Bioscience Reports, Vol. 10, No. 1, 1990
Effect of Haloperidol Withdrawal on
Somatostatin Level and Binding in Rat Brain
E. Perez-Oso, ~ M. P. L o p e z - R u i z I and E. ArU]a 1'2
Received February 10, 1989; revised version June 26, 1989
The effects of withdrawal on the level and specific binding of somatostatin in the frontoparietal cortex
and hippocampus of the rat after chronic haloperidol treatment were examined using 12SI-Tyrll
somatostatin as tracer. One week after haloperiodol withdrawal the number of specific somatostatin
receptors in both brain areas returned to control values, after having decreased as the result of chronic
administration. Neither administration of haloperidol nor withdrawal of it affected the levels of
somatostatin-like immunoreactivity (SLI) in the two brain areas studied. The return of the
somatostatin receptor number to control values after haloperidol withdrawal may be related to the
motor side-effects that are clinically observed when the haloperidol treatment is terminated.
KEY WORDS: somatostatin receptors; haloperidoi withdrawal; frontoparietal cortex; hippocampus;
rat.
INTRODUCTION
The neuroleptic haloperidol is widely used as antipsychotic agent (1). A
characteristic feature of haloperidol withdrawal after chronic administration is the
appearance of motor abnormalities which are associated with an increase in the
number of dopamine receptors in rat brain (2-5). Nevertheless, the motor
abnormalities seen after terminating the haloperidol treatment might not necessarily be due to changes in the dopaminergic system exclusively, since other
neurotransmitters such as somatostatin (5-8) and TRH (9) also influence motor
control. Haloperidol administration has recently been shown to decrease the level
of somatostatin-like immunoreactivity (SLI) both in striatum and nucleus
accumbens (10-11) and in the number of specific somatostatin receptors in the rat
cerebral cortex and hippocampus (12). However, nothing is known of the effect
on somatostatin binding of the withdrawal from chronic haloperidol administration in rat brain.
The purpose of the present study is to examine the effect of withdrawal after
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Alcala de
Henares, Madrid, Spain.
2 To whom correspondence should be addressed.
15
0144-8463/90/0200-0015506 00/0(~) 1990PlenumPublishingCorporation
Perez-Oso, Lopez-Ruizand Arilla
16
chronic haloperidol administration on the specific somatostatin receptors in rat
frontoperietal cortex and hippocampus. SLI levels in both brain areas were also
examined.
MATERIAL AND METHODS
Chemicals
Synthetic Tyr11-somatostatin and somatostatin tetradecapeptide were purchased from Universal Biologicals Ltd (Cambridge, U.K.): haloperidol from
Syntex Latino Laboratories, (Barcelona, Spain); bacitracin and bovine serum
albumin (fraction V) from Sigma (St. Louis, Mo U.S.A.); dextran from
Pharmacia T70 (Uppsala, Sweden); charcoal (Norit A) from Serva, Feinbiochemica (Heidelberg, F.R.G.); and carrier-free Na125-I (IMS 30, 100 mCi/ml)
from the Radiochemical Centre (Amersham, U.K.). Tyr 1~ somatostatin was
radioidonated by the chloramine-T method (13). The specific radioactivity of the
tracer was about 250 Ci/g. The rabbit antibody used in the radioimmunoassay
technique was purchased from the Radiochemical Centre (Amersham, U.K.).
This antiserum was raised in rabbits against somatostatin-14 conjugated to bovine
serum albumin and is specific for somatostatin, but since somatostatin-14
constitutes the C-terminal portions of both somatostatin-25 and somatostatin-28,
the antiserum does not distinguish between these three forms. All other reagents
were from regular commercial sources.
Experimental Animals
Male Sprague-Dawley rats, weighing 200-230 g and fed a regular diet ad
libitum were used. The animals were divided into three groups. Group 1 rats were
given daily 0.5 mg/Kg of haloperidol s.c. for 3 weeks. Group 2 rats were given
daily 0.5 mg/Kg of haloperidol s.c. for 3 weeks followed by 1 week without the
drug. Group 3 was the control and received saline injections. Rats were sacrificed
by decapitation, the brains removed and the frontoparietal cortex and hippocampus rapidly dissected (14)).
