Neuro-Psychophanacol

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Prog. Neuro-Psychophanacol. 1961, Vo1.5, pp.603-607 
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Copyright 01981 Pergamon Press Ltd. 
PROLACTIN LEVELS AND AMPHETAMINE-INDUCED BEHAVIOURAL 
CHANGES FOLLOWING FLUPHENAZINE DECANOATE ADMINISTRATION 
2. MERALI 
Department of Pharmacology and 
School of Psychology 
University of Ottawa 
Ottawa, Ontario KIN 9A9, Canada 
1. 
2. 
3. 
4. 
Abstract 
A single injection of fluphenazine decanoate (FD) antagonized effects of acute d-amphet- 
amine (2.5 mg/kg) for a variable period of time (4 to 28 days), depending upon the dos- 
age of the neurolcptic used (2.5 or 5.0 mg/kg) and the behavioural parameter(s) monitor- 
ed. 
Locomotion and rearing were antagonized for a longer duration than was sniffing. Normal 
locomotor response to amphetamine was attained 12 and 28 days following the administrat- 
ion of 2.5 and 5.0 mg/kg FD, respectively. However, the 2.5 mg/kg FD group displayed 
significantly more locomotor activity on days 20 and 24 post-treatment. A similar super- 
sensitive response was not demonstrable with the higher dose of FD (5.0 mg/kg), or with 
the other behavioural measures. 
The prompt and pronounced elevation of serum prolactin in response to the neuroleptic 
returned to within the normal range by days 4 and 14, following administration of 2.5 
and 5.0 mg/kg FD, respectively. 
These results indicate that the behavioural paradigm is more sensitive in monitoring the 
effects of FD and could serve as a useful model in investigating the dose- and time- 
related effects of other long-acting neuroleptics. 
Key words: antagonism of amphetamine-induced behavioural changes, fluphenazine decanoate, 
prolactin, supersensitivity 
Introduction 
It is believed that neuroleptics disinhibit prolactin release from the anterior pituitary 
by blocking the dopamine rcccptors functionally influenced by the activity of the tuberoin- 
fundibular tract (Meites and Clemens, 1972; Meltzer et al., 1976). Thus the study of serum 
prolactin alterations in response to neuroleptic drugs has the potential of reflecting the 
extent of dopamine receptor blockade on the tuberoinfundibular-pituitary dopamine system, 
which could possibly serve as a convenient model of monitoring indirectly the extent of neur- 
oleptic blockade on other dopaminergic systems, such as the mesolimbic and nigrostriatal 
tracts (Meltzer and Fang, 1976). 
Amphetamine elicits a behavioural syndrome characterized by purposeless and repetitive 
movements which in the rodents take the form of stereotyped locomotion, sniffing, rearing 
and biting. According to current views, these stereotyped behaviours result from stimulation 
of dopamine receptors of the nigrostriatal and the mesolimbic systems (Iversen and Koob, 
1977; Morelli et al., 1980; Mumford et al., 1979). The objectives of the present investi- 
gation were to study the time-course and dose-effect of fluphenazine decanoatc (FD) on 1) 
the tuberoinfundibular-pituitary dopamine system (as reflected by changes in serum prolactin) 
and 2) the nigrostriatal and/or mesolimbic dopamine systems (as reflected by antagonism of 
amphetamine-induced behavioural changes). 
603 
604 2. Merali 
Method 
Animals. Male Sprague-Dawley 
innenvironment maintained 
to 6 p.m.). 
rats (224fll 
at 24’C, 60% 
g) with free access to food and water, were housed 
relative humidity and with 12 hr of light (6 a.m. 
Behavioural Testing. This portion of the study consisted of two experiments in which the 
antagonistic effects of two doses of FD (2.5 mg/kg and 5.0 mg/kg) upon behavioural changes 
induced by d-amphetamine (2.5 mg/kg), were monitored. The experimental chambers for behav- 
ioral observation were identical to the rats ’ home cage (clear polycarbonate; 43 x 23 x 15 
cm) except for the following modifications: the floor was elevated, marked off into six 
equal sections and had seven holes (2.5 cm diameter). 
