Grafting
of fetal substantia nigra to striatum reverses
behavioral deficits induced by MPTP in primates:
a
comparison with other types of grafts as
controls
Taylor JR, Elsworth JD, Roth RH,
Sladek JR Jr, Collier TJ, Redmond DE
Jr.
Neurobehavior Laboratory,
Yale University School of Medicine, New
Haven
Abstract
Fetal substantia nigra (SN) cells were
transplanted into the caudate nucleus (CN) of
four vervet monkeys (Cercopithecus aethiops
sabaeus) that had been treated with
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP). MPTP treatment appears to produce a
syndrome similar to that observed in patients
with idiopathic Parkinson's disease. Normal and
parkinsonian behaviors were quantitated by
trained observers 5 days/week. Twenty-eight
behaviors based on previous factor analyses were
individually scored and rated. Parkinsonian
signs included freezing, head and limb tremor,
difficulty in eating, delayed initiation of
movement, poverty of movement, tremor that
stopped with intention, decreased response to
threats, and lying immobile in the cage.
These signs were combined to give an overall
rating of parkinsonism. A summary measure of
'normal' healthy behavior was also examined,
including such behaviors as yawning,
scratching, self-grooming, shifting, and eating.
Overall ratings of parkinsonism increased and
those of healthy behavior decreased after MPTP.
In the 4 monkeys grafted with fetal SN cells
into the CN, behavior returned to pre-treatment
levels by the time of sacrifice (2, 5, or 7.5
months after grafting). Three control subjects
were transplanted with either SN cells into an
inappropriate brain site (cortex) or
inappropriate, non-dopaminergic, cells
(cerebellar) into the CN. Subjects were also
compared with three control animals that did not
receive MPTP but received cryopreserved or fresh
SN and other cells into the CN. Only
MPTP-treated subjects that received SN cells
into the CN showed evidence of a reversal of the
MPTP syndrome after transplantation. In
addition, grafting in animals that were not
MPTP-treated did not appear to affect behavior.
This paper reports the specific behavioral
effects of severe MPTP toxicity that were or
were not reversed after transplantation and
suggests that only fetal SN cells grafted into
the CN may be able to reverse behavioral
deficits in MPTP-treated monkeys.
Introduction
Transplantation of fetal neural tissue to
rodents and to non-human primates has been
reported to alter behavior, biochemistry, and
morphology of the host brain (Bj6rklund and
Stenevi 1979, 1984; Bj6rklund et al. 1981; Lund
and Hauschka 1976; Perlow et al. 1979; Gash et
al. 1980; Sladek et al. 1986, 1987a,b). The
specific destruction of dopamine systems by the
neurotoxin 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) in human
(Langston et al. 1983; Stern et al. 1985) and
non-human primates (Bankiewicz et al. 1986;
Burns et al. 1983, 1985; Chiueh et al. 1984,
1985; Degryse and Colpaert 1986; Elsworth et al.
1987a,b, 1989; Kitt et al. 1986; Jenner and
Marsden 1986; Langston et al. 1984; Mitchel et
al. 1986) and the similarities with the
neuropathology of Parkinson's disease have
provided a valuable model for studying the
ability of transplanted dopamine-containing
fetal tissue to modify behavior.
Few reports have quantitatively assessed the
behavioral deficits after MPTP-induced
parkinsonism in monkeys although motor deficits
akin to those observed in patients with
idiopathic Parkinson's disease and after
accidental MPTP ingestion (Langston et al. 1983)
have been documented. MPTP has effects on motor
performance that vary within and between species
(Eidelberg et al. 1986; Di Paolo et al. 1986;
Jenner and Marsden 1986; Elsworth et aL 1987a,
1989). In fact in mildly affected subjects,
there may be some behavioral recovery over time
after initial impairments (Eidelberg et al.
1986; Nomoto et al. 1986; Schneider et al.
1987), even though striatal dopamine levels are
markedly decreased (Elsworth et al. 1989).
Moreover residual behavioral deficits may be
hard to detect in these mildly affected animals
even when detailed observational analyses are
used. It is necessary therefore to be able to
quantitate and compare the MPTPimpairments with
other subjects before and after intervention
aimed at ameliorating parkinsonism such as
transplantation.
We examined the behavioral consequences of
transplanting fetal substantia nigra (SN) cells
into the caudate nucleus (CN) of monkeys that
become moderately to severely affected by MPTP
treatment and compared these subjects with
control subjects. Because the behavioral effects
of MPTP administration can vary between animals
and some improvements may occur spontaneously,
we used sensitive observational analyses and
compared transplanted monkeys with control
grafted subjects. Two types of control grafted
MPTP-treated subjects were studied. Those with
inappropriate grafts of SN tissue into cortex
and with inappropriate cerebellar donor tissue
into CN were used to examine the 1) non-specific
effects of surgery and dopaminergic tissue
implantation, and 2) the effects of surgical
damage to the CN and non-dopaminergic tissue
implantation. In addition, subjects that were
not given MPTP were also given transplants of SN
and other cells into CN to determine whether or
not dopaminergic and other grafted tissue could
modify normal behavior or generate abnormal
behavior.
