Retinal axon regeneration in peripheral nerve, tectal, and muscle grafts in adult rats.

This study examined whether prior regenerative growth through peripheral nerve (PN) bridging grafts influenced the specificity with which lesioned adult rat retinal ganglion cell (RGC) axons grew into co-grafts of developing target tissue (fetal superior colliculus). Growth into nontarget (muscle) tissue was also examined. Autologous PN was grafted onto the transected optic nerve. After 14 days, the distal ends of the PNs were placed next to, or inserted into, embryonic tectal tissue or into autologous muscle grafts placed in frontal cortex cavities. Host retinal projections were examined 3-8 months later using anterograde and retrograde tracing techniques. In rats in which there was good apposition between PN and tectal tissue, small numbers of RGC axons were observed growing into the tectal grafts (maximum distance of 180 microm). No evidence of specific innervation of appropriate target regions within tectal grafts was detected, even though such regions (identified by acetylcholinesterase histochemistry) were often located close to the PN grafts. In rats with PN/muscle co-grafts, the extent of retinal axon outgrowth was greater (up to 465 microm from the PN tip) and labelled profiles that resembled motor endplates were seen contacting muscle fibres. Previous studies have shown that spontaneously regenerating RGC axons consistently and selectively innervate appropriate target areas in fetal tectal tissue grafted directly into optic tract lesion cavities. Together, the data suggest that exposure to a PN environment may have reduced the extent of adult retinal axon growth into fetal tectal transplants and affected the way regenerating axons responded to specific developmental cues expressed by target cells in the co-grafted tissue.

Astrocytes, oligodendroglia, extracellular space volume and geometry in rat fetal brain grafts.

Fetal neocortex or tectum transplanted to the midbrain or cortex of newborn rats develops various degrees of gliosis, i.e. increased numbers of hypertrophied, glial fibrillary acidic protein-positive astrocytes. In addition, there were patches or bundles of myelinated fibres positive for the oligodendrocyte and central myelin marker Rip, and increased levels of extracellular matrix molecules. Three diffusion parameters--extracellular space volume fraction alpha (alpha = extracellular volume/total tissue volume), tortuosity lambda (lambda = square root(D/ADC), where D is the free and ADC is the apparent tetramethylammonium diffusion coefficient) and non-specific uptake k'--were determined in vivo from extracellular concentration-time profiles of tetramethylammonium. Grafts were subsequently processed immunohistochemically to compare diffusion measurements with graft morphology. Comparisons were made between the diffusion parameters of host cortex and corpus callosum, fetal cortical or tectal tissue transplanted to host midbrain ("C- and T-grafts") and fetal cortical tissue transplanted to host cortex ("cortex-to-cortex" or C-C-grafts). In host cortex, alpha ranged from 0.20 +/- 0.01 (layer V) to 0.21 +/- 0.01 (layers III, IV and VI) and lambda from 1.59 +/- 0.03 (layer VI) to 1.64 +/- 0.02 (layer III) (mean +/- S.E.M., n = 15). Much higher values were found in "young" C-grafts (81-150 days post-transplantation), where alpha = 0.34 +/- 0.01 and lambda = 1.78 +/- 0.03 (n = 13), as well as in T-grafts, where alpha = 0.29 +/- 0.02 and lambda = 1.85 +/- 0.04 (n = 7). Further analysis revealed that diffusion in grafts was anisotropic and more hindered than in host cortex. The heterogeneity of diffusion parameters correlated with the structural heterogeneity of the neuropil, with the highest values of alpha in gray matter and the highest values of lambda in white matter bundles. Compared to "young" C-grafts, in "old" C-grafts (one year post-transplantation) both alpha and lambda were significantly lower, and there was a clear decrease in glial fibrillary acidic protein immunoreactivity throughout the grafted tissue. In C-C-grafts, alpha and lambda varied with the degree of graft incorporation into host tissue, but on average they were significantly lower (alpha = 0.24 +/- 0.01 and lambda = 1.66 +/- 0.02, n = 8) than in young C- and T-grafts. Well-incorporated grafts revealed less astrogliosis, and alpha and lambda values were not significantly higher than those in normal host cortex. The observed changes in extracellular space diffusion parameters could affect the movement and accumulation of neuroactive substances and thus impact upon neuron-glia communication, synaptic and extrasynaptic transmission in the grafts. The potential relevance of these observations to human neuropathological conditions associated with acute or chronic astrogliosis is considered.

