Locomotor hyperactivity induced by psychotomimetic drugs, such as amphetamine and phencyclidine, is widely used as an animal model of psychosis-like behaviour and is commonly attributed to an interaction with dopamine release and N-methyl-D-aspartate (NMDA) receptors, respectively. However, what is often not sufficiently taken into account is that the pharmacological profile of these drugs is complex and may involve other neurotransmitter/receptor systems. Therefore, this study aimed to assess the effect of three antagonists targeting different monoamine pathways on amphetamine- and phencyclidine-induced locomotor hyperactivity. A total of 32 rats were pre-treated with antagonists affecting dopaminergic, noradrenergic and serotonergic transmission: haloperidol (0.05 mg/kg), prazosin (2 mg/kg) and ritanserin (1 mg/kg), respectively. After 30 min of spontaneous activity, rats were injected with amphetamine (0.5 mg/kg) or phencyclidine (2.5 mg/kg) and distance travelled, stereotypy and rearing recorded in photocell cages over 90 min. Pre-treatment with haloperidol or prazosin both reduced amphetamine-induced hyperactivity although pre-treatment with ritanserin had only a partial effect. None of the pre-treatments significantly altered the hyperlocomotion effects of phencyclidine. These findings suggest that noradrenergic as well as dopaminergic neurotransmission is critical for amphetamine-induced locomotor hyperactivity. Hyperlocomotion effects of phencyclidine are dependent on other factors, most likely NMDA receptor antagonism. These results help to interpret psychotomimetic drug-induced locomotor hyperactivity as an experimental model of psychosis.
Psychosis is a common clinical manifestation of many psychiatric conditions including schizophrenia, bipolar disorder and depression [1]. Amphetamine and phencyclidine are psychotomimetic drugs, extensively used to study the pathophysiology of psychosis as they produce behavioral changes in animals that can model psychosis-like behaviors in humans [2]. The behavioral and biochemical effects of amphetamine and phencyclidine in animals have close parallels in humans. For example, acute administration of amphetamine and phencyclidine results in increased locomotor activity in animals and psychomotor agitation in humans [2, 3]. In many studies, amphetamine-induced hyperactivity is used as a model of dopamine release [2] whereas the action of phencyclidine is usually assumed to be related to N-methyl-D-aspartate (NMDA) receptor antagonism [4, 5]. However, the literature on the pharmacology of these drugs is quite inconsistent reflecting multifaceted complexities associated with psychotomimetic drug molecular actions that remain unresolved.
Some studies conclude that amphetamine-induced locomotor hyperactivity is
critically dependent on dopamine release in the nucleus accumbens and can
therefore be used to model a psychosis-like hyperdopaminergic state [6, 7]. These
studies must be interpreted with caution as other evidence points to a variety of
independent mechanisms involved in the effect of amphetamine on dopamine
transmission. For example, amphetamine can reduce the release of dopamine, it can
block vesicular monoamine transporter activity and activate dopamine D
In contrast to amphetamine, it is generally accepted that the action of
phencyclidine on psychosis-like behaviors is due to NMDA receptor hypofunction
[4, 5, 12]. Acute phencyclidine administration in rats produces hyperlocomotion
that has translational relevance to positive symptoms in humans [12, 13].
However, there is evidence attributing at least part of phencyclidine’s action to
dopaminergic, noradrenergic and serotonergic transmission [14, 15]. Several
studies suggest dopaminergic involvement in hyperlocomotion effects of
phencyclidine [16, 17, 18, 19] and, according to one of these studies, phencyclidine is
able to cause dysregulation in frontal dopamine release [17]. Moreover,
serotonergic neurotransmission seems to be critical for the regulation of
phencyclidine-induced locomotor hyperactivity [20], and phencyclidine-induced
glutamate efflux in frontal cortical regions is modulated by serotonin 5HT
The aim of this study was to re-evaluate dopaminergic, noradrenergic and serotonergic involvement in hyperlocomotion induced by amphetamine or phencyclidine, specifically by examining the inhibitory effects of haloperidol, prazosin and ritanserin, which target dopaminergic, noradrenergic and serotonergic activity, respectively [22, 23, 24].
