Experimental neurotrophic factor therapy leads to cortical synaptic ...

REVIEW PAPER

Experimental Neurotrophic Factor Therapy Leads to Cortical Synaptic Remodeling and Compensates for Behavioral Deficits A Claudio Cuello Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada Submitted: October 18, 1996 Accepted: October 31, 1996

This brief review discusses experimental therapy with neurotrophic factors in a model of central nervous system (CNS) neural atrophy and synaptic loss resulting from unilateral cortical infarctions. It discusses the trophic factor protection of the cholinergic phenotype of neurons belonging to the forebrain-to-neocortex projection, as well as the capacity of trophic therapy to elicit synaptogenesis in the cerebral cortex of adult animals. Finally, it addresses the behavioral consequences of trophic factor-induced synaptic remodeling of the neocortex in this model.

Key Words: neurotrophic factors, cholinergic system, cortical synaptogenesis

synapses,

behavior, neural regeneration,

INTRODUCTION

This paper was originally presented on the occasion of my being awarded the 1995 Heinz Lehmann Award from the Canadian College of Neuropsychopharmacology. One ofthe key threads of Dr Lehmann's research has been his interest in integrating modem cellular and molecular neurobiology with neurological and psychiatric disorders. It is my hope that this review will contribute to the ongoing success of this important integration of the basic and clinical sciences. Over the past decade, our knowledge concerning the role and function of neurotrophic factors (NTFs) has grown by

leaps and bounds. A fairly large number of new NTFs have been described, each ofwhich displays remarkable effects on the morphology and biochemistry of neurons both in vitro and in vivo. Classically, these are large polypeptide molecules that play a fumdamental role in the development of the nervous system and are generally produced by either glial cells or neurons. They are synthesized (and assumed to be released) in a well-regulated manner. While most of our knowledge about the neurobiology of NTFs has been gathered in vitro, it has become evident that these molecules are important in the maintenance of phenotypic characteristics Based on the Heinz Lehmann Award Lecture presented by Dr of neurons in the central and peripheral nervous system Cuello at the 18th Annual Meeting of the Canadian College of Neuropsychopharmacology, June 4-7, 1995, Vancouver, British (Lindsay and others 1994; Thoenen 1995). From a medical standpoint, these substances have become ofpotential theraColumbia, Canada. interest because of evidence that NTFs can protect or peutic Address for correspondence: Dr AC Cuello, Department of the repair of degenerating neurons in a variety of facilitate Pharmacology and Therapeutics, McGill University, 3655 DrumH3G IY6. laboratory experimental models. Some obvious therapeutic mond, Suite 1325, Montreal, QC JPsychiatry Neurosci, Vol 22, No 1, 1997

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NTF therapy and cortical synaptic repair

Table 1 NTFs with potential therapeutic applications Receptors Acronym High affinity Low affinity Effects, potential applications

The neutrophins Nerve Growth Factor

p75NTR

Rescues forebrain cholinergic neurons; de novo synaptogenesis. Alzheimer's disease? Diabetes neuropathology? Protects developing embryonic

Factor

dopaminergic neurons; rescues

Neurotrophin 3

axotomized cortical pyramidal neurons. Parkinson's disease? Stroke? Rescues axotomized cortical pyramidal

Brain-Derived Neurotrophic

Neurotrophin 4/5 Other NTFs Ciliary Neurotrophic Factor acidic-Fibroblast Growth

NGF

TrkA

p75NTR

BDNF

TrkB

NT3

TrkC

p75NTR

NT4/5

TrkB

p75NTR

CNTF

CNTFRa,

a-FGF

LIFR gpl 30 FGFR2 and others

Factor

Heparan sulfate

protoglycan (HSP)

basic-Fibroblast Growth Factor

b-FGF

FGFR1 and others

HSP

neurons Rescues motoneurons. Amiotrophic Lateral Sclerosis (ALS)?