Mast cell number and maturation in the central nervous system: influence of tissue type, location and exposure to steroid hormones.

While it is well established that brain mast cells are usually associated with the cerebral vasculature, in ring doves mast cells lie directly in the neuropil of the medial habenula. During normal development mast cells enter the habenula and complete their differentiation in situ. In the present study, we asked what characteristics of the medial habenula contribute to mast cell entry and differentiation. Grafts of embryonic habenula or control optic tectal grafts were placed in the lateral ventricle or anterior chamber of the eye. Transplantation alters the location of the habenula as well as its neural and vascular connections. Three groups of hosts were used for the ventricular grafts: four-month-old and killed three months after transplantation; four-month-old and killed seven months later, and two- to three-year-old gonadectomized males killed three months later. Hosts for the intraocular grafts were four months of age and killed three months later. Mast cells were present in the habenular grafts but not in the control tissue. Mast cells in three- and seven-month-old grafts were phenotypically immature when compared to those of hosts. They contained fewer metachromatic granules, fewer granules immunoreactive to an antiserum against gonadotropin-releasing hormone, and no highly-sulphated proteoglycans. As previously described, gonadectomized adults had fewer mast cells in their medial habenula than did intact animals, but there was no change in mast cell number in habenular grafts. The current experiments indicate that the occurrence and survival of mast cells can occur within the microenvironment of the medial habenula, but that maturation of these cells requires the normal connections of this nucleus. Furthermore, gonadectomy appears to alter mast cell number in the medial habenula by generating a secondary signal which the transplanted tissue is incapable of receiving or processing.

Schwann cells and fetal tectal tissue cografted to the midbrain of newborn rats: fate of Schwann cells and their influence on host retinal innervation of grafts.

Schwann cells transplanted into the adult central nervous system (CNS) can exert powerful growth-promoting effects on damaged axons. An important issue is whether central axons induced to regrow by Schwann cells retain the capacity to recognize and selectively innervate their appropriate target cells. To examine how Schwann cells may influence the specificity of neuron-neuron interactions in CNS neuropil, we cultured neonatal rat Schwann cells and mixed them with dissociated fetal tectal cells. In some instances, Schwann cells were prelabeled with Hoechst dye 33342. Schwann cells comprised between 2.5 and 15% of the combined cell population. After reaggregation, cografts were injected onto the midbrain of newborn rats. One to 6 months later, grafts were examined for the presence of Schwann cells and the pattern and density of host retinal innervation of the cografts was assessed. Immunohistochemical studies showed that areas of the transplants containing large numbers of surviving Hoechst-labeled Schwann cells were strongly immunoreactive for the low-affinity nerve growth factor receptor (p75), S-100, GFAP, and laminin. Very little peripheral (Po positive) myelin was seen. As in pure fetal tectal grafts, host retinal axons were sometimes observed to innervate superficial, localized areas in the cografts known to be homologous to the retinorecipient layers of the superior colliculus. Unlike pure tectal grafts, however, optic axons were not confined to these regions and fibers were often dispersed within the cograft neuropil. Dense growth was seen in association with Hoechst-labeled Schwann cells and, in some cases, optic axons were observed to grow toward Schwann cells and away from nearby target areas. These observations suggest that, under certain circumstances, Schwann cells can stimulate retinal axons to grow into inappropriate (nontarget) regions in the CNS, presumably by producing growth promoting factors which mask or compete with signals released from the target neurons themselves.

Rostrocaudal polarity formation of chick optic tectum.

