Primate nucleus basalis of meynert p75NGFR-containing cholinergic neurons are protected from retrograde degeneration by the ganglioside GM1

Neuroscience Vol. 53, No. I, pp. 4!&56, 1993 Great Britain

0306-4522/93

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PRIMATE NUCLEUS BASALIS OF MEYNERT NEURONS ARE P75 NGFR-CONTAINING CHOLINERGIC PROTECTED FROM RETROGRADE DEGENERATION BY THE GANGLIOSIDE GM, E. P. PIORO,* D. MAYSINGER,* F. R. ERVIN,~ G. DESYPRIS* and A. C. CIJEZLLO*$ *Department of Pharmacology and Therapeutics, McGill University, McIntyre Medical Sciences Building, 3655 Drummond Street, Montreal, Quebec, Canada H3G IY6 tNeurobehaviora1 Unit, Department of Psychiatry, McGill University, Allan Memorial Hospital, Montreal, Quebec, Canada Abstract-The effects of unilateral devascularizing lesions of the neocortex in primates (Cercopithecus aethiops) on the immunoreactivity of choline acetyltransferase and the low-affinity nerve growth factor receptor (p75NGFR)were investigated in cell bodies of the nucleus basalis of Meynert. Choline acetyltransferase enzymatic activity was measured in the dissected ipsi- and contralateral nucleus basalis of Meynert as well as in the remaining cortex adjacent to the lesion. Cortically lesioned animals displayed a shrinkage of p75 NGFR-immunoreactive cholinergic cell bodies in only the intermediate portion of the nucleus basalis of Meynert as well as a depletion of choline acetyltransferase activity in this cellular complex. In contrast, cortically lesioned monkeys treated with monosialoganglioside did not reveal a significant loss of choline acetyltransferase activity or shrinkage of nucleus basalis of Meynert cholinergic neurons, but rather a modest hypertrophy. These results are discussed in relation to a possible use of putative trophic agents in the repair of the damaged central nervous system.

been an interest in these compounds because of their effect on neural developmenti and their neuroprotective properties in various rodent models of lesioning.5,10~‘1~‘5 Although the mechanisms of action of gangliosides are not well understood, their effects have been demonstrated in various neuronal populations, including catecholaminergic and cholinergic systems (for review, see Ref. 5). The demonstration of retrograde degeneration in the rat nbm after cortical lesioning and its prevention after GM, administration prompted us to investigate whether similar changes would occur in the nonhuman primate. Two basic questions were addressed in this preliminary study: do choline@ magnocellular neurons of the monkey nucleus basalis of Meynert (nbM) undergo retrograde degeneration after neocortical infarction as revealed by neuronal shrinkage and decreased ChAT activity? If so, can this degeneration be prevented by exogenous monosialoganglioside as in the rodent? The demonstration of such neuronal responses in the nonhuman primate is of great importance before proposing any clinical application of neurotrophiclike factors (e.g. GM,) in the treatment of neurologically compromised patients.

Retrograde degeneration of cholinergic neurons in the nucleus basalis magnocellularis (nbm) of adult rats is known to occur after cortical devascular-

ization. This is evidenced by a reduction in both neuronal cross-sectional area” and choline acetyltransferase (ChAT) activity in the microdissected nbm.” Actual loss of ChAT and low affinity nerve growth factor (NGF) receptor (p75NGFR)immunoreactivity in neurons of the nbm ipsilateral to the infarcted neocortex may also occur with more prolonged survival.** Functionally, animals with such unilateral cortical lesions display behavioral abnormalities, including learning and retention difficulties as shown in passive avoidance and Morris water maze paradigms.’ These immunocytochemical, biochemical and behavioral changes can be prevented in the rat up to a one-month survival by the short-term administration of the monosialoganglioside, GM,.4 Monosialoganglioside, a ganglioside with one sialic acid residue, is part of a family of ubiquitous intramembranous glycosphingolipids which are particularly prevalent in neural tissue.5,‘5,28There has $To whom correspondence should be addressed. ChAT, choline acetyltransferase; DAB, 3,3’diaminobenzidine tetrahydrochloride; IR, immunoreactive; p7SNGFR, low-affinity nerve growth factor receptor; GM,, monosialoganglioside; NGF, nerve growth factor; NGFrS; nerve growth factor receptor 5; nbm, nucleus basalis magnocellularis; nbM, nucleus basalis of Meynert; PAP, peroxidase-antiperoxidase; PB, phosphate buffer; PBS; phosphate-buffered saline.