Tissue Extraction and Radioimmnnoassay of Somatostatin
Somatostatin was extracted from both cerebral cortex and hippocampus
following the method of (Patel and Reichlin (15)). Somatostatin recovery was
similar at both brain areas since the yield of the overall extraction procedure was
about 85% in the two brain areas studied. Protein was determined by the method
of Lowry et al. (16). Immunoreactive somatostatin levels were measured by a
modified specific radioimmunoassay method (15), with a sensitivity limit of
10 pg/ml. Incubation tubes prepared in triplicate contained 100 pl samples of
unknown or standard solutions of 0-500 pg cyclic somatostatin tetradecapeptide
diluted in phosphate buffer (0.05M. pH7.2 containing 0.3% bovine serum
Somatostatin Receptors After Haioperidol Withdrawal
17
albumin. 0.01 M EDTA), 200 #1 appropriately diluted anti-somatostatin serum,
100/A freshly prepared 125I-Tyral somatostatin diluted in buffer to give
6,000 c.p.m. (equivalent to 5-10 pg), and enough buffer to give a final volume of
0.8 ml. All reagents and assay tubes were kept chilled in ice before incubating for
48h at 4~ Separation of bound and free hormone was accomplished by addition
of 1 ml dextran-coated charcoal (dextran 0.2% w/v: charcoal 2% w/v). Dilution
curves for each brain area were parallel to the standard curve. The intra-assay
and inter-assay variation coefficients were 6.3% and 8.5% respectively.
Binding Assay on Membrane Preparations
Synaptosomal membranes from frontoparietal cortex and hippocampus were
prepared as described by Reubi et al. (17). Protein was determined by the method
of Lowry et al. (16). The assay for specific binding of 125I-Tyr11 somatostatin to
synaptosomal membranes from cerebral cortex and hippocampus was performed
according to the modified method of Czernik and Petrack (18). Briefly, the
binding assay was conducted under standard conditions at 30~ in 250/~1 of a
medium consisting of 50mM Tris-HC1 buffer (pH7.5), 5 mM MgCI2, 0.2%
bovine serum albumin and 0.1 mg/ml bacitracin with 250 pM 1251-Tyrll somatostatin (up to 10 nM). The reaction was initiated by the addition of synaptosomal
membranes (1.5mg protein/ml). After 60rain incubation, membrane-bound
peptide was separated by centrifugation and radioactivity determined. Nonspecific binding was obtained from the amount of radioactivity bound in the
presence of 1 0 - 7 M somatostatin and represents about 25% of the binding
observed in the absence of unlabelled peptide. This non-specific component was
substracted from the total bound radioactivity in order to obtain the corresponding specific binding. The inactivation of 125I-TyrH somatostatin in the incubation
medium after exposure to membranes was studied by observing the ability of the
peptide to rebind to fresh membranes (19).
Statistical Analysis
Individual experiments were performed in duplicate. All results are given as
the mean + S.E.M., and the statistical significance of the differences between
groups was calculated by Student's t test.
RESULTS
As shown in Fig. 1, administration of haloperidol for 3 weeks produced no
changes in SLI levels in any brain area studied. Following withdrawal from
haloperidol administration the SLI levels did not differ from control values,
either. A single control group was therefore considered sufficient to express these
results.