Rescues motoneurons and placode sensory neurons. ALS? Rescues nbm cholinergic neurons. Improved behavioural performance after cortical infarctions? Stroke? Rescues septal cholinergic neurons; protects mature dopaminergic neurons.

Parkinson's disease? GDNF c-ret Glial-Derived Neurotrophic Rescues adult dopaminergic cell bodies; stimulates dopamine turnover. Parkinson's GDNFa Factor disease? This table summarizes current data on experimentally demonstrated neuroprotective-neuroreparative effects of NTFs when applied exogenously in a variety of animal models and the receptors through which these actions are elicited. The data were extracted from the reports and reviews cited in this communication. (-) indicates that no receptor has been identified as yet.

targets for these new families of molecules include trauma and degenerative diseases of the nervous system. From the basic sciences perspective, much work is still needed in order to understand the potential therapeutic opportunities ofNTFs, as well as their limitations or undesirable effects, before full-scale clinical trials can commence (Cuello and Thoenen 1995). Some clinical applications have already been assayed, however, with varying degrees of success. Thus recent attempts to apply purified mouse Nerve Growth Factor (NGF) in Alzheimer's disease (AD) showed some benefits but had to be discontinued because of weight loss and cefalalgias (Sieger and others 1993). This undesirable effect could be due to the direct sensory, proinflammatory effects of NGF in excessive amounts (Lewin and Mendell 1993; McMahon and others 1995; McMahon and Priestley 1995). In our own laboratory, we have found that the overexpression of chicken NGF secreted by oligodendrocytes as a transgene provokes anomalous ectopic synaptic formation

of sensory peptide-containing terminals within the spinal cord white matter of transgenic mice (Ma and others 1995). Will trophic therapy, therefore, be undesirable in AD? I would like to propose that NTFs (NGF or others) have a defined opportunity in AD therapy provided that some basic conditions are met. These should be, first, early treatment, that is, at a stage when there is still sufficient regenerative capacity left in the diseased brain to respond to NTFs. This possibility is currently hampered by a lack of presymptomatic biological diagnoses for the disease. Second, we need to learn about the range of specificity of diverse NTFs, their interactions and the dosages required to render neuroprotection or neuroregeneration with minimal undesirable side effects. Third, the field anxiously awaits a suitable animal model for AD and other neurodegenerative conditions where these potential therapies could be properly and exhaustively investigated.

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Neurotrophins in the maintenance of phenotypic characteristics of cholinergic neurons

C)

Figure 1. Schematic representation of the basalocortical lesion model. The area circled with broken lines in (a) indicates the extent of the unilateral, partial, devascularizing cortical lesion. Figure la is a cartographic representation of the rat neocortex extended from the cingulate to piriform cortex. Figure lb indicates the relative position of the cholinergic basocortical pathway from the nucleus basalis magnocellularis (NBM) to neocortex (c). Figure lc represents the characteristic depletion in ChAT in the microdissected NBM 30 d after an ipsilateral cortical infarction. Figure ld represents the degree of atrophy observed in ChATimmunoreactive cell bodies and dendrites of the nucleus basalis resulting from the cortical lesion. (Camera lucida drawing from immunocytochemical preparations. Reproduced with permission from Cuello 11994J.)

A veritable revolution has occurred in recent years with the advancement in our knowledge ofNTFs and the demonstration of their efficacy in protecting or repairing a variety of CNS neuronal systems. This knowledge is bound to produce a profound impact on neurology and psychiatry in the next century. New information continues to evolve at a very rapid pace. For example, the high-affinity receptor molecules for GDNF have been identified only recently (Durbec and others 1996; Treanor and others 1996; Trupp and others 1996). The features of some ofthe most promising NTFs with putative neuroreparative actions are summarized in Table 1.

