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Behavioural and biochemical consequences of persistent overstimulation of mesolimbic dopamine systems in the rat
CopyrIght
002%3YO8;82,040327-05’603 00.0 Q 1982 Prrgamon Press Ltd
BEHAVIOURAL AND BIOCHEMICAL CONSEQUENCES PERSISTENT OVERSTIMULATION OF MESOLIMBIC DOPAMINE SYSTEMS IN THE RAT B. COSTALL, A. M. DOMENEY and Postgraduate
School
of Studies Bradford
OF
R. J. NAYLOR
in Pharmacology, University BD7 lDP, England
of Bradford.
(Accepted 21 October 1981) separated into high and low activity responders on the basis of their response, in terms of hyperactivity, to peripherally administered (-)N-n-propylnorapomorphine [( -)NPA] were subject to chronic infusion of dopamine or its solvent bilaterally into the mesolimbic system (nucleus accumbens) for 13 days via Alzet osmotic minipumps. Both high and low activity responders exhibited enhanced spontaneous locomotor activity during the infusion of dopamine (but not solvent) whether measurements were made over a 180-min period in individual photocell cages or over 24 hr via Automex activity meters in a grouped situation. The ability of (-)NPA to stimulate locomotor activity was virtually abolished in both high and low activity responders from the second day of the infusion of dopamine to its termination. This reduction continued for up to 42 days following withdrawal of dopamine from the high activity animals. In contrast, the animals initially classified as low activity responders gave markedly enhanced activity when challenged with (-)NPA 2-3 weeks after withdrawal from dopamine. Hyperactivity, caused both by infused dopamine and by peripherally administered (-)NPA, was shown to be selectively antagonized by neuroleptic agents. Solvent infusion did not alter behavioural responding from control either during or after infusion. Radioligand binding assays usingC3H]NPA showed three clear differences between the two selected groups of rats. First, there were significantly more [3H]NPA binding sites in the mesolimbic tissue of normal high activity responders than normal low activity animals; secondly, chronic dopamine infusion increased C3H]NPA binding sites for low activity animals at a time when their responses to (-)NPA were markedly enhanced; thirdly, chronic infusion of dopamine decreased the numbers of [3H]NPA binding sites in high activity responders at a time when their responses to (-)NPA were markedly reduced. Summary-Rats,
The involvement of the mesolimbic dopamine system in the control of motor behaviour is well established in experimental animals, and it has been suggested that an enhanced dopaminergic activity within this system may be involved in psychomotor disease states in man [see reviews by Carlsson (1978) and Hornykiewicz (1978)]. Knowledge on the importance of dopamine in the mesolimbic system for motor performance of animals is derived from data on the effects of acute drug-neurotransmitter treatment, but it is difficult to envisage a disease process in man as reflecting a single acute or transitory neurotransmitter disturbance. If psychomotor disease states do involve an overactivity of dopamine in the mesolimbic system, this may be more realistically considered in terms of a persistent neurotransmitter change. In the present study, therefore, the behavioural and biochemical changes consequent on a persistent overstimulation of mesolimbic dopamine systems in the rat brain were investigated. METHODS
The studies utilized male Sprague-Dawley (CD) rats weighing 300 + 25 g at the time of initial stereotaxic surgery. Before surgery, the rats were selected into two groups, low and high activity responders (see Results section), on the basis of their locomotor 327
hyperactivity response to challenge with 0.05 mg/kg of (-)N-n-propylnorapomorphine [( -)NPA.HCI, Research Biochemicals Inc., prepared in O.l’j; sodium metabisulphite] given subcutaneously (Costall, Hui and Naylor, 1980). Locomotor activity was assessed using individual, screened perspex cages (25 x 15 x 15cm high), each fitted with one photocell unit placed off-centre. Interruptions of the light beams were recorded electromechanically and the level of locomotor activity expressed as a count per 5 min period. In addition to the measurement of hyperactivity, the rats were observed visually for the development of stereotyped behaviour which was simply scored as &absent. l-periodic sniffing, 22continuous sniffing, 3-periodic biting, 4--continuous biting. Scores were summated at each time of assessment for the construction of graphs of intensity (summated score) against time. To facilitate comparisons between the behavioural profiles plotted in this manner. the areas under the curves were determined for both stereotypy and hyperactivity. Since stereotypy graphs were prepared from a summated score, the number of animals per group was kept constant for comparative studies in which the significance of changes was assessed using the MannWhitney U-test. Hyperactivity data was normally distributed and the significance of changes assessed using the Student’s t-test.
