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Chapter 34 Seeking axon guidance molecules in the adult rat CNS
L. McI~erracher, G. Doucet and S. Rossignol (Eds.) Progress in Brain Research, Vol. 137 © 2002 Elsevier Science B.V. All rights reserved
CHAPTER 34
Seeking axon guidance molecules in the adult rat CNS Guy Doucet * and Audrey Petit D~partement de Pathologie et Biologie Cellulaire and Centre de Recherche en Sciences Neurologiques, Universit~ de Montrfal, C.P. 6128, succursale Centre-ville, Montreal, QC H3C 3J7, Canada
Introduction
The last decade has been particularly rich in developments in the field of neural regeneration, as discussed in several chapters of this volume. Most of the efforts - - and the most fruitful, up to now - - were devoted to the identification of inhibitory factors responsible for growth limitations, and possible means to overcome them (McKeon et al., 1991; Fawcett, 1994; McKerracher et al., 1994; Mukhopadhyay et al., 1994; Smith-Thomas et al., 1994; Fitch and Silver, 1997; Chen et al., 2000; GrandPre et al., 2000). This craze for inhibitory factors left in the lurch the search for cellular and molecular factors, other than the neurotrophins, that might stimulate and direct axonal growth in a context of regeneration. Now, in spite of the progress in the identification of inhibitory molecules, blocking the action of these molecules has yielded only partial regeneration of lesioned axonal pathways and incomplete functional recovery. However, axonal regeneration is probably not limited solely by inhibitory molecules in the environment of the mature CNS. Oriented axonal growth, during development, results from several factors that attract, repulse or impede the movement of neuronal processes and thereby determine the
* Correspondence to: G. Doucet, Dfpartement de Pathologie et Biologie Cellulaire, Universit6 de Montrfal, C.P. 6128, succursale Centre-ville, Montrfal, QC H3C 3J7, Canada. Tel.: +I-514-343-6255; Fax: +1-514-343-5755; E-mail: guy.doucet @umontreal.ca
adopted direction. Regenerative growth would likely depend on similar vectorial combination of forces. Neurons being living elements, the driving forces are intrinsic to the growing cells, not in their environment. It is the neurons themselves which express the whole motor machinery, constituted by their cytoskeleton (microtubules microfilaments, neurofilaments and their associated molecules) and their energy metabolism. The elaboration of the axonal cytoskeleton in any given direction is influenced by signals present in the external environment - - extracellular matrix molecules, cell adhesion molecules, attractant and repellant, short- or long-range molecules (see Tessier-Lavigne and Goodman, 1996) -but recognition of all these signals depends on the expression of receptors at the surface of the growing cell (the response to netrins, for example, see Manitt and Kennedy, 2002, this volume). It is then the state of the intracellular signaling pathways and their action on the cytoskeleton that determines whether any given signal will have attractant, repellant or stabilizing effects (see Qiu et al., 2002, this volume; Manitt and Kennedy, 2002, this volume; Ellezam et al., 2002, this volume). It is therefore essential to identify the molecules that are susceptible to guide axon growth in the adult CNS, as well as the involved receptors and intracellular signaling pathways, in order to better understand, and eventually manipulate the potential for regeneration in the adult CNS. The search and study of guidance molecules is naturally hindered by the preponderance of axon growth inhibition in the adult CNS. However, substantial experimentation over the last
454 four decades, using neural lesions and transplantation, has demonstrated that axon guidance cues still exist in the adult CNS. The task consists henceforth of identifying these molecular cues, which might be molecules that serve the same ends during development. In this paper, we will first review succinctly lesion and transplantation experiments showing the existence of axon guidance cues in the adult CNS, as well as the capacity of mature neurons to recognize and respond to such cues in the mature CNS, or in grafted peripheral or fetal neural tissues. We will then describe our own experimental model, which will hopefully lead to the identification of axon guidance molecules for mature 5-HT neurons.
Axon guidance in the adult CNS Most of the lesion experiments demonstrating anatomical plasticity in the adult CNS were conducted before the 1980s (reviewed by Bj6rklund and Stenevi, 1979; Kiernan, 1979; Bj6rklund et al., 1981; Cotman et al., 1981). The anatomical plasticity thus disclosed consisted generally in a local sprouting of the lesioned axons or of unlesioned homo- or heterotypic pathways. These studies demonstrated that the mature CNS was not immutable and that its axons could still respond appropriately, but sometimes also aberrantly, to local guidance cues. Nevertheless, some lesion experiments, using specific cytotoxic drugs (5,6- or 5,7-dihydroxytryptamine) and producing less structural damage, demonstrated that serotoninergic (5-HT) neurons were capable of longdistance regeneration - - down to the lumbar spinal cord or up to the forebrain (reviewed in Bj6rklund and Stenevi, 1979; Bj6rklund et al., 1981). In the 1970s-1980s, neural transplantation was used extensively to study anatomical plasticity in the CNS (reviewed in Bj6rklund and Stenevi, 1979, 1984). Since adult CNS neurons do not survive transplantation, these studies used transplants of fetal nervous tissue in adult animals, either to test for the presence, in the mature CNS, of guidance cues that could be recognized by fetal neurons or, inversely, to examine the response of adult host neurons to tropic molecules present in neural grafts. Most of these studies demonstrated that grafted fetal neurons could establish specific projections in
the adult host brain, but usually with only shortdistance projections (see Bj6rklund and Stenevi, 1984). More recently, however, long-distance projections were observed in xenograft experiments - human or porcine fetal neural tissue in adult rat CNS (Wictorin et al., 1990; Wictorin and Bj6rklund, 1992; Wictorin et al., 1992; Isacson and Deacon, 1996, 1997). These projections were specific for their usual targets, even after ectopic placement. For example, fetal human dopamine neurons implanted in the adult rat substantia nigra, following a 6-hydroxydopamine lesion of the host dopamine neurons, extended axons exclusively in the rostral direction, towards the striaturn (Wictorin et al., 1992); confirming the existence of long-range guidance in the adult CNS. Moreover, some of these results could best be interpreted as axonal responses to long-range attractant, since ectopic nigral graft-derived axons could reach their normal target, the thalamus, by taking unusual pathways (Isacson and Deacon, 1996). It remains to be seen whether intact or severed adult neurons could also respond to such cues. Recent experiments with endogenous adult neural stem cells, have also demonstrated the persistence of axon guidance signals in the adult hippocampus, allowing for the integration of newly generated neurons into its circuitry (Markakis and Gage, 1999). Although mature axons rarely grow for long distances in the adult CNS environment, their responses to neural grafts indicate that they can recognize some guidance molecules. Indeed, work by Bj6rklurid and colleagues (see Bj6rklund and Stenevi, 1979) showed that noradrenaline neurons of the locus coeruleus could reproduce the sympathetic innervation pattern of the iris implanted into the mature brain. The brain-derived cholinergic innervation of the implanted iris being different, these observations suggested the existence of guidance signals in the grafted iris, to which monoamine or cholinergic axons were able to react. Moreover, 5-HT neurons could also innervate the implanted iris with the sympathetic pattern, if there had been a prior destruction of the host noradrenaline neurons of the locus coeruleus; suggesting that CNS neurons of different types were able to respond to the same guidance molecules expressed in the iris, although with different affinities. Several other studies with fetal neural tissues implanted into the adult CNS