The optic tectum receives retinal fibers in a topographically ordered manner. For the formation of the precise connections, the tectum is believed to be positionally specified by gradients of molecules along axes. Rostrocaudal polarity of the tectum is first detectable at embryonic day 2 (E2) in the chick, by the caudorostral gradient of en expression, then by the rostrocaudal gradient of cytoarchitectonic development. Tectum rotation experiments showed that tectum rostrocaudal polarity is not determined at around 10-somite stage, but is fixed on E3. Ectopic tectum was produced in the diencephalon by transplanting the mesencephalic alar plate heterotopically. In the ectopic tectum, en expression was weakest at the caudal (nearest to the host diencephalo-mesencephalon junction) and strongest at the rostral end. Consequently, the pattern of en expression in the host and ectopic tecta was nearly a mirror image. Retinal fibers projected to the ectopic tectum in a topographic order in accordance with the inverted gradient of the en expression pattern. Ectopic tecta was also produced by heterochronal transplantations between E3 host and E2 donor, where the en pattern was preserved. Retinotectal projection pattern was also preserved, suggesting that en expression patterns are followed by retinotopic order with regard to rostrocaudal polarity.

The distribution of astrocytes, oligodendroglia and myelin in normal and transplanted rat superior colliculus: an immunohistochemical study.

Immunohistochemical methods have been used to determine the distribution of macroglia and myelin in the normal rat superior colliculus (SC) and in grafts of fetal tectal tissue. The fetal tissue was derived from 15 day-old (E15) rat embryos and was transplanted onto the midbrain of newborn host rats of the same (PVG/c) strain. Antibodies to glial fibrillary acidic protein (GFAP) and carbonic anhydrase II (CAII) were used to visualize astrocytes and oligodendroglia respectively. Myelin was immunostained with antibodies to either proteolipid protein (PLP) or myelin basic protein (MBP). In the normal SC, GFAP positive astrocytes were found scattered throughout the SC, particularly in the superficial layers. They were especially prominent at the pial surface, around major blood vessels and at the midline between the two colliculi. CAII immunoreactive oligodendroglia and associated myelin were also found throughout the SC; by far the lowest density was seen in the stratum griseum superficiale (SGS). Both types of macroglia cell were found in abundance in tectal transplants, indicating that the precursors of these glial types were present in the E15 rat mesencephalon. In mature grafts, large numbers of fibrous astrocytes were found throughout the neuropil and the level of GFAP immunoreactivity was consistently greater than in host SC. Astrocytes seemed to be maintained in a reactive, perhaps immature state within the grafted tissue. Tectal transplants possessed large numbers of fully differentiated CAII-positive oligodendroglia and the grafts contained a dense network of myelinated axons. However the distribution of CAII and PLP immunoreactivity was not homogeneous; there were localized, well-defined regions that contained few oligodendroglia and relatively little myelin. These areas stained intensely for acetylcholinesterase (AChE) and were almost certainly homologous to the SGS of normal SC. The relative lack of oligodendroglia in the AChE stained patches in grafts and in SGS in situ suggests that local factors influencing the proliferation and distribution of oligodendroglia in normal SC may have been operating in a similar manner within the tectal transplant neuropil.

Spontaneous regeneration of adult rat retinal ganglion cell axons in vivo.

The superior brachial region of the left optic tract was lesioned in adult rats and foetal tectal tissue was implanted into the lesion site. The retinal projection from the contralateral (right) eye was examined 2 to 8 months later. In the majority of animals, retinal ganglion cell (rgc) axons were found to regenerate through cellular membranes which formed over the lesion. Axon growth could extend for up to 5 or 6 mm. Surviving tectal grafts were identified in all host rats. In animals in which regrowing rgc axons contacted tectal grafts, axons were found to innervate selectively their appropriate target regions within the graft neuropil.

The morphology and connectivity of dissociated and reaggregated fetal tectal tissue transplanted to the midbrain of newborn rats.