32x
B. COSTALL ct trl. /PP25
140-45mml
a=
injection
b= guide
unit,
cannula,
12.5mm
I0~30mmdiameterl
9.0
IO.65
mm
guide tips. After the 14 days, the animals were lightly anaesthetized with ether for the subcutaneous implantation into the back neck region of two Alzet@ osmotic minipumps each attached via 3-4 mm PP25 and 4&45 mm PP60 polythene tubing to stainless steel injection units (0.3 mm diam.) which were made to fit permanently into the previously implanted guides in place of the stylets, but terminating 3.5 mm below the guide tips at the centre of the nucleus accumbens (see Fig. 1). The pumps had been previously filled with dopamine solution (4.16 pg/Ltl. dopamine,HCI, Koch Light, prepared in N, bubbled solution of O.l”,, sodium metabisulphite). or its solvent, and the entire injection unit primed for between 5 and 8 hr at 37 C. The design of the intracerebral injection units allowed the procedure to continue with no obvious disturbance to the free movement and behavioural repertoire of the animal. Complications were absent when the pumps were filled and implanted using a careful aseptic technique; there were no obvious signs of tissue reaction to the pumps when these were removed after 13 days. Maintenance of good health also depended on consistent post-operative care. The pumps delivered dopamine or its solvent at a constant rate of 0.48 $/hr from the time of implantation, and thus provided a dose of dopamine into the accumbens of 50~~ over a 24.hr period. Although the minipumps were designed to deliver solution at a constant rate for I4 days. removal on day 13 precluded any “fall-off” effect when the dose may not be accurately determined. The present experiments utilized three groups of low and high activity responders which were each further subdivided into (1) normal. untreated animals, (2) animals receiving infusion of dopamine, (3) animals receiving infusion of solvent. One group of normal~dopamineesolvent rats was used to assess changes in spontaneous locomotor activity both during the delivery of dopaminc solvent and after its discontinuation. a second group was used to follow the possible changes in the locomotor hyperactivity and stereotypy responses to (-)NPA both during and after infusion, whilst the third group was used to charactcrisc the mechanism underlying the change in spontaneous locomotion during the dopamine infusion. The latter studies used haloperidol (Janssen, prepared in loo lactic acid), a-fluphenthixol. HCI (Lundbeck, distilled water). propranolol.HCI (I.C.I.. distilled water). piperoxan.HCI (Roche, distilled water) and methysergide hydrogen maleinate (Sandoz. distilled water) given intraperitoneally 30 min before the animals were placed in individual photocell cages for assessment of spontaneous locomotor activity (counts recorded electromechanically every IO min but cumulated over 60 min to give a measure of spontaneous locomotion). Behavioural experiments were continued for 42 days after removal of the osmotic minipumps. Animals used in tests of responsiveness to (-)NPA or which had received the antagonists were then sacrithe
13-4mml
mm diametetl
Fig. 1. Diagrammatic
representation of the construction of a guide cannula and an intracerebral injection unit attached to an Alzet osmotic minipump (although shown as single units, implantations were always bilateral). The 9.0-mm-long guide cannulae. held in perspex holders. were implanted chronically. When the injection umta were inserted, 9.5 mm stylets (0.30 diam. stainless steel) were removed. The injection units. of similar construction. were made to extend 3.5 mm below the guide tips (termination at the centre of the nucleus accumbens): a length of 0.65 mm diam. stainless steel tubing acted as a stop. Bcforc surgery. the latter was angled at approx. 45 : at surgery. this angle was readjusted with fine pliers to approx. 30 with the underlying perspcx of the cannula holder drilled away to allow a correctly angled placement to be individually tailored for each animal (firmly fixed with acrylic cement). The angled tubing was attached via a short length of PP25 polythene tubing (3 4 mm) and via 40 45 mm of PP60 tubing to an Alzet osmotic minipump. The entire unit, pump attached via polythene tubmg to angled stop and injection unit. was primed with solvent or dopamine solution before surgery. and at the time of implantation the commencement of pumping was shown by the appearance of a fluid dl-oplct at the tip of the injection unit.
In addition to (-)NPA-induced hyperactivity, spontaneous locomotion of both high and low activity responding animals was measured before and after dopamine infusion into the mesolimbic region using both the photocell technique for individual rats and Automcx activity meters for rats housed in groups of five. The general plan throughout the experiments was to place housing cages permanently on the Automex activity meters, with a record being made at 60-min intervals on automatic print-out facilities. and to remove animals for a fixed 180-min period per day (09.0&12.00 hr) for assessment of responses in the photocell cages. Significance in the change in Automex counts was assessed using the Student’s t-test. Both housing and behavioural testing rooms were maintained at a temperature of 21 k 2 C. The selected animals were subject to standard stereotaxic techniques for the implantation of chronically indwelling guide cannulae for subsequent bilateral intracerebral injection at the centre of the nucleus accumbens [Ant. 9.4, Vert. 0.0, Lat. + 1.6, (De Groot, 1959)]. Rats were anaesthetized with chloral hydrate (300 mg/kg i.p.) and placed in a Kopf stereotaxic instrument. Implanted guides were constructed of stainless steel. 0.65 mm diam., held bilaterally in perspex holders. Guides terminated 3.5 mm above the centre of the nucleus accumbens and were kept patent for a 14-day recovery period using stainless steel stylets, 0.3 mm diam.. which extended 0.5 mm beyond
Chronic
intra-accumhens
dopamine
infusion
329
2 x 5 ml ice cold Tris-HCl buffer: the bound radioactivity was determined. Specific binding was defined as the difference between C3H]NPA binding in the presence or absence of 10 pM ADTN (2-amino-6,7-dihydroxytetralin) and under optimal conditions accounted for approx. 607; of total C3H]NPA binding. RESULTS Sqwatinn
,013
.025
,05
.l mg/kg S.C. I-INPA
Fig. 2. Dose-dependency of hyperactivity to (-)NPA of animals separated into activity groups (A) high (n = 12), (B) intermediate 01 = 22). IC) low ()J = 21) and (D) zero (II = 5). Hyper~~ctivity was measured in photocell cages as counts:5 min. Example SEMs given.