Abbrektions:

EXPERIMENTAL PROCEDURES Animals

Twenty-two healthy adult male monkeys (Cercopithecus St Kitts) weighing 4.55.5 kg, with age range approx. lo-15 years, were used for this study and were obtained from the Caribbean Primate facility on St Kitts,

aethiops,

49

f-. I’. P1rrao Cl ‘Ii.

50

E. Caribbean. All surgeries and perioperative handling ol the animals were carried out in accordance with the Guidelines of Canadian council of animal care and McGill University Animal Care Committee. After being randomly divided into three groups: sham-operated (seven), iesioned (eight) and lesioned/GM,-treated (seven), they underwent craniotomy, neocortical devascularization and placement of gelatin films (see below). Following surgery, the animals were housed in individual cages and monitored daily for signs of distress. They had free access to water and were regularly fed fruit and High Protein Monkey Chow (Purina Mills, Inc.). After a six-month survival, they were processed either for biochemistry: sham-operated (three), lesioned (four) and lesioned/GM,-treated (three) or for immunocytochemistry: sham-operated (four), lesioned (four) and lesion~~GM,-treated (four). Preparation

of gelatin

,films

Sterile gelatin (7.5 g; Sigma, St Louis) was dissolved in double-distilled water (36.5 ml) with gentle heating and ultra pure glycerol (5 g; Bethesda Research Laboratories) was added. The dosage of GM, (Fidia Research Laboratories, Abano Terme, Italy) incorporated into the gelatin films was extrapolated from the amount given i.c.v. to the rat over a seven-day period.4 Based on estimated differences in brain volume between adult rat and monkey, the average was 175 mg GM,iaei. The comDound was dissolved in 1.5 ml of donbi~-distiii~ water and added to the gelatin solution with gentle stirring to avoid air bubbles. Blank gelatin films used in sham-operated animals did not contain GM,. The warm solution was poured onto a flat glass surface to produce a uniform layer approximately 1 mm thick. After hardening of the gelatin. rectangular-shaped pieces were cut to 2.5 x 4.5 cm and stored in a sterile container at 4-C until use within two weeks, Surgeries

After induction of general anesthesia with an intramuscular (i.m.) injection of a ketamine sulfate (Ke.etaiar, Park Davis; 15-20 mg/kg~xyi~ine (Rompun, 3ayer Agrochem. U.K.; 1.5-2.0 mg/kg) mixture, the scalp was cleaned and shaved; anesthesia was maintained intraoperatively with intermittent i.m. administration of the same mixture (l&i5 mg/kg per 30 min). Ketalar possesses intrinsic analgesic characteristics and when combined with xyiazine produces a more profound anesthetic effect. Int~mu~ular injections of dexamethasone (0.2mgikg) and penicillin G (40,000 U/kg) were given pre-operatively. The monkey’s head was secured in a Kopf stereotaxic head frame and the left hemicranium was washed with iodine and alcohol. Following periosteal infiltration with 2% xyiocaine for greater analgesia, a wide inverted U-shaped incision was made in the scalp on either side of the left ear. The underlying temporalis muscle was reflected inferiorly and a 3.5 x 5.0 cm craniectomy was made with a dental drill. The skull flap was elevated and the underlying dura mater was opened with iridectomy microscissors and reflected inferiorly. This craniectomy consistently exposed a wide area of the left cerebral hemisphere including posterior frontal, superior temporal, parietai and anterior occipital cortices (Fig. 1). In lesioned animals, piai blood vessels supplying the gyri in these regions were coagulated using a Maiis bipolar cautery device (Valley Forge Scientific Inc. Valley Forge, PA). Cortical vessels of sham-operated monkeys were not injured and those of the primary motor cortex (Brodmann Area 4) were not disturbed in lesioned animals. Adequate obliteration of vessel lumina was evidenced by the resultant pallor of the subjacent cortical parenchyma. The pre-cut gelatin film (see above) was then placed onto the exposed area of neocortex, a covering piece of sterile Geifoam (Upjohn, K~am~oo) securing it in place. After repositioning of the dura and suturing of temporalis muscle into place. the skin was’ closed with interrupted sutures. The monkeys

im, injection of dexamethasone portopel atively (0.2 mg/kg) as well as a one-week course of daily penicillin G im. injections (40,000 Ulkg). Motor and sensory deficits were documents and animals m~~nifestingsigns of distress were given i.m. analgesics received another