Discussion
Fetal SN neurons grafted into the CN that
survived at least 7.5 months appeared to be
associated with amelioration of the moderate to
severe behavioral impairments induced by MPTP
treatment. Examination of these subjects using
parkinsonian summary scores and healthy behavior
scores allowed us to quantify and compare the
incidence of abnormal behaviors and the changes
in the normal repertoire of behavior for this
primate species following the administration of
MPTP. Subjects given MPTP that received
inappropriate fetal cells or placements did not
show evidence of either an increase in normal
healthy behaviors or a decrease in overall
parkinsonian symptoms. In non MPTP-treated
subjects, there was no evidence of adverse
effects of the grafting procedure on normal
behavior.
These results suggest that transplantation
of fetal dopaminergic SN cells into the CN may
reverse experimental parkinsonism in monkeys
treated with M PTP. Although surgical
intervention itself does not appear to be
responsible for behavioral changes (since
inappropriately grafted subjects that were
severely affected by the toxin did not show
signs of improvement) we cannot determine
whether less severely affected subjects grafted
inappropriately would also have shown some
behavioral improvements. Nevertheless, three of
the four SN-CN subjects were severely affected
by MPTP administration as classified by
parkinsonian summary score, and all subjects
showed sustained recovery after grafting for
their respective periods studied before
sacrifice.
Previous studies have demonstrated a
behavioral deficit induced by MPTP treatment
that is characterized by decreased motor
activity as measured by activity counts or
decreased movement (Bankiewicz et al. 1986;
Burns et al. 1983, 1985; Chiueh et al. 1984,
1985; Degryse and Colpaert 1986; Jenner and
Marsden 1986; Langston et al. 1984). More
quantitative assessment of motor function during
arm movements or simple reaching tasks has also
been examined in MPTP-treated monkeys (Doudet et
al. 1985, 1986; Schultz et al. 1985; Schneider
et al. 1988). This study confirms the presence
of motor impairments and extends this
characterization to include specific
parkinsonian-like motor deficits that were
observed after MPTP, such as poverty of
movement, immobility, freezing, delayed
initiation of movement, tremor, and tremor that
decreases with intentional movement. Several of
these impairments may correspond to akinesia,
bradykinesia, resting tremor and other motor
deficits that are clinically used to describe
aspects of idiopathic Parkinson's disease. The
behavioral rating and scoring methodology
employed here quantified these behavioral
impairments and allowed us to determine discrete
changes in abnormal and normal behavior over
time. Overall changes in behavior also could be
assessed and measured by parkinsonian summary
scores and healthy behavior scores. The precise
behavioral consequences of MPTP toxicity as well
as the overall condition of the subjects provide
valuable data that support the similarity of the
MPTP syndrome to Parkinson's disease. In
addition, by detailed analysis of the effects of
MPTP administration on normal and parkinsonian
behavior it is possible to determine whether
either fetal SN cells grafted into the CN or
control grafting can reverse or even subtly
alter the MPTP-induced behavioral deficits.
The present results suggest that
MPTP-induced deficits can be gradually reversed
in the months after grafting of SN cells into
the CN. Overall ratings of parkinsonism, which
provide assessment of behavior similar to that
used to determine impariments in patients with
Parkinson's disease, indicated that these
subjects were not behaviorally different from
non MPTP-treated subjects at the time of their
sacrifice (up to 2, 6 or 7.5 months).
MPTP-induced impairments such as freezing,
immobility, and poverty and delayed initiation
of movement were abolished following grafts of
SN-CN by the time of sacrifice. However,
analyses of individual behavior indicated that
head and limb tremor, although reduced, was
still present in the two animals sacrificed at
7.5 months after SN-CN grafting: Of course, we
could not determine whether or not tremor would
have eventually disappeared if these subjects
had not been sacrificed. In addition, healthy
behavior scores in the SN-CN group remained
slightly below baseline values at sacrifice.
Although categories of behavior associated with
anxiety (yawn, chew, scratch, and
self-groom) returned to normal, behavior
associated with arousal (shift, tail flag,
bipedal lookout and vertical climb) remained
decreased after placement of appropriate grafts
in MPTPtreated subjects.
Behavioral recovery may have been due to
presence of dopamine neurons within the graft.
Monkeys that showed sustained behavioral
recovery from MPTP possessed welldefined grafts
of fetal tissue that contained cells that
stained positively for TH, indicating the
presence of dopamine neurons. These neurons
often appeared in dense clusters at various
locations in the grafts, generally at the
interface with the host brain. Although the
grafts appeared to survive without precavitation
surgery and the cells chronologically were still
relatively immature, some possessed extensive
neuritic ramifications that coursed throughout
the neuropil of the graft and occasionally
traversed relatively large distances into the
host brain surrounding the grafted tissue.
