REM SLEEP DEPRIVATION IN AN EXPERIMENTAL
MODEL OF PARKINSON'S DISEASE
LUIZ A. F. ANDRADE * JOSÉ G. C. LIMA * SÉRGIO TUFIK **
PAULO H. F. BERTOLUCCI * E. A. CARLINI **
Parkinson's disease (PD) is a common neurological condition all over the
world. Most of the cases are considered to be the result of a degenerative
process affecting mainly nigrostriatal dopamine neurons, leading to a marked
dopamine deficiency in the striatum. Other neurotransmitter abnormalities occur
in several different regions of the brain, but, by far, the dopamine depletion
is the more striking feature of the disease. Twenty years after the introduction
of L-Dopa as a treatment for PD and after other new pharmacological
strategies, mainly using direct dopamine agonists (bromocriptine, lisuride,
pergolide) this condition still offers a great treatment challenge. Ferguson &
Dement
6, Alves et al.l and Carlini & Lindsey
3showed that REM sleep deprived
(REM SD) rats displayed aggressive behavior after receiving amphetamine.
Non-deprived control animals which received a similar dose of the drug did not
display aggressiveness. Carlini & Lindsey
3had observed that REM SD rats
receiving apomorphine (a potent direct dopamine receptor agonist) also showed
the same aggressive behavior, whereas non-deprived animals did not. This
effect could not be blocked by pretreatment with alpha-MPT, a tyrosine-hydro
¬xilase inhibitor
1 5or by administration of L-Dopa, and was enhanced by using
bromocriptine and piribedil (direct receptor agonists)i5. These findings suggest
that REM SD may be acting by producing supersensitivity of the post-synaptic
dopamine receptors, both in the striatum and cortical-limbic areas. Pflug &
T o l l e d made the first attempt at using total SD in depressed patients,
reporting improvement. Since then many others have been using the same
p r o c e d u r e
2 , 4 , 9 , 1 6. Endogenous depression has been considered as the
conse-quence of monoamine decreased function in CNS. Although noradrenaline and
serotonin may be the major neurotransmitters involved, dopamine undoubtly
plays a role in the pathophysiology, since improvement of this condition is
D e p a r t m e n t s o f N e u r o l o g y a n d N e u r o s u r g e r y * a n d P s y c h o b i o l o g y **, E s c o l a P a u l i s t a
d e M e d i c i n a , S ã o P a u l o , B r a z i l . W o r k p a r t i a l l y s u p p o r t e d b y C N P q a n d F I N E P . P . H . F . B . w a s a f e l l o w f r o m F A P E S P ( P r o c . 8 3 / 1 3 0 7 - 5 ) . T h i s w o r k r e c e i v e d t h e
brought by the use of drugs with action on the dopaminergic system, such as
nomifensine
8, mianserine, butriptiline, maprotiline and i p r i n d o l e
7 , 1 3.
All these data support the possibility that post-synaptic dopaminergic
receptors become supersensitive after REM SD. This led us to the application
of this procedure in an experimental model of PD obtained in the rat.
M A T E R I A L . A N D M E T H O D S
T h e e x p e r i m e n t w a s m a d e u s i n g m a l e a l b i n o W i s t a r r a t s f r o m o u r o w n c o l o n y ,
w e i g h t i n g f r o m 3 0 0 t o 4 0 0 g , a n d w i t h a g e r a n g i n g f r o m 4 t o 5 m o n t h s . B e f o r e t h e
e x p e r i m e n t t h e a n i m a l s w e r e k e p t i n g r o u p s o f 3 i n a m e t a l c a g e ( 3 0 x 1 8 x 1 6 c m ) a n d
i n i n d i v i d u a l l a r g e w o o d e n c a g e s , a f t e r t h e s u r g i c a l p r o c e d u r e . D u r i n g a l l t h e e x p e r i m e n t
t h e a n i m a l s w e r e k e p t a t a t e m p e r a t u r e o f 23o C ± 1 ° C, i n c o n t r o l l e d c o n d i t i o n s
o f l i g h t a n d d a r k n e s s a n d w i t h w a t e r a n d f o o d a d l i b .