455 have demonstrated that monoamine and cholinergic neurons could respond more readily to graft-derived signals than other types of CNS neurons (Bjrrklund et al., 1981; Bjrrklund and Stenevi, 1984; Nothias et al., 1988; Wictorin et al., 1988, 1989b; Nothias et al., 1990; Labandeira-Garcia et al., 1991; Triarhou et al., 1992). Other lightly myelinated axons, such as the primary dorsal root ganglion afferents expressing CGRP, also grow readily into neural grafts; suggesting that myelination may be an important factor in this phenomenon (Tessler et al., 1988; Itoh and Tessler, 1990; Nothias and Peschanski, 1990; Itoh et al., 1992). Other types of point-to-point CNS projection neurons could also innervate neural grafts, but only when there had been a prior lesion of their endogenous target (Bjrrklund and Stenevi, 1984; Armengol et al., 1989; T~nder et al., 1989; Wictorin and Bjrrklund, 1989; Field et al., 1991; Labandeira-Garcia et al., 1991; Girman, 1993; Schulz et al., 1993; Girman, 1994), of parallel projections (SCrensen et al., 1986), or of peripheral branches (Erzurumlu and Ebner, 1988; Ebner et al., 1989). In such cases, either the development of the grafts was modified by lesions of the host brain, so as to make them produce more attractant molecules, or the lesions changed the response of the host target-deprived neurons, which then became more responsive to signals from the grafts (by inducing the expression of receptors or changing the state of intracellular signaling pathways). In any case, these experiments show that mature neurons can respond, or can be induced to respond, to guidance cues. At present the identity of these signals of the adult CNS, or of the neural grafts, that are acting on adult neurons is unknown. Adult and fetal neurons can have divergent responses to the same signals (e.g., Debellard et al., 1996; Fawcett, 1997; Borisoff et al., 2000). Even during neural development, the neuronal response to axon guidance signals changes along the way to the final destination of the axon tip (e.g., Stein and Tessier-Lavigne, 2001). It will therefore be important to define in space and time the specific guidance cues that may be acting on selective groups of axons, in order to promote their functional regeneration. Indeed, many of the above-mentioned lesion or grafting experiments have disclosed the formation of aberrant connections, particularly after ectopic placement of neural grafts (Wiklund and M~llghrd,
1979; Hallas et al., 1980; Oblinger and Das, 1982; Itoh and Tessler, 1990; Bosler et al., 1992; Zwimpfer et al., 1992); a situation that may have some similarity with that of lesioned axons, following trauma. Indeed, lesioned axons growing in the adult CNS must find their targets along pathways that greatly differ from those they had followed during development. Extensive knowledge of the molecules expressed in the intact or lesioned adult CNS that could be used by growing axons as guidance cues would help to manipulate their expression in order to minimize aberrant connections and to optimize those appropriate for recovery of function. There has been immense progress lately in the identification of axon guidance molecules acting during neural development (e.g., Tessier-Lavigne, 1994; Tessier-Lavigne and Goodman, 1996; Brose and Tessier-Lavigne, 2000; Stein and Tessier-Lavigne, 2001). What is still missing is the knowledge of their expression, distribution and roles in the adult CNS. We need to know if these molecules can serve to guide the axon growth of adult neurons, i.e., if they might promote or, inversely, hinder regeneration; and also how they might be affected by CNS injury. Considering the number of genes that are still unknown (Bork and Copley, 2001; Hogenesch et al., 2001), it is also likely that a fair number of such molecules remain to be identified. Most of the currently known guidance molecules have been discovered by studying experimental models in which axon growth from defined populations of neurons could be manipulated in vivo or in vitro. Many of the above transplantation experiments showing axon guidance activities might contribute to the discovery of new guidance molecules, if analyzed further. Combined with the recent advances in genomic (DNA chips) and proteomic (2D gel electrophoresis and mass spectrometry) approaches, such models could contribute to the progress in the field of CNS injury and regeneration. The experiments described below represent an attempt in this direction. Use of neural transplants into the striatum as a model to study axon guidance in adult rat brain
To determine whether adult host neurons could innervate ectopic grafts, we have first studied the in-
456 nervation by host brain neurons of ventral mesencephalic (VM) grafts implanted into the neostriatum of adult rats - - in collaboration with Anders BjOrklund and Patrick Brundin (Doucet et al., 1989). We examined the projections from the frontal cortex (anterograde transport of PHA-L), the neostriatum (DARPP-32 immunohistochemistry and P H A - L anterograde transport) and the mesencephalic raphe (5-HT immunohistochemistry), i.e., the major afferent projections c o m m o n to both transplanted (VM) and recipient (neostriatum) target regions. It was expected that 5-HT axons would be the most profuse inside the grafts, since they project densely to the substantia nigra, and since most of the dorsal raphe 5-HT neurons projecting to the nigra have collaterals into the neostriatum. However, the densest host projections into the core of VM grafts were those from the frontal cortex. Neostriatal projections were also profuse, but remained confined within 100-300 Ixm of the inner graft border; whereas 5-HT axons showed very little growth into the grafts: only few axons were detected inside the graft, essentially near the graft-host border (Fig. 1). This contrasted with the observation of
profuse 5-HT innervations into grafts of fetal striatal tissue implanted into the ibotenic acid-lesioned neostriatum (Wictorin et al., 1988). Thus, in spite of their so-called 'diffuse' mode of projection, adult 5-HT axons appear to display anatomical selectivity in their reaction to a new innervation target. It was therefore of particular interest to define the conditions in which they would or would not grow.
Serotonin innervation of VM and striatal transplants into the neostriatum These experiments also demonstrated that the grafted VM tissue was not inhibitory to 5-HT axon growth, at least for immature 5-HT axons. Indeed, when VM grafts included even very few co-grafted 5HT perikarya (4-5 per 50-txm-thick section), they were filled with a very dense 5-HT axonal network (Doucet et al., 1989; Mounir et al., 1994). This dense 5-HT innervation was present even when the host 5-HT neurons had been destroyed by 5,7DHT (Mounir et al., 1994), confirming that it took origin in the grafted 5-HT neurons. We therefore examined the hypothesis that immature 5-HT axons
Fig. 1. Diagram summarizing the results of axonal tracing from the host cerebral cortex, striatum and dorsal raphe (serotonin) into intrastriatal ventral mesencephalic grafts. The most profuse projections from the host brain came from the frontal cortex, with axons innervating all areas of the grafts. Host striatal axons remained essentially confined within a 100-300-1xm-largeband in the peripheral zones of the grafts, whereas serotonin axons were virtually absent from the core of the grafts and only occasional in the periphery (see Doucet et al., 1989).