The experimental protocol was carried out in 32 male Sprague-Dawley rats
(Department of Pathology, University of Melbourne). The rats were housed under
standard conditions in groups of 2–3, with free access to food and water. They
were maintained on a 12 h: 12 h light/dark cycle (lights on at 0700 h) at a
constant temperature of 21
D-amphetamine sulfate (Sigma Chemical Co., St. Louis, MO, USA; 0.5 mg/kg) and phencyclidine HCl (PCP, Sigma; 2.5 mg/kg) were dissolved in 0.9% saline and injected subcutaneously (s.c.) in the nape of the neck using an injection volume of 1 mL/kg of body weight. Haloperidol (Serenace®, 5 mg ampoules, Searle Laboratories, Crows Nest, NSW, Australia) was diluted to the required doses (0.05 mg/kg) in saline; vehicle treatment was saline. Prazosin (Sigma) was dissolved in hot water and then diluted to 2 mg/kg in saline, while ritanserin (Sigma) was dissolved in DMSO (1%) and then diluted in saline to give a dose of 1 mg/kg; vehicle treatment consisted of half the rats receiving saline and half receiving 1% DMSO in saline. The three drugs were administered intraperitoneally (i.p.) in an injection volume of 1 mL/kg of body weight. The dose selection for each drug was based on the unpublished and published work completed in our laboratory [25, 26, 27] as well as the literature [23, 24].
All experiments were carried out in the morning between 08:30 and 11:30. To
minimise the impact of circadian rhythms on drug action, the same time of the
light phase i.e., morning was chosen for the study. The study included 4 groups
of n = 8 rats/group; 2 groups were treated with amphetamine and 2 groups with
phencyclidine. Using a repeated-measures design, these groups were randomly
pre-treated with either (1) vehicle and haloperidol (0.05 mg/kg), or (2) vehicle,
prazosin (2 mg/kg) and ritanserin (1 mg/kg), before being treated with
amphetamine or phencyclidine, with 3 days clearance allowed between each
pre-treatment. Rats were pre-treated i.p. with either vehicle or drug 15 min
before being placed in the photocell cages. After 30 min of spontaneous activity
in the photocell cages, rats were injected with either 0.5 mg/kg amphetamine or
2.5 mg/kg phencyclidine and behavioral responses recorded over a further 90 min.
Behavioral responses such as distance travelled, stereotypy and vertical
counts/rearing were monitored using eight automated photocell cages (43
Data were expressed as the mean
When analyzing the time course of distance travelled, there was a significant
pre-treatment
Distance travelled after amphetamine (top panels: A, C, E) or
phencyclidine (bottom panels: B, D, F) in rats pre-treated with vehicle (white
bars) or psychotomimetic-drug (black bars; A, B: haloperidol 0.05 mg/kg, C, D:
prazosin 2 mg/kg, E, F: ritanserin 1 mg/kg). Spontaneous activity (‘Baseline’)
was recorded for 30 min before rats were injected with amphetamine (0.5 mg/kg) or
phencyclidine (2.5 mg/kg) and activity recorded for a further 90 min (time
blocks: 0–30 min, 30–60 min, 60–90 min). Bars represent total distance
travelled in 30 min (group average; cm)
Analysis of stereotypic scores supported the distance travelled findings. There
was a pre-treatment
Amphetamine | Phencyclidine | |
Vehicle | 2060 |
710 |
Haloperidol | 1452 |
503 |
Vehicle | 1730 |
726 |
Prazosin | 697 |
575 |
Ritanserin | 1702 |
957 |
Rats were pre-treated with vehicle, haloperidol (0.05 mg/kg), prazosin (2 mg/kg)
or ritanserin (1 mg/kg) and vertical counts/rearing were obtained during 90 min
after injection of amphetamine (0.5 mg/kg) or phencyclidine (2.5 mg/kg).