ficed for histological confirmation of site of infusion. The infusion site for dopamine was located as an area of deposition of the oxidative products of dopamine which invariably extended from the area of the nucleus accumbens to the tuberculum olfactorium: in no experiment could the absence or presence or degree of deposition of oxidative products of dopamine be correlated with any behavioural or biochemical change. The site of infusion of the solvent was located. at the time of sacrifice, from the site of termination of the guide cannulae tracks. Animals which had been tested only for changes in spontaneous locomotion were sacrificed for biochemical determination of any change in the binding of C3H]NPA caused by dopamine-solvent infusion. For the radioligand binding assays, tissue was taken from each of the three groups of high and low activity animals. Concentrations of C3H]NPA (0.1.-2.0 nM) were selected to demonstrate a saturable binding (see Costall, Fortune, Law, Naylor, Neumeyer and Nohria, 1980) and allow Scatchard analyses; the limited amount of mesolimbic tissue available precluded repeated experiments. Mesohmbic tissue. nucleus accumbens plus tuberculum olfactorium, approx. weight 10 mg per side, was dissected out over ice and homogenized in 15 mM Tris-HCl buffer with a Polytron homogenizer (setting “5” for 10 set). The homogenate was centrifuged twice (10 min, 5000 g) at 4°C with resuspension in fresh buffer. After incubation at 37‘C for 1Omin the homogenate was again centrifuged twice with resuspension in fresh buffer. Final resuspension was in Tris-HCI buffer (pH 7.4 at 25°C) containing 5 mM Na,EDTA. Each assay tube contained 5mg wet wt mesoiimbic tissue [equivalent to approx. 3oOpg protein, measured by the method of Lowry, Rosebrough, Farr and Randall (1951)] in a total volume of 1.1 ml. After incubation (20min, 25°C) with C3H]NPA (58.5Ci/mmol, New England Nuclear) samples were rapidly filtered over Whatman GF/B filters and rinsed rapidly with
@‘rats into high nnd k~w nctirity respondrrs
Within any group of nOrId, non-selected animals challenged with 0.05 mgjkg S.C. (-)NPA (a dose selected from a previous study; Costall rt ul., 19X0), a certain proportion always gave high intensity locomotor hyperactivity responses (60-80 counts/5 min, total), others a “low intensity” response l&20”/ (l&25 counts/5 min. 3@40”;, total). others responded as an “intermediate” group (30-60 counts/S min, 3@40’:: total) whilst others, 10-20’;& total, gave no locomotor hyperactivity response at all. Animals initially divided into the four groups on the basis of their locomotor responses to O.O5mg/kg (-)NPA maintained the distinctions in motor performance when subject to a full f -)NPA dose response analysis (Fig. 2). The intermediate group and zero activity group were eliminated from subsequent studies and the other two groups, demonstrating the extreme differences in locomotor activity, selected as the “low activity” and “high activity” responders. There were no significant differences between the time courses and intensities of the stereotyped responses of these two groups of rats (Fig. 3) (indeed there was no difference between any of the four groups when subjected to a full dose range of (-)NPA, [email protected] mg,/kg, results not shown). The differential time courses for the development of locomotor hyperactivity and stereotypy allowed a separation: thus at 0.05 mg/kg S.C. (-)NPA (Fig. 3) stereotypy developed rapidly after administration with a maximum apparent at 20-30min whilst locomotor activity developed after a delay of some 60min (high activity group) with a maximum intensity apparent at 9&150min. It is emphasized that the development of stereotyped behaviour does not necessarily obscure the development of hyperactivity at the doses used: thus. during the 6@-90min after (-)NPA injection the rats demonstrated an increasing hyperactivity against a backgrollnd of moderate-intense stereotypy and this hyperactivity was then only slightly increased, notwithstanding a marked decrease in stereotYPY. The spontaneous locomotor activity levels of both of rats were indistinguishable whether groups measurements were made by housing animals individually in the photocell cages or in groups on the Automex activity meters (Fig.4). It is particularly noticeable from the data obtained from the Automex meters that the marked diurnal variations were closely followed by rats from both activity groups (Fig. 4).
330
I
1
10 20
,
40
60
80 Time
Low
~~f,v,fy
100 I min
120
140
160
180
I
responder
Es!Highacttvityresponder Fig. 3. Different time courses for hyperactivity and stereotyped behaviour induced by 0.05 mg/kg (se.) of (-)NPA. Hyperactivity, measured in photocell cages as counts/5 min, is shown for both low (em) and high (m----H) activity responders and similarly, stereotyped behaviour, scored and presented in histogram form as summated scores, is shown for both low and high activity responders. Time is given in minutes following the administration of (-)NPA. n = 5. SEMs 5.22135%.
Efkt of i~&sion qf dopamine into thr accumbens on locomotor rrsp0n.sivfnes.s spontaneous locomotor activity was When measured in individual photocell cages over a 60-min period between 9 a.m. and 12 noon a distinctive pattern of increased locomotion could be seen in both the high and low activity responders receiving dopamine infusion into the accumbens. Peaks of
Photocell
Measure
m
s : ._ E
8
hyperactivity were apparent on days 3-6 of the infusion and again on days 10 and 11 (Fig. 5). There were no significant differences between the increases in spontaneous locomotion caused by the dopamine infusion in either the low or high activity responders (P > 0.05). Solvent infusion into the accumbens did not significantly alter the spontaneous locomotor responses of either groups of rats as compared with control values prior to infusion (Fig. 5).
AutomexMeasure
8r
2.304.306.308.3010.3Opm 12.302.304.306.306.3010.30am t Lights
out
Fig. 4. Spontaneous locomotor activity of rats selected into high (HA) and tow (LA) activity responders on the basis of their responses to (-)NPA. Spontaneous locomotion was measured both in individual photocell cages over a 60-min period (expressed as counts/60 min, SEMs shown, n = 5) and by housing in groups of five and placing the cage for measurement of locomotor reactivity by Automex activity meters (automatic printout at 60-min intervals, O-0 low and m--_-E high activity responders).
Chronic
1 +
2
t
3
intra-accumbens
I
‘
I
4
5
6
dopamine
J
789
I
*
10
11
days of infusion
implantation
Of pumps
infusion
*
12
i
13 t Pumps removed
331
1Y---i%86 days after infusion
Fig. 5. Photocell measure (individual cages for a 60-min period, shown in countsj60min. measured between 09.00 and 12.00 am. each day) of changes in spontaneous locomotor activity of rats caused by the slow, discrete infusion of dopamine (50 ,xg delivered over a 24-hr period at a rate of 0.48 .nl:hr) into the mesolimbic tissue of rats selected as e--O low and m--m high activity responders. The spontaneous locomotion of low and high activity animals receiving infusion of dopamine solvent (0.48 &hr) is shown as -0 and ii&--U respectively. Responses are shown for the 13 days of infusion and 38 days post-infusion. Day 1 shows responses obtained in the morning before pump implantation in the afternoon. Postoperative-anaesthetic effect is shown in the reduced readings from the morning of day 2. n = 7. SEMs 2.5145”;. ,”
Readings taken over 24hr periods using the Automex activity meters and grouped rats also showed dopamine that infused into the accumbens, but not solvent, raised the level of spontaneous locomotor activity responding in both the low and high
‘ 0
1
*
1
3 1 2 4 implantation of pumps
*
4
*
‘
*
? 8 days of infusion
g
5
*
6
(
groups of rats (P < 0.01-P < 0.001, Fig. 6). Although there was a tendency for the development of peaks of hyperactivity on days 3 and 6. particularly in the rats classified as high activity responders. the clear biphasic development of hyperactivity observed with
activity
L ‘0
* ”
‘
t
i2 i3 Pumps removed
-*,b-J 7 14 26 38 ’ days after infusion
Fig. 6. Automex measure (from groups of five rats; readings taken at 60-mitt intervals were plotted for a 24-hr period and the area under the curve determined, values given here in arbitrary units) of changes in spontaneous locomotor activity of rats caused by the slow, discrete infusion of dopamine (S0/1g delivered over a 24-hr period at a rate of 0.48 &hr) into the mesolimbic tissue of rats selected as C---m low and m---m high activity responders. The spontaneous locomotion of low and high activity animals receiving infusion of dopamine solvent (0.48 &hr) is shown as O---O and U--U respectively. Responses are shown for the 13 days of infusion and 38 days post-infusion. 0 indicates the responses of animals during the 24 hr prior to implantation of pumps. Postoperativeeanaesthetic motor depression is shown clearly for the first day following pump impl~tation~ the depression of locomotion was also clear during the morning of day 2 (as detected using the photocell technique, see Fig. 4) but was somewhat masked as activity increased during the night period.