457 would have a higher affinity than adult ones for VM tissue, by quantifying host-derived 5-HT axons in VM grafts following implantation into immature and adult recipients. Transplants were done in newborn (postnatal days 5-7 or P5-P7), juvenile (P15) and adult (body weight 200-250 g) rats and examined 2 months later by 5-HT immunohistochemistry. We then found a much denser innervation of the grafts in newborn recipients than in juvenile or adult ones. Indeed, with recipient ages P15 and above at the time of implantation, there were practically no 5-HT axons in the core of VM grafts (Mounir et al., 1994), in keeping with our earlier observations (Doucet et al., 1989). The 5-HT innervation of fetal striatal - - but not VM - - grafts had been reported to be less dense after implantation in intact rather than ibotenic acidlesioned striatum (Labandeira-Garcia et al., 1991; Lu et al., t991). To rule out the possibility that the differences in 5-HT innervation we had observed between striatal and VM grafts be due to the prior excitotoxic lesion, we compared the 5-HT innervation of VM and striatal grafts following implantation into intact neostriatum. Since no data were available on the 5-HT innervation of striatal grafts implanted in newborns, the study included VM or striatal grafts implanted in neonatal, juvenile and adult rat striatum. To quantify the innervation, we used a method based on the uptake of tritiated 5-HT, in vitro, followed by autoradiography and image analysis, 6 months after transplantation (Doucet and Descarries, 1993). Brain slices were incubated with 10 -6 M [3H]5-HT, in the presence of a monoamine oxidase inhibitor and of an inhibitor of monoamine uptake by dopaminergic neurons. These slices were then fixed with glutaraldehyde, embedded in epoxy resin and sectioned at a thickness of 4 Ixm for autoradiography. Serotonin axon varicosities were then counted within the grafts (see Fig. 2B,D). Adjacent semi-thin sections from the same brain slices were processed by post-embedding immunohistochemistry for tryptophan hydroxylase (TPH), tyrosine hydroxylase (TH, Fig. 2A) or dopamine- and adenosine-regulated phosphoprotein-32 (DARPP-32, Fig. 2C) (Ouimet et al., 1984). Sections immunostained for TPH confirmed the absence of 5-HT neurons inside the grafts, whereas TH or DARPP-32 staining was used to assess the distribution of 5-HT fibers in relation to that
of dopamine or striatal neurons, in VM or striatal grafts (Wictorin et al., 1989a), respectively. The quantitative analyses demonstrated that the DARPP-32-positive patches, representing the true striatal compartments of 'striatal' grafts, were three to four times more densely innervated by host 5-HT axons than VM grafts, or DARPP-32immunonegative (non-striatal) regions of 'striatal' grafts. In all types of grafts (VM as well as DARPP32-positive and DARPP-32-negative regions of striatal grafts), the amount of 5-HT innervation decreased by 75-80% between grafts implanted in neonatal versus adult rats (and examined 6 months later) (Pierret et al., 1998). Therefore, the proportion of 5-HT innervation in VM and striatal grafts remained unchanged between newborn and adult recipients. These results demonstrated that the relative affinity of the 5-HT axons present in the host striatum for VM or striatal grafts remained constant between newborns and adults. The differences between adult and neonatal 5-HT innervations of the grafts was likely the result of an intrinsic developmental decrease in the growth capacity of the mature host 5-HT neurons (Goldberg et al., 2001). Nevertheless, the 5-HT innervation of striatal grafts decreased gradually in juvenile and adult recipients, whereas the amount of 5-HT innervation of VM grafts in juvenile recipients was as low as that in adult recipients, as in our previous study (Mounir et al., 1994). Therefore, the intrinsic developmental decrease in growth capacity for a given type of neuron may not follow the same time course for different targets, i.e., in their response to different guidance molecules. It follows from this idea that different signaling pathways involved in axon growth and guidance might mature at different rates in a given neuron. It would then be imaginable to selectively reactivate one signaling pathway, in order to promote the regeneration of specific projections. Influence of the glial scar or chondroitin sulfate proteoglycans
A difference in the glial scar, or in the expression of axon growth inhibitory molecules, such as chondroitin sulfate proteoglycans (CSPG), might have explained the difference in 5-HT innervation between VM and striatal grafts. We therefore ex-
458 amined the glial scar and expression of CSPG by immunohistochemistry at different time points after transplantation of V M tissue in newborn, juvenile or adult rats, as well as of striatal tissue in adults (Petit et al., 2002). Ten days after transplantation, immunohistochemistry for GFAP showed the presence of astrocytes inside and around the grafts, forming a glial scar, in all cases. This glial scar appeared only slightly less dense around V M grafts implanted in newborns. Also, the distribution of GFAP-immunostained elements was more heterogeneous in striatal grafts, as previously reported by others (Gates et al., 1996). The expression of CSPG was very similar in all types of grafts and in recipients of any age, at 10 days. Serotonin immunoreactive axons were already profuse in VM grafts implanted in newborns, but rare in VM and striatal grafts implanted in adults. Therefore, the presence of a glial scar or CSPG had no negative impact on the ingrowth of immature 5-HT axons. At longer time points, GFAP and CSPG immunoreactivities subsided gradually in striatal grafts implanted in adults, but remained highly heterogeneous, such that patches of intense staining remained present between 10 days and 2 months. Serotonin axons gradually invaded these striatal grafts and were clearly more profuse in DARPP-32-positive patches, although many were also present in DARPP-32negative zones. Interestingly, the DARPP-32-positive patches corresponded to GFAP- and CSPG-negative zones of these grafts (the latter two being then largely overlapping), 2 months after transplantation. Thus, for striatal grafts, the time course of appearance and distribution of 5-HT axons was in inverse correlation with those of GFAP and CSPG. Nevertheless, a similarly heterogeneous 5-HT innervation of striatal grafts, inversely correlated with GFAP, had also been demonstrated following implantation in newborn rats (Pierret et al., 1998). Therefore,
since neonatal 5-HT axons were not inhibited by the glial scar or CSPG around or in the VM grafts, as stated above, this heterogeneity in 5-HT innervation of striatal grafts could not be explained by the complementary distribution of GFAP or CSPG. Other elements - - attractant or repellant - - in the different compartments of striatal grafts must be responsible of the differential affinity of host 5-HT axons. The situation was different for VM grafts. Serotonin axons remained rare in these grafts, even after 2 months, as observed in our previous studies; even though GFAP immunoreactive elements were greatly reduced and the expression of CSPG was totally abolished. It was then concluded that the absence of 5-HT innervation inside VM grafts could not be attributed to the astrocyte scar or the expression of CSPG in the graft. Our interpretation was that either V M grafts contained molecules that were inhibitory to the ingrowth of 5-HT axons or, else, that striatal grafts (and particularly their DARPP-32-positive zones) contained molecules that were attractant for these axons. To settle this issue, it was crucial to determine the type of cells that expressed the molecules responsible for the repellent or attractant activity.
Role of astrocytes in the graft In view of the complexity of the grafted tissue, it is not easy to identify molecules that may attract 5-HT axons into striatal grafts, or repel them from VM grafts. A more simple model was needed for that purpose. Because astrocytes had been shown in several instances to have different regional phenotypes that could affect the growth of dopamine or 5-HT neurons (Denis-Donini et al., 1983, 1984; Chamak et al., 1987; Liu and Lander, 1992a), it was proposed that they might be the cells that influenced 5-HT axon ingrowth into the grafts.
Fig. 2. Ventral mesencephalic (A,B) and striatal (C,D) grafts in juvenile (P15) rats. The animals were sacrificed 6 months after transplantation. (A) Semi-thin section from a brain slice incubated with tritiated 5-HT. The section was treated for post-embedding TH-immunohistochemistry, showing grafted dopamine neurons and processes. (B) An adjacent section of the same brain slice, but exposed to a nuclear emulsion, for autoradiography of 5-HT nerve terminals, showing that serotonin axon terminals were very sparse in the graft. (C) A section treated in post-embedding DARPP-32-immunohistochemistry, showing the striatal neurons. Part of the graft contains true striatal tissue, and part of it contains non-striatal tissue (DARPP-32-negative). (D) Adjacent section showing that the DARPP-32-positive area is more densely innervated by host 5-HT axons. From Pierret et al. (1998). Reproduced with permission.
