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Basal forebrain lesions facilitate adult host fiber ingrowth into neocortical transplants
Brain Research. 448 (1988) 53-66 Elsevier
53
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Basal forebrain lesions facilitate adult host fiber ingrowth into neocortical transplants C.F. H6hmann
I and F.F. Ebner 2
t Department of Child Psychiatry, The Johns Hopkins University Medical School, Baltimore, MD (U.S.A.) and 2Division of Biology and Medicine, Brown University, Providence, RI (U.S.A.)
(Accepted 20 October 1987) Key words: Neocortex; Transplant; Basal nucleus; Plasticity; Regeneration
The ability of mature host thalamic neurons to innervate embryonic (E19) cortex when implanted into the cortex of adult hosts was compared in normal and basal forebrain lesioned mice. The ingrowth of mature horseradish peroxidase-labeled thalamic axons into the transplants is facilitated by prior basal forebrain lesions. We discuss the possible reasons for the lesion-induced enhancement of axonal ingrowth, including the possibility that the enhanced ingrowth of thalamic fiber systems may be related to the loss of cortical innervation by extrathalamic brainstem inputs, especially cholinergic afferent fibers. The results support the interpretation that extrathalamic inputs to cortex play a modulatory role in regulating the growth and connections of specific sensory fiber systems during brain responses to injury. INTRODUCTION Cholinergic cell bodies located in an extensive region of the basal telencephalon give rise to projections that innervate all of cortex 3°'33'42'6°. Cholinergic mechanisms have been shown to modulate cortical cell firing in response to electrical and natural stimulation both in the developing kitten c o r t e x 63'64 and in the adult brain 3~'4°'72. Thus, the cholinergic inputs to cerebral cortex may influence cortical plasticity in ways that potentially are of great importance for some types of cortical function. It is in this context that we would like to understand the various influences that cholinergic fibers can exert in the cerebral cortex. Brain transplantation has been used to study the capacity of mature central neurons to reorganize their connections in response to brain injury. For example, under certain conditions, the axons of adult neurons can invade immature central nervous system (CNS) t i s s u e grafts 36"48"52"56'58"59'70"71 or use grafted peripheral nerve tissue as a substrate for axonal elongation 57. Such interconnections are more extensive
when host as well as donor tissue are immature 9"2L 31.5L52, supporting the conclusion that some types of regenerative capacity decrease with age. Axonal elongation in t h e mature CNS leading to the establishment of new connections at distances over 1 m m away is especially recalcitrant, even when the substrate available for innervation consists of implanted cells ranging in maturity from dividing neuroepithelial cells to postmitotic neurons that are forming synapses for the first time. Thus host and transplant interactions display an age dependent 'critical' or 'sensitive' period in the ease with which new connections can be established that is comparable to some features of normal postnatal development 5'61'62. Host fiber ingrowth into or through transplants can be viewed as a 'plastic' response of host cortex, an attempt to adjust to the damage inflicted during implantation of new potential 'target' cells. All fiber systems in cortex are not equal however, in their ability to reform new connections. For example, we have documented repeatedly the rapid ingrowth of cholinergic fibers into transplants which were not innervated by thalamic fibers during the
Correspondence: F.F. Ebner, Division of Biology and Medicine, Brown University, Providence, RI, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
54 same period. In each case, thalamic axons can be demonstrated in abundance up to the border of, but not within, the transplants. The idea we wanted to test in this study was whether the growth of host axons, especially the thalamic fibers, into transplants may be negatively influenced by the striking regenerative capacity of cholinergic fiber systems. In previous studies we have examined the characteristic morphology and histochemistry of perinatal donor cortex transplanted into adult neocortex s" 14.15.65-69 the levels of muscarinic cholinergic receptors 25"27, as well as the time course of acetylcholinesterase (AChE)-labeled host fiber ingrowth 24"53. Transplants of prenatal donor cortex receive a normal level of ch,:Jiinerg~c innervation by the adult host brain within 1-2 months after surgery. Removal of cholinergic projections to the host cortex will remove most of the cholinergic fibers available to regrow into the transplant 28. Interconnections between host and transplant can then be studied under conditions of markedly reduced cholinerg:tc fiber influence. In the present paper we compare transplants of embryonic donor cortex into normal adult hosts with transplants into mature hosts previously depleted of cholinergic inputs. The results indicate that ingrowth of adult host thalamic axons into the transplants is facilitated by prior basal forebrain lesions. MATERIALS AND METHODS Young adult (2-4 months) BALB/c mice reared in our own breeding colony were used as host animals in all experiments. Cortex removed for implantation was obtained from timed-pregnancy animals by caesarean section at 19-20 days of gestation (E19-20) after vaginal plug detection in the mother. In BALB/c mice P20 is usually the last day of gestation. Host animals that had received basal nucleus lesions were allowed to recover from this surgery for 3-10 weeks before receiving a transplant into the
hemisphere ipsilateral to the lesion. Transplantation into both lesioned and normal animals was performed in age-matched hosts. Where possible (availability of donor tissue was a limiting factor) surgery on the lesioned and normal host group was performed on the same day with donor tissue derived from embryos of the same litter. Six to 12 weeks after transplantation all animals received injections of wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP) centered on the ventrobasal nucleus (VB) of the thalamus ipsilateral to the hemisphere containing the transplant. The tissue was then processed using tetramethylbenzidine (TMB) as a substrate for HRP. In all lesioned animals, and about half of all unlesioned cases, 3 parallel sets of sections were processed; one for the H R P reaction, one for AChE histochemistry and one for Cresyl violet cell staining. In the remainder of the animals TMBreacted sections were lightly counterstained with Neutral red. Basal forebrain lesions were performed according to the methods described in H6hmann et al. 28. In brief, an electrode mounted on a stereotaxic device was advanced parallel to the base of the brain in an anterior to posterior direction and electrothermic lesions were made by passing DC current (1.7 mA for 30 s) in several locatiors along the electrode tract. Transplantation was carried out using a procedure described in detail in previous publications 15'68. We transplant a slab of parietal cortex measuring approximately 0.7 by 3.0 mm by nearly the entire thickness of the donor cortex. The donor tissue was inserted in a posterolateral to anteromedial direction in parallel with the pial surface of host parietal cortex and all transplants were contained entirely within the 6 layers of the host cortex. That is, none of the cases included in this study contained transplants that had any direct centact with the lateral ventricle. Immediately prior to insertion of the donor tissue a piece of host parietal cortex of approximately the same size
Fig. 1. Coronal sections through different levels of mouse forebrain stained for HRP using the TMB chromagen (A) and for AChE (B). A: the WGA-HRP injection site (asterisk in A) is confined to the thalamus. This injection site belongs to case 7 in which the transplant is still visible in cortex at this section level (open arrow in A). TMB reaction product is visible in cortex in the vicinityof the transplant. B: the typical appearance of parietal cortex, stained for ACHE, one week after a lesion of the basal nucleus area. Note the marked diminution in the concentration of histochemically detectable AChE in the hemisphere (arrowheads point to pial surface) ipsilateral to the lesion (clear area around electrode track, (asterisk)).