332
B.
et ul,
COSTALL
Duringinfusion
After
Infusion
O.?r
OL III
c2
I
I
I
I
4
6
8
10
a,
12 +
im~antatjon
Pumps
of pumps
removed
I
*
I
7
14
21
I
26
1
35
I
42days
Fig. 7. Locomotor hyperactivity and stereotypy responses to (-)NPA.
(0.05 mg/kg SC.), both during the 13 days of infusion with dopamine or its solvent and for 42 days after discontinuation of infusion. O---O indicates the response to (-)NPA of low activity animals receiving dopamine, O-O low activity animals receiving solvent, B--M high activity animals receiving dopamine and U---O high activity animals receiving solvent. Hyperactivity responses to (-)NPA were measured in counts;5 min. stereotypy responses were scored; this data was plotted graphically from onset to termination and the overall responses measured as areas under the hyperactivity and stereotypy curves; areas representing the hyperactivity or stereotypy response are expressed in arbitrary units. tz = IO. SEMs 4.5 I I.Yo
single rats between 9 a.m. and 12 noon was not apparent when measurements were taken over 24-hr periods from grouped rats (Fig. 6).
The ability of (-)NPA to stimulate locomotor activity was virtually abolished (P < 0.001) in both low and high activity responders from the second day of dopamine infusion to its termination. this being most marked in the group of rats which normally gave a marked response to (-)NPA (Fig, 7). Thirteen days of solvent infusion did not at any time significantly alter the responses to (-)NPA of either high or low activity responders (P > 0.05, Fig. 7). Stereotyped responding to f-)NPA was markedly reduced by the dopamine infusion, but not by the solvent (P < 0.001, Fig. 7). After discontinuation of infusion of solvent all animals maintained levels of hyperactivity and stereotypy responses to (-)NPA which were indistingLlishable from control values obtained before infusion (Fig. 7). The reduced stereotypy responses observed in both groups of animals receiving chronic dopamine returned towards normal over the 5-week period after discontinuing the infusion, although a reduction below solvent values was still apparent by the fifth
week (P < 0.01). In contrast, animals initially selected as low hyperactivity responders to (-)NPA showed markedly enhanced hyperactivity responses (P < 0.001) which developed between 2 and 3 weeks after dopamine withdrawal and then persisted at the level of normal high activity responders to the termination of the experiment. In further contrast, the markedly reduced responses to (-)NPA of animals which initially gave high responses remained below or approximating to the normal responsiveness of rats initially classified as low activity responders (reduction in responding highly significant, P < 0.001). Effects qf‘ potential antagonists 011 the incrrusrd sponlocomotor uctirity caused by the intro-orrumhens in~usiurlof (~opurnin~, taneous
Of the potential antagonists tested, only pretreatment with haloperidol and a-Hupenthixol was shown to antagonize the increased locomotion caused by the dopamine infusion, both during days 3-5 and 10 and 11; propranolol, piperoxan and methysergide were without effect (Table 1). @%cts C$ chronic ~l~.~~~jn~hi~ dopnmine irtfision WI the number of‘ [31-I)NPA hinding sites in mesolimhic tissue Three clear differences could be identified between groups of animals from the binding studies. First, that there were significantly more [‘H]NPA binding sites
Chronic Table
I. Selective locomotion
intra-accumbens
dopamine
antagonism by neuroleptics of the enhanced spontaneous caused by dopamine infusion into the accumbens Hyperactivity Dose (mg/kg i.p.)
Antagonist
tissue low
than
795 and that
normal 190 fmol/mg
chronic
of normal activity
protein,
infusion
of
high
activity
(B,,,
animals respectively),
dopamine
134 129 110 149 141
ani-
values
secondly,
increased
the
in animals initially classified as low activity responders but showing a markedly enhanced response at the time of sacrifice (from B,,, 190 to 415 fmol/mg protein) and, thirdly, that the chronic dopamine infusion can, conversely, decrease the C3H]NPA binding sites in mesolimbic tissue of rats classified initially as high activity responders but exhibiting a markedly reduced response to (-)NPA at the time of sacrifice (from B,,, 795 to 485 fmol/mg protein) (Fig. 8). Whilst Scatchard analysis revealed clear changes in the number of binding sites, the similarity in K, values 0.760.96 nM indicated no change in receptor affinity.