Differences between pre-treatments were analyzed by ANOVA. Data are expressed as
total counts (group average) |
Stereotypic counts after amphetamine (top panels: A, C, E) or
phencyclidine (bottom panels: B, D, F) in rats pre-treated with vehicle (white
bars) or psychotomimetic-drug (black bars; A, B: haloperidol 0.05 mg/kg, C, D:
prazosin 2 mg/kg, E, F: ritanserin 1 mg/kg). Spontaneous activity (‘Baseline’)
was recorded for 30 min before rats were injected with amphetamine (0.5 mg/kg) or
phencyclidine (2.5 mg/kg) and activity recorded for a further 90 min (time
blocks: 0–30 min, 30–60 min, 60–90 min). Bars represent total stereotypic
counts in 30 min (group average)
Analysis of the time course of distance travelled revealed a main effect of time
(F
Analysis of distance travelled showed a significant pre-treatment
There were significant main effects of prazosin pre-treatment (F
There were significant main effects of prazosin pre-treatment (F
Analysis of stereotypy scores showed a significant pre-treatment
When analyzing distance travelled, there was a significant pre-treatment
Analysis of stereotypy scores revealed a main effect of time (F
Analysis of distance travelled revealed a main effect of time (F
Analysis of stereotypy scores revealed a pre-treatment
In this study, we used three different antagonists to evaluate their inhibitory effects on amphetamine- and phencyclidine-induced locomotor hyperactivity. The principal findings of this study were that: (1) amphetamine-induced locomotor hyperactivity was attenuated by pre-treatment with haloperidol and prazosin; (2) amphetamine-induced locomotor hyperactivity was partially reduced by ritanserin; (3) phencyclidine-induced locomotor hyperactivity was not affected by either antagonist. These findings suggest that dopaminergic as well as noradrenergic neurotransmission is critical for the regulation of hyperlocomotion effects of amphetamine while phencyclidine-induced hyperactivity is dependent on other factors, most likely NMDA receptor antagonism or other not yet known mechanisms but, importantly, not dopaminergic or noradrenergic mechanisms.
Psychotomimetic drug-induced locomotor hyperactivity is generally attributed to limbic-striatal modulation of brainstem motor circuits [2]. Classic micro-injection and lesion studies have highlighted the role of dopamine in the nucleus accumbens (ventral striatum) in modulating the ambulatory locomotor response to amphetamine, while dopamine in the caudate putamen (dorsal striatum) is instead involved in the stereotypy/rearing induced by amphetamine [28, 29, 30, 31]. The nucleus accumbens is an important regulatory interface between limbic and motor systems in driving adaptive behavior through inputs from the prefrontal cortex, hippocampus and amygdala, and outputs to the ventral pallidum and substantia nigra [32]. Recent advances in human neuroimaging techniques call into question the involvement of the mesolimbic system in relation to psychotic symptoms and instead point to the importance of dopaminergic nigrostriatal pathways, specifically in the dorsal striatum [33, 34].
Haloperidol, the predominantly dopamine D
Our findings suggest a complex overlap of dopaminergic D
The effect of ritanserin via 5-HT
In contrast to amphetamine, phencyclidine-induced hyperactivity was not affected
by any of the three antagonists used. Given that dopamine D
There are several limitations of this study. Firstly, only male rats were used in this study. Given that we and others have shown sex differences in psychotomimetic-induced behaviors in animals [48, 49], future studies should include both male and female rats. Second, future studies should consider circadian rhythms and whether testing should occur during the light or dark phase. However, doses of amphetamine (0.6 mg/kg) similar to that used in this study were found to increase locomotor activity regardless of the large difference in baseline activity between the light and dark phases [50]. Third, the locomotor photocell system automatically measured ‘stereotypy’ as any repetitive beam breaks within a virtual box, however the exact behaviour within the virtual box, such as circling and/or head weaving, was not well-defined. Finally, we have not directly examined NMDA receptor antagonism in phencyclidine-induced behaviours, hence, at this stage we can only assume that NMDA receptor hypoactivity is involved. Future research should use selective NMDA receptor agonists and antagonists to dissociate the role of glutamatergic NMDA receptors in phencyclidine-induced locomotor hyperactivity.
This study shows that the noradrenergic, as well as the dopaminergic system, is
involved in mediating amphetamine- but not phencyclidine-induced locomotor
hyperactivity. In addition to dopamine D
ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HCl, hydrochloric acid; NMDA, N-methyl-D-aspartate.
SK and MvdB designed the research study. SK performed the research. AG analyzed the data. SK and AG wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
Animals were raised and handled at the Department of Pathology, the University of Melbourne, Melbourne, Australia until 5 weeks of age. They were then transferred to the Behavioral Neuroscience Laboratory at the Mental Health Research Institute in Melbourne (Parkville, VIC Australia) where all experiments were conducted. The experimental protocol was approved by the Animal Experimentation Ethics Committee of the University of Melbourne, Melbourne, Australia (AEEC #01159). All scientific procedures using animals were carried out in accordance with the Prevention of Cruelty to Animals Act 1986 and the Australian code of practice for the care and use of animals for scientific purposes (1997).
We thank all the reviewers for their constructive feedback.
This research was part-funded by the National Health and Medical Research Council of Australia (AG CDF 1108098, Project Grant 509234). The Florey Institute of Neuroscience and Mental Health acknowledges the funding from the Victorian Government’s Operational Infrastructure Support.
The authors declare no conflict of interest.