doses (4-12 mg/kg/day) of d-amphetamine over a period of. 35 days. Three phases of behavioural change were discerned: phase I during which animals ...
Psychopharmacology
Psychopharmacology(1982) 78:245 251
9 Springer-Verlag 1982
Behavioural and Biochemical Effects of Chronic Amphetamine Treatment in the Vervet Monkey R. M. Ridley, H. F. Baker, F. Owen, A. J. Cross, and T. J. Crow Division of Psychiatry, Clinical Research Centre, Watford Road, Harrow, Middlesex, England Abstract. Five vervet monkeys were administered increasing
doses ( 4 - 1 2 mg/kg/day) of d-amphetamine over a period of 35 days. Three phases of behavioural change were discerned: phase I during which animals exhibited repetitive stereotyped action sequences with rapid head movements, occasional abnormal grooming, picking at the cage, hand-staring and snatching; phase 2 in which behaviour became progressively more restricted and animals became markedly unresponsive to auditory, visual and tactile stimuli; phase 3 was characterised by the abrupt development of gross over-responsiveness to environmental stimuli, ataxia and tremor. At post-mortem, by comparison with controls, amphetamine-treated monkeys showed marked depletions of the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in corpus striatum and cerebral cortex and reductions in the activities of tyrosine hydroxylase and dopa decarboxylase in striatum. Turnover of these monoamines, assessed by high-performance liquid chromatography determinations of their respective metabolites, was also reduced. These findings are interpreted as evidence of monoamine neurone destruction, most severely in the case of DA neurones. Though there was a non-significant reduction in 3H-spiperone binding (reaching almost 50 % in nucleus accumbens), numbers of receptors for the monoamines NA and 5-HT were not significantly changed, and the activities of the enzymes choline acetyltransferase and glutamine decarboxylase were similar in experimental and control animals. The contrast of these findings with those seen in post-mortem brains in schizophrenia is discussed. Key words: d-Amphetamine chronic treatment - Dopamine
- Noradrenaline - 5-HT Schizophrenia - Vervet monkey
Receptor Binding
-
Schi~brring 1971). In primates, acute amphetamine administration frequently induces rapid head movements (checking) and idiosyncratic stereotypies, although its effects on locomotion appear to be species-dependent (Ridley et al. 1980). Pharmacologically, acute amphetamine administration enhances the release of dopamine (DA) and noradrenaline (NA) from nerve terminals (Moore 1977). The evolution of behavioural patterns during chronic treatment leads one to suspect further neurochemical changes. Multiple daily injections of amphetamine in rodents lead to an augmentation in behavioural response characterised by a more rapid onset and heightened magnitude of stereotypy (Segal and Mandell 1974). Similar effects are seen with continuous administration of amphetamine from implanted slow-release pellets (Ellison et al. 1978). We have observed augmentation of certain behaviours, but tolerance in other behaviours, in the marmoset after chronic oral administration (Ridley et al. 1979). In the present experiment we have been able to observe the behavioural effects of increasing doses of amphetamine in the vervet monkey and their subsequent neurochemical effects (together with those of undrugged control animals) in postmortem brain. In particular we have studied the effects of chronic amphetamine administration on central dopaminergic, noradrenergic and serotonergic mechanisms. Since it has been suggested that chronic amphetamine results in a general inhibition of protein synthesis in brain (Trulson and Jacobs 1980), we have assayed the activities of choline acetyltransferase (CAT) and glutamate decarboxylase (GAD), enzymes associated with neurotransmitter systems not considered to be directly affected by amphetamine administration, but each of which, it has been suggested, may be involved in the pathophysiology of schizophrenia (Friedhoff and Alpert 1973; Roberts 1972). Materials and Methods
It is well established that chronic amphetamine administration may lead to a paranoid psychosis in humans (Connell 1958; Griffiths et al. 1972) and to severe and progressive behavioural disturbances in animals (Ellinwood et al. 1973; Ellinwood and Kilbey 1975). Although the effects of chronic amphetamine on certain aspects of brain biochemistry have been reported in rodents (Wagner et al. 1980; Ellison et al. 1978) and in cats (Trulson and Jacobs 1979a, b, 1980), the effects on central neurotransmitter metabolism in the primate have not been comprehensively investigated. When given acutely to rodents, amphetamine induces locomotion and stereotyped sniffing and gnawing (Fog 1970; Offprint requests to: H. F. Baker