Procedure. Twelve rats received FD (5.0 mg/kg; i.m.) whereas the control group (12 rats) were 
given an equal volume (0.1 ml/100 g) of the vehicle(sesame oil). Each rat was observed con- 
secutively for 1 min duration, evey 6 min, for the entire observation session. The behav- 
ioural parameters being monitored were operationally defined as follows: 1) Sniffing: nose 
and whiskers moving; 2) poking: rat’s nose visibly protruding through one of the holes on 
the floor; 3) Rearing: standing on hindlegs, with forelimbs off the floor; 4) Locomotion: 
hindlegs crossing into another section of the floor; 5) Gnawing: rats biting on anything in 
the cage. Once in their experimental chambers, the rats were allowed 0.5 hr of acclimatiz- 
at ion period, following which all rats received a saline (placebo) injection and were obser- 
ved and rated for the subsequent 0.5 hr period. Immediately following this all rats were 
injected with d-amphetamine (2.5 mg/kg; i.p.) and the behaviours rated for 2.5 hr, as descri- 
bed above.Asimilar experiment was conducted using a smaller dose of FD (2.5 mg/kg). 
Prolactin Studies. Separate experiments were performed to assess the effects of two doses of 
FD (2.5 and 5.0 mg/kg) on serum prolactin levels. Fourty-eight rats were injected with FD 
(5.0 mg/kg; i.m.) and 18 control rats with an equal volume (0.1 ml/100 g) of the vehicle 
(sesame oil). Six hours after the first injection (day 0), groups of control (n=6) and FD- 
treated (n=G) rats were decapitated, with care being taken not to unduly stress the animals. 
Trunk blood was collected and plasma separated out, and stored frozen until subsequent anal- 
ysis. Plasma samples from groups of 6 neuroleptic-treated rats were similarly obtained on 
days 1, 4. 7, 14, 21 and 35 following the administration of fluphenazine decanoate. Samples 
from 2 additional groups of control animals were obtained about mid-way through (day 21, n=6) 
and at the end of the experiment (day 35, n=6). The above procedure was repeated on addit- 
ional 66 rats, using a different dose of FD (2.5 mg/kg). 
Prolactin Assay. Prolactin levels were determined in the serum samples in duplicate by a 
double antibody radioimmunoassay method, using reagents kindly supplied by Dr. A.F. Parlow, 
through the NIAMDD Rat Pituitary Hormone Distribution Program. 
Results 
Behavioural Experiments. The raw data of the individual behaviours was collapsed over 0.5 hr 
intervals, and subjected to analysis of variance with repeated measures. 
Globally, the poking and grooming behaviours occurred with extremely low frequency in both 
the groups, and were excluded from subsequent statistical analysis. 
Administration of d-amphetamine markedly stimulated the locomotor activity of the control 
rats, which diminished gradually over the 2.5 hr observation period. The locomotor response 
of the neuroleptic-treated rats was significantly lower than that of controls on test days 4, 
7, 11, 14, 18, 21 and 28. Although initially the amphetamine-induced locomotor activity re- 
mained blunted at every 0.5 hr test interval, with the progression of days significant block- 
ade was only demonstrable during the first and/or second post-amphetamine 0.5 hr bins. Sim- 
ilarly, the frequency of amphetamine-induced rearing was markedly suppressed for up to 28 
days following the administration of FD. Amphetamine-induced sniffing was also suppressed by 
pretreatment with FD. However, this suppression (in contrast to that of locomotion and rear- 
ing) was present for a shorter duration (14 days). 