Tectal tissue from E15 or E16 Wistar rat embryos was dissociated and reaggregated (DR) prior to transplantation on to the midbrain of newborn host rats. We wished to determine how complete disruption of the donor tissue (i) affected the subsequent morphological development of the grafts in the host brain, and (ii) whether this procedure affected the selectivity with which host retinal axons innervated target regions in the tectal transplants. Forty-three to 135 days after transplantation, host rats received binocular injections of wheatgerm agglutinin-conjugated horseradish peroxidase. After perfusion, frozen sections of the grafts and underlying host brainstem were cut and reacted with tetramethylbenzidine to identify retinal projections, or stained for either acetylcholinesterase (AChE), Nissl or neurofibrils. All host brains contained identifiable DR grafts. Each brain contained at least one large transplant and numerous smaller pieces of graft tissue. The fragmentation of DR grafts was greater than that seen in direct, undissociated tectal transplants; however the morphology of individual DR grafts was markedly similar to direct grafts. Of particular interest was the presence in DR grafts of localized, often oval or circular regions, that possessed high AChE activity and contained mostly small (5 to 10 microns) close-packed neurons. AChE-dense patches were found both superficially and deep within DR grafts and appeared identical to those seen in direct transplants. These regions are thought to be homologous to the superficial, retinorecipient layers of normal superior colliculus (SC) and it is likely that the formation of these localized areas resulted from the selective association of presumptive SGS neurons within the reaggregating neuropil. In almost all cases, host retinal input to DR grafts was confined to the localized AChE-dense patches, suggesting that despite the dissociation procedure, specific retinal innervation of regions containing at least some of the appropriate target cells was maintained in DR tectal grafts.

Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity.

In the E4 (embryonic day 4) chick tectal primordium, engrailed expression is strong at the caudal end and gradually weakens toward the rostral end. We used quail-chick chimeric tecta to investigate how the caudorostral gradient of engrailed expression is established and whether it is correlated with the subsequent rostrocaudal polarity of tectal development. To examine the positional value of the tectal primordium, we produced ectopic tecta in the diencephalon by transplanting a part of the mesencephalic alar plate heterotopically. In the ectopic tectum, the gradient of the engrailed expression reversed and the strength of the expression was dependent on the distance from the mes-diencephalon junction; the nearer the ectopic tectum was to the junction, the weaker the expression was. Consequently, the pattern of the engrailed expression in the host and ectopic tecta was nearly a mirror image, suggesting the existence of a repressive influence around the mes-diencephalon junction on the engrailed expression. We examined cytoarchitectonic development in the ectopic tecta, which normally proceeds in a gradient along the rostrocaudal axis; the rostral shows more advanced lamination than the caudal. In contrast, the caudal part of the ectopic tecta (near to the mes-diencephalon junction) showed more advanced lamination than the rostral. In both the host and ectopic tecta, advanced lamination was observed where the engrailed expression was repressed, and vice versa. Next we studied the correlation between engrailed expression and retinotectal projection from a view of plasticity and rigidity of rostrocaudal polarity in the tectum. We produced ectopic tecta by anisochronal transplantations between E3 host and E2 donor, and showed that there is little repressive influence at E3 around the mes-diencephalon junction. We then made chimeric double-rostral tectum (caudal half of it was replaced by rostral half of the donor tectum) or double-caudal tectum at E3. The transplants kept their original staining pattern in hosts. Consequently, the chimeric tecta showed wholly negative or positive staining of engrailed protein on the grafted side. In such tecta retinotectal projection pattern was disturbed as if the transplants retained their original position-specific characters. We propose from these heterotopic and anisochronal experiments that the engrailed expression can be a marker for subsequent rostrocaudal polarity in the tectum, both as regards cytoarchitectonic development and retinotectal projection.

Regeneration of retinal axons in grafts of peripheral and central nervous tissue in the adult rat.

Peripheral nerves were autologously grafted to the retinae of adult rats. The distal end of the nerves was inserted into fetal tectal tissue placed within cortical lesion cavities. Two to 8 months later, anterograde pathway tracing using horseradish peroxidase demonstrated retinal axons regenerating along the length of the PNS nerve graft in 87% of animals with viable PNS and fetal tectal grafts. Of these animals, retinal axons grew into the tectal neuropil in 23% of cases but specific innervation of appropriate target regions was not detected.