Nine vervet monkeys (six male, three female Cercopithecus aethiops) weighing 3 - 5 kg each, were individually housed in cages (70 x 70 x 80 cm) for 2years prior to and during this experiment. Animals had undergone brief intermittent pairing for breeding and had limited visual contact. This situation was beyond our control and, although it limited the variety of behaviour which might have occurred and precluded assessment of social interaction, it did apply equally to control and experimental groups and did permit the assessment of each animal individually. Drug Administration. Five previously undrugged monkeys were administered 4 mg/kg/day d-amphetamine sulphate for
0033-3158/82/0078/0245/$ 01.40
246 11 days, followed by 6 mg/kg for 9 days, 8 mg/kg for 7 days, 10mg/kg for 5 days and 12mg/kg for 3 days. The drug was administered at 10:30 AM to each animal in 500ml blackcurrant-flavoured drinking water each day. Three control animals were given 500 ml blackcurrant-flavoured drinking water each day. All or almost all the fluid was consumed by the animals each day, although animals did not appear to be seriously fluid deprived. Typically, the water was consumed in small quantities throughout the day. Acceptance of the fluid did not alter during the course of treatment even at high drug doses.
Behavioural Observations. Behavioural observations could not be made on one undrugged animal owing to the rigid interpretation of government regulations. Observations of the other animals were made in the early afternoon for 4 days prior to drug administration and for 26 of the 34-day drug treatment. Each animal was observed by two observers who had previously established a high interobserver correlation during simultaneous observation. Observations were made at a short distance from each cage, since circumstances did not permit observation from an obscured position. The observers independently observed each animal for 90 s on each observation day and classified the animals' behaviour every 1.5 s (using a metronome) into the following six mutually exclusive categories: 1) Checking (rapid movement of the head relative to the torso) resembled the rapid changes of head or eye orientation observed in macaques and attributed to hypervigilance by Garver et al. (1975). We do not assume that checking, in the stereotyped form induced by amphetamine, retains a vigilance function. We have shown that while normal checking is compatible with visual observing responses, amphetamineinduced checking is not (Ridley et al. 1980). 2) Bobbing (rapid vertical movement of head and shoulders) was usually directed towards other animals or the observers. It was thought to be a communicative signal, since bobbing by the observers generally elicited this behaviour. Observers took care not to move their heads while taking behavioural readings. 3) Pivoting (rapid movement of only part of the torso) usually consisted of rapid sideways movements of the head and shoulders in a lowered position while the haunches were kept high and stationary, but could also consist of rocking while in a sitting position. We believe this to be a form of cage stereotypy brought about by the long period of capacity. Similar pacing and rocking may be seen in many zoo animals (Ridley and Baker 1982). 4) Activities (behaviours including eating, drinking, grooming, gnawing and fingering parts of the cage) were, as far as possible, recorded individually and then summed to give on overall activity assessment. Although all the actions in this category could be construed as purposeful, we do not imply that they retained their original purpose when performed in a stereotyped or repetitive way. 5) Locomotion (movement of the whole body) was limited by the size of the cage. 6) Inactivity was indicated by no discernible movement. Neurochemical Assays. On the morning following the last day of drug administration, brains were removed for subsequent biochemical analysis. Each animal was sedated with 1 mg/kg ketamine IM and then heavily anaesthetised by injecting 1.5 ml Nembutal (60mg/ml, Abbott, Queensborough, UK) deep into the right thoracic cavity. The cranium was opened
and the brain rapidly removed. All samples were frozen immediately and stored at - 4 0 ~ until dissection. After dissection the samples were stored over liquid nitrogen until analysed.