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Dissection and culture of neonatal astrocytes Ventral Mesencephalon
Neostriatum
Co-implantation of cultured astrocytes with ventral mesencephalic (or striatal) tissue
or _. i or
Cerebral Cortex
7 Fig. 3. Technique used for co-transplantation of astrocytes cultured from the neonatal ventral mesencephalon, neostriaturn or cerebral cortex, and then mixed with dissociated fetal (E14-E15) ventral mesencephalicor striatal tissue, before implantation into the striatum of adult rats. From Petit et al. (200l). Reprinted with permission To test this hypothesis, cultured astrocytes from three different brain regions were co-implanted with fetal VM or striatal tissue into the intact striatum of adult rats (Petit et al., 2001) (see Fig. 3). The same autoradiographic technique, described above, was used to determine the density of 5-HT innervation in each type of graft. This quantitative analysis clearly showed that co-grafts of VM containing striatal or neocortical astrocytes were three to four times more densely innervated by host 5-HT axons than control VM grafts or co-grafts of VM enriched with cultured VM astrocytes (Fig. 4). Moreover, co-grafts of striatal tissue with VM astrocytes were innervated as profusely as single striatal grafts, demonstrating that VM astrocytes were not inhibitory to 5-HT axon ingrowth. To ensure that this difference in the density of 5-HT innervation resulting from the addition of cortical or striatal astrocytes was not due to some indirect effect on the glial scar or on the expression of CSPG in the graft, we also examined the different types of grafts by GFAP and CSPG immunohistochemistry (Petit et al., 2001). No difference between graft types were found in the distribution of these molecules. The density of CSPG immunostaining, as well as the surface area of GFAP immunoreactive processes in the core of the grafts, at the graft-host border, and in the adjacent host striatal tissue were also quantified. The results showed no difference in
CSPG immunoreactivity. As for GFAP, all types of co-grafts contained more astrocytes than single VM grafts, as expected for grafts that had been enriched with astrocytes; but there was no difference between the groups of co-grafts, including VM grafts with VM astrocytes which were as poorly innervated by 5-HT axons as single VM grafts. It was concluded that the differences in 5-HT innervation between cografts were not due to a reduction in the glial reaction or in the expression of CSPG. Altogether, the latter observations rather indicated that cortical and striatal astrocytes attracted adjacent host 5-HT axons normally fated to innervate the striatum. Interestingly, some of these axons might have been collaterals of axons innervating the ventral midbrain (van der Kooy and Hattori, 1980; Imai et al., 1986), which might account for the few axons that were found in control VM grafts.
Influence of co-grafted astrocytes on other graft afferents To test the possibility that these effects of astrocytes on 5-HT axons represented a general growthpromoting action on all surrounding host axons, we also examined the influence of the different populations of astrocytes on the other types of host afferents that had previously been demonstrated into the VM grafts, i.e., cortical and striatal afferents (see above).
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Fig. 4. Grafts of ventral mesencephalic tissue, with or without co-grafted astrocytes, into the striatum of adult rats. Pictures are from 4-1xm-thick sections from 200-1xm-thick brain slices processed in vitro for tritiated serotonin uptake and autoradiography, 2 months after transplantation. (A) Single ventral mesencephalic graft. (B) Co-graft containing ventral mesencephalic astrocytes. (C) Co-graft with striatal astrocytes. (D) Co-graft with cortical astrocytes. (E) The complete section of the graft in A, to show the size of the pictures in A - D (white rectangle). In all cases the left part displays host striatum and the right part the graft. Arrows point to silver grain aggregates representing labeled serotonin varicosities. From Petit et al. (2001). Reprinted with permission.
462 We found no increases or decreases in the number of labeled axons following multiple injections of biotinylated dextran in the ffontoparietal cortex or after DARPP-32 immunohistochemistry. Therefore, it appears that the growth-promoting effects of striatal or cortical astrocytes were specific for 5-HT axons.
Advantages of the model for the search of axon guidance molecules Now, since we have three highly purified populations of astrocytes in culture, with different effects on 5-HT axons, it becomes possible to use a comparison of gene and protein expression between them, in the hope of finding candidate molecules with the same biological activity on 5-HT axons as in cografts. Some molecules with known action on 5-HT axons are among such candidates, such as brainderived neurotrophic factor (BDNF), glial cell linederived neurotrophic factor (GDNF) or S100~ (Liu and Lander, 1992b; Beck et al., 1996; Mamounas et al., 2000; Nishi et al., 2000), but the probability is high that the observed effects were due to unknown molecules. Indeed, recent estimates of the total human genome are in the order of 30,00060,000 genes (Bork and Copley, 200l; Hogenesch et al., 2001), and the total number of genes in the rat or mouse genomes are probably near the same order of magnitude. Among these genes, the number of those with a known protein product is about 15,000, which represents 25-50% of the total estimate. Therefore, a newly detected activity has approximately a 50-75% chance of being due to an unidentified molecule. We are currently running experiments with gene chips and 2D gel electrophoresis, comparing gene and protein expression in different astrocyte populations in culture. Preliminary results already ruled out BDNF, GDNF and S10013 as candidates for the effects. They also indicate that the number of gene products expressed differently among astrocyte populations from the cortex or VM is relatively low, in the order of a few hundred. Taking into account the numbers of proteins involved in structural or metabolic functions, and the structure expected from presumed intercellular signaling molecules, such numbers should be reducible to a manageable number of candidates to be tested with appropriate tests in vitro.
We are also developing tests in vitro that will reproduce the features of the transplantation model described above; based on the collagen gel explant technique of Tessier-Lavigne (Tessier-Lavigne et al., 1988; Kennedy et al., 1994). Serotoninergic axons innervating neostriatal explants will thus be challenged with VM tissue, with or without added cortical or striatal astrocytes. Such in vitro models should help to further characterize the nature of the molecules involved (membrane or matrix-bound, diffusible, etc.), allowing the number of criteria that candidate molecules will need to fulfil to account for the observed activity to be increased. These in vitro characterizations will thus be useful to reduce the number of molecules to be tested and the same techniques will then serve to test the candidate molecules generated from genomic and proteomic data.
Conclusion Several inhibitory molecules have been identified as being responsible for the poor regenerative capacities of CNS neurons. However, a reduction in the level of growth-promoting or axon guidance molecules might also contribute in turning the balance towards inhibition. Lesion and transplantation experiments suggested that guidance molecules are still present in the adult CNS, but adult neurons might require some priming to respond to these signals. A better knowledge of growth-promoting or guidance molecules might allow their manipulation to turn back the balance towards appropriately directed axon growth. Several models suggesting tropic activities in the adult CNS exist, but need to be developed further in vitro to allow identification of the molecules that underlie the observed effects. A better knowledge of 5-HT axon guidance molecules would certainly be relevant for spinal cord injury, in view of the role of 5-HT in the control of locomotion in brain and the spinal cord (Jacobs and Fornal, 1995; Schmidt and Jordan, 2000; see also Rossignol et al., 2002, this volume).
Acknowledgements We thank Dr. Laurent Descarries for his critical revision of this manuscript. This work was supported by the Canadian Institutes for Health Re-
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s e a r c h ( C I H R ) , t h e F o n d s d e la r e c h e r c h e e n s a n t 6 d u Q u 6 b e c ( F R S Q ) , t h e F o n d s p o u r la f o r m a t i o n d e s c h e r c h e u r s e t l ' a i d e ~t la r e c h e r c h e ( F C A R ) a n d G r o u p e d e r e c h e r c h e s u r le s y s t & n e n e r v e u x c e n t r a l (FCAR Center).