55
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56 was removed from the site of transplantation to make room for the graft. For the HRP injections into the thalamus, the head of deeply anesthetized animals was immobilized in a stereotaxic apparatus with ear bars and incisor bar in the same horizontal plane. An opening was made in the bone over the cerebellum and a 10 l~! Hamilton syringe filled with a 5% solution of WGA-HRP was advanced into the VB according to the following stereotaxic coordinates: vertical = 3 mm below 0, lateral = 1.5 mm to the right and posterior = 2.4 mm posterior to zero. Injections of 0.25 F~lof the WGA-HRP were made at this location and the animals were sacrificed 24 h later. Brains were processed according to the protocol of Mesulam 49. Briefly, animals were perfused with saline solution followed by a perfusate containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer. Brains were postfixed in this fixative for 3-4 h, incubated overnight in the 0.1 M phosphate buffer containing 10% sucrose, and subsequently cut on a freezing microtome at 50 /~m. The tissue was collected in phosphate buffer and rinsed 3 ~imes with fresh buffer prior to the TMB reaction. The tissue was then incubated for 20 min in a solution containing: 1% sodium nitroferricyanide dissolved in a 0.01 M acetate buffer (pH 3.3) and 0.5% TMB (dissolved in ethanol). Hydrogen peroxide at a concentration of 0.3% was added at the end of the preincubation and the tissue was incubated in this solution for another 20 min. The incubation was terminated by repeatedly rinsing the tissue in the 0.01 M acetate buffer. The tissue was then immediately mounted onto glass slides from the acetate buffer solution. The mounted tissue sections were blotted and dried before immersion into concentrated methyl salicylate for 2 min to stabilize the reaction product, defatted in xylene and coverslipped. Sections that were counterstained with Neutral red were defatted after the first methyl salicylate rinse, briefly run through decreasing concentrations of ethanol into water, stained, and resubmerged in methyl salicylate for another 2 min before they were coverslipped frem xylene. AChE bistochemistry was per~armed according to the protocol of Hardy et al.2° controls for AChE enzyme specificity and greater detail of the method is given in H6hmann and Ebner 26.
RESULTS
General characteristics of the transplant Several criteria had to be met by each transplant case in order to be included in the study. First, the transplant had to be confined to cortex, contain healthy appearing cells throughout, and be well integrated but clearly distinguishable from host cortex. Second, the WGA-HRP injection site, as visualized by TMB chromagen, had to be confined to the thalamus (although not to VB) and anterograde WGAHRP transport restricted to the thalamic projection areas (Fig. 1 shows one injection site). Cases which showed spillover into the basal forebrain or cortically projecting brainstem areas were not included. (Using the horizontal approach to the thalamus, the electrode never transversed the cortex.) Third, anterograde transport of WGA-HRP in cortex had to be present near the transplant. Fourth, the lesion in the basal forebrain-lesioned hosts had to be verified histologically to be within the boundaries previously shown to result both in a 30-70% decrease of choline acetyltransferase (CHAT) activity and a depletion of AChE staining in cortex 2s. The typical appearance of AChE histochemistry in a coronal section through the forebrain of a basal nucleus lesioned mouse is illustrated in Fig. lB. Fig. 2 shows a drawing depicting the extent of the basal nucleus lesions in the cases included in this study. Fifteen cases met above criteria in the present study. Seven transplants were located in basal forebrain lesioned hosts and eight transplants in normal adult hosts (see Table I). There was considerable variability in size and shape among these transplants. Some cases showed, in coronal sections, the typically rounded, trilaminar appearance that has been reported for E18-19 donor transplants in normal hosts 14'67'68. However, many cases, especially those located in lesioned hosts, were smaller and less regular in shape. None of the transplants in normal hosts had any indication of host thalamocortical fiber ingrowth (Fig. 3D). All normal host cases showed good anterograde and retrograde transport of WGA-HRP into the vicinity of the transplant. In many cases labeled host fibers were present up to the host-transplant border, but never crossed over this border into the graft. Anterogradely labeled fibers found within a few
57
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Fig. 2. Projection drawings of the anterior (level A) to posterior (level E) extent of the basal forebrain lesion in all cases included in this study. The solid outline depicts a lesion that was reacted for AChE one week postoperatively and displayed the same degree of AChE disappearance as that shown in Fig. lB. The dashed lines outline the lesions in the transplant cases. Note that the lesions vary in size but overlap in the region of the ventromedial globus pallidus which contains the cholinergic projection neurons to parietal cortex. AC, anterior commissure; CC, corpus callosum; CCtx, cingulate cortex; EP, entopeduncular nucleus; FCtx, frontal cortex; GP, globus pallidus; Ht, hypothalamus; LPA, lateral preoptic area; OCtx, occipital cortex; PCtx, parietal cortex; St, striatum; SI, substantia innominata; S, septum; Vb, ventrobasal complex; ZI, zona incerta.
hundred microns of the transplant were fewer in number in normal hosts than in lesioned hosts. Retrogradely labeled cell bodies could be seen, however, in comparable numbers in both lesioned and unlesioned hosts suggesting that the differences in anterograde transport were not due to differences in size and location of the W G A - H R P injection site. All transplants in basal forebrain lesioned hosts showed some ingrowth of WGA-HRP-positive fibers into the transplant. The n u m b e r of fibers within the transplants of lesioned hosts varied considerably. In most cases a few distinct fibers could be followed
across the host-transplant border and into the transpk,.lt for several hundred microns (Figs. 4A,B and 5 A - C ) . Some transplants, however, contained a multitude of HRP-filled fibers which could be seen crossing the host-transplant interface to enter the transplant in several locations (Fig. 3 A - C ) . W G A HRP-iabeled fiber ingrowth did not always occur throughout the transplant yet in all cases fibers were evident in more than 3 consecutive sections. On the other hand, fiber ingrowth into the transplant never approached the density of anterograde fiber labeling in the surrounding host cortex.
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59 TABLE I Summary of similarities and differences in the preparation and the results for all 15 cases
The symbols describing AChE intensity and amount of HRP filled fibel ingrowth are ratings on a relative scale. The density of AChE in normal hosts would be rated as + + q-; anterograde WGA-HRP transport in the host animal ranged from + + + to + + + +. PostLSN, time after lesion; post-XPL, time after transplantation; wks, weeks; (E), gestational day; n.d.. not done; * HRP injection 2 days prior to sacrifice. Xplant number
Lesion AChE in Xplant Post-LSN time (wks.) Post-XPL time (wks.) Dot.or age (E) HR v fibers in Xplant
I
2
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12 20
8 19
7 19
7 19
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There were some differences in the experimental conditions among the transplants, such as (1) animals were sacrificed at several different post-transplantation survival times, (2) there could have been up to 24 h variation in donor age because plugs were checked only in the morning, (3) the basal nucleus lesioned host animals sustained two operations and an H R P injection compared to one operation plus an HRP injection in the normal host animals and (4) transplantation occurred at different postlesion times depending on the health of the basal nucleus lesioned anima!s. Since these variables may have a bearing on host-transplant interactions we compared the similarities and differences in case preparation with the amount of H R P labeled fiber ingrowth in Table I. The only variable which consistently correlated with HRP fiber ingrowth into the transplant was the presence of the basal forebrain lesion. No correlation appeared to exist between donor age, survival time or lesion-transplantation interval and H R P labeled fiber density (see Table I). Fibers labeled by thalamic W G A - H R P injection are seen in the transplant by 6 weeks after transplantation, the earliest time investigated, and they persist at least as long as 12 weeks post-transplantation. Lesions performed between 3 and 10 weeks prior to transplantation were associated with fiber ingrowth into transplants. Labeled cells in the transplant
On 3 occasions we observed labeled cells in a trans-
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plant. Each cell was the only one of its kind in the transplant and all 3 cell bearing transplants were located in basal nucleus lesioned hosts. One of the 3 cells was located within 50/tin of the host-transplant border and thus may have been located in the host, considering our section thickness of 50/zm (see Fig. 5C). The other two labeled cells were located deep within the transplant and therefore could be cells retrogradeiy labeled by the thalamic injection of W G A - H R P (Fig. 5 C - E ) . A C h E stain in the transplant
The amount of A C h E reaction product contained in the lesioned host transplants appeared to correlate with both the extent of the lesion and the postlesion survival time. Some regrowth of AChE-positive fibers into the depleted host cortex and consequently into the transplant had occurred in all transplants by the time of sacrifice. Fig. 4C shows the typical appearance of such AChE-positive fibers after presumed regrowth into depleted host cortex and transplant. The largest number of AChE-positive fibers was found in transplants number 4, 6 and 7 (see Table I) which had either received a smaller than average lesion (case 4) or had long postlesion survival times. AChE-stained attd WGA-HRP-Iabeled fibers from the thalamic injections always coexisted in the transplants of basal nucleus lesioned hosts, but no consistent relationship could be observed between the distribution of the two different fiber groups.