This review focuses mainly on our experience in the repair of CNS cholinergic neurons of the nucleus basalis magnocellularis. These neurons supply the bulk (probably all in humans) of the acetylcholine released in the cerebral cortex. This CNS "basalocortical" pathway is particularly interesting because of its vulnerability to cortical lesions (Pearson and others 1983; Sofroniew and others 1983; Liberini and others 1994) and its involvement in AD pathology (Bowen and others 1976; Davies and Maloney 1976; Whitehouse and others 1982). Indeed, some of the cognitive deficiencies in AD can be attributed to the significant compromise of cholinergic neurons projecting to the neocortex (Bartus and others 1982). Beyond their possible relevance to disease states, these neurons also provide an excellent opportunity to investigate the limits and circumstances that pennit the repair of CNS neurons. These neurons form part of the so-called basal forebrain cholinergic neuron system, which also includes the pathway from the medial septum to the hippocampus. These 2 pathways may be seen as prototypes of other NGF-sensitive neuronal systems. NGF, the best-known member of the neurotrophin family, has been shown to be retrogradely transported by basal forebrain cholinergic neurons (Seiler and Schwab 1984). The level of expression of the peptide and its corresponding messenger ribonucleic acid (mRNA) signal closely correspond to the degree of CNS cholinergic innervation (Korsching and others 1985). This has led us to believe that these cholinergic neurons receive their trophic stimulation from NTFs offered to nerve terminals, and it is the basis for the postulate that this molecule acts as a "target-derived" trophic substance (Lucidi-Phillipi and Gage 1993). The clarification of the receptor systems through which the neurotrophins act on neurons has been the focus of much of the research in the field. Briefly, radioautographic and immunocytochemical studies have demonstrated that basal forebrain cholinergic neurons are well endowed with both low- (p75LNGFR) and high-affinity (pl4OrkA) receptors for NGF (Richardson and others 1986; Raivich and Kreutzberg 1987; Springer and others 1987; Pioro and Cuello 1990; Pioro and others 1990; Steininger and others 1993; Gibbs and Pfaff 1994; Sobreviela and others 1994; Anderson and others 1995; Muragaki and others 1995). Extensive unilateral cortical lesions in this brain area led to a marked atrophy of the nucleus basalis magnocellularis cholinergic neurons, typically studied by the degree ofreduction ofthe cross-sectional area of choline acetyltransferase (ChAT) immunoreactive (IR) neurons as measured by image analysis technology (Garofalo and Cuello 1994). These changes are accompanied by an equally marked depletion of ChAT enzymatic activity (Stephens and others 1985; Cuello and others 1989) (Figure 1). In this experimental paradigm, the mRNA steadystate levels for both the low- (p75) and high-affinity (trkA)