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CHAPTER 34
Seeking axon guidance molecules in the adult rat CNS Guy Doucet * and Audrey Petit D~partement de Pathologie et Biologie Cellulaire and Centre de Recherche en Sciences Neurologiques, Universit~ de Montrfal, C.P. 6128, succursale Centre-ville, Montreal, QC H3C 3J7, Canada
Introduction
The last decade has been particularly rich in developments in the field of neural regeneration, as discussed in several chapters of this volume. Most of the efforts - - and the most fruitful, up to now - - were devoted to the identification of inhibitory factors responsible for growth limitations, and possible means to overcome them (McKeon et al., 1991; Fawcett, 1994; McKerracher et al., 1994; Mukhopadhyay et al., 1994; Smith-Thomas et al., 1994; Fitch and Silver, 1997; Chen et al., 2000; GrandPre et al., 2000). This craze for inhibitory factors left in the lurch the search for cellular and molecular factors, other than the neurotrophins, that might stimulate and direct axonal growth in a context of regeneration. Now, in spite of the progress in the identification of inhibitory molecules, blocking the action of these molecules has yielded only partial regeneration of lesioned axonal pathways and incomplete functional recovery. However, axonal regeneration is probably not limited solely by inhibitory molecules in the environment of the mature CNS. Oriented axonal growth, during development, results from several factors that attract, repulse or impede the movement of neuronal processes and thereby determine the
* Correspondence to: G. Doucet, Dfpartement de Pathologie et Biologie Cellulaire, Universit6 de Montrfal, C.P. 6128, succursale Centre-ville, Montrfal, QC H3C 3J7, Canada. Tel.: +I-514-343-6255; Fax: +1-514-343-5755; E-mail: guy.doucet @umontreal.ca
adopted direction. Regenerative growth would likely depend on similar vectorial combination of forces. Neurons being living elements, the driving forces are intrinsic to the growing cells, not in their environment. It is the neurons themselves which express the whole motor machinery, constituted by their cytoskeleton (microtubules microfilaments, neurofilaments and their associated molecules) and their energy metabolism. The elaboration of the axonal cytoskeleton in any given direction is influenced by signals present in the external environment - - extracellular matrix molecules, cell adhesion molecules, attractant and repellant, short- or long-range molecules (see Tessier-Lavigne and Goodman, 1996) -but recognition of all these signals depends on the expression of receptors at the surface of the growing cell (the response to netrins, for example, see Manitt and Kennedy, 2002, this volume). It is then the state of the intracellular signaling pathways and their action on the cytoskeleton that determines whether any given signal will have attractant, repellant or stabilizing effects (see Qiu et al., 2002, this volume; Manitt and Kennedy, 2002, this volume; Ellezam et al., 2002, this volume). It is therefore essential to identify the molecules that are susceptible to guide axon growth in the adult CNS, as well as the involved receptors and intracellular signaling pathways, in order to better understand, and eventually manipulate the potential for regeneration in the adult CNS. The search and study of guidance molecules is naturally hindered by the preponderance of axon growth inhibition in the adult CNS. However, substantial experimentation over the last
454 four decades, using neural lesions and transplantation, has demonstrated that axon guidance cues still exist in the adult CNS. The task consists henceforth of identifying these molecular cues, which might be molecules that serve the same ends during development. In this paper, we will first review succinctly lesion and transplantation experiments showing the existence of axon guidance cues in the adult CNS, as well as the capacity of mature neurons to recognize and respond to such cues in the mature CNS, or in grafted peripheral or fetal neural tissues. We will then describe our own experimental model, which will hopefully lead to the identification of axon guidance molecules for mature 5-HT neurons.
Axon guidance in the adult CNS Most of the lesion experiments demonstrating anatomical plasticity in the adult CNS were conducted before the 1980s (reviewed by Bj6rklund and Stenevi, 1979; Kiernan, 1979; Bj6rklund et al., 1981; Cotman et al., 1981). The anatomical plasticity thus disclosed consisted generally in a local sprouting of the lesioned axons or of unlesioned homo- or heterotypic pathways. These studies demonstrated that the mature CNS was not immutable and that its axons could still respond appropriately, but sometimes also aberrantly, to local guidance cues. Nevertheless, some lesion experiments, using specific cytotoxic drugs (5,6- or 5,7-dihydroxytryptamine) and producing less structural damage, demonstrated that serotoninergic (5-HT) neurons were capable of longdistance regeneration - - down to the lumbar spinal cord or up to the forebrain (reviewed in Bj6rklund and Stenevi, 1979; Bj6rklund et al., 1981). In the 1970s-1980s, neural transplantation was used extensively to study anatomical plasticity in the CNS (reviewed in Bj6rklund and Stenevi, 1979, 1984). Since adult CNS neurons do not survive transplantation, these studies used transplants of fetal nervous tissue in adult animals, either to test for the presence, in the mature CNS, of guidance cues that could be recognized by fetal neurons or, inversely, to examine the response of adult host neurons to tropic molecules present in neural grafts. Most of these studies demonstrated that grafted fetal neurons could establish specific projections in
the adult host brain, but usually with only shortdistance projections (see Bj6rklund and Stenevi, 1984). More recently, however, long-distance projections were observed in xenograft experiments - human or porcine fetal neural tissue in adult rat CNS (Wictorin et al., 1990; Wictorin and Bj6rklund, 1992; Wictorin et al., 1992; Isacson and Deacon, 1996, 1997). These projections were specific for their usual targets, even after ectopic placement. For example, fetal human dopamine neurons implanted in the adult rat substantia nigra, following a 6-hydroxydopamine lesion of the host dopamine neurons, extended axons exclusively in the rostral direction, towards the striaturn (Wictorin et al., 1992); confirming the existence of long-range guidance in the adult CNS. Moreover, some of these results could best be interpreted as axonal responses to long-range attractant, since ectopic nigral graft-derived axons could reach their normal target, the thalamus, by taking unusual pathways (Isacson and Deacon, 1996). It remains to be seen whether intact or severed adult neurons could also respond to such cues. Recent experiments with endogenous adult neural stem cells, have also demonstrated the persistence of axon guidance signals in the adult hippocampus, allowing for the integration of newly generated neurons into its circuitry (Markakis and Gage, 1999). Although mature axons rarely grow for long distances in the adult CNS environment, their responses to neural grafts indicate that they can recognize some guidance molecules. Indeed, work by Bj6rklurid and colleagues (see Bj6rklund and Stenevi, 1979) showed that noradrenaline neurons of the locus coeruleus could reproduce the sympathetic innervation pattern of the iris implanted into the mature brain. The brain-derived cholinergic innervation of the implanted iris being different, these observations suggested the existence of guidance signals in the grafted iris, to which monoamine or cholinergic axons were able to react. Moreover, 5-HT neurons could also innervate the implanted iris with the sympathetic pattern, if there had been a prior destruction of the host noradrenaline neurons of the locus coeruleus; suggesting that CNS neurons of different types were able to respond to the same guidance molecules expressed in the iris, although with different affinities. Several other studies with fetal neural tissues implanted into the adult CNS