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61 ACHE- and WGA-ttRP-labeled fibers neither avoided each others target territory nor did they preferentially occupy the same space within the transplant (Fig. 4 compare A and B with C). There was no obvious relationship between the extent of AChE fiber regrowth into a transplant and the amount of thalamic fiber ingrowth (see Table I). DISCUSSION The present results show a clear correlation between the presence of a basal forebrain lesion in the host, and subsequent presence of host axons in the transplant that could be labeled by thalamic injection. Such ingrowth did not correlate with any other variable in the experiments. Thus it appears that basal forebrain lesions changed the propensity of other, (thalamic) host fibers to grow into the transplants.
Origin of WGA-HRP-labeled elements in the transplant Our results support the interpretation that many of the sprouting fibers arise from thalamic nuclei or from recurrent collaterals of corticothalamic neurons or both. The selection of cases according to location of the injection site makes sources other than the reciprocal interconnections very unlikely as the origin of the labeled fiber ingrowth. It is also unlikely that all iiqet~tions into the basal forebrain lesioned hosts involved extrathalamic structures while all injections into unlesioned hosts did not. Furthermore, the pattern of transported WGA-HRP within host cortex corresponded to the terminal pattern of thalamic nuclei 23'75. Labeling was not seen in areas such as hippocampus and superficial cortical layers of frontal cortex that have been identified as projection areas for brainstem catecholaminergic and serotoninergic nuclei 13'17'18'35'42.It is unlikely that we labeled catecholaminergic projections into the transplant vicinity while sparing all other projection areas. A cholinergic origin of WGA-HRP-Iabeled fibers in the transplant is unlikeiy since the distribution pattern of residual AChE stain in the transplants never matched the pattern of labeled fiber ingrowth into any transplant. The possibility that we are labeling sprouted recurrent collaterals of ia~,'er VI neurons requires further study to estimate the probability of thi~ interpretation.
Effect of basal forebrain lesions on plasticity The absence of adult host fiber ingrowth into cortical transplants has been reported by other investigators 48. While adult host cholinergic and in many cases also catecholaminergic (CA) fibers readily grow into transplants 4.16.36.37.59, specific, precisely projecting, mature fiber systems always are more reluctant to invade transplant tissue 4s'52"55"56"71.A considerable difference in response to injury between specific fiber systems and ACh and CA fibers can also be seen in the normal adult animal 4. Thus, the adult cortex clearly has reduced its ability to mount as much of a plastic response to transplantation as the immature host. We have shown here that the basal forebrain lesion is capable of relaxing the constraints on this condition in some as yet unspecified manner. Since the purpose of the basal forebrain lesion was to destroy the basal nucleus cholinergic input to neocortex, the present results are consistent with the current speculations that ACh has an influence on the plasticity of cortical connections during normal development.
Is fiber ingrowth into the transplant a consequence of A Ch depletion ? Depletion of ACh is not the only consequence of a basal forebrain lesion made by thermal coagulation and the possibility cannot be ruled out that the enhanced ingrowth is related to factors other than or in addition to the disruption of cholinergic mechanisms. Recently it has been shown that injury to the CNS induces the production of substances which have a general effect on neuronal growth promotion and survival45.5°. The basal forebrain lesion could potentially result in the release of such a non-specific trophic factor in cortex which then promotes fiber ingrowth into the transplant. However, the basal forebrain lesion occurs at a site remote from cerebral cortex, the factor would need to be transported by the damaged afferent axons or generated by neurons in cortex responding to the brain injury or deafferentation. In general, degeneration of axons following a lesion appears to be a sufficient stimulus for the production of these trophic factors only for a relatively short period of time and only when the degenerating system constitutes a major input to the deafferented area. In ti'ansplantation by 3 - l 0 weeks a~d still enhance tha-
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63 lamic fiber ingrowth into the transplant. Injury-induced release of trophic factors as late as 10 weeks after the lesion seems unlikely to maintain levels at growth-promoting concentrations, since available evidence suggests that this neuronotrophic activity had decreased to almost baseline values before the end of the third week after the lesion s°. However, a more specific trophic factor activity, as recently indicated by the work of Gage ~9, deserves further attention in relation to the fiber sprouting observed in our cases. Another possible consequence of basal forebrain lesions could be the destruction of non-cholinergic ascending systems such as the serotonin and catecholamine fibers that travel in the medial forebrain bundle. As can be seen in Fig. 2, our lesions varied in size, and while all lesions destroyed cortically projecting cholinergic neurons, only some of them encroached on the medial forebrain bundie. There was no correlation between the mediolateral position of the lesion and the extent of fiber ingrowth into the transplant. Further experiments are necessary to test the possibility of noradrenergic or serotoninergic involvement in thalamocortical fiber sprouting. Several precedents exist for the idea that sprouting can be induced as a consequence of cholinergic depletion n'12a9'39'44. Both septal lesions and basal forebrain lesions have been shown to result in sympathetic fiber sprouting into their respective target areas 11'12'43. In the case of the septal lesion paradigm considerable evidence exists that the sprouting of sympathetic fibers into the hippocampus is the consequence of cholinergic depletion 12'37'43. At least one of the paradigms of CNS to CNS transplantation, which did result in considerable amounts of transplant innervation by adult host-specific fibers involved host brain areas that contained little or no ACh. Oblinger and Das reported that embryonic transplants to the adult cerebellum received innervation from several different host fiber systems 52. The adult cerebellum contains barely detectable levels of CHAT, the lowest in brain 34'39. Transplam~ of embryonic neocortex inserted into the tecturn of adult hosts which had undergone prior optic tract section have shown regeneration of optic tract fibers into the transplan¢ 8. The possibility that the cholinergic input to the recipient rectum is partially interrupted under these experimental conditions has
not been studied.
Possible mechanism for fiber ingrowth into the transplants induced by basal forebrain lesions of the host If depletion of cholinergic fibers is directly responsible for thalamic fiber ingrowth into the transpiant, then there are two possible mechanisms that could account for such a response; one is that cholinergic innervation actively inhibits the ability of thalamocortical fibers to elongate due to cholinergic receptor mediated effects, and the other is that the cholinergic influence induces a response such as synapse formation which stabilizes the elongating fibers. Specific thalamic nuclei show rapid and dramatic atrophy following the removal of their cortical target area 2'22'46. Thus, the high density of labeled thalamocortical fibers in the vicinity of the transplant could be viewed as an effect of the cholinergic depletion. The transplant procedure removed the original cortical target of these fibers in and around the transplant location. However the presence of immature CNS transplants may be a sufficient stimulus for the survival of some fibers and cells, which would otherwise atrophy 7"22. The sprouting fibers found in the transplant may originate from areas of cortex sufficiently distant from the site of transplantation to have escaped any damage 9. On the other hand, we did observe a considerable difference between normal and lesioned cases in regard to the number of anterogradely labeled fibers in close proximity to the transplant. Sunde and Zimmer have argued that adult specific fiber systems in the host do not usually invade transplant tissue since all synaptic sites in the graft are occupied by intrinsic fibers TM. The premise was that host-specific fibers do not grow fast enough to reach the transplant in time to find vacated synaptic sites. Based on this logic the observed ingrowth of host ACh fibers into the neocorticai grafts could be attributed to the greater growth potential and faster growth speed of these fiber systems (discussed in ref. 4). It could thus be argued that basal forebrain lesions act through making synaptic sites available in the transplant which then can be occupied by specific cortical afferents. Unless AChE-positive fibers operate under different rules, they should not sprout back into the transplant as they eventually do. There should be no space left for these late sprouting fibers after intrinsic graft fibers and host specific afferents
64 have claimed their territory. Yet, regrowing AChE stained fibers appeared in the transplant in densities comparable to the surrounding host cortex. Further, we have not observed any sign of mutual avoidance of the AChE and the HRP fiber pattern. Clearly, this particular type of interaction between two fiber systems is not a competitive synaptic replacement paradigm as it occurs, for example, in hippocampal plasticity44, but synaptogenesis could exert a transient influence at several stages during the response to injury. Previous studies have documented that sprouting in the adult CNS requires the stimulus of vacated synaptic sites44's4. The possibility exists that ACh may decrease plasticity in the CNS both during normal development and after brain injury, The very late maturation of cholinergic markers and the widespread morphological distribution of these fibers in the developing cortex would enable the cholinergic system to regulate and ultimately terminate plasticity26'27. The developing neocortex shows most plasticity at times when synapses are still being formed and while synapse numbers are equal to or higher than in adulthood 1"6"29.Thus ACh may act during normal development anuior under transplant conditions to restrict the number of connections that can be made. Cholinergic mechanisms have been implicated in the etiology of several mental and neurological diseases, the most prominent being Alzheimer's dementia. Patients with Alzheimer's disease suffer from a progressive loss of memory and cognitive ability and at autopsy their brains have shown a consistent decrease of cortically projecting cholinergic basal forebrain neurons with a concomitant deficiency in the levels of cortical cholineacetyltransferase (CHAT)
REFERENCES 1 Adams, C.E., Mihailoff, G.A. and Woodward, D.J., A transient component of the corticospinal tract arises in the visual cortex. Neurosci. Leu., 36 (1983) 243-248. 2 Barron, K.D., Means, E.D. and Larsen, E., Ultrastructure of retrograde deg. in the thalamus of rat, J. Neuropathol. Exp. Neurol, 32 (1973) 218-244. 3 Bartus, R.T., Dean III, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-416. 4 Bj6rklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervoussystem, Physiol. Rev., 59 (1979) 62-100.