T h e e x p e r i m e n t a l m o d e l c o n s i s t e d o f b i l a t e r a l e l e c t r o l y t i c l e s i o n s o f t h e n i g r o s t r i a t a l
p a t h w a y i n t h e l a t e r a l h y p o t h a l a m i c a r e a . T h e s e l e s i o n s w e r e o b t a i n e d b y a s t e r e o t a x i c
a p p a r a t u s . T h e s t e r e o t a x i c p a r a m e t e r s w e r e a s f o l l o w s : t h e e l e c t r o d e t i p w a s p u t 3 m m
b e h i n d t h e b r e g m a , 2 m m l a t e r a l l y t o t h e m e d i a l l i n e o n b o t h s i d e s a n d 8 . 5 m m b e l l o w
t h e d u r a , t h e b r e g m a a n d l a m b d a b e i n g p u t a t t h e s a m e l e v e l . A t t h i s p o i n t o n
e i t h e r s i d e o f t h e b r a i n t h e e x p e r i m e n t a l a n i m a l s r e c e i v e d a c a t h o d i c e l e c t r i c a l c u r r e n t
o f 2 m A f o r 1 0 s e c o n d s . T h e c o n t r o l a n i m a l s r e c e i v e d s h a m o p e r a t i o n s i n w h i c h a l l
p r o c e d u r e s w e r e t h e s a m e , b u t nr* e l e c t r i c c u r r e n t w a s a p p l i e d t o t h e e l e c t r o d e . A l l
a n i m a l s r e c e i v e d b a r b i t u r a t e a n e s t h e s i a f o r t h e s u r g i c a l p r o c e d u r e ( s o d i u m p e n t o b a r b i t a l
— 5 0 m g / k g ) . R E M S D w a s a c h i e v e d b y u s i n g t h e i n v e r t e d f l o w e r p o t t e c h n i q u e (s).
A c c o r d i n g t o t h i s m e t h o d t h e a n i m a l w a s p l a c e d i n a s m a l l s q u a r e w o o d p l a t f o r m
h a v i n g 6 c m o f s i d e s u r r o u n d e d b y w a t e r . T h e s q u a r e p l a t f o r m s , i n s t e a d o f t h e u s u a l
r o u n d o n e s , w e r e e m p l o y e d i n o r d e r t o f a c i l i t a t e t h e m a i n t e n a n c e o f t h e o p e r a t e d
a n i m a l s o n t h e t o p . T h i s w a t e r t a n k h a d a d e v i c e f o r f e e d i n g . T h e l e n g h t o f R E M
S D w a s 7 2 h o u r s . A l l r e s u l t s w e r e a n a l y s e d u s i n g o n e - w a y a n a l y s i s o f v a r i a n c e
( A N O V A ) a n d p o s t e r i o r l y S t u d e n t ' s t t e s t f o r d e p e n d e n t o r i n d e p e n d e n t s a m p l e s . T h e
m i n i m a l a d m i t t e d s i g n i f i c a n c e l e v e l w a s 5 % ( p < 0 . 0 5 ) .