455 have demonstrated that monoamine and cholinergic neurons could respond more readily to graft-derived signals than other types of CNS neurons (Bjrrklund et al., 1981; Bjrrklund and Stenevi, 1984; Nothias et al., 1988; Wictorin et al., 1988, 1989b; Nothias et al., 1990; Labandeira-Garcia et al., 1991; Triarhou et al., 1992). Other lightly myelinated axons, such as the primary dorsal root ganglion afferents expressing CGRP, also grow readily into neural grafts; suggesting that myelination may be an important factor in this phenomenon (Tessler et al., 1988; Itoh and Tessler, 1990; Nothias and Peschanski, 1990; Itoh et al., 1992). Other types of point-to-point CNS projection neurons could also innervate neural grafts, but only when there had been a prior lesion of their endogenous target (Bjrrklund and Stenevi, 1984; Armengol et al., 1989; T~nder et al., 1989; Wictorin and Bjrrklund, 1989; Field et al., 1991; Labandeira-Garcia et al., 1991; Girman, 1993; Schulz et al., 1993; Girman, 1994), of parallel projections (SCrensen et al., 1986), or of peripheral branches (Erzurumlu and Ebner, 1988; Ebner et al., 1989). In such cases, either the development of the grafts was modified by lesions of the host brain, so as to make them produce more attractant molecules, or the lesions changed the response of the host target-deprived neurons, which then became more responsive to signals from the grafts (by inducing the expression of receptors or changing the state of intracellular signaling pathways). In any case, these experiments show that mature neurons can respond, or can be induced to respond, to guidance cues. At present the identity of these signals of the adult CNS, or of the neural grafts, that are acting on adult neurons is unknown. Adult and fetal neurons can have divergent responses to the same signals (e.g., Debellard et al., 1996; Fawcett, 1997; Borisoff et al., 2000). Even during neural development, the neuronal response to axon guidance signals changes along the way to the final destination of the axon tip (e.g., Stein and Tessier-Lavigne, 2001). It will therefore be important to define in space and time the specific guidance cues that may be acting on selective groups of axons, in order to promote their functional regeneration. Indeed, many of the above-mentioned lesion or grafting experiments have disclosed the formation of aberrant connections, particularly after ectopic placement of neural grafts (Wiklund and M~llghrd,
1979; Hallas et al., 1980; Oblinger and Das, 1982; Itoh and Tessler, 1990; Bosler et al., 1992; Zwimpfer et al., 1992); a situation that may have some similarity with that of lesioned axons, following trauma. Indeed, lesioned axons growing in the adult CNS must find their targets along pathways that greatly differ from those they had followed during development. Extensive knowledge of the molecules expressed in the intact or lesioned adult CNS that could be used by growing axons as guidance cues would help to manipulate their expression in order to minimize aberrant connections and to optimize those appropriate for recovery of function. There has been immense progress lately in the identification of axon guidance molecules acting during neural development (e.g., Tessier-Lavigne, 1994; Tessier-Lavigne and Goodman, 1996; Brose and Tessier-Lavigne, 2000; Stein and Tessier-Lavigne, 2001). What is still missing is the knowledge of their expression, distribution and roles in the adult CNS. We need to know if these molecules can serve to guide the axon growth of adult neurons, i.e., if they might promote or, inversely, hinder regeneration; and also how they might be affected by CNS injury. Considering the number of genes that are still unknown (Bork and Copley, 2001; Hogenesch et al., 2001), it is also likely that a fair number of such molecules remain to be identified. Most of the currently known guidance molecules have been discovered by studying experimental models in which axon growth from defined populations of neurons could be manipulated in vivo or in vitro. Many of the above transplantation experiments showing axon guidance activities might contribute to the discovery of new guidance molecules, if analyzed further. Combined with the recent advances in genomic (DNA chips) and proteomic (2D gel electrophoresis and mass spectrometry) approaches, such models could contribute to the progress in the field of CNS injury and regeneration. The experiments described below represent an attempt in this direction. Use of neural transplants into the striatum as a model to study axon guidance in adult rat brain
To determine whether adult host neurons could innervate ectopic grafts, we have first studied the in-
456 nervation by host brain neurons of ventral mesencephalic (VM) grafts implanted into the neostriatum of adult rats - - in collaboration with Anders BjOrklund and Patrick Brundin (Doucet et al., 1989). We examined the projections from the frontal cortex (anterograde transport of PHA-L), the neostriatum (DARPP-32 immunohistochemistry and P H A - L anterograde transport) and the mesencephalic raphe (5-HT immunohistochemistry), i.e., the major afferent projections c o m m o n to both transplanted (VM) and recipient (neostriatum) target regions. It was expected that 5-HT axons would be the most profuse inside the grafts, since they project densely to the substantia nigra, and since most of the dorsal raphe 5-HT neurons projecting to the nigra have collaterals into the neostriatum. However, the densest host projections into the core of VM grafts were those from the frontal cortex. Neostriatal projections were also profuse, but remained confined within 100-300 Ixm of the inner graft border; whereas 5-HT axons showed very little growth into the grafts: only few axons were detected inside the graft, essentially near the graft-host border (Fig. 1). This contrasted with the observation of
profuse 5-HT innervations into grafts of fetal striatal tissue implanted into the ibotenic acid-lesioned neostriatum (Wictorin et al., 1988). Thus, in spite of their so-called 'diffuse' mode of projection, adult 5-HT axons appear to display anatomical selectivity in their reaction to a new innervation target. It was therefore of particular interest to define the conditions in which they would or would not grow.
Serotonin innervation of VM and striatal transplants into the neostriatum These experiments also demonstrated that the grafted VM tissue was not inhibitory to 5-HT axon growth, at least for immature 5-HT axons. Indeed, when VM grafts included even very few co-grafted 5HT perikarya (4-5 per 50-txm-thick section), they were filled with a very dense 5-HT axonal network (Doucet et al., 1989; Mounir et al., 1994). This dense 5-HT innervation was present even when the host 5-HT neurons had been destroyed by 5,7DHT (Mounir et al., 1994), confirming that it took origin in the grafted 5-HT neurons. We therefore examined the hypothesis that immature 5-HT axons
Fig. 1. Diagram summarizing the results of axonal tracing from the host cerebral cortex, striatum and dorsal raphe (serotonin) into intrastriatal ventral mesencephalic grafts. The most profuse projections from the host brain came from the frontal cortex, with axons innervating all areas of the grafts. Host striatal axons remained essentially confined within a 100-300-1xm-largeband in the peripheral zones of the grafts, whereas serotonin axons were virtually absent from the core of the grafts and only occasional in the periphery (see Doucet et al., 1989).