number
of binding
min)
After antagonist
Before antagonist
n = 5. SEMs are given. *Reduction significant to P < 0.001 (Student’s
mals
(counts/5
-
0.1 0.05 5.0 10.0 2.5
Haloperidol a-Flupenthixol Propranolol Piperoxan Methysergide
in the mesolimbic
333
infusion
sites for C3H]NPA
f f jI & f
7 6 12 12 21
52 37 137 168 153
f 12’ + 5* * 9 f 11 17
r-test)
action in rats of differing sensitivities, It is emphasized that there was no difference in the hyperactivity response to dopamine between high and low activity groups. The prolonged infusion of dopamine into the nucleus accumbens led to deposition of oxidative products of dopamine in both the areas of the nucleus accumbens and tuberculum olfactorium. This possibly
DISCUSSION
The present study was initially directed to the development of a simple method to allow dopamine to be infused chronically into cerebral tissue. The technique described, of coupling intracerebral injection units to Alzet osmotic minipumps proved entirely satisfactory for the injection of dopamine into a mesolimbic area of the rat brain. Using this technique the effects of persistent dopamine stimulation from the area of the nucleus accumbens on (a) spontaneous motor behaviour and (b) dopamine agonist induced behavioural effects of hyperactivity and stereotypy were determined, with particular regard to the importance of basal locomotor activity levels for determining drug action. (-)N-n-propylnorapomorphine [( -)NPA] proved to be a most useful dopamine agonist for causing both dose-related hyperactivity and dose-related stereotypy which can be dissociated on a temporal basis (Costall et al., 1980). The present use of (-)NPA allowed a clear subdivision of rats into two groups, those that responded with a marked hyperactivity and those which did not. The stereotypy response was the same in all animals. This subdivision of rats into “high” and “low” activity responders was the essential basis of the attempt to distinguish differences in drug
0
100
200
300
400
500
600
f mol bound
700
/mg
800 protein
Fig. 8. Scatchard analyses of effects of dopamme infusion into the accumbens on the binding of C3H]NPA to mesolimbic tissue. Data is given for low activity animals subject to infusion of vehicle (0) or dopamine (0) and high activity animals subject to vehicle (0) or dopamine (m). The vehicle treatments did not significantly alter the r3H]NPA binding from normal, the normal control values for low and high activity animals being K, = 0.94 nM, B,,, = 205 fmol;mg protein and K, = 0.88 nM. E,,, = 730 fmoljmg protein respectively. At the time of sacrifice (6 weeks after discontinuing infusion) the dopamine-treated rats showed maximally changed locomotor hyperactivity responding to (-)NPA, low activity animals showing a markedly enhanced response. high activity animals a reduced response. n = 6 (the limited amount of mesolimbic tissue precluding replicate experiments). SEMs < IO”,,.
334
B. COSTALL et al.
reflects a diffusion of dopamine from the deposition site to the tuberculum olfactorium and, therefore, the discussion is confined to the effects of dopamine infusion into the “mesolimbic area” of the rat brain. During such an infusion the rats exhibited enhanced locomotor responsiveness, measured both as individual activity in the photocell cages and as grouped activity on the Automex activity meters. Using the photocell technique of measurement, the increased locomotion over the 13-day period of infusion was shown to assume a biphasic pattern, peak responses being obtained on days 3-5 and again on days 10 and 11 of infusion. This biphasic pattern was not apparent from the less precise measure of group “reactivity”, although this technique clearly showed a raised level of locomotor responding during the 13-day period of infusion. That discrete injections of dopamine into the mesolimbic area of the rat brain can increase locomotor activity has been shown in numerous studies using the acute intracerebral injection technique (Pijnenburg and Van Rossum. 1973; Costall and Naylor, 1975), although these have frequently assessed the dopamine response after monoamine oxidase inhibition; here it is demonstrated that the chronic infusion of dopamine into mesolimbic tissue could enhance locomotor activity in the absence of monoamine oxidase inhibition. During this stage of infusion when locomotor activity-reactivity was enhanced, the responsiveness of rats to both the hyperactivityand stereotypy-inducing actions of the dopamine agonist, ( -)NPA, was markedly reduced, which may be behavioural reflections of reduced receptor “sensitivity”, a term used here in its broadest sense, during the period of persistent dopamine stimulation. It is interesting that animals initially selected as low and high activity responders to challenge with (-)NPA were indistinguishable in tests for spontaneous locomotion before, during, and after the period of dopamine infusion, and were indistinguishable in their non-responsiveness to (-)NPA during infusion. The only differentiation between these two groups, other than in their initial selection. was in their changed responsiveness to the hyperactivity component of the action of (-)NPA after termination of dopamine infusion. As with observations made before and during infusion, there were no differences in the stereotypy patterns of the two groups of rats during the weeks following infusion; the ability of (-)NPA to induce stereotypy reappeared simultaneously and to the same extent in both groups, slowly returning towards control values (although, even 5 weeks after dopamine withdrawal, this behavioural component was still of reduced intensity). In complete contrast, the two groups of high and low activity rats did give differential hyperactivity responses to (-)NPA after withdrawal of dopamine, but the nature of the change in responsiveness of the two groups was virtually to reverse the initial classification Thus. animals initially classified as high ac-
tivity responders to (-)NPA maintained the low activity response, established during infusion, for the entire 5-week post-infusion period, whilst those animals initially classified as low activity responders demonstrated, within 2-3 weeks of ceasing the dopamine infusion, a persistent high activity response to (-)NPA which approximated in intensity to responses of control high activity animals. Observations made at the behavioural level were mirrored by the biochemical changes, although the use of [3H]NPA to label dopamine receptors (see Creese, Padgett, Fazzini and Lopez, 1979; Titeler and Seeman, 1979) only allowed correlations to be made with the activity component of behavioural responses to (-)NPA. Thus, normal or animals treated with control solvent which were shown to give a high hyperactivity response to (-)NPA challenge were found to possess more binding sites for (-)NPA in their mesolimbic areas than normal or control animals giving low activity responses to (-)NPA. After 13 days of continuous dopamine infusion, when high activity animals exhibited markedly reduced responses to (-)NPA, their normal number of mesolimbic C3H]NPA binding sites was shown to be markedly reduced whilst the numbers of [3H]NPA binding sites in the mesolimbic tissue of initially low activity responders were significantly increased above control values. It was of interest that the affinity of the receptors for [“H]NPA did not change. That the behavioural and biochemical consequences of chronic mesolimbic dopamine infusion can correlate with the basal locomotor responsiveness of rats to a dopamine agonist is the most important finding of the present study. In order to detect the differential changes caused by persistent overstimulation in the mesolimbic dopamine system, not only is careful preselection of rats essential, but this must be further selective in terms of motor response studied (in the present situation, hyperactivity and not stereotypy) and careful in terms of methodology of behavioural measure (for hyperactivity, individual or grouped responses). The changes which occur as a consequence of persistent mesolimbic dopamine stimulation would appear to involve an interaction with dopamine receptors since both the response to the dopamine itself and to the (-)NPA, which is used to characterize the behavioural change, were specifically antagonized by the neuroleptic agents haloperidol and r-flupenthixol. Whilst the present data are preliminary in that further doses of dopamine and other agonists are clearly required to establish the specificity of action, the concept is proposed that overstimulation of dopamine mechanisms may finally function to reduce an “active” motor state or increase the activity of an “underactive” state, and this would appear particularly pertinent to an understanding of behavioural change consequent on exaggerated mesolimbic dopamine function, or on peripheral administration of a dopamine agonist to modify motor function.