Biochemical Assays. Noradrenaline bitartrate (NA), dopamine (DA), 5-hydroxytryptamine creatine sulphate (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol(HMPG), tyrosine, glutamic acid, choline bromide, pyridoxal phosphate and catalase were obtained from Sigma (Poole, UK). Lysergic acid diethylamide (LSD) was obtained from Sandoz (Feltham, UK), pargyline from Abbott (Queensborough, UK), 6,7dimethyl-5,6,7,8-tetrahydropterine (DMPH4) from Koch Light (Haverhill, UK), propranolol from ICI (Alderly Edge, UK) and the isomers of butaclamol from Ayerst (Farnborough, UK). All other reagents were obtained from BDH (Poole, UK). Details of radioactive compounds (Radiochemical Centre, Amersham) are as follows: [phenyl4AH]spiperone (25Ci/mmol): 1-[propyl-2,3AH]-dihydroalprenolol (60 Ci/mmol); 3H-WB4101 (19.7 Ci/mmol), [23H]LSD (15 Ci/mmol); [1-14C]acetylcoenzyme A (55.4 mCi/mmol); 5-adenosyl-I.-[methyl-3H]methionine (12.9 Ci/mmol); L-3,4-dihydroxy[ring 2,5,6AH]phenylalanine (28 Ci/mmol); L-[U-14C]-glutamic acid (285mCi/ mmol); L-[3,5-3H]tyrosine (45 Ci/mmol). [4- 3H]Clonidine hydrochloride (22.2 Ci/mmol) was obtained from New England Nuclear (Southampton, UK). The number of assays carried out was limited, in some instances, by the size of the samples available from some anatomical regions. DA and NA concentrations were determined by the radioenzymatic method described by Coyle and Henry (1973). 5-HT levels were determined by the radiometric technique of Saavedra et al. (1973). Brain levels of DOPAC, HVA, HMPG and 5-HIAA were measured by high-performance liquid chromatography with electrochemical detection, using the technique of Cross and Joseph (1981). As outlined in our earlier paper (Owen et al. 1981), tyrosine hydroxylase (TH) activity was assayed by the tritium-release method of Lerner et al. (1977) and dopa decarboxylase (DDC) activity by the technique of McCamen et al. (1972). CAT and GAD activities were measured using the radiometric techniques of Fonnum (1975) and Waddington and Cross (1978) respectively. The methods used for high-affinity binding of ligands to DA, NA and 5-HT receptors are outlined in Table 1. Results
Behavioural Changes. It was our overall impression that the amphetamine-treated animals progressed through three stages of behavioural deterioration (Fig. 1). Phase 1, which began during the first few days of treatment and lasted about 7 days, was characterised by long, complicated and repetitive sequences of actions and an increase in rapid head movements. Although the behaviour was idiosyncratic for each animal, it typically consisted of sequences drawn from their normal behavioural repertoire such as grooming, picking at parts of the cage, chewing debris and sawdust and moving around the cage in an ordered pattern. However, also included were some unusual features such as staring at the hands, snatching at the air as if catching insects and excessively meticulous grooming.
247 Table
l. Methods used for high-affinity binding of ligands to DA, NA and 5-HT receptors
Ligand
Displacing agent
Reference
aH-Spiperone (0.5 nM) 3H-DHA (1.0 nM) 3H-WB4101 (1.0 nM) 3H-Clonidine (1.0 nM) 3H-LSD (3.0 nM)
(+) Butaclamol (0.1 gM) Propranolol (i.0 pM) NA (100 pM) NA (lOpM) LSD (1.0 I-tM)
Owen et al. (1978) Bylund and Snyder (1976) U'Pritchard and Snyder (1979) U'Pritchard and Snyder (1979) Bennett and Snyder (1975)
Motor
I--Tremor I-Poor balance }--Ataxia t-Responsive to touch
activity
I--- Cramped posture Unresponsive to touch
Stereotypies
]
0 i
5O
E
I
l
I
I
I
I
5
i0
15
20
25
30
i
0
i
4
i
6
i
8
, p