457 would have a higher affinity than adult ones for VM tissue, by quantifying host-derived 5-HT axons in VM grafts following implantation into immature and adult recipients. Transplants were done in newborn (postnatal days 5-7 or P5-P7), juvenile (P15) and adult (body weight 200-250 g) rats and examined 2 months later by 5-HT immunohistochemistry. We then found a much denser innervation of the grafts in newborn recipients than in juvenile or adult ones. Indeed, with recipient ages P15 and above at the time of implantation, there were practically no 5-HT axons in the core of VM grafts (Mounir et al., 1994), in keeping with our earlier observations (Doucet et al., 1989). The 5-HT innervation of fetal striatal - - but not VM - - grafts had been reported to be less dense after implantation in intact rather than ibotenic acidlesioned striatum (Labandeira-Garcia et al., 1991; Lu et al., t991). To rule out the possibility that the differences in 5-HT innervation we had observed between striatal and VM grafts be due to the prior excitotoxic lesion, we compared the 5-HT innervation of VM and striatal grafts following implantation into intact neostriatum. Since no data were available on the 5-HT innervation of striatal grafts implanted in newborns, the study included VM or striatal grafts implanted in neonatal, juvenile and adult rat striatum. To quantify the innervation, we used a method based on the uptake of tritiated 5-HT, in vitro, followed by autoradiography and image analysis, 6 months after transplantation (Doucet and Descarries, 1993). Brain slices were incubated with 10 -6 M [3H]5-HT, in the presence of a monoamine oxidase inhibitor and of an inhibitor of monoamine uptake by dopaminergic neurons. These slices were then fixed with glutaraldehyde, embedded in epoxy resin and sectioned at a thickness of 4 Ixm for autoradiography. Serotonin axon varicosities were then counted within the grafts (see Fig. 2B,D). Adjacent semi-thin sections from the same brain slices were processed by post-embedding immunohistochemistry for tryptophan hydroxylase (TPH), tyrosine hydroxylase (TH, Fig. 2A) or dopamine- and adenosine-regulated phosphoprotein-32 (DARPP-32, Fig. 2C) (Ouimet et al., 1984). Sections immunostained for TPH confirmed the absence of 5-HT neurons inside the grafts, whereas TH or DARPP-32 staining was used to assess the distribution of 5-HT fibers in relation to that
of dopamine or striatal neurons, in VM or striatal grafts (Wictorin et al., 1989a), respectively. The quantitative analyses demonstrated that the DARPP-32-positive patches, representing the true striatal compartments of 'striatal' grafts, were three to four times more densely innervated by host 5-HT axons than VM grafts, or DARPP-32immunonegative (non-striatal) regions of 'striatal' grafts. In all types of grafts (VM as well as DARPP32-positive and DARPP-32-negative regions of striatal grafts), the amount of 5-HT innervation decreased by 75-80% between grafts implanted in neonatal versus adult rats (and examined 6 months later) (Pierret et al., 1998). Therefore, the proportion of 5-HT innervation in VM and striatal grafts remained unchanged between newborn and adult recipients. These results demonstrated that the relative affinity of the 5-HT axons present in the host striatum for VM or striatal grafts remained constant between newborns and adults. The differences between adult and neonatal 5-HT innervations of the grafts was likely the result of an intrinsic developmental decrease in the growth capacity of the mature host 5-HT neurons (Goldberg et al., 2001). Nevertheless, the 5-HT innervation of striatal grafts decreased gradually in juvenile and adult recipients, whereas the amount of 5-HT innervation of VM grafts in juvenile recipients was as low as that in adult recipients, as in our previous study (Mounir et al., 1994). Therefore, the intrinsic developmental decrease in growth capacity for a given type of neuron may not follow the same time course for different targets, i.e., in their response to different guidance molecules. It follows from this idea that different signaling pathways involved in axon growth and guidance might mature at different rates in a given neuron. It would then be imaginable to selectively reactivate one signaling pathway, in order to promote the regeneration of specific projections. Influence of the glial scar or chondroitin sulfate proteoglycans
A difference in the glial scar, or in the expression of axon growth inhibitory molecules, such as chondroitin sulfate proteoglycans (CSPG), might have explained the difference in 5-HT innervation between VM and striatal grafts. We therefore ex-
458 amined the glial scar and expression of CSPG by immunohistochemistry at different time points after transplantation of V M tissue in newborn, juvenile or adult rats, as well as of striatal tissue in adults (Petit et al., 2002). Ten days after transplantation, immunohistochemistry for GFAP showed the presence of astrocytes inside and around the grafts, forming a glial scar, in all cases. This glial scar appeared only slightly less dense around V M grafts implanted in newborns. Also, the distribution of GFAP-immunostained elements was more heterogeneous in striatal grafts, as previously reported by others (Gates et al., 1996). The expression of CSPG was very similar in all types of grafts and in recipients of any age, at 10 days. Serotonin immunoreactive axons were already profuse in VM grafts implanted in newborns, but rare in VM and striatal grafts implanted in adults. Therefore, the presence of a glial scar or CSPG had no negative impact on the ingrowth of immature 5-HT axons. At longer time points, GFAP and CSPG immunoreactivities subsided gradually in striatal grafts implanted in adults, but remained highly heterogeneous, such that patches of intense staining remained present between 10 days and 2 months. Serotonin axons gradually invaded these striatal grafts and were clearly more profuse in DARPP-32-positive patches, although many were also present in DARPP-32negative zones. Interestingly, the DARPP-32-positive patches corresponded to GFAP- and CSPG-negative zones of these grafts (the latter two being then largely overlapping), 2 months after transplantation. Thus, for striatal grafts, the time course of appearance and distribution of 5-HT axons was in inverse correlation with those of GFAP and CSPG. Nevertheless, a similarly heterogeneous 5-HT innervation of striatal grafts, inversely correlated with GFAP, had also been demonstrated following implantation in newborn rats (Pierret et al., 1998). Therefore,
since neonatal 5-HT axons were not inhibited by the glial scar or CSPG around or in the VM grafts, as stated above, this heterogeneity in 5-HT innervation of striatal grafts could not be explained by the complementary distribution of GFAP or CSPG. Other elements - - attractant or repellant - - in the different compartments of striatal grafts must be responsible of the differential affinity of host 5-HT axons. The situation was different for VM grafts. Serotonin axons remained rare in these grafts, even after 2 months, as observed in our previous studies; even though GFAP immunoreactive elements were greatly reduced and the expression of CSPG was totally abolished. It was then concluded that the absence of 5-HT innervation inside VM grafts could not be attributed to the astrocyte scar or the expression of CSPG in the graft. Our interpretation was that either V M grafts contained molecules that were inhibitory to the ingrowth of 5-HT axons or, else, that striatal grafts (and particularly their DARPP-32-positive zones) contained molecules that were attractant for these axons. To settle this issue, it was crucial to determine the type of cells that expressed the molecules responsible for the repellent or attractant activity.
Role of astrocytes in the graft In view of the complexity of the grafted tissue, it is not easy to identify molecules that may attract 5-HT axons into striatal grafts, or repel them from VM grafts. A more simple model was needed for that purpose. Because astrocytes had been shown in several instances to have different regional phenotypes that could affect the growth of dopamine or 5-HT neurons (Denis-Donini et al., 1983, 1984; Chamak et al., 1987; Liu and Lander, 1992a), it was proposed that they might be the cells that influenced 5-HT axon ingrowth into the grafts.
Fig. 2. Ventral mesencephalic (A,B) and striatal (C,D) grafts in juvenile (P15) rats. The animals were sacrificed 6 months after transplantation. (A) Semi-thin section from a brain slice incubated with tritiated 5-HT. The section was treated for post-embedding TH-immunohistochemistry, showing grafted dopamine neurons and processes. (B) An adjacent section of the same brain slice, but exposed to a nuclear emulsion, for autoradiography of 5-HT nerve terminals, showing that serotonin axon terminals were very sparse in the graft. (C) A section treated in post-embedding DARPP-32-immunohistochemistry, showing the striatal neurons. Part of the graft contains true striatal tissue, and part of it contains non-striatal tissue (DARPP-32-negative). (D) Adjacent section showing that the DARPP-32-positive area is more densely innervated by host 5-HT axons. From Pierret et al. (1998). Reproduced with permission.