Chronic
intra-accumbens
Ackno~~lrdyements This work was supported by the Wellcome Trust. The authors are grateful for gifts of drugs from I.C.I., Janssen Pharmaceutics, Lundbeck, Research Biochemicals Inc.. Roche and Sandoz. REFERENCES Carlsson A. (1978) Antipsychotic drugs, neurotransmitters, and schizophrenia. Am. J. Psychiat. 135: 164173. Costall B.. Fortune D. H., Law S-J.. Naylor R. J., Neumeyer J. L. and Nohria V. (1980) (-)N-(Chloroethyl)norapomorphine inhibits striatal dopamine function via irreversible receptor binding. Nature, Lond. 285: 571-573. Costall B.. Hui S.-C. G. and Naylor R. J. (1980) Denervation in the dopaminergic mesolimbic system: funtional changes followed using (-)N-n-propylnorapomorphine depend on the basal activity levels of rats. Neuropharmacolog_r 19: 1039-1048. Costall B. and Naylor R. J. (1975) The behavioural effects of dopamine applied intracerebrally to areas of the mesolimbic system. Eur. J. Pharmac. 32: 87792.
dopamine
infusion
335
Creese I., Padgett L., Fazzini E. and Lopez F. (1979) [‘H]N-n-propyl-norapomorphine: a novel agonist ligand for central dopamine receptors. Eur. .I. Pharmac. 56: 411-412. De Groot J. (1959) The rat forebrain in stereotaxic coordinates Verh. K. Ned. Akad. Wet. 52: 1439. Hornykiewicz 0. (1978) Psychopharmacological implications of dopamine and dopamine antagonists: a critical evaluation of current evidence. Neuroscience 3: 773-783.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. hiol. Chem. 193: 2655275. Pijnenburg A. J. J. and Van Rossum J. M. (1973) Stimulation of locomotor activity following injection of dopamine into the nucleus accumbens. J. Pharm. PharMUC. 25:
1003~1005.
Titeler M. and Seeman P. (1979) Selective labelling of different dopamine receptors by a new agonist C3H]ligand: [3H]N-propylnorapomorphine. Eur. J. Pharmac. 56: 29
I -292.
002%3YO8;82,040327-05’603 00.0 Q 1982 Prrgamon Press Ltd
BEHAVIOURAL AND BIOCHEMICAL CONSEQUENCES PERSISTENT OVERSTIMULATION OF MESOLIMBIC DOPAMINE SYSTEMS IN THE RAT B. COSTALL, A. M. DOMENEY and Postgraduate
School
of Studies Bradford
OF
R. J. NAYLOR
in Pharmacology, University BD7 lDP, England
of Bradford.
(Accepted 21 October 1981) separated into high and low activity responders on the basis of their response, in terms of hyperactivity, to peripherally administered (-)N-n-propylnorapomorphine [( -)NPA] were subject to chronic infusion of dopamine or its solvent bilaterally into the mesolimbic system (nucleus accumbens) for 13 days via Alzet osmotic minipumps. Both high and low activity responders exhibited enhanced spontaneous locomotor activity during the infusion of dopamine (but not solvent) whether measurements were made over a 180-min period in individual photocell cages or over 24 hr via Automex activity meters in a grouped situation. The ability of (-)NPA to stimulate locomotor activity was virtually abolished in both high and low activity responders from the second day of the infusion of dopamine to its termination. This reduction continued for up to 42 days following withdrawal of dopamine from the high activity animals. In contrast, the animals initially classified as low activity responders gave markedly enhanced activity when challenged with (-)NPA 2-3 weeks after withdrawal from dopamine. Hyperactivity, caused both by infused dopamine and by peripherally administered (-)NPA, was shown to be selectively antagonized by neuroleptic agents. Solvent infusion did not alter behavioural responding from control either during or after infusion. Radioligand binding assays usingC3H]NPA showed three clear differences between the two selected groups of rats. First, there were significantly more [3H]NPA binding sites in the mesolimbic tissue of normal high activity responders than normal low activity animals; secondly, chronic dopamine infusion increased C3H]NPA binding sites for low activity animals at a time when their responses to (-)NPA were markedly enhanced; thirdly, chronic infusion of dopamine decreased the numbers of [3H]NPA binding sites in high activity responders at a time when their responses to (-)NPA were markedly reduced. Summary-Rats,
The involvement of the mesolimbic dopamine system in the control of motor behaviour is well established in experimental animals, and it has been suggested that an enhanced dopaminergic activity within this system may be involved in psychomotor disease states in man [see reviews by Carlsson (1978) and Hornykiewicz (1978)]. Knowledge on the importance of dopamine in the mesolimbic system for motor performance of animals is derived from data on the effects of acute drug-neurotransmitter treatment, but it is difficult to envisage a disease process in man as reflecting a single acute or transitory neurotransmitter disturbance. If psychomotor disease states do involve an overactivity of dopamine in the mesolimbic system, this may be more realistically considered in terms of a persistent neurotransmitter change. In the present study, therefore, the behavioural and biochemical changes consequent on a persistent overstimulation of mesolimbic dopamine systems in the rat brain were investigated. METHODS
The studies utilized male Sprague-Dawley (CD) rats weighing 300 + 25 g at the time of initial stereotaxic surgery. Before surgery, the rats were selected into two groups, low and high activity responders (see Results section), on the basis of their locomotor 327
hyperactivity response to challenge with 0.05 mg/kg of (-)N-n-propylnorapomorphine [( -)NPA.HCI, Research Biochemicals Inc., prepared in O.l’j; sodium metabisulphite] given subcutaneously (Costall, Hui and Naylor, 1980). Locomotor activity was assessed using individual, screened perspex cages (25 x 15 x 15cm high), each fitted with one photocell unit placed off-centre. Interruptions of the light beams were recorded electromechanically and the level of locomotor activity expressed as a count per 5 min period. In addition to the measurement of hyperactivity, the rats were observed visually for the development of stereotyped behaviour which was simply scored as &absent. l-periodic sniffing, 22continuous sniffing, 3-periodic biting, 4--continuous biting. Scores were summated at each time of assessment for the construction of graphs of intensity (summated score) against time. To facilitate comparisons between the behavioural profiles plotted in this manner. the areas under the curves were determined for both stereotypy and hyperactivity. Since stereotypy graphs were prepared from a summated score, the number of animals per group was kept constant for comparative studies in which the significance of changes was assessed using the MannWhitney U-test. Hyperactivity data was normally distributed and the significance of changes assessed using the Student’s t-test.