Biochemistry

After the six-month survival, monkeys were verv deeply anesthetized with ketamine--HCl (Ketalar 20 mg;kg) and perfused intracardiaiiy with cold phosphate buffer (PB: pH 7.4). Brains were quickly removed and areas of interest dissected bilaterally and frozen on dry ice. The region of the nbM (Ch4)” removed en bloc was between the level of the anterior commissure, anteriorly and origin of the optic tracts, posteriorly; these levels were approximately represented on the ventral surface of the brain between the anterior and posterior borders of the optic chiasm. This dissection, therefore, included the anterior and intermediate portions of the nbM but not its posterior extent.” Additionally, a lip of healthy-appearing neocortex adjacent to the devascuiarized lesion was dissected with a width of approximately 5-10 mm: an equivalent region of cortex was dissected from the uninjured contralateral hemisphere. ChAT enzymatic activity was determined in 2911aliquots maintained at - 70°C utilizing a radioenzymatic procedure as described by Fonnum.8 Protein content was determined in 5-iii aiiquots by dye binding.’ All determinations were performed in duplicate, with experimental and corresponding vehicle control samples.

After the monkeys were very deeply anesthetized with ketamine.-HCi (20 mg/kg), the descending aorta was clamped and systemic vascuiature was flushed through the ascending aorta with 500ml of PB (pH 7.4). The solution was then changed to buffered 4% -formaidehydeJJ.05% plutaraldehvde/O. 1 M PB (aH 7.4) with 1000 ml infused over 30 min. Brains were &mediately removed, put into 10% sucrose-PB (pH 7.4) and stored at 4°C for up to two weeks. After blocking the brain to include the entire nbM, the hemispheres were sagittally divided and sectioned on a sledge microtome (Reichert) equipped with a freezing stage (Baldwin, Inc., Cambridge, U.K.). Fifty-micrometer-thick sections were collected serially in PB and stored at 4°C until antibody incubation. Alternate sections were incubated overnight at 4’ C in either rabbit anti-ChAT antisera (A; Chemicon Intl Inc.) or anti-p7SNGFRmonoclonai antibody [&; NGF receptor 5 (NGFrS)] which recognizes the low-affimty NGF receptor in primates. The specificities of the anti-ChAT3 and NW&” antibodies have been previously described. Ail subsequent steps were carried out at room temperature using phosphate-buffered saline (PBS; pH 7.4) containing 0.2% Triton X-100 for washes, dilution of antibodies and 3,3’diaminobenzidine tetrahydrochloride (DAB) reaction. After washing, sections were incubated for 1 h either in goat anti-rabbit serum {$: ?&ma) for anti-ChAT antisera or in rabbit anti-mouse serum& Sigma) for NGFr5 and then washed again. Subsequently, tissue was incubated for 2 h in either rabbit anti-goat peroxidase-antiperoxidase (PAP; $6; Sigma) for anti-ChAT or monoclonal mouse PAP ,(f: Medicorp, Canada) for NGFrS. After thorough washmg, sections were incubated in 0.06% solution of DAB for 15 min and a subsequent IO-15 min in the same solution containing 0.01% hydrogen peroxide. After final washing, sections were mounted on subbed slides and were dehydrated, cleared and coverslipped. Alternate sections where anti-ChAT or NGFr.5 antibodies were omitted served as negative controls and were processed in parallel as described above. Select brain levels were stained with Cresyi Violet for nuclear identification. 1”

nbM p75NGFRcontainingcholinergic neurons

51

Analysis of immunocytochemistry

Cross-sectional area measurements of &AT- and P75NGFR-immunoreactive(IR) neurons were made in representative sections from the anterior, mid and posterior Portions of the nbM. These levels correspond relatively well to previously described subdivisions within the primate nbM of choline+ neurons: anterior (Ch4a), intermediate (Chrli) and posterior (Chrlp), respectively, which have been shown to have distinct, although overlapping cortical projection fields.” Such IR cells were visualized with a Polyvar (Reichert Jung) microscope equipped with a 10 x objective lens and transferred to a semi-automated image analysis system (Quantimet 920, Cambridge Instruments) using a 12.5 x projection lens. Two fields located in the representative sections were analysed by two independent observers at each level of the nbM (anterior, intermediate and posterior) from each of four animals and the average cell body crosssectional areas were obtained for each field. The number of neurons measured per nbM region ranged between 100 and 200 of the sham, lesioned and lesioned/GM,-treated animals. Cell bodies which appeared distorted due to inadequate fixation or freezing artifact were edited out of the quantitative analysis. However, the majority of sections selected for analysis of neuronal size was free of such changes. The image analysis recognition of ChAT-IR cell bodies is illustrated in Fig. 2. Statistical significance was tested between the three groups using ANOVA and high stringency (Tukey) post hoc analysis.

a.