activity3.t°,74. Animal models of Alzheimer's disease, employing lesions to cholinergic basal forebrain neurons have been used to study the impact of cholinergic depletion on cerebral cortex t2'32'33'41'47'73. The present results are consistent with the notion that a decrease in cholinergic basal forebrain neurons may lead to significant long-term changes in cortical organization. In summary, we have reported sprouting of thalamic afferents into cortical transplants in response to basal forebrain lesions in the adult host animals after prior basal forebrain lesions. We discuss the possibility that the enhanced ingrowth of specific fiber systems may be related to the loss of cortical innervation by cholinergic basal forebrain inputs. The mechanism by which host fiber ingrowth into the transplant is facilitated remains unknown, but simp!e replacement of AChE-labeled fibers by thalam~c fibers in the transplant appears unlikely. The present results are, to our knowledge, the first morpholc,gical indication that ACh may influence the growth and connections of non-cholinergic CNS fiber syste,las. An interesting question for the future is whether similar cholinergic influences can be shown to control thalamocortical fiber distribution and synaptogenesis during normal development.
ACKNOWLEDGEMENTS This research was supported by NIH Grant NS13031. A preliminary report of these data has been given elsewhere 68. This research was used in partial fulfillment of the thesis requirements for the PhD degree (C.F.H.).
5 Biakemore, C., Maturation and modificatien in the developing visual system. In R. Held, H.W. Leibowitz and H.-L. Teuber (Eds.), Handbook of Sensor)' Physiology, Vol. 8, 1978,pp. 377-433. 6 Blue, M.E. and Parnaveles, J.G., The formation and maturation of synapsesin the visual cortex of the rat. I. Qualitative analysis, II. Quantitative analysis, J. Neurocytol., 12 (1983) 599-616 and 697-712. 7 Bregman, B.S. and Reier, P.J., Neural tissue transplants in repair of injured immature mammalian spinal cord, Eric Fernstrom Symposium on Transplantation in the Mammalian CNS, Lund, Sweden, 1984.
8 Busalacchi, P. and Ebner, F.F., The vascularizationof neocortical transplants, Soc. Neurosci. ,4bstr., 8 (1982)247.
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receptor binding in normal and transplanted cortex, Dev. Brain Res., 23 (1985) 243-253. 28 H6hmann, C.F., Bear, M.F. and Ebner, F.F., Glutamic acid decarboxylase activity decreases in mouse neocortex after lesions to the basal forebrain, Brain Research, 333 (1985) 165-168. 29 Ivy, G.O. and Killackey, H.P., Ontogenetic changes in the projection of neocortical neurons, J. Neurosci., 2 (1982) 735-743. 30 Jacobowitz, D.M. and Creed, J., Cholinergic projection sites of the nucleus of tractus diagonalis, Brain Res. Bull., 10 (1983) 365-372. 31 Jaeger, C.B. and Lurid, R.D., Transplantation of embryonic occipital cortex into the tectal region of newborn rats: a light-microscopic study of organization and connectivity of the transplant, J. Comp. Neurol., 194 (1980) 571-597. 32 Johnston, M.V., McKinney, M. and Coyle, J.T., Evidence for a cholinergic projection to neocortex from neurons in basal forebrain, Proc. Natl. Acad. Sci. U.S.A,, 76 (1979) 5392-5396. 33 Johnston, M.V., McKinney, M. and Coyle, J.T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Neurol., 61 (1981) 479-484. 34 Kobayashi, R.M., Brownstein, M., Saavedra, J.M. and Palkovits, M., Choline acetyltransferase content in discrete regions of the rat brainstem, J. Neurochem., 24 (1975) 637-640. 35 Koda, L.Y. and Bloom, F.E., A light and electron microscopic study of noradrenergic terminals in the rat dentate gyrus, Brain Research, 120 (1977) 327-335. 36 Kromer, L.F., Bj6rklund, A. and Stenevi, U., Regeneration of the septohippocampal pathway in adult rats is promoted by utilizing embryonic hippocampal implants as bridges, Brain Research, 210 (1981) 173-200. 37 Kromer, L.F., Cholinergic axons from delayed septal implants and cholinergic fibers co-exist in the denervated dentate gyrus, Brain Res. Bull., 9 (1982) 539-544. 38 Krnjevic, K. and Phillis, J.W., Acetylcholine sensitive cells in cerebria cortex, J. Physiol. (Lond.), 166 (1963) 296-327. 39 Kuczenski, R., Segal, D.S. and Mandell, A.J., Regional and subcellular distrubition and kinetic properties of rat brain choline-acetyltransferase m some functional considerations, J. Neurochem., 24 (1975) ~9-45. 40 Lamour, Y., Dutar, P. and Jobert, A., Exictatory effect of acetylcholine on different types of neurons in the first somatosensory neocorte~: of the rat: laminar distribution and pharmacological characteristics, Neuroscience, 7 (1982) 1483- 1494. 41 Lehmann, J., Nagy, J.I., Atmadja, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 42 Lidov, H.G.W. and Molliver, M.E., An immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal fields, Brain Res. Bull., 8 (1982) 389--430. 43 Loy, R., Mi!ner, T.A. and Moore, R.Y., Sprouting of sympathetic axons in the hippocampal formation: conditions necessary top elicit ingrowth, Exp. Neurol., 67 (1980) 399-411. 44 Lynch, G., Rose, G., Gall, C. and Cotman, C.W., The response of the dentate gyrus to partial deafferentiation. In
66 M. Santini (Ed.), Proceedings of the Golgi Centennial Symposium, Raven, New York, 1975. 45 Manthorpe, M., Nieto-Sampedro, M., Skaper, D.S., Lewis, E.R., Barbin, G., Longo, F.M., Cot,an, C.W. and Varon, S., Neuronotrophic activity in brain wounds of the developing rat. Correlation with implant survival in the wound cavity, Brain Research, 267 (1983) 47-56. 46 Matthews, M.A., Death of the central neuron: an electron microscopic study of thalamic retrograde degeneration following cortical ablation, J. Neurocytol., 2 (1973) 265-288. 47 McKinney, M. and Coyle, J.T., Regulation of neocortical muscarinic receptors: effect of drug treatment and lesion, J. Neurosci., 2 (1982) 97-105. 48 McLoon, S.C. and Lund, R.D., Development of fetal retina, tectum and cortex transplanted to the superior colliculus of adult rats, J. Comp. Neurol., 217 (1983) 379-389. 49 Mesulam, M.M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neuronal efferents and afferents, J. Histochem. Cytochem., 26 (1978) 106-117. 50 Nieto-Sampetro, M., Manthorpe, M., Barbin, G., Varon, S. and Cotman, C.W., Injury induced neuronotrophic activity in adult rat brain: correlations with survival of delayed implants in the wound cavity, J. Neurosci., 3 (1983) 2219-2229. 51 OblingeL M.M., Hallas, B.H. and Das, G.D., Neocortical transplants in cerebellum of rat: their afferents and efferents, Brain Research, 189 (1980) 228-232. 52 Oblinger, M.M. and Das, G.D., Connectivity of neural transplants in adult rats: analysis of afferents aud efferents of neocortical transplants in the cerebellar hemisphere, Brain Research, 249 (1982) 3 i-49. 53 Park, J., Clinton, R.J. and Ebner, F.F., The growth of catecholamine and AChE containing fibers irlto neocortical transplants, Soc. Neurosc/. Abstr., 10 (1984) 1083. 54 Raisman, G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. 55 Raisman, G. and Fields, P.M., A quantitative investigation of the development of collateral innervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241-264. 56 Raisman, G. and Ebner. F.F., Mossy fiber projections into and out of hippocampal transplants, Neuroscience, 9 (1983) 783-801. 57 Richardson, P.M., McGuinness, U.M. and Aguayo, A.J., Axons from CNS neurons regenerate into PNS grafts, Nature (Lond.), 284 (1980) 264-265. 58 Ross, R. and Das, G.D., Adult rat optic tract axons sprout into neocortical transplants, Anat. Rec., 199 (1981) 217A. 59 Rothman-Schonfeld, A. and Katzman, R., Autoradiographic demonstration of the central origin of regenerating fibers into iris tissue implants in the rat mesencephalon, Brain Research, 197 (1980) 335-363. 69 Saper, C.B., Organization of cerebral cortical afferent sys-