Brain plasma membranes from the two experimental groups and the control
group bound 125I-Tyr 11 somatostatin in a time-dependent fashion; an apparent
equilibrium was observed between 50-180 min at 30~ (data not shown). All
subsequent binding studies were therefore conducted at 30~ for 60 min. To rule
out the possibility of different somatostatin-degrading activities in the membrane
Perez-Oso, Lopez-Ruiz and Arilla
18
[--1 Control
J~l Haloperidol
10
.m
P
e'~
3
E
rr--
..J
u1
0
Frontopofietol cortex
Hippocompus
Fig. 1. Effect of haloperidol administration and
withdrawal on somatostatin-like immunoreactivity
(SLI) level in rat frontoparietal cortex and hippocampus. In each of the experiments, determinations were
made in duplicate. No statistically significant
differences are obtained when compared with the
control animals.
preparations that could affect results interpretation, peptide degradation was
determined (19). Membranes from both brain areas showed a similar peptide
degradation capacity and their values were about 10% in all the experimental
groups.
Frontoparietol cortex
6
o
0.06
W
0
C
:3
3
ff
0o3
"1"1
0
I
oo 11
10 9
8
[SSI, -IogM
0
I
0.02
Bound nM
0.04
Fig.1 2. Left panel: Competitive inhibition of specific 125 I-Tyr 11 somatostatin ( 125 I Tyr 1-SS, 250 pM) binding to membranes of the frontoparietal cortex by unlabelledsomatostatin. Membranes (1.5 mg protein/ml) were incubated for 60 rain at 30~ in
the presence of 250 pM lzSI-Tyrlt-SS and increasing concentrations of native peptide.
Points correspond to control (O) haloperidol-treated (A) and haloperidol withdrawn
animals (C)). Values are expressed as the mean + SEM of five replicate experiments.
Right panel: Scatchard analysis of the same data.
Somatostatin Receptors After Haloperidol Withdrawal
19
-0.06
9.
f17
0
..~
c"
0.03
0
.0
00
A
1
!.
I
0
-0
[
oo 11
I
I
10
9
[SS].'IogM
I
8
I
0
I
0.02
Bound,nM
I
0.04
Fig. 3, Left panel: competitive inhibition of specific tzSI-Tyr'~ somatostatin (tzSI-Tyrl~-SS, 250 pM)
binding to membranes of the hippocampus by unlabelled somatosta,n. Membranes (1.5mg
protein/ml) were incubated for 60 rain at 30~ in the presence of 250 pM lZSl-Tyr~-SS and increasing
concentrations of native peptide. Points correspond to control ( 0 ) haloperidol-treated (A) and
haloperidol withdrawn animals (O). Values are expressed as the mean + SEM of five replicate
experiments. Right panel: Scatchard analysis of the same data.
Increasing concentrations of unlabelled somatostatin competitively inhibited
the specific binding of laSI-Tyr'l somatostatin to brain membrane in the
preparations of all the experimental groups (Fig. 2-3, left panels). However, the
specific binding of the tracer to membranes prepared from the frontoparietal
cortex and hippocampus in the haloperidol-treated group was significantly lower
than in the control animals in both the absence and the presence of unlabelled
Table 1. Effect of haloperidol administration and withdrawal on specific somatostatin receptors in the frontoparietal cortex and hippocampus of the rat.
Frontoparietal
cortex
Hippocampus
CONTROLS
Kd nM
Bmax
0.435 + 0.04
219 • 15
0.273 + 0.03
206 • 12
HALOPER1DOL
Kd nM
Bmax
0.469 + 0.14
122 • 11(*)
0.224.0.05
131 • 16(*)
0.422 + 0.07
241 • 32
0.341 + 0.03
239 • 26
H A L O P E R 1 D O L W1THDRA W N
Kd nM
Bmax
Binding parameters were obtained by Scarchard (20) analysis of data from Figs. 2-3,
right panels. Bmax. Binding capacity (femtomoles of somatostatin bound per mg
protein). Values represent the mean •
of five rats in each group. *p < 0.005 vs.
control.
Perez-Oso, Lopez-Ruizand Arilla
20
somatostatin throughout the whole range of concentrations studied. One week
after haloperidol withdrawal the specific binding of the tracer to membranes of
both brain areas returned to control values. Scatchard analysis (20) of these
results indicates that haloperidol treatment decreased the number of somatostatin
receptors without changing the affinity constant while haloperidol withdrawal
reversed the haloperidol-induced decrease in somatostatin receptors (Figs. 2-3
and Table 1).
