ANTHONY G. PHILLIPS*, DAVID A. CARTER, and HANS C. FIBIGER. Department of Psychology and Division of Neurological Science, Department of ...
Psychopharmacology49, 23- 27 (1976)
Psycho pharmacology 9 by Springer-Verlag1976
Differential Effects of para-Chlorophenylalanine on Self-Stimulation in Caudate-Putamen and Lateral Hypothalamus ANTHONY G. PHILLIPS*, DAVID A. CARTER, and HANS C. FIBIGER Department of Psychologyand Divisionof Neurological Science, Department of Psychiatry,Universityof British Columbia, Vancouver (Canada)
Abstract. Rats were prepared with chronic bipolar
electrodes aimed at either the "caudate-putamen or lateral hypothalamus and those displaying consistent self-stimulation were given additional training at half-maximal current intensities. All subjects received an intragastric injection of para-chlorophenylalanine (400 mg/kg) and self-stimulation tests continued until pre-injection rates were re-established. Responding in both brain areas was suppressed 24 h after drug treatment. The next day, self-stimulation rates in the hypothalamus increased to 115 % of pre-drug levels reaching a level of 180 % by the third day of post-drug testing. In contrast, self-stimulation of sites in the neostriatum continued to decline, with minimal levels reaching 48 % of control on the fourth post-drug day. Self-stimulation rates in both groups had returned to control levels by post-drug day 6. These data indicate that the role of serotonergic mechanisms in brain stimulation is locus specific, and that the specific nature of this role may be determined by interaction with other neurochemical systems. The possible interaction between dopaminergic and serotinergic mechanisms in the neostriatum is discussed as a model of self-stimulation in this region of the brain. Key words." Intracranial self-stimulation - Caudateputamen - Hypothalamus - Serotonin - Dopamine - Para-chlorophenylalanine - Rat.
INTRODUCTION Recent attempts to define the neurochemical and neuroanatomical substrates of brain-stimulation reward have focussed almost exclusively on brain * Offprint requests to: Dr. A. G. Phillips,Departmentof Psychology, University of British Columbia, Vancouver, V6T IW5, Canada
catecholamines (German and Bowden, 1974) with particular emphasis being placed on two of the ascending catecholamine systems identified by histochemical fluorescence (Ungerstedt, 1971 ; Lindvall and Bjourklund, 1974). One of the systems arises from noradrenergic cell bodies of the A6 cell group in the locus coeruleus and electrode placements in the vicinity of this nucleus and its ascending efferent projection the "dorsal tegmental bundle", support intracranial self-stimulation (ICS) (Crow et al., 1972; Ritter and Stein, 1973, 1974). The second system, the dopaminergic nigrostriatal bundle (NSB) originates in the A9 region of the pars compacta of the substantia nigra (Ungerstedt, 1971) and ICS can be elicited along its trajectory, from the substantia nigra (Crow, 1972; Routtenberg and Malsbury, 1969), through the dorsolateral hypothalamus, medial limb of the internal capsule (Prado-Alcala et al., 1975), and from its termination in the caudate putamen (CPU) (Phillips et al., 1976). Although lesion studies have failed to confirm a role for the dorsal tegmental bundle in brain-stimulation reward (Clavier and Routtenberg, 1974, 1976; Clavier et al., 1976), a similar approach has provided evidence for the involvement of the dopaminergic NSB in ICS (Phillips et al., 1976). In the latter study rats with ICS electrodes in CPU were treated with desipramine to protect noradrenergic neurons prior to unilateral destruction of the A9 dopamine cell bodies by intracranial injections of 6-hydroxydopamine. This treatment eliminated ICS in the ipsilateral CPU, while producing only a temporary disruption in ICS at sites in the contralateral neostriatum. The failure to obtain brain-stimulation reward in the dopamine denervatcd neostriatum clearly suggests an important role for dopamine. However it would be an oversimplification to argue for an exclusive role. The neostriatum also contains moderate concentrations of serotonin (Bogdanski et al., 1957), which has