Fetal tectum grafted as a cell suspension into the adult rat inferior colliculus.

The known structural and functional features of the inferior colliculus provided an advantageous substrate for examining the outcome of neural transplantation. In the present study, tissue from the midbrain tectum of Sprague-Dawley rat fetuses was removed at 15, 16 and 19 days of gestation (E15, E16 and E19), incubated as a dissociated cell suspension for 12 to 15 h in a medium containing True Blue for prelabeling, and injected into the central nucleus of the inferior colliculus (CNIC) of 28 normal male adult conspecifics. Viable tectal grafts were found in 57.5% of host animals with 65.2% located in CNIC. Grafts of E16 tissue had a survival rate of 84.2%, which was twice that of E15 or E19 donors, and there was no difference in graft survival rate in hosts sacrificed up to 6 months after the implantation procedure. Neurons prelabeled with True Blue and counterstained with thionin were identified as uniquely green stained cells in brightfield illumination and brilliant opaque cells in darkfield microscopy. Grafts were organized in tightly packed clusters of cells including large rounded polygonal and smaller cells similar in size to normal CNIC large and small multipolar neurons. Cell morphology of the larger ovoid and fusiform grafted neurons most closely resembled that of typical adult CNIC cell populations; other implanted cell types appeared immature even when host post-implant survival was extended. At longer post-implant intervals graft and host cells further intermingled, and both cellular and fibrous bridges were observed at the interface between host and graft tissue. The present work demonstrates the viability and development of homotypic cells grafted into a central auditory structure utilizing a simple method of prelabeling donor tissue with a fluorescent dye.

Transplant of embryonal nervous tissue preserves the responses of rat retinal ganglion cells after section of the optic nerve.

We have investigated the effectiveness of embryonal tectal tissue transplants in preserving the physiological activity of lesioned ganglion cells by recording the visual responses from the adult rat retina after section of the optic nerve, with or without transplants of embryonal nervous tissue on the stump. We have found that transplant of embryonal nervous tissue at the level of the optic nerve section has dramatic effects in preserving visual retinal responses to patterned stimuli for times as long as five months after surgery. By this time retinal responses to patterned stimuli have almost completely disappeared in control animals with optic nerve section alone.

Regeneration of axons from the adult rat optic nerve: influence of fetal brain grafts, laminin, and artificial basement membrane.

After transection of the optic nerve of adult rats, most of the axons in the proximal stump die and the surviving ones are unable to regenerate into the distal optic nerve. Since the fetal brain has an inherent capacity to regenerate axons, we investigated whether fetal (E16) target regions of optic axons (thalamus and tectum) transplanted to the completely transected optic nerve of adult rats would promote axon regeneration. In control operated rats, axon growth beyond the site of transection was restricted to a few fibers that grew irregularly within the connective tissue scar. By contrast, in grafted animals directed outgrowth of optic axons toward the transplant started at 6 days postoperation (p.o.) and reached its maximum 15 days p.o. and later, when numerous single optic fibers and small axon fascicles had grown toward and into the graft, where they formed arborizations and terminal varicosities. Regenerating optic axons were further advanced than GFAP-positive strands of astroglia that emanated from the proximal optic nerve stump. Laminin immunoreactivity appeared at 6 days p.o. in the zone of reactive astroglia in the terminal part of the optic nerve stump. Later it showed a distribution complementary to the pattern of GFAP immunoreactivity, which it seemd to circumscribe. There was no unequivocal codistribution of laminin immunoreactivity with regenerating axons. In further experiments, target regions from different ontogenetic stages (E14 to neonate and adult) and nontarget regions (E16, cerebral cortex or spinal cord) were grafted to the optic nerve stump. With the exception of the adult grafts, all transplants had effects on axon regeneration comparable to those of E16 target regions. In order to test the effects of extracellular matrix molecules on axon regeneration, a basement membrane gel reconstituted from individual components of the Engelbreth-Holm-Sarcoma (EHS) sarcoma was implanted between proximal and distal optic nerve stumps. No axons were induced to regenerate by this matrix. Likewise, laminin adsorbed to nitrocellulose paper and implanted at the lesion site did not stimulate axon growth from the proximal optic nerve stump. These results indicate that fetal brain is able to induce and direct regrowth of axons from the optic nerve toward the graft across a substrate that is not composed of astroglia or basement membrane components like laminin. The directed growth of axons in the absence of a preformed substrate implies a chemotactic growth response along a concentration gradient mediated by neurotropic molecules released from the graft.