With the lower dose of FD (2.5 mg/kg), a similar over-all pattern in the blockade of amph- 
etamine-induced locomotor (Fig. l), rearing and sniffing activity, could be seen. However, 
these effects were less pronounced and of a shorter duration than those resulting from the 
Fluphenazine and amphetamine - induced behavioural changes 605 
Eiil VEHICLE t d-AMP (2.5 q/kg) &i$ FPZ DECANOATE (2.5 mg/kg) t d-AMP (2.5 mg/kg) 
C 4 8 12 16 20 24 28 
DAYS AFTER ADMINISTRATION 
Fig. 1. Time-course of fluphenazine-decanoate (2.5 mg/kg) effect on d-amphetamine-induced lo- 
comotor activity. Tridimensional figure: On the ordinate: frequency of the quantified acti- 
vity; on the horizontal abscissa: days following the administration of fluphenazine decanoate 
; on the oblique abscissa: time after d-amphetamine administration. The cumulative profile 
of vehicle-treated controls is represented by the unshaded area on the extreme left and pro- 
files of fluphenazine decanoate-treated groups are represented by the subsequent shaded 
areas. n=6 for each test session. *Significantly different with respect to controls at ~~0.05 
higher dose of FD. Amphetamine-induced locomotor activity was partially antagonized by FD 
(2.5 mg/kg) on days 4, 8 and 12. However, on days 20 and 24, the neuroleptic treated rats 
displayed significantly more locomotor activity (see Fig. 1). With rearing behaviour, a par- 
tial blockade could also be seen for the same duration (up to 12 days), following which a re- 
instatement and maintenance of normal responsiveness was apparent. As in the case with the 
larger dose, amphetamine-induced sniffing was antagonized for the shortest period. In this 
instance, antagonism of sniffing was demonstrable mainly on the first day (day 4) and on the 
second half hour of the next test session (day 8). 
Prolactin Studies. There was no statistically significant difference in the prolactin levels 
of 3 control groups (n=6) sacrificed at the beginning (day 0), the middle (day 21) and the 
end of the experiment (day 35). Administration of FD (5.0 mg/kg) resulted in a pronounced 
elevation of prolactin (1447% of control) within 6 hours of the injection (day 0). The pro- 
lactin level remained significantly elevated, albeit by a smaller magnitude, on sampling days 
1, 4 and 7 (see Table 1). By day 14, prolactin level had returned to within the normal range 
and remained there for the duration of the experiment. With the lower dose of FD (2.5 mg/kg) 
prolactin concentration increased significantly (by 352%) within 6 hours of administration 
(day 0). However, this elevation was of a much smaller magnitude than that following the 
larger dose of the neuroleptic, and was detectable only up to day 1 of sampling. 
Discussion 
The present results demnstrate that a single injection of FD can antagonize behavioural 
606 2. ?Qrali 
Table 1 
Effect of fluphenazine decanoate (FD) on plasma prolactin 
Days Following FD Administration 
C 0 1 4 7 14 21 35 
Prolactin levels (rig/ml) 
FD 25. mg/kg 10.7t2.6 37.8-Cl2.2 24.727.5 8.420.9 9.621.6 12.3t2.0 10.3k1.7 12.9t1.7 
(100%) (352%) * (230%) * (78%) (893) (115%) (97%) (120%) 
FD 5.0 mg/kg 7.6t1.8 109.929.3 2O.Ok2.3 24.Ot2.6 16.3t1.6 8.9t2.3 6.821.5 7.7k2.0 
(100%) (1447%)* (263%)* (316%)* (215%) * (116%) (89%) (102%) 
*Significantly different with respect to controls at ~~0.05. 
effects of acute d-amphetamine (2.5 mg/kg) for a variable period of time (4 to 28 days), dep- 
ending upon the dosage of the neuroleptic used (2.5 or 5.0 mg/kg) and the behavioural parame- 
ter(s) monitored. Globally, the magnitude of behavioural antagonism and its time course were 
greater with the higher dose of the neuroleptic. 
At the level of individual behavioural components, there seemed to be a differential anta- 
gonistic effect engendered by this neuroleptic: locomotion and rearing behaviours were anta- 
gonized for a much longer duration than was the sniffing response. This differential antag- 
onistic effect may be related to the involvement of distinct neurochemical and/or neuroanat- 
omical mechanisms in the mediation of d-amphetamine-induced behavioural changes. Indeed, in 
recent years it has become evident that various components of stereotypy do not arise from 
the same brain area; locomotion seems to depend on stimulation of dopaminergic mechanisms in 
the mesolimbic system while licking and gnawing arise from the striatum (Iversen and Koob, 
1977; Morelli et al., 1980; Kelly et al., 1975). It is thus tempting to suggest that,at the 
doses employed in this experiment, FD may be exerting a preferential effect on limbic over 
striatal dopaminergic neurotransmission. An alternative explanation may be gleaned from rec- 
ent evidence implicating the existence of at least two distinct types of dopamine receptors 
(Cools, 1977; Titeler et al., 1978). It is thus also possible that the sniffing behaviour 
may depend more on the stimulation of the dopamine receptors relatively insensitive to fluph- 
enazine effects. Following the apparent reinstatement of normal locomotor response, the 2.5 
mg/kg FD group displayed significantly more locomotor activity on days 20 and 24 post-treat- 
ment . This reversal in activity lends further credence to the hypothesis that dopamine rece- 
ptors become supersensitive during withdrawal from chronic exposure to receptor blocking ag- 
ents. It is of interest to note that a similar supersensitive response was not demonstrable 
with the higher dose of FD (5.0 mg/kg). This could possibly be attributed to the experiment- 
al span and testing intervals, which may not have coincided with the supersensitive state, or 
perhaps this response may have reversed before effective levels of fluphenazine were gone 
(Wheeler and Roth, 1980). In contrast to the locomotor activity, supersensitive response was 
not reflected in rearing and sniffing behaviours. These results also indicate that the moni- 
toring of discrete behavioural components might be more informative than single stereotypy 
scores obtained from the constellation of behavioural repertoires. 