BEsULTs

Animals

b.

The postoperative course was uncomplicated except for mild right hemiparesis’ hemianesthesia and visual field deficit. They did not appear distressed and were able to feed themselves. The motor weakness was virtually undetectable within 10-14 days of surgery. Although formal evaluation of the sensory deficits was not performed, these seemed to persist longer. Gross anatomy of lesioned neocortex There was no detectable remnant of the gelatin or Gelfoam overlying the lesioned neocortex which had undergone moderate atrophy. The most convex aspects of the cortical gyri were predominantly affected, appearing yellow-brown with small foci of cavitation. The primary motor cortex (Area 4), whose surface arterioles had not been damaged, appeared less affected while the remaining neocortex outside the circumscribed area of lesioning appeared intact. The area most affected by the lesion, which included

Fig. 1.

Fig. I. (a) Pictorial representation of the procedure used to obliterate the pial blood vessels overlying the superficial gyri in Cercopithecus aethiops. A pair of jeweller’s forceps are connected to a bipolar cautery device. (b) Schematic dorsal view of the C. aethiops brain. The broken lines over the left hemisphere indicate the area of dural opening and approximate limits of the devascularixing lesion. (c) Pictorial representation of a coronal section of the primate brain at the level of intermediate nbM showing the hemispheric areas affected by the devascularixing lesion. Note that there is some involvement of subcortical white matter but the deep gyri remain largely intact.

of ChAT-IR cell bodies (asterisks) at relatively high magnification in the arthiops nbM as visualized on the image analysis system screen. cell body recognition by the image analysis system of the same field. Scale bar= 15&m.

Fig. 2. (a) Representation

intermediate region of the Ccwopithecus (b) Semi-automated

some involvement of subcortical represented in Fig. 1.

white matter,

is

Compared to ChAT activity in the nbM of sham-operated animals which averaged 120.5 + 6.8 mnol/mg protein per h (100 + 6%), enzyme activity in nbM ipsilateral to the cortical devascularizing lesion was 69 + 5% of control in untreated lesioned animals and 92 rf: 5% of control in monkeys receiving GM, (Table 1). ChAT activity in nbM of the contralateral hemisphere remained unchanged between the sham, lesioned and lesion&/GM,treated groups (results not shown). Additionally, there was no statistically significant difference in ChAT activity in the strip of neocortex surrounding the lesioned area when comparing the different groups of monkeys (Table 1).

Fifty-micrometer-thick sections throughout the anteroposterior extent of the nbM, which were stained for either ChAT- or p7SNGFR-immuno-

reactivity, demonstrated numerous magnocellular neurons. The specific distribution of such ChAT- and P75 NGFR-IR neurons within anterior, intermediate and posterior regions of the nbM, as well as their size and morphological appearance, was consistent with previous primate reports.‘3*‘7.‘x Measurement of cross-sectional area of these neurons from each of the three aforementioned regions of the nbM ipsilateral to the cortical infarction revealed a significant shrinkage of the ChAT- and ~75~o~~-IR neurons only in its intermediate portion (Table 2; ‘Table 1. Choline acetyltransferase activity ipsilateral to the neocortieat lesion, expressed in nmoi/mg protein per h nbM**

Cortex***

Group

n

ChAT activity

ChAT activity

Sham Lesioned Les~on~~GM,

3 4 3

120.5 & 6.8 83.7 f 3.8* 110.7 + 3.5

31.4 + 4.4 33.4 + 5.4 26.9 & 3.1

*Significantly different from sham-operated animals at P < 0.05; **includes its anterior and intermediate portions; ***includes a 5%lo-mm-wide lip of healthy appearing neocortex adjacent to the devascularized lesion.

nbM ~75~~~~-containing chohnergic neurons Table 2. Cross-sectional areas of choline acetyltransferaseimmunoreactive neurons in the nucleus basalis of Meynert ipsilateral to neocortical lesion, expressed in pm2 Anterior nbM