24 been implicated in brain-stimulation reward (Miliaressis etal., 1975; Poschel and Ninteman, t 97~). Nerve terminals in the neostriatum containing serotonin arise partly if not exclusively from the dorsal raphe nucleus (Miller et al,, 1975). These authors have also shown that stimulation of the dorsal raphe nucleus reliably inhibited the discharge of cells in the CPU. Behavioral pharmacological analysis of the neostriaturn has suggested the existence of a dopamine-sensi:tive area and a serotonin-sensitive area within the caudate nuclei of cats (Cools and van Rossum, 1970; Cools, 1973). Furthermore the behavioral syndrome induced by activation of the dopamine-sensitive area may be dependent upon the integrity of the serotoninsensitive region to Which it is hypothetically linked by a cholinergic mechanism (Cools, 1974). Cools (1974) 9has shown that behaviors correlated with activation of dopamine receptors in the neostriatum were blocked by intracranial injections of-para-chlorophenylalanine (PCPA), a drug which inhibits the biosynthesis of serotonin (Koe and Weissman, 1966). If ICS in the neostriatum is subserved by a transsynaptic relationship between dopamine and serotonin similar to that proposed by Cools (1974), it should be attenuated by pretreatment with PCPA. Support for this hypothesis is contradictory, as treatment with P C P A can either increase or decrease current intensities thresholds for ICS (Gibson et al., 1970). Furthermore, similar treatment has been shown to facilitate ICS at sites in the lateral hypothalamus (Blum and Geller, 1969; Poschel and Ninteman, 1971) as has neurotoxic destruction of serotonin neurones (Poschel et al,, 1974). Electrode placement appears to be a critical variable, as Miliaressis et al. (1975) have recently reported inhibition of brain-stimulation reward at sites in median raphe after treatment with PCPA. As a test of the hypothesis that decreased synthesis of serotonin should inhibit ICS in the CPU, animals in the following experiment were treated with PCPA. To provide additional data on the possible locus specific effects of this treatment, a group of animals with electrodes in the lateral hypothalamus was also included.
METHODS
Subjects. Tbe subjects were male Wistar rats (Woedlyn Laboratories, Guelph Ontario), weighing 280-320 g at the time of surgery. Animals were housed individually in stainless steel cages, located in a climatically controlled colony room with a 12 h light/ dark cycle. Food and water were available ad libitum. Surgery and Histology9 Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.), placed in a stereotaxic apparatus (David Kopf) and small diameter bipolar nichrome electrodes (Plastic Products Co. MS 303- 0.005 in) werechronicallyimplanted
Psychopharmacology49 (1976) according to standard procedure. Electrodes were aimed at either the anterior CPU or LH. The co-ordinates, accordingto Pellegrino and Cushman (1967), were (A) CPU: A + 9.0 ram, L + 2.5 ram, V - 5.0- 6.0 mm from dura, and (B) LH : A + 4.5 mm, L + 1.8 ram, V -8.5 mm from dura. At the completion of the experiment, all subjects were asphixiated with CO2, their brains rapidly removed and stored in 10~o formalin. Brains were frozen, sectioned at 40 ~t and sections containing electrode tracts were mounted and stained with Luxol fast blue and counterstained with thionin.