32x
B. COSTALL ct trl. /PP25
140-45mml
a=
injection
b= guide
unit,
cannula,
12.5mm
I0~30mmdiameterl
9.0
IO.65
mm
guide tips. After the 14 days, the animals were lightly anaesthetized with ether for the subcutaneous implantation into the back neck region of two Alzet@ osmotic minipumps each attached via 3-4 mm PP25 and 4&45 mm PP60 polythene tubing to stainless steel injection units (0.3 mm diam.) which were made to fit permanently into the previously implanted guides in place of the stylets, but terminating 3.5 mm below the guide tips at the centre of the nucleus accumbens (see Fig. 1). The pumps had been previously filled with dopamine solution (4.16 pg/Ltl. dopamine,HCI, Koch Light, prepared in N, bubbled solution of O.l”,, sodium metabisulphite). or its solvent, and the entire injection unit primed for between 5 and 8 hr at 37 C. The design of the intracerebral injection units allowed the procedure to continue with no obvious disturbance to the free movement and behavioural repertoire of the animal. Complications were absent when the pumps were filled and implanted using a careful aseptic technique; there were no obvious signs of tissue reaction to the pumps when these were removed after 13 days. Maintenance of good health also depended on consistent post-operative care. The pumps delivered dopamine or its solvent at a constant rate of 0.48 $/hr from the time of implantation, and thus provided a dose of dopamine into the accumbens of 50~~ over a 24.hr period. Although the minipumps were designed to deliver solution at a constant rate for I4 days. removal on day 13 precluded any “fall-off” effect when the dose may not be accurately determined. The present experiments utilized three groups of low and high activity responders which were each further subdivided into (1) normal. untreated animals, (2) animals receiving infusion of dopamine, (3) animals receiving infusion of solvent. One group of normal~dopamineesolvent rats was used to assess changes in spontaneous locomotor activity both during the delivery of dopaminc solvent and after its discontinuation. a second group was used to follow the possible changes in the locomotor hyperactivity and stereotypy responses to (-)NPA both during and after infusion, whilst the third group was used to charactcrisc the mechanism underlying the change in spontaneous locomotion during the dopamine infusion. The latter studies used haloperidol (Janssen, prepared in loo lactic acid), a-fluphenthixol. HCI (Lundbeck, distilled water). propranolol.HCI (I.C.I.. distilled water). piperoxan.HCI (Roche, distilled water) and methysergide hydrogen maleinate (Sandoz. distilled water) given intraperitoneally 30 min before the animals were placed in individual photocell cages for assessment of spontaneous locomotor activity (counts recorded electromechanically every IO min but cumulated over 60 min to give a measure of spontaneous locomotion). Behavioural experiments were continued for 42 days after removal of the osmotic minipumps. Animals used in tests of responsiveness to (-)NPA or which had received the antagonists were then sacrithe
13-4mml
mm diametetl
Fig. 1. Diagrammatic
representation of the construction of a guide cannula and an intracerebral injection unit attached to an Alzet osmotic minipump (although shown as single units, implantations were always bilateral). The 9.0-mm-long guide cannulae. held in perspex holders. were implanted chronically. When the injection umta were inserted, 9.5 mm stylets (0.30 diam. stainless steel) were removed. The injection units. of similar construction. were made to extend 3.5 mm below the guide tips (termination at the centre of the nucleus accumbens): a length of 0.65 mm diam. stainless steel tubing acted as a stop. Bcforc surgery. the latter was angled at approx. 45 : at surgery. this angle was readjusted with fine pliers to approx. 30 with the underlying perspcx of the cannula holder drilled away to allow a correctly angled placement to be individually tailored for each animal (firmly fixed with acrylic cement). The angled tubing was attached via a short length of PP25 polythene tubing (3 4 mm) and via 40 45 mm of PP60 tubing to an Alzet osmotic minipump. The entire unit, pump attached via polythene tubmg to angled stop and injection unit. was primed with solvent or dopamine solution before surgery. and at the time of implantation the commencement of pumping was shown by the appearance of a fluid dl-oplct at the tip of the injection unit.
In addition to (-)NPA-induced hyperactivity, spontaneous locomotion of both high and low activity responding animals was measured before and after dopamine infusion into the mesolimbic region using both the photocell technique for individual rats and Automcx activity meters for rats housed in groups of five. The general plan throughout the experiments was to place housing cages permanently on the Automex activity meters, with a record being made at 60-min intervals on automatic print-out facilities. and to remove animals for a fixed 180-min period per day (09.0&12.00 hr) for assessment of responses in the photocell cages. Significance in the change in Automex counts was assessed using the Student’s t-test. Both housing and behavioural testing rooms were maintained at a temperature of 21 k 2 C. The selected animals were subject to standard stereotaxic techniques for the implantation of chronically indwelling guide cannulae for subsequent bilateral intracerebral injection at the centre of the nucleus accumbens [Ant. 9.4, Vert. 0.0, Lat. + 1.6, (De Groot, 1959)]. Rats were anaesthetized with chloral hydrate (300 mg/kg i.p.) and placed in a Kopf stereotaxic instrument. Implanted guides were constructed of stainless steel. 0.65 mm diam., held bilaterally in perspex holders. Guides terminated 3.5 mm above the centre of the nucleus accumbens and were kept patent for a 14-day recovery period using stainless steel stylets, 0.3 mm diam.. which extended 0.5 mm beyond
Chronic
intra-accumhens
dopamine
infusion
329
2 x 5 ml ice cold Tris-HCl buffer: the bound radioactivity was determined. Specific binding was defined as the difference between C3H]NPA binding in the presence or absence of 10 pM ADTN (2-amino-6,7-dihydroxytetralin) and under optimal conditions accounted for approx. 607; of total C3H]NPA binding. RESULTS Sqwatinn
,013
.025
,05
.l mg/kg S.C. I-INPA
Fig. 2. Dose-dependency of hyperactivity to (-)NPA of animals separated into activity groups (A) high (n = 12), (B) intermediate 01 = 22). IC) low ()J = 21) and (D) zero (II = 5). Hyper~~ctivity was measured in photocell cages as counts:5 min. Example SEMs given.