459
460
Dissection and culture of neonatal astrocytes Ventral Mesencephalon
Neostriatum
Co-implantation of cultured astrocytes with ventral mesencephalic (or striatal) tissue
or _. i or
Cerebral Cortex
7 Fig. 3. Technique used for co-transplantation of astrocytes cultured from the neonatal ventral mesencephalon, neostriaturn or cerebral cortex, and then mixed with dissociated fetal (E14-E15) ventral mesencephalicor striatal tissue, before implantation into the striatum of adult rats. From Petit et al. (200l). Reprinted with permission To test this hypothesis, cultured astrocytes from three different brain regions were co-implanted with fetal VM or striatal tissue into the intact striatum of adult rats (Petit et al., 2001) (see Fig. 3). The same autoradiographic technique, described above, was used to determine the density of 5-HT innervation in each type of graft. This quantitative analysis clearly showed that co-grafts of VM containing striatal or neocortical astrocytes were three to four times more densely innervated by host 5-HT axons than control VM grafts or co-grafts of VM enriched with cultured VM astrocytes (Fig. 4). Moreover, co-grafts of striatal tissue with VM astrocytes were innervated as profusely as single striatal grafts, demonstrating that VM astrocytes were not inhibitory to 5-HT axon ingrowth. To ensure that this difference in the density of 5-HT innervation resulting from the addition of cortical or striatal astrocytes was not due to some indirect effect on the glial scar or on the expression of CSPG in the graft, we also examined the different types of grafts by GFAP and CSPG immunohistochemistry (Petit et al., 2001). No difference between graft types were found in the distribution of these molecules. The density of CSPG immunostaining, as well as the surface area of GFAP immunoreactive processes in the core of the grafts, at the graft-host border, and in the adjacent host striatal tissue were also quantified. The results showed no difference in
CSPG immunoreactivity. As for GFAP, all types of co-grafts contained more astrocytes than single VM grafts, as expected for grafts that had been enriched with astrocytes; but there was no difference between the groups of co-grafts, including VM grafts with VM astrocytes which were as poorly innervated by 5-HT axons as single VM grafts. It was concluded that the differences in 5-HT innervation between cografts were not due to a reduction in the glial reaction or in the expression of CSPG. Altogether, the latter observations rather indicated that cortical and striatal astrocytes attracted adjacent host 5-HT axons normally fated to innervate the striatum. Interestingly, some of these axons might have been collaterals of axons innervating the ventral midbrain (van der Kooy and Hattori, 1980; Imai et al., 1986), which might account for the few axons that were found in control VM grafts.
Influence of co-grafted astrocytes on other graft afferents To test the possibility that these effects of astrocytes on 5-HT axons represented a general growthpromoting action on all surrounding host axons, we also examined the influence of the different populations of astrocytes on the other types of host afferents that had previously been demonstrated into the VM grafts, i.e., cortical and striatal afferents (see above).
461
Fig. 4. Grafts of ventral mesencephalic tissue, with or without co-grafted astrocytes, into the striatum of adult rats. Pictures are from 4-1xm-thick sections from 200-1xm-thick brain slices processed in vitro for tritiated serotonin uptake and autoradiography, 2 months after transplantation. (A) Single ventral mesencephalic graft. (B) Co-graft containing ventral mesencephalic astrocytes. (C) Co-graft with striatal astrocytes. (D) Co-graft with cortical astrocytes. (E) The complete section of the graft in A, to show the size of the pictures in A - D (white rectangle). In all cases the left part displays host striatum and the right part the graft. Arrows point to silver grain aggregates representing labeled serotonin varicosities. From Petit et al. (2001). Reprinted with permission.
462 We found no increases or decreases in the number of labeled axons following multiple injections of biotinylated dextran in the ffontoparietal cortex or after DARPP-32 immunohistochemistry. Therefore, it appears that the growth-promoting effects of striatal or cortical astrocytes were specific for 5-HT axons.
Advantages of the model for the search of axon guidance molecules Now, since we have three highly purified populations of astrocytes in culture, with different effects on 5-HT axons, it becomes possible to use a comparison of gene and protein expression between them, in the hope of finding candidate molecules with the same biological activity on 5-HT axons as in cografts. Some molecules with known action on 5-HT axons are among such candidates, such as brainderived neurotrophic factor (BDNF), glial cell linederived neurotrophic factor (GDNF) or S100~ (Liu and Lander, 1992b; Beck et al., 1996; Mamounas et al., 2000; Nishi et al., 2000), but the probability is high that the observed effects were due to unknown molecules. Indeed, recent estimates of the total human genome are in the order of 30,00060,000 genes (Bork and Copley, 200l; Hogenesch et al., 2001), and the total number of genes in the rat or mouse genomes are probably near the same order of magnitude. Among these genes, the number of those with a known protein product is about 15,000, which represents 25-50% of the total estimate. Therefore, a newly detected activity has approximately a 50-75% chance of being due to an unidentified molecule. We are currently running experiments with gene chips and 2D gel electrophoresis, comparing gene and protein expression in different astrocyte populations in culture. Preliminary results already ruled out BDNF, GDNF and S10013 as candidates for the effects. They also indicate that the number of gene products expressed differently among astrocyte populations from the cortex or VM is relatively low, in the order of a few hundred. Taking into account the numbers of proteins involved in structural or metabolic functions, and the structure expected from presumed intercellular signaling molecules, such numbers should be reducible to a manageable number of candidates to be tested with appropriate tests in vitro.
We are also developing tests in vitro that will reproduce the features of the transplantation model described above; based on the collagen gel explant technique of Tessier-Lavigne (Tessier-Lavigne et al., 1988; Kennedy et al., 1994). Serotoninergic axons innervating neostriatal explants will thus be challenged with VM tissue, with or without added cortical or striatal astrocytes. Such in vitro models should help to further characterize the nature of the molecules involved (membrane or matrix-bound, diffusible, etc.), allowing the number of criteria that candidate molecules will need to fulfil to account for the observed activity to be increased. These in vitro characterizations will thus be useful to reduce the number of molecules to be tested and the same techniques will then serve to test the candidate molecules generated from genomic and proteomic data.
Conclusion Several inhibitory molecules have been identified as being responsible for the poor regenerative capacities of CNS neurons. However, a reduction in the level of growth-promoting or axon guidance molecules might also contribute in turning the balance towards inhibition. Lesion and transplantation experiments suggested that guidance molecules are still present in the adult CNS, but adult neurons might require some priming to respond to these signals. A better knowledge of growth-promoting or guidance molecules might allow their manipulation to turn back the balance towards appropriately directed axon growth. Several models suggesting tropic activities in the adult CNS exist, but need to be developed further in vitro to allow identification of the molecules that underlie the observed effects. A better knowledge of 5-HT axon guidance molecules would certainly be relevant for spinal cord injury, in view of the role of 5-HT in the control of locomotion in brain and the spinal cord (Jacobs and Fornal, 1995; Schmidt and Jordan, 2000; see also Rossignol et al., 2002, this volume).
Acknowledgements We thank Dr. Laurent Descarries for his critical revision of this manuscript. This work was supported by the Canadian Institutes for Health Re-
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s e a r c h ( C I H R ) , t h e F o n d s d e la r e c h e r c h e e n s a n t 6 d u Q u 6 b e c ( F R S Q ) , t h e F o n d s p o u r la f o r m a t i o n d e s c h e r c h e u r s e t l ' a i d e ~t la r e c h e r c h e ( F C A R ) a n d G r o u p e d e r e c h e r c h e s u r le s y s t & n e n e r v e u x c e n t r a l (FCAR Center).
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