Mid nbM

Posterior nbM

Group

n

Cell area

Cell area

Cell area

Sham Lesioned Lesioned/GM,

4 4 4

374 * 51 453 + 56 451 * 43

418 + 30 302f30* 525 f 30*

425 k 39 426+31 410 + 35

n, number of animals. *Significantly different from shamoperated animals at P < 0.05. Fig. 3). ChAT-IR neurons in lesioned animals diminished in size to approximately 70% of those in sham-operated animals (Table 2). The apparent shrinkage of p75NoFR-containing neurons in nbM of lesioned monkeys (which likely represent the same choline+ population’3) was more modest but still evident when compared to those in sham-operated animals (Fig. 3; Table 3). There was no significant change in neuronal size in the anterior and posterior portions. The ordinarily prominent p75NGFR-IR neuropil observed in sham-operated nbM at relatively high magnification was noticeably sparse in lesioned animals (Fig. 3d, e). This shrinkage in cross-sectional area of both ChAT- and ~75 NGFR-IRneurons in the intermediate region of the nbM was completely prevented by the administration of GM, (Tables 2, 3; Fig. 3). Not only were the immunostained cell bodies of treated monkeys significantly larger than those of lesioned animals but they were hypertrophic compared to their counterparts in sham-operated animals (Tables 2, 3). Neuritic processes in the nbM of GM,-treated animals formed a much more extensive network even when compared to non-lesioned controls. This was particularly evident in the p75NGFR-IR neuropil (Fig. 3). DISCUSSION

Lesioned animals

In this study, we have demonstrated that magnocellular ChAT- and p75 NGFR-IRneurons of the nonhuman primate nbM, as in the rodent nbM,4’4,26 undergo retrograde degeneration after ipsilateral neocortical ischemic injury. This pathological change is evident both biochemically and immunocytochemitally. An earlier report had described atrophic changes in the Nissl-stained neurons of the macaque monkey and human nbM after extensive cortical lesions,” a finding consistent with a secondary cholinergic cell atrophy in Alzheimer’s disease”s2’ The 30% decrease of ChAT activity in the monkey nbM demonstrated here six months after neocortical lesioning is significant but not as dramatic as that seen in the rat nbm one month after cortical devascularization which approximated 40%.4,25 There are several possible explanations for this. The value may represent an averaged ChAT activity in the entire

53

nbM because its en bloc dissection would include the anterior division whose cholinergic neurons have been shown in this study to undergo little or no atrophy. Therefore, a more significant decrease of ChAT activity in the intermediate nbM where P75 NGFR-containing cholinergic neurons show significant degeneration would be masked. Secondly, the extent of neocortical injury may be insufficient to produce a greater degree of retrograde degeneration of the cholinergic nbM neurons due to the relatively widespread cholinergic projections in the primate neocortex.‘8 Alternatively, this ChAT activity may represent a partially recovered value after the protracted (six-month) postoperative survival. In the young and adult rat nbm, ChAT activity decreases maximally by 30 days postlesioning with subsequent spontaneous increase to near control levels until at least four months of survival.26 Finally, although less likely, it may represent a difference in cholinergic neuronal response to injury between the two species. Microscopic analysis revealed significant shrinkage of ChAT- and ~75 NGFR-IRcell bodies only in the mid portion of the nbM and not in its anterior or posterior regions. This correlates well with the lesioned neocortical areas being the terminal fields of axons whose lesioned choline@ cell bodies are known to be primarily in the intermediate nbM.” However, the overall degree of retrograde degeneration in the intermediate nbM may have been limited by not lesioning the primary motor cortex to minimize postoperative hemiparesis because cell bodies here project to at least the frontal opercular region.” There was no significant difference in the degree of shrinkage between ChAT- and p7SNGFR-IR nbM neurons. This is not surprising because colocalization studies in rodents’s6 and primatesI have shown these antigens to be present in largely the same cell population. Monosialoganglioside-treated

animals

The complete protection of ChAT activity in the decorticated rat nbm after monosialoganglioside treatment4 is recapitulated in the nonhuman primate nbM. Sham-operated levels of ChAT activity are retained in lesioned animals treated with GM, as are the cross-sectional areas of ChAT- and p7SNGFR-IR nbM neurons. In these experiments, ChAT- and p75NGFR-IR neurons of the monkey nbM became significantly hypertrophied after treatment unlike their counterparts in the rat nbm. This was particularly true in the intermediate region of the basalis where such cells seem most vulnerable to neocortical injury. Although the cause of this relative cellular hypertrophy is unclear, it is probably not simply an injury-related reaction of the neurons which in the rat nbm was shown to be transient.2’ The possibility of this change being artifactual is slight but requires further study. The accompanying prominence neuritic processes in of p75NoFR-immunostained lesioned/GM,-treated monkeys is also of note and