tems in the rat. II. Magnocellular basal nucleus, J. Comp. Neurol., 222 (1984) 313-332. 61 Schneider, G.E., It is really better to have your brain lesion early? A revision of the 'Kennard Principle', Neuropsychologia, 17 (1979) 557-583. 62 SiUito, A.M., Plasticity in the visual cortex, Nature (Lond.), 303 (1983) 477-478. 63 Singer, W., Central-core control of visual cortex functions. In F.O. Schmitt and E.G. Warden (Eds.), The Neurosciences Fourth Study Program, MIT, Cambridge, MA, 1979, pp. 1093-2003. 64 Singer, W. and Rauschecker, J.P., Central core conh'ol of developmental plasticity in the kitten visual cortex: II Electrical activation of mesencephalic and diencephalic projections, Exp. Brain Res., 47 (1982) 223-233. 6.5 Smith, L.M. and Ebner, F.F., The fine structure of embryonic neocortex transplanted into adult mouse neocortex, Soc. Neurosci. Abstr., 8 (1982) 247. 66 Smith, L.M., Bear, ~A.F., Schmechel, D.E. and Ebner, F.F., GAD immunoreactivity in transplanted mouse neocortex, Soc. Neurosci. Abstr., 8 (1983) 226. 67 Smith, L.M. and Ebner, F.F., Effect of donor age on cell composition of neocortical transplants, Eric Fernstrom Symposium on Transplantation in the Mammalian CNS, Lund, Sweden, 1984. 68 Smith, L.M., Hohmann, C.F. and Ebner, F.F., The development of specific cell types and connectivity in neocortical transplantation. In A. Bj0rklund and U. Stenevi (Eds.), Proceedings of the Eric Fernstrom S'~;nposium on Transplantation in the Mammalian CNS, Lund, Sweden, 1984. 69 Smith, L.M. and Ebncr, F.F., The expression of GFA synthesis iu donor and host astrocytes following transplantation, Soc. Neurosci. Abstr., 10 (!984) 983. 70 Sunde, I~.A. and Zimmer, J., Transplantation of central nervous tissue, Acta Neurol, Scand., 63 (1981) 232-335. 71 Sunde, N.A. and Zimmer, J., Cellular, histochemical and connective organization of the hilo/-ncampus and fascia dentata transplanted to different regions of immature and adult rat brain, Dev. Brain Res., 8 (1983) 165-191. 72 Waterhouse, B.D., Moises, H.C. and Woodward, D.J., Alpha-receptor mediated facilitation on somatosensory cortical neuronal responses to exotatory synaptic inputs and iontophoretically applied acetylcholine, Neuropharmacology, 20 (1981) 907-920. 73 Wenk, H,, Bigl, V. and Meyer, U., Cholinergic projections from magnoceilular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Rev., 2 (1980) 295-31o. 74 Whitehouse, P.J., Price, D.L., Struble, R.G.. Clark, A.W., Coyle, J.T. and DeLong, M.R., Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain, Science, 215 (1982) 1237-1238. 75 Wise, S.P., The laminar organization of certain afferent and efferent fiber systems in the rat somatosensory cortex, Brain Research, 90 (1975) 139-141.
/
/
53
BRE 13492
Basal forebrain lesions facilitate adult host fiber ingrowth into neocortical transplants C.F. H6hmann
I and F.F. Ebner 2
t Department of Child Psychiatry, The Johns Hopkins University Medical School, Baltimore, MD (U.S.A.) and 2Division of Biology and Medicine, Brown University, Providence, RI (U.S.A.)
(Accepted 20 October 1987) Key words: Neocortex; Transplant; Basal nucleus; Plasticity; Regeneration
The ability of mature host thalamic neurons to innervate embryonic (E19) cortex when implanted into the cortex of adult hosts was compared in normal and basal forebrain lesioned mice. The ingrowth of mature horseradish peroxidase-labeled thalamic axons into the transplants is facilitated by prior basal forebrain lesions. We discuss the possible reasons for the lesion-induced enhancement of axonal ingrowth, including the possibility that the enhanced ingrowth of thalamic fiber systems may be related to the loss of cortical innervation by extrathalamic brainstem inputs, especially cholinergic afferent fibers. The results support the interpretation that extrathalamic inputs to cortex play a modulatory role in regulating the growth and connections of specific sensory fiber systems during brain responses to injury. INTRODUCTION Cholinergic cell bodies located in an extensive region of the basal telencephalon give rise to projections that innervate all of cortex 3°'33'42'6°. Cholinergic mechanisms have been shown to modulate cortical cell firing in response to electrical and natural stimulation both in the developing kitten c o r t e x 63'64 and in the adult brain 3~'4°'72. Thus, the cholinergic inputs to cerebral cortex may influence cortical plasticity in ways that potentially are of great importance for some types of cortical function. It is in this context that we would like to understand the various influences that cholinergic fibers can exert in the cerebral cortex. Brain transplantation has been used to study the capacity of mature central neurons to reorganize their connections in response to brain injury. For example, under certain conditions, the axons of adult neurons can invade immature central nervous system (CNS) t i s s u e grafts 36"48"52"56'58"59'70"71 or use grafted peripheral nerve tissue as a substrate for axonal elongation 57. Such interconnections are more extensive
when host as well as donor tissue are immature 9"2L 31.5L52, supporting the conclusion that some types of regenerative capacity decrease with age. Axonal elongation in t h e mature CNS leading to the establishment of new connections at distances over 1 m m away is especially recalcitrant, even when the substrate available for innervation consists of implanted cells ranging in maturity from dividing neuroepithelial cells to postmitotic neurons that are forming synapses for the first time. Thus host and transplant interactions display an age dependent 'critical' or 'sensitive' period in the ease with which new connections can be established that is comparable to some features of normal postnatal development 5'61'62. Host fiber ingrowth into or through transplants can be viewed as a 'plastic' response of host cortex, an attempt to adjust to the damage inflicted during implantation of new potential 'target' cells. All fiber systems in cortex are not equal however, in their ability to reform new connections. For example, we have documented repeatedly the rapid ingrowth of cholinergic fibers into transplants which were not innervated by thalamic fibers during the
Correspondence: F.F. Ebner, Division of Biology and Medicine, Brown University, Providence, RI, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
54 same period. In each case, thalamic axons can be demonstrated in abundance up to the border of, but not within, the transplants. The idea we wanted to test in this study was whether the growth of host axons, especially the thalamic fibers, into transplants may be negatively influenced by the striking regenerative capacity of cholinergic fiber systems. In previous studies we have examined the characteristic morphology and histochemistry of perinatal donor cortex transplanted into adult neocortex s" 14.15.65-69 the levels of muscarinic cholinergic receptors 25"27, as well as the time course of acetylcholinesterase (AChE)-labeled host fiber ingrowth 24"53. Transplants of prenatal donor cortex receive a normal level of ch,:Jiinerg~c innervation by the adult host brain within 1-2 months after surgery. Removal of cholinergic projections to the host cortex will remove most of the cholinergic fibers available to regrow into the transplant 28. Interconnections between host and transplant can then be studied under conditions of markedly reduced cholinerg:tc fiber influence. In the present paper we compare transplants of embryonic donor cortex into normal adult hosts with transplants into mature hosts previously depleted of cholinergic inputs. The results indicate that ingrowth of adult host thalamic axons into the transplants is facilitated by prior basal forebrain lesions. MATERIALS AND METHODS Young adult (2-4 months) BALB/c mice reared in our own breeding colony were used as host animals in all experiments. Cortex removed for implantation was obtained from timed-pregnancy animals by caesarean section at 19-20 days of gestation (E19-20) after vaginal plug detection in the mother. In BALB/c mice P20 is usually the last day of gestation. Host animals that had received basal nucleus lesions were allowed to recover from this surgery for 3-10 weeks before receiving a transplant into the
