaining of entorhinodentate axons transplant, this cannot be the case for the restoration of Thy- 1.1 by entorhinal grafts into the entorhinallydeafferented dentate gyrus of a Thy-l.2 host:25 the only way that Thy-l. 1 can appear in a Thy-l.2 host is by ingrowth from the graft. While we have not directly demonstrated at the cellular level that this is due to specific Thy-l .l-positive donor entorhinal axons, there is good evidence from a number of systemslo,*’ that Thy-l is an axonal antigen. By analogy, therefore, we consider that the OM restoration by the grafts is most likely to be due to the ingrowth of donor entorhinal axons. As with transplant-induced reconstruction of other hippocampal afferents,24 the restoration of OM immunoreactivity only occurs when the graft is in direct contact at some point with the denervated target tissue. The fibres will not cross even short intervening distances of inapprop~ate tissue to reach the target, but in those cases where the graft does make direct contact at one point with the correct target, the projection will spread from that point (like a spot of ink on blotting paper) for considerable distances within the denervated terminal field, and at some levels even reinnervate the entire field.* The restricted distribution of OM-1 immunoreactivity in the transplanted cases is puzzling. In normal animals, OM-1 immunoreactivity extends evenly throughout the full thickness of the outer two-thirds of the dentate molecular layer. In the reinnervate material, the OM-1 immunoreactivity accurately reconstructs the boundary between the inner one-third (commissural/association zone) and the middle one-third, but it decreases progressively in intensity in the distal direction along the dendrites, and (unlike OM-2 to OM-4 immunoreactivity) never fully reoccupies the outer one-third. We feel it is unlikely that the exclusion of OM-1 from the outer one-third of the molecular layer is due to a residual host projection, surviving because of incompleteness in the removal of the host entorhinal area. In a number of the cases with the selective OM-1 restoration to the middle layer (e.g. EG-81, .._ *With this material we are unable to reproduce the long, pathway-type axonal outgrowth observed in several recent studies using suspensions of early (e.g. E13-E14) neuroblasts,‘ss’ga difference which may be due to the much later age of the donor tissue (postmitotic neurons) in

our material.

81

Fig. 4), the completeness of the entorhinal deafferentation is beyond question. The failure of the OM-1 reinnervation to fill the entire “radial” thickness of the outer two-thirds of the dentate molecular layer (about 1~15O~m) could hardly be due to inadequate numbers of fibres, since the same pattern recurs in all 14 cases, and the restricted middle band of OM-1 immunoreactivity (like that of OM-2 to OM-4) is able to spread “laterally” throughout the entire extent of the supra- and infrapyramidal blades of the dentate gyrus (along a distance of around 6 mm). Why the transplanted entorhinal axons can regularly restore part, but never all, of the full OM-1 distribution pattern of the normal dentate outer molecular layer will perhaps be clarified when we are able to understand the cellular distribution of OM-1 and OM-2 to OM-4 immunoreactiviti~. As would be expected from the evidence that they recognize epitopes on the same antigen2i the OM-2, OM-3 and OM-4 immunostaining patterns are always identical in distribution (although not necessarily in intensity). OM-1 recognizes a different antigen, and the question arises whether it is present in the same axons as OM-2 to OM-4 (but distributed differently at the subcellular level within the reinnervating axons), or whether it is present in different axons from those containing the OM-2,OM-3 and OM-4 immunoreactive antigens. In this context it should also be recalled that the middle and outer thirds of the dentate molecular layer, while both normally receiving an entorhinal input through the perforant path, differ sharply in the origin of this projection (medial versus lateral entorhinali2), and in their Timm staining pattern.26 Finally, it should be noted that the discovery of cell-type-specific surface glycoprotein antigens, such as OM-I-OM-4, is not only useful as an experimental “label” for studying reinnervation, but may also offer clues to the molecular signals which the tissue itself uses to direct and restrict the distribution of entorhinal projection fibres to the outer two-thirds of the dentate granule cell dendrites in normal development and after transplantation. are grateful to Mr D. J. Atkinson for assistance with the grafting experiments, and to Dr P. M. Field for helpful discussion. Dr B. Anderton generously donated ascites of the RT97 antibody. Acknowledgements-We

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(Accepted I July 1991)