DISCUSSION
The present study shows that following withdrawal from chronic haloperidol
administration the number of specific somatostatin receptors in both brain areas
returned to control values, although it had previously been decreased as a result
of chronic administration. The level of SLI was not affected either by chronic
haloperidoi treatment or by subsequent haloperidol withdrawal.
The levels of SLI and the binding parameters of specific somatostatin
receptors in frontoparietal cortex and hippocampus of control rats were similar to
those previously reported by other authors (21-23). It should be mentioned that
the Scatchard analysis demonstrated the existence of only one type of somatostatin receptor. This feature agrees with some studies (18, 21, 24), but differs from
others (22, 25). It is conceivable that use of small somatostatin analogs (22) or
their labeling with very different isotopes (25) might explain this discrepancy.
The lack of modifications in the level of SLI in the frontoparietal cortex and
hippocampus both after withdrawal and chronic haloperidol-treatment agrees
with Radke et al. (11), and Beal and Martin (10) respectively.
The molecular mechanism by which the number of specific somatostatin
receptors return to control values seven days after cessation of chronic haloperidol administration is not known. The primary effect of haloperidol is through
blockage of dopamine receptors in the central nervous system (26-28). When the
blockage has expired, supersensitivity to dopamine agonists develops and persists
for varying periods of time (2-4, 29). Burt et al. (5) reported that rats treated
chronically with haloperidol showed enhanced brain dopamine receptor binding
at 5 and 12 days after termination of drug treatment. Several studies have shown
anatomical and functional interconnections between dopaminergic and somatostatinergic systems (30-33). What is more, there is experimental data that suggest
the existence of dopamine receptors on somatostatin nerve terminals (4).
These earlier findings together with the present results, have inclined us to
speculate that dopamine receptors could somehow control the expression of
somatostatin receptors.
The recovery of the number of somatostatin receptors in both of the brain
regions examined with respect to the control values 1 week after withdrawing
haloperidol may be related to the motor side-effects that are observed clinically
when the neuroleptic treatment is terminated (2-4). It is possible that since the
number of somatostatin receptors returns to control values while the dopamine
receptors remain supersensitive (2-5), an imbalance may develop between this
Somatostatin Receptors After Haloperidol Withdrawal
21
n e u r o p e p t i d e a n d t h e d o p a m i n e s y s t e m , w h i c h w o u l d l e a d to m o t o r a b n o r malities. T h e s e results p r o v i d e f u r t h e r e v i d e n c e for i n t e r a c t i o n b e t w e e n d o p a m i n e
n e u r o n s a n d s o m a t o s t a t i n n e u r o n s in t h e f r o n t o p a r i e t a l c o r t e x a n d h i p p o c a m p u s ,
a n d suggest t h a t p e p t i d e r g i c n e u r o n s m a y b e i m p o r t a n t in t h e clinical m o t o r
a b n o r m a l i t i e s s e e n a f t e r t h e n e u r o l e p t i c t r e a t m e n t is t e r m i n a t e d . F u r t h e r e x p e r i m e n t s a r e r e q u i r e d to c o n f i r m this h y p o t h e s i s .
ACKNOWLEDGEMENTS
This w o r k w a s s u p p o r t e d b y g r a n t s f r o m t h e C o m i s i o n I n t e r m i n i s t e r i a I d e
C i e n c i a y T e c n o l o g i a (PB87-0753) a n d t h e F o n d o d e I n v e s t i g a c i o n e s S a n i t a r i a s d e
la S e g u r i d a d Social o f S p a i n (88/0903). T h e a u t h o r s t h a n k C a r o l F. W a r r e n , f r o m
t h e A l c a l a d e H e n a r e s U n i v e r s i t y I n s t i t u t e o f E d u c a t i o n Sciences for h e r e d i t o r i a l
help.
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