hemisphere ipsilateral to the lesion. Transplantation into both lesioned and normal animals was performed in age-matched hosts. Where possible (availability of donor tissue was a limiting factor) surgery on the lesioned and normal host group was performed on the same day with donor tissue derived from embryos of the same litter. Six to 12 weeks after transplantation all animals received injections of wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP) centered on the ventrobasal nucleus (VB) of the thalamus ipsilateral to the hemisphere containing the transplant. The tissue was then processed using tetramethylbenzidine (TMB) as a substrate for HRP. In all lesioned animals, and about half of all unlesioned cases, 3 parallel sets of sections were processed; one for the H R P reaction, one for AChE histochemistry and one for Cresyl violet cell staining. In the remainder of the animals TMBreacted sections were lightly counterstained with Neutral red. Basal forebrain lesions were performed according to the methods described in H6hmann et al. 28. In brief, an electrode mounted on a stereotaxic device was advanced parallel to the base of the brain in an anterior to posterior direction and electrothermic lesions were made by passing DC current (1.7 mA for 30 s) in several locatiors along the electrode tract. Transplantation was carried out using a procedure described in detail in previous publications 15'68. We transplant a slab of parietal cortex measuring approximately 0.7 by 3.0 mm by nearly the entire thickness of the donor cortex. The donor tissue was inserted in a posterolateral to anteromedial direction in parallel with the pial surface of host parietal cortex and all transplants were contained entirely within the 6 layers of the host cortex. That is, none of the cases included in this study contained transplants that had any direct centact with the lateral ventricle. Immediately prior to insertion of the donor tissue a piece of host parietal cortex of approximately the same size
Fig. 1. Coronal sections through different levels of mouse forebrain stained for HRP using the TMB chromagen (A) and for AChE (B). A: the WGA-HRP injection site (asterisk in A) is confined to the thalamus. This injection site belongs to case 7 in which the transplant is still visible in cortex at this section level (open arrow in A). TMB reaction product is visible in cortex in the vicinityof the transplant. B: the typical appearance of parietal cortex, stained for ACHE, one week after a lesion of the basal nucleus area. Note the marked diminution in the concentration of histochemically detectable AChE in the hemisphere (arrowheads point to pial surface) ipsilateral to the lesion (clear area around electrode track, (asterisk)).
55
.6,
R'.L
.4 i
13
56 was removed from the site of transplantation to make room for the graft. For the HRP injections into the thalamus, the head of deeply anesthetized animals was immobilized in a stereotaxic apparatus with ear bars and incisor bar in the same horizontal plane. An opening was made in the bone over the cerebellum and a 10 l~! Hamilton syringe filled with a 5% solution of WGA-HRP was advanced into the VB according to the following stereotaxic coordinates: vertical = 3 mm below 0, lateral = 1.5 mm to the right and posterior = 2.4 mm posterior to zero. Injections of 0.25 F~lof the WGA-HRP were made at this location and the animals were sacrificed 24 h later. Brains were processed according to the protocol of Mesulam 49. Briefly, animals were perfused with saline solution followed by a perfusate containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer. Brains were postfixed in this fixative for 3-4 h, incubated overnight in the 0.1 M phosphate buffer containing 10% sucrose, and subsequently cut on a freezing microtome at 50 /~m. The tissue was collected in phosphate buffer and rinsed 3 ~imes with fresh buffer prior to the TMB reaction. The tissue was then incubated for 20 min in a solution containing: 1% sodium nitroferricyanide dissolved in a 0.01 M acetate buffer (pH 3.3) and 0.5% TMB (dissolved in ethanol). Hydrogen peroxide at a concentration of 0.3% was added at the end of the preincubation and the tissue was incubated in this solution for another 20 min. The incubation was terminated by repeatedly rinsing the tissue in the 0.01 M acetate buffer. The tissue was then immediately mounted onto glass slides from the acetate buffer solution. The mounted tissue sections were blotted and dried before immersion into concentrated methyl salicylate for 2 min to stabilize the reaction product, defatted in xylene and coverslipped. Sections that were counterstained with Neutral red were defatted after the first methyl salicylate rinse, briefly run through decreasing concentrations of ethanol into water, stained, and resubmerged in methyl salicylate for another 2 min before they were coverslipped frem xylene. AChE bistochemistry was per~armed according to the protocol of Hardy et al.2° controls for AChE enzyme specificity and greater detail of the method is given in H6hmann and Ebner 26.
RESULTS
General characteristics of the transplant Several criteria had to be met by each transplant case in order to be included in the study. First, the transplant had to be confined to cortex, contain healthy appearing cells throughout, and be well integrated but clearly distinguishable from host cortex. Second, the WGA-HRP injection site, as visualized by TMB chromagen, had to be confined to the thalamus (although not to VB) and anterograde WGAHRP transport restricted to the thalamic projection areas (Fig. 1 shows one injection site). Cases which showed spillover into the basal forebrain or cortically projecting brainstem areas were not included. (Using the horizontal approach to the thalamus, the electrode never transversed the cortex.) Third, anterograde transport of WGA-HRP in cortex had to be present near the transplant. Fourth, the lesion in the basal forebrain-lesioned hosts had to be verified histologically to be within the boundaries previously shown to result both in a 30-70% decrease of choline acetyltransferase (CHAT) activity and a depletion of AChE staining in cortex 2s. The typical appearance of AChE histochemistry in a coronal section through the forebrain of a basal nucleus lesioned mouse is illustrated in Fig. lB. Fig. 2 shows a drawing depicting the extent of the basal nucleus lesions in the cases included in this study. Fifteen cases met above criteria in the present study. Seven transplants were located in basal forebrain lesioned hosts and eight transplants in normal adult hosts (see Table I). There was considerable variability in size and shape among these transplants. Some cases showed, in coronal sections, the typically rounded, trilaminar appearance that has been reported for E18-19 donor transplants in normal hosts 14'67'68. However, many cases, especially those located in lesioned hosts, were smaller and less regular in shape. None of the transplants in normal hosts had any indication of host thalamocortical fiber ingrowth (Fig. 3D). All normal host cases showed good anterograde and retrograde transport of WGA-HRP into the vicinity of the transplant. In many cases labeled host fibers were present up to the host-transplant border, but never crossed over this border into the graft. Anterogradely labeled fibers found within a few
57
.w°'),
E •
x . . '
•
D
[I
Fig. 2. Projection drawings of the anterior (level A) to posterior (level E) extent of the basal forebrain lesion in all cases included in this study. The solid outline depicts a lesion that was reacted for AChE one week postoperatively and displayed the same degree of AChE disappearance as that shown in Fig. lB. The dashed lines outline the lesions in the transplant cases. Note that the lesions vary in size but overlap in the region of the ventromedial globus pallidus which contains the cholinergic projection neurons to parietal cortex. AC, anterior commissure; CC, corpus callosum; CCtx, cingulate cortex; EP, entopeduncular nucleus; FCtx, frontal cortex; GP, globus pallidus; Ht, hypothalamus; LPA, lateral preoptic area; OCtx, occipital cortex; PCtx, parietal cortex; St, striatum; SI, substantia innominata; S, septum; Vb, ventrobasal complex; ZI, zona incerta.