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NGF receptor are modulated in a differential manner, such that p75 tends to be upregulated during the 1 st day following lesion only to diminish thereafter, while trkA mRNA levels fall initially to be moderately upregulated at later stages when the cholinergic neuron becomes atrophic (Figueiredo and others 1995b). These changes in mRNA can be further regulated by the administration of NGF, which prevents the loss of p75 mRNA and further stimulates trkA mRNA (Figure 2). This observation is particularly interesting, as it indicates that atrophic neurons close to cell death put up sufficient neurotrophin receptors to capture, presumably, whatever neurotrophic stimulation is available. This explains why even the late administration of NTFs can rescue already atrophic neurons, even in circumstances in which they are barely visible (Hagg and others 1988; Garofalo and Cuello 1995). Since it was I st demonstrated that exogenously applied NGF rescued basal forebrain cholinergic neurons (Hefti 1986), a great many studies have confirmed this biological activity in a number of species, including the primate CNS (Liberini and Cuello 1994). The relevance of these findings cannot be emphasized enough: they provide the rationale for a new therapeutic strategy for treating degenerating CNS neurons, something that only a decade ago was deemed unrealistic. Currently, the common wisdom on the role of endogenously produced NTFs is that they modulate the neuronal phenotype. The steady trophic stimulation of the neuronsbelieved to be largely "target-derived" but with evidence pointing to the existence of autocrine and paracrine mechanisms as well regulates the level of biochemical activity responsible for the maintenance of the neuronal morphological features and even, perhaps, their synaptic contacts (see below). This role in maintaining the shape and biochemistry of these neurons has been shown for NGF-which remains the prototypical NTF-where the application of anti-NGF antibodies in neonatal rats produced a diminution of cholinergic markers in the septohippocampal system, whereas the application ofNGF led to the upregulation of these markers (Vantini and others 1989). Negation of the target-derived offering of NTFs by ablation ofthe hippocampus resulted in the atrophy, but not death, of the cholinergic septal neurons (Sofroniew and others 1993), which indicates that NTFs are not essential for the survival ofthese neurons in the adult state but that they are responsible for their shape (and presumably their biochemistry). Knockout transgenic models have illustrated the importance of having the corresponding neurotrophin present and have also demonstrated the more decisive role of the corresponding high-affinity receptor for the survival and phenotypic appearance of NGF-sensitive neurons during developmental stages (Klein and others 1993, 1994; Crowley and others 1994; Enfors and others 1994; Smeyne and others 1994; Snider 1994). Studies negating the influence of NTFs during adulthood, that is, after completion of developmental stages, are hindered by difficulties in administering anti-NTF antibodies at that point, as well as by the lack of suitable, well-tested antagonists. The recent generation of

lesionW (Day 0)

aY

-----

----

->

a

I.!

Days after lesion

Figure 2. Differential expression of p75 and trkA mRNA steady-state levels (quantitative in situ hybridization) after cortical lesion (solid lines). Values normalized (100%) to signals from control NBM neurons. Broken line indicates values for trkA after lesions and NGF treatment. Data from Figueiredo and others (1995b).

mimetic peptides that are antagonistic to NGF has raised the prospect of new experimental strategies, but the potential of these peptides has yet to be fully explored (LeSauteur and others 1995). Trophic factors and cortical synaptogenesis in experimental animals As should be expected, NTFs are responsible for inducing synaptogenesis during development (Lewin and Barde 1996). Our work at McGill has illustrated that-most importantly in the context of putative neurologicalneuropsychiatric therapies-exogenously applied NTFs in the mature and fully differentiated CNS are capable of generating new synapses in the cerebral cortex of adult animals (Cuello 1994). These studies were fueled by the observation that the application of NGF in cortically lesioned animals, besides rescuing the biochemical and anatomical features of cholinergic cell bodies, led to a robust and unusual increment in the enzymatic activity for the acetylcholine synthesizing enzyme, ChAT (Cuello and others 1989). This profound change in the capacity of synthesizing acetylcholine led us to question whether it was the result of a biochemical modulation or whether it represented a structural change in the

so

Journal of Psychiaby & Neuroscience

d .~~~~~~~~~~~

40

q'I~~~~~~~~~~~~~~~~~~~~ 0.3001

01000.200 ~fl ~ Contra[ Cot + NCF

vo

NeOfJq

VeNlec

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60-1

lion *NCF

Figure 3. Electron micrographs of cortical ChAT-IR boutons from layer V of the rat somatosensory cortex. Representative profiles of cholinergic presynaptic boutons of control (a), lesioned vehicle-treated (b), and lesioned 2.5S NGF-treated (c) animals. Arrows indicate synapses. Inset of Figure 3c: Details of a cortical cholinergic synapse from a lesioned NGF-treated rat. Scale bars = 0.5 gm. Figure 3d shows a cross-sectional area of ChAT-IR boutons in cortical layer V; 3e the percentage of varicosity profiles quantified with visible synaptic contacts. *P < 0.01 from control (ANOVA, post hoc Tukey test; operator was blinded to treatment groups.) Reproduced from Garofalo and others (1992).