Procedure. Following a 7-day post operation recovery period, all animals were screened for ICS in five identical plexigtass chambers (46 cm x 30 cm x 24 cm). Depression of a small 2.5 cm wide bar activated an AC constant current stimulator which delivered a variable intensity (1 -200 oA) 60 Hz sine wave stimulus of fixed duration (0.2 s) through a flexible cable to the chronic electrode assembly. Screening of subjects for ICS utilized the procedure described by Phillips et al. (1976). Eight animals with CPU electrodes, and 6 with hypothalamic placements were selected for the experimental groups and all were given at least 7 days additional testing for ICS. In preparation for drug tests, the current intensity was set individually to produce bar pressing at half the maximal rates9 Testing continued for 15 min per day with these current intensities, until ICS performance had stabilized (i.e. less that 10 variation on 3 consecutive days). When all subjects had displayed stable ICS rates, each animal was placed under light ether anesthesia prior to intragastric intubation of a 400 mg/kg dose of PCPA, as a suspension in H20. Post-drug tests for ICS began 24 h later and continued for 15min per day until pre-drug performance was re-established.
RESULTS
Histology. All animals in the C P U group had electrodes placements in the medial aspect of the anterior neostriatum between AP planes 8 . 8 - 9 . 4 (Pelligrino and Cushman, 1967). The 6 electrode placements aimed at the lateral hypothalamus were in the appropriate region o f the brain with slight variation in the dorso-ventral plane (see Fig. 1). Intracranial Self-Stimulation.
As may be seen in Figure 2, treatment with P C P A produced either a facilitation or inhibition of ICS, depending on electrode placement. Responding in both brain areas was suppressed 24 h after drug treatment, but by 48 h ICS in the lateral hypothalamus showed a 15 increase over pre-drug levels and peaked at 180~o of control, 72 h after PCPA treatment. Rates declined from this level to return to control levels on post-drug days 5 and 6. In contrast, the same dose of P C P A resulted in a decline in ICS at CPU sites, with minimal levels reaching 48 ~o of control on post-drug day 4. The raw data were analyzed by a 2 way analysis of variance which revealed a significant main effect (F = 5.03, df 1, 12, P < 0.05). Simple main effects were also observed for each group; i.e. CPU, (F = 3.01, df6.72, P < 0.05), hypothalamus, (F = 5.96, df6.72, P < 0.05), Multiple comparisons were made utilizing the Tukey procedure (Winer, 1971) in order to identify post-drug scores that were significantly different from
A. G. Phillips et al. : Para-Chlorophenylalanine and Self-Stimulation
pre-drug controls. This analysis revealed that PCPA produced a significant increase in hypothalamic ICS on post-drug day 3 and a significant decrease in CPU-ICS on post-drug day 4.
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DISCUSSION The present findings suggest that brain stimulation reward in the neostriatum can be influenced by neural systems in addition to the dopaminergic NSB (Phillips et al., 1976). A serotonergic mechanism is clearly implicated by the temporary suppression of ICS at neostriatal sites following treatment with PCPA. Although this drug has been shown to have other effects in addition to its action on serotonergic neurons the disruption of noradrenaline biosynthesis appears to have a shorter time span, in the order of 24 h (Welch and Welch, 1967) whereas the lowest levels of serotonin are reached 3 days after drug treatment, with levels returning to approximately 5 0 ~ of normal values by day seven (Koe and Weisman, 1966). The initial suppression of ICS at both hypothalamic and CPU placements, 24 h after injection of PCPA may well reflect the inhibition of catecholamine synthesis. This finding is similar to the inhibitory effect of PCPA on hypothalamic ICS, as reported by Stark and Fuller (1972). On the other hand, the more dramatic facilitatory effect on hypothalamic ICS occurs 3 days after drug treatment, at a time which correlates well with the maximal depletion of serotonin levels. In this regard, the present data confirm the earlier observations of Poschel and Ninteman (1971). It may appear inconsistent that ICS in both hypothalamus and CPU should return to control when the biochemical levels are still significantly less than normal. However, recent experiments on disruption of feeding and drinking following damage to catecholamine systems suggest that deple-
Fig. 1. Location of maximal penitration of electrodes tracts from subjects in the caudate-putamen group (N = 8) and lateral hypothalamus group (N = 6). Coronal sections are redrawn from the atlas of Pelligrino and C u s h m a n (1967)
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LH
175-
150-
A
125-