hundred microns of the transplant were fewer in number in normal hosts than in lesioned hosts. Retrogradely labeled cell bodies could be seen, however, in comparable numbers in both lesioned and unlesioned hosts suggesting that the differences in anterograde transport were not due to differences in size and location of the W G A - H R P injection site. All transplants in basal forebrain lesioned hosts showed some ingrowth of WGA-HRP-positive fibers into the transplant. The n u m b e r of fibers within the transplants of lesioned hosts varied considerably. In most cases a few distinct fibers could be followed
across the host-transplant border and into the transpk,.lt for several hundred microns (Figs. 4A,B and 5 A - C ) . Some transplants, however, contained a multitude of HRP-filled fibers which could be seen crossing the host-transplant interface to enter the transplant in several locations (Fig. 3 A - C ) . W G A HRP-iabeled fiber ingrowth did not always occur throughout the transplant yet in all cases fibers were evident in more than 3 consecutive sections. On the other hand, fiber ingrowth into the transplant never approached the density of anterograde fiber labeling in the surrounding host cortex.
58
, : .:. 2 o.
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~ ~ .~. o
•.~ = ~
.~~ 0
e~ee
•
I.,
==~E.:•
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59 TABLE I Summary of similarities and differences in the preparation and the results for all 15 cases
The symbols describing AChE intensity and amount of HRP filled fibel ingrowth are ratings on a relative scale. The density of AChE in normal hosts would be rated as + + q-; anterograde WGA-HRP transport in the host animal ranged from + + + to + + + +. PostLSN, time after lesion; post-XPL, time after transplantation; wks, weeks; (E), gestational day; n.d.. not done; * HRP injection 2 days prior to sacrifice. Xplant number
Lesion AChE in Xplant Post-LSN time (wks.) Post-XPL time (wks.) Dot.or age (E) HR v fibers in Xplant
I
2
3
4
5
6
7
8
10
11
+
+
+
+
+
+
+
+/-
+
+/-
++
+
++
+
. . . ++ ++
. ++
. ++
15
12
9
9
15
20
22
/
/
/
6 20
6 20
6 20
6 19
9 20
8 19
12 20
8 19
7 19
7 19
+
++
++
+
+
+
+++ .
There were some differences in the experimental conditions among the transplants, such as (1) animals were sacrificed at several different post-transplantation survival times, (2) there could have been up to 24 h variation in donor age because plugs were checked only in the morning, (3) the basal nucleus lesioned host animals sustained two operations and an H R P injection compared to one operation plus an HRP injection in the normal host animals and (4) transplantation occurred at different postlesion times depending on the health of the basal nucleus lesioned anima!s. Since these variables may have a bearing on host-transplant interactions we compared the similarities and differences in case preparation with the amount of H R P labeled fiber ingrowth in Table I. The only variable which consistently correlated with HRP fiber ingrowth into the transplant was the presence of the basal forebrain lesion. No correlation appeared to exist between donor age, survival time or lesion-transplantation interval and H R P labeled fiber density (see Table I). Fibers labeled by thalamic W G A - H R P injection are seen in the transplant by 6 weeks after transplantation, the earliest time investigated, and they persist at least as long as 12 weeks post-transplantation. Lesions performed between 3 and 10 weeks prior to transplantation were associated with fiber ingrowth into transplants. Labeled cells in the transplant
On 3 occasions we observed labeled cells in a trans-
9
.
.
.
13
14
15
++
. n.d.
n.d.
n.d.
/
/
/
/
/
7 19
8-9 19
8 20
10 20
10 20
.
12
.
.
.
.
.
plant. Each cell was the only one of its kind in the transplant and all 3 cell bearing transplants were located in basal nucleus lesioned hosts. One of the 3 cells was located within 50/tin of the host-transplant border and thus may have been located in the host, considering our section thickness of 50/zm (see Fig. 5C). The other two labeled cells were located deep within the transplant and therefore could be cells retrogradeiy labeled by the thalamic injection of W G A - H R P (Fig. 5 C - E ) . A C h E stain in the transplant
The amount of A C h E reaction product contained in the lesioned host transplants appeared to correlate with both the extent of the lesion and the postlesion survival time. Some regrowth of AChE-positive fibers into the depleted host cortex and consequently into the transplant had occurred in all transplants by the time of sacrifice. Fig. 4C shows the typical appearance of such AChE-positive fibers after presumed regrowth into depleted host cortex and transplant. The largest number of AChE-positive fibers was found in transplants number 4, 6 and 7 (see Table I) which had either received a smaller than average lesion (case 4) or had long postlesion survival times. AChE-stained attd WGA-HRP-Iabeled fibers from the thalamic injections always coexisted in the transplants of basal nucleus lesioned hosts, but no consistent relationship could be observed between the distribution of the two different fiber groups.
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61 ACHE- and WGA-ttRP-labeled fibers neither avoided each others target territory nor did they preferentially occupy the same space within the transplant (Fig. 4 compare A and B with C). There was no obvious relationship between the extent of AChE fiber regrowth into a transplant and the amount of thalamic fiber ingrowth (see Table I). DISCUSSION The present results show a clear correlation between the presence of a basal forebrain lesion in the host, and subsequent presence of host axons in the transplant that could be labeled by thalamic injection. Such ingrowth did not correlate with any other variable in the experiments. Thus it appears that basal forebrain lesions changed the propensity of other, (thalamic) host fibers to grow into the transplants.
Origin of WGA-HRP-labeled elements in the transplant Our results support the interpretation that many of the sprouting fibers arise from thalamic nuclei or from recurrent collaterals of corticothalamic neurons or both. The selection of cases according to location of the injection site makes sources other than the reciprocal interconnections very unlikely as the origin of the labeled fiber ingrowth. It is also unlikely that all iiqet~tions into the basal forebrain lesioned hosts involved extrathalamic structures while all injections into unlesioned hosts did not. Furthermore, the pattern of transported WGA-HRP within host cortex corresponded to the terminal pattern of thalamic nuclei 23'75. Labeling was not seen in areas such as hippocampus and superficial cortical layers of frontal cortex that have been identified as projection areas for brainstem catecholaminergic and serotoninergic nuclei 13'17'18'35'42.It is unlikely that we labeled catecholaminergic projections into the transplant vicinity while sparing all other projection areas. A cholinergic origin of WGA-HRP-Iabeled fibers in the transplant is unlikeiy since the distribution pattern of residual AChE stain in the transplants never matched the pattern of labeled fiber ingrowth into any transplant. The possibility that we are labeling sprouted recurrent collaterals of ia~,'er VI neurons requires further study to estimate the probability of thi~ interpretation.