cortical cholinergic network. These investigations led to the demonstration that NGF given as "therapeutic drug" resulted in synaptogenesis in the CNS of adult animals subject to partial, unilateral cortical infarctions (strokes) in the cerebral cortex. In this animal model, a loss of the cholinergic fiber network and ChAT-IR varicosities is observed ipsilateral to the areas suffering strokes. Under NGF treatment, however, the cholinergic network in these animals expands, as does the number of cholinergic varicosities (Garofalo and others 1992, 1993), thus explaining the supranormal levels ofChAT activity in NGF-treated rats. These light microscopy investigations were complemented by high-resolution immunocytochemical investigations of ChAT-IR boutons, which revealed that individual cholinergic presynaptic boutons became hypertrophic in the cerebral cortex of lesioned and NGF-treated rats and that the number of synaptic membrane differentiations in ChAT-IR boutons (presynaptic elements) rose

to twice the number observed in the naive controls

(Garofalo and others 1992) (Figure 3). Furthermore, when

Vol 22, No 1, 199 7

the tridimensional reconstruction of these boutons was undertaken, their volume and synaptic area was seen to be substantially expanded (Garofalo and others 1993). These observations are of great relevance to our understanding of the function of the cerebral cortex, as these animals display evident improvement in behavioral tasks when compared with nontreated infarcted animals (see below). In the present model, NGF therapy has a proven effect on the size and extent ofthe synaptic area that should result in synaptic efficiency, as recently discussed by Pierce and Lewin (1994), who proposed an association between the size of presynaptic elements and synaptic efficiency. We hypothesize, therefore, that NTF therapy could lead to higher synaptic efficiency in the cortical cholinergic network, and probably in other systems as well. Since the number of cortical synapses is bound to affect higher functions, would a therapy geared to modulating the number ofcortical synapses find an application in neurology and psychiatry? More research is still required to answer these questions, but there is strong evidence that the loss of cortical presynaptic elements is best correlated with the deterioration of mental functions in AD (DeKosky and Scheff 1990; Masliah and others 1991; Terry and others 1991). The idea that synaptic attrition in AD is linked to its neuropathology is reinforced by the recent observation of Games and others (1995) that the formation of diffuse A-8 plaques in the cerebral cortex and hippocampus is accompanied by the loss of presynaptic elements (synaptophysinimmunoreactive) in transgenic mice carrying a minigen coding for the valine 717 mutation of the amyloid precursor protein. If indeed this is the evolution of AD, it is reasonable to assume that synaptic attrition is an early component of its neuropathology, something which could be redressed by the timely application of NTFs in the absence of therapeutics capable of arresting plaque and tangle formation. The NGF-induced synaptic changes in the cerebral cortex are consistent with the finding that this neurotrophin facilitates the invasion of cholinergic fibers and the formation of proximal cholinesterase positive synapses in the hippocampus with the axotomized fornix (Kawaja and others 1992) and the biochemical indication of upregulation of presynaptic cholinergic markers in the partially deafferentated hippocampus (Lapchak and Hefti 1991). These experimental therapies have been shown to facilitate cortical acetylcholine (ACh) output after lesions of the nucleus basalis (Scali and others 1994) and the release of endogenous ACh in vivo in the cortex adjacent to stroke lesions (Maysinger and others 1992). Furthermore, NGF provokes a dose-dependent upregulation of both ChAT activity and the high-affinity choline uptake in synaptosomes obtained from cortically lesioned rats (Garofalo and Cuello 1995). Will a given trophic factor affect only a given type of synapse or will it have broader effects? This is a matter of current interest. Thus BDNF or neurotrophin-3 (NT3), but not NGF, disrupts the synaptic patterning in the visual cortex