Morphological and functional studies of rat tectal transplants.

Donor tectal tissue from fetal rats was transplanted to the tectal region of intact host newborn rats. Histological and ultrastructural characteristics of mature tectal grafts were examined with special emphasis placed on glial elements and the interface between transplant and host brain. Most transplants survived, proliferated, and differentiated to mature neurons and glial cells. Mature transplants were found to contain small and medium sized neurons which formed clusters along the transplant border. Neurites traversed the interface between transplant and host brain. There was an absence of phagocytosis, inflammatory reactions and chromatolytic neurons throughout the transplant. Classical staining techniques and GFAP immunoreactive stains revealed only mild focal astrogliosis confined mostly to the subpial and perivascular regions of the transplant-host interface. Ultrastructural examination of the transplant revealed numerous axodendritic synapses with aggregates of synaptic vesicles and well formed postsynaptic membranes. The absence of a leptomeningeal membrane at several points along the transplant-host interface may allow neurites from both sides to establish functional synaptic contacts. Single unit recording combined with anterograde WGA-HRP neuronal tract tracing was used to determine the presence and extent of host retinal innervation of tectal transplants.

The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study.

The distribution of choline acetyltransferase was determined in normal and transplanted rat superior colliculus with choline acetyltransferase immunohistochemistry. This distribution was compared to the pattern of histochemically detected acetylcholinesterase activity. To determine cholinergic input to the superficial superior colliculus, double labelling experiments combining retrograde tracing methods and choline acetyltransferase immunohistochemistry were carried out. No choline acetyltransferase-containing neurons were observed in the rat superior colliculus. A dense network of choline acetyltransferase-immunoreactive fibres and terminals was seen in the intermediate layers of the normal superior colliculus. The distribution was patchy and very similar to the pattern of acetylcholinesterase activity. Occasional fibres and terminals were seen in the deep tectal laminae. The superficial layers contained a low number of choline acetyltransferase-stained fibres and terminals but a comparatively high level of acetylcholinesterase activity. Following a unilateral injection of a tracer into the superficial superior colliculus, retrogradely labelled choline acetyltransferase-immunoreactive neurons were found in the dorsal and ventral subnuclei of the ipsilateral parabigeminal nucleus. As in the normal superior colliculus, choline acetyltransferase-positive neurons were not found in tectal transplants. However, choline acetyltransferase-immunoreactive fibres and terminals were seen in grafts but only in those which had extensive connections with the host midbrain. The pattern of staining most closely resembled that seen in the intermediate layers of the normal superior colliculus. The similar arrangement of choline acetyltransferase and acetylcholinesterase activity in the intermediate layers of normal rat superior colliculus provides further evidence for cholinergic innervation to these layers, probably originating in the dorsal and pedunculopontine tegmental nuclei. The data from the double labelling experiments indicate that the choline acetyltransferase-immunoreactive terminals observed in the superficial layers represent the terminal field of an ipsilateral cholinergic projection from the parabigeminal nucleus. Tectal grafts receive cholinergic innervation from the host. The evidence suggests that much of this input derives from the cholinergic nuclei in the brainstem tegmentum which normally project to the intermediate tectal layers.

Tectal tissue grafted to the midbrain of newborn rats: effect of donor age on the survival, growth and connectivity of transplants.