Effect of basal forebrain lesions on plasticity The absence of adult host fiber ingrowth into cortical transplants has been reported by other investigators 48. While adult host cholinergic and in many cases also catecholaminergic (CA) fibers readily grow into transplants 4.16.36.37.59, specific, precisely projecting, mature fiber systems always are more reluctant to invade transplant tissue 4s'52"55"56"71.A considerable difference in response to injury between specific fiber systems and ACh and CA fibers can also be seen in the normal adult animal 4. Thus, the adult cortex clearly has reduced its ability to mount as much of a plastic response to transplantation as the immature host. We have shown here that the basal forebrain lesion is capable of relaxing the constraints on this condition in some as yet unspecified manner. Since the purpose of the basal forebrain lesion was to destroy the basal nucleus cholinergic input to neocortex, the present results are consistent with the current speculations that ACh has an influence on the plasticity of cortical connections during normal development.
Is fiber ingrowth into the transplant a consequence of A Ch depletion ? Depletion of ACh is not the only consequence of a basal forebrain lesion made by thermal coagulation and the possibility cannot be ruled out that the enhanced ingrowth is related to factors other than or in addition to the disruption of cholinergic mechanisms. Recently it has been shown that injury to the CNS induces the production of substances which have a general effect on neuronal growth promotion and survival45.5°. The basal forebrain lesion could potentially result in the release of such a non-specific trophic factor in cortex which then promotes fiber ingrowth into the transplant. However, the basal forebrain lesion occurs at a site remote from cerebral cortex, the factor would need to be transported by the damaged afferent axons or generated by neurons in cortex responding to the brain injury or deafferentation. In general, degeneration of axons following a lesion appears to be a sufficient stimulus for the production of these trophic factors only for a relatively short period of time and only when the degenerating system constitutes a major input to the deafferented area. In ti'ansplantation by 3 - l 0 weeks a~d still enhance tha-
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63 lamic fiber ingrowth into the transplant. Injury-induced release of trophic factors as late as 10 weeks after the lesion seems unlikely to maintain levels at growth-promoting concentrations, since available evidence suggests that this neuronotrophic activity had decreased to almost baseline values before the end of the third week after the lesion s°. However, a more specific trophic factor activity, as recently indicated by the work of Gage ~9, deserves further attention in relation to the fiber sprouting observed in our cases. Another possible consequence of basal forebrain lesions could be the destruction of non-cholinergic ascending systems such as the serotonin and catecholamine fibers that travel in the medial forebrain bundle. As can be seen in Fig. 2, our lesions varied in size, and while all lesions destroyed cortically projecting cholinergic neurons, only some of them encroached on the medial forebrain bundie. There was no correlation between the mediolateral position of the lesion and the extent of fiber ingrowth into the transplant. Further experiments are necessary to test the possibility of noradrenergic or serotoninergic involvement in thalamocortical fiber sprouting. Several precedents exist for the idea that sprouting can be induced as a consequence of cholinergic depletion n'12a9'39'44. Both septal lesions and basal forebrain lesions have been shown to result in sympathetic fiber sprouting into their respective target areas 11'12'43. In the case of the septal lesion paradigm considerable evidence exists that the sprouting of sympathetic fibers into the hippocampus is the consequence of cholinergic depletion 12'37'43. At least one of the paradigms of CNS to CNS transplantation, which did result in considerable amounts of transplant innervation by adult host-specific fibers involved host brain areas that contained little or no ACh. Oblinger and Das reported that embryonic transplants to the adult cerebellum received innervation from several different host fiber systems 52. The adult cerebellum contains barely detectable levels of CHAT, the lowest in brain 34'39. Transplam~ of embryonic neocortex inserted into the tecturn of adult hosts which had undergone prior optic tract section have shown regeneration of optic tract fibers into the transplan¢ 8. The possibility that the cholinergic input to the recipient rectum is partially interrupted under these experimental conditions has
not been studied.
Possible mechanism for fiber ingrowth into the transplants induced by basal forebrain lesions of the host If depletion of cholinergic fibers is directly responsible for thalamic fiber ingrowth into the transpiant, then there are two possible mechanisms that could account for such a response; one is that cholinergic innervation actively inhibits the ability of thalamocortical fibers to elongate due to cholinergic receptor mediated effects, and the other is that the cholinergic influence induces a response such as synapse formation which stabilizes the elongating fibers. Specific thalamic nuclei show rapid and dramatic atrophy following the removal of their cortical target area 2'22'46. Thus, the high density of labeled thalamocortical fibers in the vicinity of the transplant could be viewed as an effect of the cholinergic depletion. The transplant procedure removed the original cortical target of these fibers in and around the transplant location. However the presence of immature CNS transplants may be a sufficient stimulus for the survival of some fibers and cells, which would otherwise atrophy 7"22. The sprouting fibers found in the transplant may originate from areas of cortex sufficiently distant from the site of transplantation to have escaped any damage 9. On the other hand, we did observe a considerable difference between normal and lesioned cases in regard to the number of anterogradely labeled fibers in close proximity to the transplant. Sunde and Zimmer have argued that adult specific fiber systems in the host do not usually invade transplant tissue since all synaptic sites in the graft are occupied by intrinsic fibers TM. The premise was that host-specific fibers do not grow fast enough to reach the transplant in time to find vacated synaptic sites. Based on this logic the observed ingrowth of host ACh fibers into the neocorticai grafts could be attributed to the greater growth potential and faster growth speed of these fiber systems (discussed in ref. 4). It could thus be argued that basal forebrain lesions act through making synaptic sites available in the transplant which then can be occupied by specific cortical afferents. Unless AChE-positive fibers operate under different rules, they should not sprout back into the transplant as they eventually do. There should be no space left for these late sprouting fibers after intrinsic graft fibers and host specific afferents
64 have claimed their territory. Yet, regrowing AChE stained fibers appeared in the transplant in densities comparable to the surrounding host cortex. Further, we have not observed any sign of mutual avoidance of the AChE and the HRP fiber pattern. Clearly, this particular type of interaction between two fiber systems is not a competitive synaptic replacement paradigm as it occurs, for example, in hippocampal plasticity44, but synaptogenesis could exert a transient influence at several stages during the response to injury. Previous studies have documented that sprouting in the adult CNS requires the stimulus of vacated synaptic sites44's4. The possibility exists that ACh may decrease plasticity in the CNS both during normal development and after brain injury, The very late maturation of cholinergic markers and the widespread morphological distribution of these fibers in the developing cortex would enable the cholinergic system to regulate and ultimately terminate plasticity26'27. The developing neocortex shows most plasticity at times when synapses are still being formed and while synapse numbers are equal to or higher than in adulthood 1"6"29.Thus ACh may act during normal development anuior under transplant conditions to restrict the number of connections that can be made. Cholinergic mechanisms have been implicated in the etiology of several mental and neurological diseases, the most prominent being Alzheimer's dementia. Patients with Alzheimer's disease suffer from a progressive loss of memory and cognitive ability and at autopsy their brains have shown a consistent decrease of cortically projecting cholinergic basal forebrain neurons with a concomitant deficiency in the levels of cortical cholineacetyltransferase (CHAT)
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activity3.t°,74. Animal models of Alzheimer's disease, employing lesions to cholinergic basal forebrain neurons have been used to study the impact of cholinergic depletion on cerebral cortex t2'32'33'41'47'73. The present results are consistent with the notion that a decrease in cholinergic basal forebrain neurons may lead to significant long-term changes in cortical organization. In summary, we have reported sprouting of thalamic afferents into cortical transplants in response to basal forebrain lesions in the adult host animals after prior basal forebrain lesions. We discuss the possibility that the enhanced ingrowth of specific fiber systems may be related to the loss of cortical innervation by cholinergic basal forebrain inputs. The mechanism by which host fiber ingrowth into the transplant is facilitated remains unknown, but simp!e replacement of AChE-labeled fibers by thalam~c fibers in the transplant appears unlikely. The present results are, to our knowledge, the first morpholc,gical indication that ACh may influence the growth and connections of non-cholinergic CNS fiber syste,las. An interesting question for the future is whether similar cholinergic influences can be shown to control thalamocortical fiber distribution and synaptogenesis during normal development.
ACKNOWLEDGEMENTS This research was supported by NIH Grant NS13031. A preliminary report of these data has been given elsewhere 68. This research was used in partial fulfillment of the thesis requirements for the PhD degree (C.F.H.).
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