Grafts (e.g. S089) that were located within
bridges of tissue that connected the corpus
callosum to the CN showed fiber growth that was
directed at the appropriate target site (i.e.,
the CN). These fibers that reached the
appropriate target site then exited the graft to
begin to ramify throughout the host brain. There
was also some evidence suggesting that
graft-induced host dopaminergic systems could be
involved after grafts of SN into CN. For
example, relatively dense TH fiber patterns were
observed in the CN and putamen of subject S147
possibly reflecting some regenerative capacity
of the partially damaged nigrostriatal system in
this mildly affected subject. However, nigral
cell losses were in the range of more severely
symptomatic subjects, and thus we cannot account
for why this subject showed this stronger TH
staining than other subjects such as S089. In
two of the subjects ($147 and $207) small
bipolar cells that stained for TH were seen in
the striatum indicating that recovery could also
have been due to induction of host dopamine
cells in the CN, or these could have been cells
that migrated from the grafts. However, we have
no clear evidence that host cells were involved
in functional dopamine release.
Subjects that were also severely affected by
MPTP that received inappropriate grafts or
placements did not show evidence of behavioral
recovery and also had substantial losses of TH
positive cells in the SN. In one of the subjects
that received SN tissue into cortex relatively
large numbers of TH positive cells were
observed, yet no behavioral recovery was found
in these inappropriately grafted subjects.
Grafts of cerebellar tissue, while of comparable
size and placement to those of nigral tissue
into the CN, revealed no TH positive cells as
expected. No evidence of graft or host induced
regeneration was observed in these subjects.
These results suggest that behavioral recovery
does not result from, 1). grafts of
mesencephalic tissue placed into cortex and
surgical trauma to the cortex overlying the CN,
or from 2). grafts of non-dopaminergic
cerebellar tissue and surgical trauma to the CN.
Subjects not treated with MPTP that received
various types of grafted tissue in various sites
continued to show healthy/ normal behaviors,
were found to have grafts that showed no signs
of rejection, and in several cases had TH
positive neurons with fiber networks.
Some evidence for the involvement of central
dopamine activity (graft or host) in the
sustained behavioral recovery observed in two of
the SN-CN subjects (S089, S147) reported here is
suggested by increased dopamine levels and
altered dopamine metabolite/dopamine ratios in
regions of the CN close to the graft but not in
regions distant from the graft (Elsworth et al.
1990). These subjects were found to have an
approximately 10 fold increase, compared with
severely impaired MPTP-treated subjects, in
dopamine concentration proximal, but not distal,
to the graft, up to 7.5 months after
transplantation. However, behavioral deficits
after MPTP were accompanied by a reduction in
(i) dopamine metabolite concentration assayed
from CSF (Taylor et al. 1990c), (ii) dopamine
concentration in cell body regions of the
mesostriatal system (Elsworth et al. 1990), and
(iii) approximately a 70% loss of TH positive
neurons in the SN in these subjects, confirming
the presence of a large MPTPinduced dopamine
lesion in the MPTP-treated compared to control
subjects.
Two of the subjects in the SN-CN group, two
in the inappropriate group and all control
subjects reported here were also investigated
using a task that has been used to examine
cognitive and subtle motor impairments (Diamond
1989; Taylor et al. 1990a, b). Only MPTP-treated
subjects that received SN cells into the CN
showed improvements in cognitive and motor
performance (Taylor et al. 1990d). We had
previously found both cognitive and subtle motor
deficits on the acquisition (or learning) of
this object retrieval/detour task in primates
mildly affected by MPTP treatment (Taylor et al.
1990a), and on the stability of these deficits
during long-term performance on this task
(Taylor et al. 1990b). In addition to the
characteristic motor deficits, which are thought
to be due mainly to loss of striatal
dopaminergic function, cognitive impairments are
also a part of the behavioral sequelae of
Parkinson's disease (Benecke et al. 1987; Reitan
and Boll 1971; Loranger et al. 1972; Lees and
Smith 1983; Stern et al. 1983; Weingartner et
al. 1984). We therefore have used a multifaceted
approach in studying MPTP-induced deficits and
the ability of fetal neural grafts to alter
behavior. A major advantage of this cognitive
and motor procedure is that it detects subtle
deficits that are not easily observed or rated.
Its disadvantage is that an animal can become
too impaired to perform at all. In the case of
very impaired animals, observational methods are
required for behavioral assessments.
The results of this study provide evidence
to support the hypotheses that MPTP produces
behavioral signs and symptoms that are
strikingly similar to those observed in patients
with idiopathic Parkinson's disease and that
behavioral recovery can be sustained for up to
7.5 months in severely debilitated subjects
after only fetal-SN cells were grafted into the
CN. Similar grafts in non-MPTPtreated subjects
did not generate abnormal behavior or affect
normal behavioral responses as assessed by the
methods described. MPTP-treated subjects grafted
with inappropriate fetal tissue or at
inappropriate implantation sites became so
impaired that they were immobile and thus few
behaviors could be documented, resulting in high
ratings of parkinsonism and low ratings of
healthy behavior, which remained stable until
sacrifice or death. Further studies in larger
numbers of subjects will be necessary to
determine the duration of behavioral recovery,
the effects sham surgery, and the possible
graftinduced and host-derived mechanisms
responsible for recovery after fetal SN
transplantation into the striatum in
MPTP-treated monkeys.