O n d a y 0 t h e a n i m a l s w e r e o p e r a t e d u p o n . T w e n t y n i n e a n i m a l s c o m p l e t e d t h e
e x p e r i m e n t a n d f o r m e d 4 g r o u p s : 6 n o n - l e s i o n e d a n d n o n - d e p r i v e d ( N L - N D ) a n i m a l s ;
9 n o n - l e s i o n e d a n d d e p r i v e d ( N L - D ) ; 6 l e s i o n e d a n d n o n - d e p r i v e d ( L - N D ) ; a n d 8
l e s i o n e d a n d d e p r i v e d ( D - D ) . O n d a y s 3 a n d 7 t h e a n i m a l s w e r e i n d i v i d u a l l y o b s e r v e d
i n t h e o p e n - f i e l d . F r o m d a y 7 t o 10 t h e a n i m a l s f r o m g r o u p s N D - D a n d D - D w e r e
R E M S D w h e r e a s t h e c o n t r o l a n i m a l s w e r e k e p t i n t h e i r o w n i n d i v i d u a l c a g e s . O n
d a y 10 a l l a n i m a l s w e r e o b s e r v e d i n t h e o p e n - f i e l d , t h e s a m e h a p p e n i n g o n d a y 14
a n d 2 1 . A s e c o n d R E M S D w a s p e r f o r m e d f r o m d a y s 2 1 t o 24, w h e n t h e l a s t o b s e r v a t i o n
i n t h e o p e n - f i e l d w a s m a d e . T h e o b s e r v a t i o n c o n s i s t e d o n r a t i n g t h e a m b u l a t i o n
( n u m b e r o f s q u a r e s i n v a d e d b y t h e a n i m a l ) , r e a r i n g ( n u m b e r o f r e a r i n g m o v e m e n t s ) ,
g r o o m i n g ( t i m e s p e n t b y t h e a n i m a l i n g r o o m i n g b e h a v i o r ) a n d l a t e n c y ( t i m e s p e n t
b y t o a n i m a l t o s t a r t t h e a m b u l a t i o n ) . T h e o b s e r v a t i o n p e r i o d w a s 3 m i n u t e s .
H a v i n g c o m p l e t e d t h e e x p e r i m e n t , t h e b r a i n s w e r e r e m o v e d , f i x e d i n 1 0 % f o r m a l i n
f o r 3 0 d a y s , c u t i n t h e f r o n t a l p l a n e i n 0 . 0 5 m m s l i c e s w h i c h w e r e e x a m i n e d t h r o u g h
R E S U L T S
F i g u r e 1 r e p r e s e n t s t h e r e s u l t s c o n c e r n i n g - t h e i t e m a m b u l a t i o n . A s c a n b e s e e n , b o t h g r o u p s o f l e s i o n e d a n i m a l s ( L - N D a n d L - D ) , b e f o r e R E M S D , w e r e s i g n i f i c a n t l y
d i f f e r e n t o f t h e t w o g r o u p s o f n o n - l e s i o n e d a n i m a l s a t t h e 3 r d d a y p o s t - s u r g e r y . T h i s d i f f e r e n c e b e c a m e l e s s s t r i k i n g a t t h e 7 t h d a y a f t e r t h e s u r g e r y . A t t h e 1 0 t h d a y ,
a f t e r t h e R E M S D ( g r o u p s N L i - D a n d L - D ) , a l a r g e i n c r e a s e ( p < 0 . 0 5 ) i n t h e
a m b u l a t o r y b e h a v i o r w a s e v i d e n t i n b o t h g r o u p s o f a n i m a l s s u b m i t t e d t o S D p r o c e d u r e c o m p a r i n g w i t h t h e o t h e r t w o g r o u p s . T h e s a m e f i n d i n g a p p e a r e d o n d a y 2 4 t h ,
a f t e r t h e s e c o n d p e r i o d o f S D . F i g u r e 2 d i s p l a y s t h e r e s u l t s f o r t h e r e a r i n g b e h a v i o r . T h e y a r e s i m i l a r t o t h e , a m b u l a t o r y b e h a v i o r , e x p r e s s i n g a l s o a l a r g e i n c r e a s e of
r e a r i n g i n t h e S D g r o u p s ( p < 0 . 0 5 ) . L a t e n c y f o r a m b u l a t i o n a n d g r o o m i n g b e h a v i o r s h o w e d d i f f e r e n t r e s u l t s o n d a y 3 a f t e r s u r g e r y , w h e n c o m p a r i n g l e s i o n e d a n d
C O M M E N T S
Motor impairment is one of the characteristic signs of Parkinson's disease.
In the operated rats a striking feature was the reduction in ambulation and
rearing observed in the first post-operative days. However, from days 3 to 7,
the animals were able to partially recover from this impairment without any
manipulation. Nonetheless, REM SD clearly improved ambulation and rearing
behavior when these two aspects of motor behavior were measured immediately
after the deprivation periods (see figures 1 and 2). In a similar animal model,
Marshall et al.u, studied some activation stimuli (ice water swimming, forced
swimming and the exposure of the rats to a colony of cats) in the motor
behavior and showed a significant increase in certain motor parameters after
the animals were exposed to these stimuli. This work was done to correlate
the results with the "paradoxical kinesia" which was observed in some PD
patients. The REM SD might share a similar mechanism to explain its action
in the animal model.