January 1997


ofneonatal cats (Cabelli and others 1995), while the application of anti-NGF antibodies can similarly interfere with the development of ocular dominance in the visual cortex (Berardi and others 1994). NGF has also been shown to produce a broad increase in synaptophysin (presynaptic sites indistinctive ofthe transmitter nature) in aged rats (Chen and others 1995). This possibility has also been raised in our studies in primates (Cercopithecus aethiops), where we found that trophic factor therapy with human recombinant NGF (hr-NGF) administered in the form of a gel for approximately 1 week rendered long-term (5 mo) biochemical and morphological preservation of the cholinergic neurons ofthe nucleus basalis of Meynert and their cortical projections (Liberini and others 1994). We also found that modulation in synaptic numbers induced by hr-NGF in the cerebral cortex occurs adjacent to the stroke lesion (Burgos and others 1995). Such a marked change in the number of overall synapses suggests that, besides the modulation of the synaptic architecture ofNGF-sensitive neuronal systems, it is possible that NTF therapy has widespread synaptic effects beyond the neuronal systems possessing the specific receptor mechanisms. One possibility that may explain NTF modulation on other (perhaps nonspecific) neuronal systems is postsynaptic activation. This is certainly a possibility in the cerebral cortex given the organization of its postsynaptic sites. Thus NGF has been shown to affect the dendritic architecture in pyramidal neurons ofold rats, preventing the "aging" ofthe dendritic network (Mervis and others 1991). These neurons are also known to respond to BDNF therapy after injury (Giehl and Tetzlaff 1996). In our previously described lesion model we have observed, in collaboration with Kolb and Gorny, that the partial dendritic atrophy induced by lesions in basilar dendrites of pyramidal neurons can be fully reverted by NGF (Kolb and others, unpublished observation) and, fuirther, that this treatment stimulates the branching of basilar dendrites and the number of spines in nonlesioned young adult rats (Kolb and others 1996). It is conceivable that these changes in synaptic architecture are consequences ofenhanced neuronal activity resulting from NGF-induced synaptogenesis. Moreover, NTFs per se are also capable of producing changes in neuronal activity that can be best defined as improving synaptic efficiency, as recently illustrated by members of the neurotrophin family in the hippocampus (Knipper and others 1994; Schuman and Madison 1994). Another possible explanation for these widespread synaptic effects ofNTFs is that they might be capable ofunleashing a "trophic factor cascade." This concept is supported by the finding that in vitro basic-Fibroblast Growth Factor (b-FGF) stimulates the production and secretion ofNGF by astrocytes (Yoshida and Gage 1992). Such phenomena are not restricted to embryonic or neonatal neural cells in vitro but also occur in the adult CNS. We have recently demonstrated with Otten and collaborators that in the remaining cortex of rats bearing unilateral stroke lesions, the application of acidic-Fibroblast

51

B

A

-OLesion+oi O~~~~~~~~~~~~~~~-

j T

I

-UW Lsuion + GM1 *-* Lesion +NW

% \

0

1

2

3 os,

4

30 31 3

3

Os

47

Figure 4. (A) Preoperative and (B) postoperative mean escape latency times for rats tested in the Morris water maze with a hidden platform. Animals were trained to find the platform as described in Garofalo and Cuello (1994). After acquisition, animals were lesioned and treated with vehicle, NGF, GM1, or NGF + GM1. All animals were retested in the task beginning 30 d postlesion (that is, 2 weeks after the end of drug treatment) for 4 consecutive days. After reacquisition, all animals were tested once more 2 weeks later (postlesion day 47). *P < 0.05, ANOVA, post hoc Newman-Keuls, n = 9 to 11 animals per group. Reproduced with permission from Garofalo and Cuello (1994).