Tectal tissue was removed from rats at embryonic ages (E) E15, E18, E20 and postnatal day 0 (P0) and grafted onto the midbrain of newborn host rats. Six to 24 weeks after transplantation we examined 1) the growth characteristics of the grafts, 2) their morphology and 3) the pattern of retinal innervation of the grafted tissue. Graft survival was markedly affected by donor age. Transplants from E15 and E18 donors showed a survival rate of 90% which decreased to 35% when tissue was taken from E20 animals. Only one graft could be definitively identified in the P0 group. The ultimate volume of the graft was inversely related to donor age; grafts taken from E15 donors grew in size and produced the largest grafts, whereas E20 grafts showed a reduction in tissue volume from the time of implantation. Host retinal input was found in surviving grafts from all fetal donor ages (E15-E20). This input was always restricted to localized areas in the grafts containing high AChE activity; these areas are believed to contain presumptive superior collicular cells from the superficial layers. Thus, in tissue taken from fetal rats, it appears that altering the donor age does not affect the selectivity with which host retinal axons grow into and innervate specific areas within tectal grafts.

Cultured fetal tectal tissue grafted to the midbrain of newborn rats: morphology of grafts and innervation by host retinal and cortical axons.

It has been shown previously that tectal tissue obtained from young embryos can be successfully transplanted to the neonatal rat brain. In the present study, tecta from E15 rat embryos were maintained as free-floating explants for 3-14 days in vitro (DIV) before being transplanted to the midbrain of newborn rats. We wished to determine how explant culture affected (i) graft survival, (ii) the subsequent morphological and histochemical development of tectal grafts and (iii) the specificity with which host retinal and cortical axons grew into and innervated the graft neuropil. Grafts were examined 6-40 weeks posttransplantation. Host retinal input was assessed by injecting the host eyes with either [3H]proline, horseradish peroxidase (HRP) or wheat-germ agglutinin conjugated HRP. The host cortical projection was examined using anterograde degeneration techniques. Frozen tissue sections were also stained for Nissl, neurofibrils or reacted for acetylcholinesterase (AChE). All 3 DIV and 7 DIV explants survived transplantation and many grew considerably in size within the host brain. 14 DIV grafts were smaller and were found in only 50% of host brains. The cellular organization, fibre architecture and pattern of AChE staining in cultured grafts was similar to that found in non-cultured tectal transplants. Furthermore, host retina and cortex projected into the grafts in a manner similar to their innervation of non-cultured tectal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex.

Retinae from embryonic mice were transplanted to the occipital cortex of neonatal rats together with their normal target regions, tectum or diencephalon, from embryonic mice or rats. In control experiments, retinae were cotransplanted with embryonic rat occipital cortex. In over 80% of the experimental animals, both transplants differentiated and grew. Ganglion cells in the retinae cotransplanted close to tectum or diencephalon survived for at least 15 weeks. Their survival was associated with the development of a distinct optic fiber layer and outgrowth of axons from the transplanted mouse retina. Specific innervation of distinct patches within the cotransplanted rat tectum or diencephalon was demonstrated by the use of an anti-mouse antibody. The innervated regions, which could be as far away as 1.3 mm from the retinae, were correlated with cytological features of the cotransplanted tectum or diencephalon. By contrast, the host cortex was never innervated by the transplanted retinae. In the control animals in which the retinae were cotransplanted with occipital cortex and in four animals in which the cotransplants lay more than 2.7 mm apart, no ganglion cells were identified and there was no evidence of an optic fiber layer, outgrowth of axons, or innervation. These results support the idea that in order to survive, retinal ganglion cells need to innervate an appropriate target region. Further, the specific innervation of regions within the cotransplanted tectum or diencephalon suggests that these target regions are able to exert a tropic influence on the axons of retinal ganglion cells, even in the absence of many of the normal structure cues.

The development and distribution of alpha-bungarotoxin binding sites in rat tectal transplants.

The development and distribution of alpha-bungarotoxin (alpha-BTX) binding sites in tectal grafts was examined autoradiographically using the radioligand [125I]-alpha-BTX. High alpha-BTX binding was observed in localized areas within grafts; these areas corresponded to regions which contained high acetylcholinesterase activity and received retinal input. Receptor differentiation also occurred in the absence of specific host afferents. The graft data show that, as in normal superior colliculus, development of high alpha-BTX binding is limited to areas containing presumptive superficial layer cells.