Fig. 3 — Histological demonstration of lesions on the nigrostriatal pathways (arrows). The lesions were induced by a 2mA electrical current with 10 seconds of duration stereotaxically directed.
procedure. There are some evidences that "denervation supersensitivity" occurs
in striatal neurons of parkinsonian patients. Lee et al.io studied the binding
of 3H-apomorphine and 3H-haloperidol to DA-receptors in the putamen and
caudate nucleus of 6 parkinsonian patients and in neurologically normal patients.
The 3H-apomorphine binding in the putamen of parkinsonian patients was
significantly decreased as compared to controls. Lee et al.
1** explained the
decreased binding of 3H-apomorphine as being the consequence of nigral cells
loss, as, according to them, this ligand would be binding predominantly to the
pre-synaptic receptors. On the other hand, the 3H-haloperidol binding, mainly
in the putamen, was significantly increased. This fact could be the result of
an increase in the number and/or in the affinity of the post-synaptic
dopami-nergic receptors, what brings support to the supersensitivity denervation theory
in PD. An alternative explanation could be given by the agonist-antagonist
concept of post-synaptic dopaminergic receptors 5,14. According to the authors
the dopaminergic receptors could exist under two inter-convertible forms: the
agonist form, in which there is a greater affinity for stimulation agents
(dopamine and apomorphine); and the antagonist form with greater affinity
for the blocking agents (haloperidol, for example). It is conceivable that under
certain circumstances (neurological disease, drugs, stress, etc.) one form could
predominate over the other and REM SD could act by favoring the presence
of the agonist form.
well known that chronic L-Dopa therapy produces a decrease of the post-synaptic
dopamine receptors, this fact being one possible cause of the progressive
dete-rioration of its effectiveness in the treatment in the long range. The SD, if
really acting as we are proposing, could induce an increase in the affinity of
these receptors to the drug.
Therefore, although we do not have as yet a satisfactory explanation for
the improvement brought about by REM SD to the lesioned rats, we feel
that a clinical trial of sleep deprivation in parkinsonian patients should be tried.
S U M M A R Y
Previous investigations have shown that REM sleep deprived (REM SD)
rats display an enhanced response to dopamine agonists. This action seems
to be mediated through a supersensitivity of dopamine post-synaptic receptors.
Accordingly, REM SD was performed on rats with an experimental model of
Parkinson's disease. The animals were bilaterally lesioned in the nigrostriatal
pathway through a stereotaxically directed electrical current. Seven days after
the surgery the animals were REM SD for 72 hours and immediatelly after
the end of this period were observed in an open field for ambulation, rearing,
grooming, and latency. In comparison with non-deprived rats there was a
significant increase in ambulation and rearing, a response that appeared again
after a second REM SD period on day 21th after the surgery. These data of
improvement of two parameters of an experimental model of Parkinson's disease
suggest that SD may be useful in this condition.
R E S U M O
Privação de sono REM em um modelo experimental da doença de Parkinson.
Investigação prévia mostrou que ratos privados de sono (REM SD)
mos-tram acentuação de resposta a agonistas dopaminérgicos. As evidências indicam
que essa ação parece ser mediada por supersensibilização de receptores
dopa-minérgicos pós-sinápticos. Com base nisso, foi feita REM SD em ratos com
modelo experimental da doença de Parkinson, nos quais foi feita lesão eletro¬
lítica bilateral de ambas as vias nigro-estriatais. Sete dias após a cirurgia os
animais eram submetidos a REM SD por 72 horas. Imediatamente após o final
deste período era feita observação em campo aberto para a ambulação,
"rearing", "grooming" e latência. Em comparação com ratos não-privados foi
observado aumento significativo na ambulação e "rearing", resposta que
reapa-receu após um segundo período de REM SD, realizado 21 dias após a cirurgia.
Estes dados, de melhora de dois parâmetros de modelo experimental da doença
de Parkinson, sugerem que a privação de sono pode ser útil nesta doença.
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