Growth Factor (a-FGF) upregulates the steady-state levels of NGF mRNA and the expression of the corresponding peptide (up to 8-fold) (Figueiredo and others 1 995a). In these circumstances, therefore, a-FGF will affect neuronal phenotypes not only of a-FGF-sensitive neurons but also of NGF-sensitive neurons. Indeed, it has been previously demonstrated that although basal forebrain cholinergic neurons are seemingly devoid of FGF receptors, they can be rescued after axotomy (Anderson and others 1988) or cortical infarcts (Figueiredo and others 1993). Behavioral consequences of NTF therapy One ofthe most provoking observations about the effects of NTFs on animal behavior has been the observation of Fischer and coworkers (1987) that the application of NGF to age-impaired rats reverts their performance in the Morris water maze. This study also reported marked cholinergic atrophy in age-impaired rats when compared with ageunimpaired rats and noted that this situation was reversed in age-impaired animals subject to NTF-therapy. Rylett and collaborators (1993) have shown that the NGF treatment of aged rats markedly increases the level of cortical ChAT activity and the number of high-affinity choline uptake sites and moderately facilitates the release ofde novo synthesized ACh. They speculate that this modest improvement in releasable ACh might explain the improved behavior. Williams and others (1993), however, point out that in the aged animal,

52


NGF treatment does not always result in beneficial effects on behavioral performance, predicting the possibility of NGFinduced weight loss or the need for prolonged treatment as the likely negative consequences ofNGF application in aged rats. The latter situation has been proved correct in the repair of cholinergic neurons in aged and CNS-lesioned rats (Garofalo 1993), where prolonged treatment with relatively high doses of the neurotrophin were required in order to regain their former synaptic architecture. Undoubtedly, more research on this front is required before the value of NTF therapy in correcting age-related neurological deficiencies can be accurately assessed. Our own experience with behavioral correlates in NTF therapy revolves around the application of NGF in the cortically lesioned rat model discussed previously. We have observed that, while most functions remain unaltered, rats receiving a unilateral cortical lesion display a marked deficit in the retention of acquired behaviors, namely passive avoidance and the ability to find the hidden platform in the Morris water maze (Elliott and others 1989). In these experiments, naive rats were trained to remain for at least 180 s in the "undesirable," illuminated side of a 2-compartment box by the administration of mild shocks when they explored the dark compartment. In the Morris water maze, rats were submitted to blocks of 4 trials per day for 4 d until they were able to find the hidden platform in an average time of 10 s. After training sessions, rats were randomly separated into 4 groups (lesioned plus vehicle, lesioned plus gg amounts of NGF for 1 week, sham operation, and no treatment) and retested some 30 d later, that is, 3 weeks after discontinuation of NGF treatment, for their ability to retain the formerly learned behaviors. The application of these moderate amounts of NGF in rats bearing cortical infarctions, comparable to those of massive stroke in humans, resulted in behavior indistinguishable from naive rats, while lesioned rats with vehicle required 4 d to reacquire the previously learned behaviors (Garofalo and Cuello 1994) (Figure 4). More recently, in collaboration with Kolb (unpublished observation), we have explored the learning capabilities of cortically lesioned rats with and without NGF treatment. In this series of experiments, we found that when rats are not treated with NGF following extensive cortical lesions, they display poor performance when exposed to new tasks, taking nearly 10 d to reach the hidden platform in the Morris water maze, for example. Lesioned animals receiving NGF treatment were moderately impeded in learning new tasks, but in an intermediate fashion between naive and lesioned nontreated animals and closer to the naive controls than to the nontreated, cortically lesioned cohort (Kolb and others 1996). We are currently exploring finer aspects ofthe behavioral performance of lesioned animals receiving NTFs in the expectation that these studies might shed some light on how much lost neurological function can be recovered by neurotrophic stimulation.

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ACKNOWLEDGEMENTS This research has been funded by grants from the Medical Research Council ofCanada, The National Institute on Aging (NIH Grant # AGI 1903-OlAl), and the Centres of Excellence, Canada. I would like to acknowledge the work of my collaborators in these efforts: BC Figueiredo, L Garofalo, B Kolb, U Otten, A Ribeiro-da-Silva, and W Tetzlaff. I would also like to thank S Parkinson for editorial assistance in preparing this manuscript.

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