54

F. P PIOKO PI Cl/.

nbM ~75~~~~-containing cholinergic neurons Table 3. Cross-sectional areas of p75NGFR-immunoreactive neurons in the nucleus basalis of Meynert ipsilateral to neocortical lesion, expressed in pm* Anterior nbM

Mid nbM

Posterior nbM

Group

n

Cell area

Cell area

Cell area

Sham Lesioned Lesioned/GM,

4 4 4

418 f. 32 448+36 454 f 30

437 f 15 364 f 18* 497*28*

399 &-29 430 + 30 422& 17

ventricular system into regions adjacent to the nbM or its effect via the nbM cortically projecting axons whose injured terminals would be subjacent to the gelatin film. In either case, the efficacy of our therapy applied at a site distant from the responsive nbM cell bodies is noteworthy and its site of action may differ from that of the intraventricular infusion of GM1 in the rat4 or NGF in the monkey.1z*29

n number of animals. *Significantly different from shamoperated animals at P < 0.05.

may result from either an up-regulation of p7SNGFR protein synthesis, alterations in axonal transport of

P75 NGFRor proliferation of neuronal processes. The underlying mechanism(s) may be common to both of these neuronal responses. As Gage and colleagues have demonstrated,’ NGF induces an hypertrophy of cholinergic neurons as well as an increase of p7SNGFRmRNA and p75NGFRprotein in rat neostriatal neurons. Whether other factors, e.g. GM,, have the same effect has not been examined. Because in our study only GM, was administered, it may have interacted with endogenous NGF because gangliosides significantly augment the neurotrophic activity of even submaximal concentrations of NGF.4 Furthermore, the endogenous levels of NGF in the primate nbM or cortex may have increased as occurs in rat nbm after cortical lesioning.16 The protection of p75NGFR-containing cholinergic nbM neurons in this experiment was accomplished using cortically-applied monosialoganglioside. As described above (see Experimental Procedures), GM, was incorporated into gelatin films which were placed over the neocortical ischemic lesion and covered with Gelfoam. It is assumed that the administered GM, would be available only as long as either the gelatin or Gelfoam persisted. An in vivo analysis of Gelfoam implants in rat brain has determined their complete degradation within 21 days.27 One can assume, therefore, that exogenous monosialoganglioside was present at the cortical surface for less than four weeks. The protection of nbM neurons whose cell bodies are not in close proximity to its site of administration suggests either a circulation of the GM, through the

55

CONCLUSION

Monosialoganglioside is able to prevent the retrograde degeneration of injured p75NGFR-IRcholinergic neurons in the primate nbM for at least six months after neocortical lesioning. Other primate studies have examined the protective effect of NGF on cholinergic ~75~~~~-containing basal forebrain neurons only one month following septohippocampal lesioning.‘2,29 Therefore, our results suggest that a relatively short period of GM, treatment may be sufficient to avert degenerative changes in cholinergic nbM neurons for an extended duration. This finding has significant implications not only for the screening of beneficial neurotherapeutic agents but also for exploring further the molecular mechanisms of neural plasticity and regeneration. Whether gangliosides will be utilized in patients with neurological deficits resulting from degenerative conditions (e.g. Alzheimer’s disease), stroke or neurotrauma remains to be seen. However, their apparent broad spectrum of action5~‘0~”makes them promising candidates in the treatment of conditions which involve multiple neuronal systems. Further primate studies are required to characterize the extent of ganglioside neuroprotection, including analysis of other neurotransmitter systems and behavioral parameters. Acknowledgements-This work was supported by the Canadian Center of Excellence for neuronal repair and Functional Recovery and in part by Fidia Research Laboratories. Dr M. Bothwell generously provided the NGFr5 monoclonal antibody. We wish to thank Dr P. Liberini and Dr R. Latt from the McGill Animal Center for valuable advice and S. Cote, M. St Louis and G. Besaccia for expert technical assistance. The editorial assistance of M. Warmuth and photographic reproductions by A. Forster are gratefully acknowledged.

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