Administration of FD resulted in a prompt and pronounced elevation of serum prolactin level 
already demonstrable at the time of the first sampling (6 hr post injection). This is conco- 
rdant with the clinical observation of Nasrallah et al. (1979) who reported that plasma pro- 
lactin concentration of patients increased (2-6 fold) between 3 and 6 hr after the injection 
of FD. These observations are also consistent with the kinetics of fluphenazine after FD ad- 
ministration and may have considerable significance for the use of FD in acute conditions 
(Curry et al., 1979). Our data also demonstrate that the effects of FD were dose dependent, 
in that the magnitude of prolactin increase and its duration were greater with the larger 
dose of the neuroleptic. 
In rats administered 2.5 and 5.0 mg/kg dose of FD prolactin levels were within the normal 
range by days 4 and 14, respectively. In male schizophrenic subjects being treated with FD, 
the prolactin levels remained elevated for up to 4 weeks (Merali and Lapierre, 1981). Furth- 
Fluphenazine and amphetamine - induced behavioural changes 607 
ermore, in the behavioural experiments reported here, effects of FD (5 mg/kg) could also be 
detected for up to 28 days. Normally, prolactin released from the pituitary is pulsatile 
and it remains possible that the change in prolactin profile (as reflected by the number and/ 
or the magnitude of prolactin peaks) may have revealed a different time-course. However, it 
is also possible that the continuous interruption of the pituitary dopamine receptors may 
have rendered them “supersensitive” to dopamine, the physiological prolactin inhibitory fact- 
or. Recently, Annunziato et al. (1980) reported that serum prolactin levels, 24 hr after the 
last neuroleptic (short-acting) injection, were significantly lower in the repeat-treated 
group than in the acute treated rats. This can be interpreted as an enhanced sensitivity of 
pituitary dopamine receptors or alternatively, the consequence of an increased turnover of 
tuberohypophyseal dopamine neurons which follows prolonged elevation of circulating prolactin 
levels (Annunziato, 1980; Naber et al., 1979). 
In conclusion, these data indicate that FD effects are reflected by changes in serum prola- 
ctin as well as by antagonism of amphetamine-induced behavioural changes. The time course of 
FD effect on the nigrostriatal and/or mesolimbic dopamine system(s) (as reflected by antagon- 
ism of amphetamine-induced behavioural changes) is longer than that on the tuberoinfundibular 
-pituitary system (as reflected by circulating prolactin levels). Thus the behavioural para- 
digm reported here seems more sensitive in detecting the effects of relatively low levels of 
FD, and suggests that this could serve as a useful model in investigating the dose- and time- 
related effects of other long-acting neuroleptics. 
Acknowledgements 
The expert tcchnicnl assistance of Ms. C. Garneau is greatly appreciated. This work was 
supported by the Ontario Mental Health Foundation (OMHF). Z. Merali is an OMHF Research 
Scholar. 
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PROLACTIN LEVELS AND AMPHETAMINE-INDUCED BEHAVIOURAL CHANGES FOLLOWING FLUPHENAZINE DECANOATE ADMINISTRATION

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PROLACTIN LEVELS AND AMPHETAMINE-INDUCED BEHAVIOURAL CHANGES FOLLOWING FLUPHENAZINE DECANOATE ADMINISTRATION

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