ficed for histological confirmation of site of infusion. The infusion site for dopamine was located as an area of deposition of the oxidative products of dopamine which invariably extended from the area of the nucleus accumbens to the tuberculum olfactorium: in no experiment could the absence or presence or degree of deposition of oxidative products of dopamine be correlated with any behavioural or biochemical change. The site of infusion of the solvent was located. at the time of sacrifice, from the site of termination of the guide cannulae tracks. Animals which had been tested only for changes in spontaneous locomotion were sacrificed for biochemical determination of any change in the binding of C3H]NPA caused by dopamine-solvent infusion. For the radioligand binding assays, tissue was taken from each of the three groups of high and low activity animals. Concentrations of C3H]NPA (0.1.-2.0 nM) were selected to demonstrate a saturable binding (see Costall, Fortune, Law, Naylor, Neumeyer and Nohria, 1980) and allow Scatchard analyses; the limited amount of mesolimbic tissue available precluded repeated experiments. Mesohmbic tissue. nucleus accumbens plus tuberculum olfactorium, approx. weight 10 mg per side, was dissected out over ice and homogenized in 15 mM Tris-HCl buffer with a Polytron homogenizer (setting “5” for 10 set). The homogenate was centrifuged twice (10 min, 5000 g) at 4°C with resuspension in fresh buffer. After incubation at 37‘C for 1Omin the homogenate was again centrifuged twice with resuspension in fresh buffer. Final resuspension was in Tris-HCI buffer (pH 7.4 at 25°C) containing 5 mM Na,EDTA. Each assay tube contained 5mg wet wt mesoiimbic tissue [equivalent to approx. 3oOpg protein, measured by the method of Lowry, Rosebrough, Farr and Randall (1951)] in a total volume of 1.1 ml. After incubation (20min, 25°C) with C3H]NPA (58.5Ci/mmol, New England Nuclear) samples were rapidly filtered over Whatman GF/B filters and rinsed rapidly with
@‘rats into high nnd k~w nctirity respondrrs
Within any group of nOrId, non-selected animals challenged with 0.05 mgjkg S.C. (-)NPA (a dose selected from a previous study; Costall rt ul., 19X0), a certain proportion always gave high intensity locomotor hyperactivity responses (60-80 counts/5 min, total), others a “low intensity” response l&20”/ (l&25 counts/5 min. 3@40”;, total). others responded as an “intermediate” group (30-60 counts/S min, 3@40’:: total) whilst others, 10-20’;& total, gave no locomotor hyperactivity response at all. Animals initially divided into the four groups on the basis of their locomotor responses to O.O5mg/kg (-)NPA maintained the distinctions in motor performance when subject to a full f -)NPA dose response analysis (Fig. 2). The intermediate group and zero activity group were eliminated from subsequent studies and the other two groups, demonstrating the extreme differences in locomotor activity, selected as the “low activity” and “high activity” responders. There were no significant differences between the time courses and intensities of the stereotyped responses of these two groups of rats (Fig. 3) (indeed there was no difference between any of the four groups when subjected to a full dose range of (-)NPA, [email protected] mg,/kg, results not shown). The differential time courses for the development of locomotor hyperactivity and stereotypy allowed a separation: thus at 0.05 mg/kg S.C. (-)NPA (Fig. 3) stereotypy developed rapidly after administration with a maximum apparent at 20-30min whilst locomotor activity developed after a delay of some 60min (high activity group) with a maximum intensity apparent at 9&150min. It is emphasized that the development of stereotyped behaviour does not necessarily obscure the development of hyperactivity at the doses used: thus. during the 6@-90min after (-)NPA injection the rats demonstrated an increasing hyperactivity against a backgrollnd of moderate-intense stereotypy and this hyperactivity was then only slightly increased, notwithstanding a marked decrease in stereotYPY. The spontaneous locomotor activity levels of both of rats were indistinguishable whether groups measurements were made by housing animals individually in the photocell cages or in groups on the Automex activity meters (Fig.4). It is particularly noticeable from the data obtained from the Automex meters that the marked diurnal variations were closely followed by rats from both activity groups (Fig. 4).
330
I
1
10 20
,
40
60
80 Time
Low
~~f,v,fy
100 I min
120
140
160
180
I
responder
Es!Highacttvityresponder Fig. 3. Different time courses for hyperactivity and stereotyped behaviour induced by 0.05 mg/kg (se.) of (-)NPA. Hyperactivity, measured in photocell cages as counts/5 min, is shown for both low (em) and high (m----H) activity responders and similarly, stereotyped behaviour, scored and presented in histogram form as summated scores, is shown for both low and high activity responders. Time is given in minutes following the administration of (-)NPA. n = 5. SEMs 5.22135%.
Efkt of i~&sion qf dopamine into thr accumbens on locomotor rrsp0n.sivfnes.s spontaneous locomotor activity was When measured in individual photocell cages over a 60-min period between 9 a.m. and 12 noon a distinctive pattern of increased locomotion could be seen in both the high and low activity responders receiving dopamine infusion into the accumbens. Peaks of
Photocell
Measure
m
s : ._ E
8
hyperactivity were apparent on days 3-6 of the infusion and again on days 10 and 11 (Fig. 5). There were no significant differences between the increases in spontaneous locomotion caused by the dopamine infusion in either the low or high activity responders (P > 0.05). Solvent infusion into the accumbens did not significantly alter the spontaneous locomotor responses of either groups of rats as compared with control values prior to infusion (Fig. 5).
AutomexMeasure
8r
2.304.306.308.3010.3Opm 12.302.304.306.306.3010.30am t Lights
out
Fig. 4. Spontaneous locomotor activity of rats selected into high (HA) and tow (LA) activity responders on the basis of their responses to (-)NPA. Spontaneous locomotion was measured both in individual photocell cages over a 60-min period (expressed as counts/60 min, SEMs shown, n = 5) and by housing in groups of five and placing the cage for measurement of locomotor reactivity by Automex activity meters (automatic printout at 60-min intervals, O-0 low and m--_-E high activity responders).
Chronic
1 +
2
t
3
intra-accumbens
I
‘
I
4
5
6
dopamine
J
789
I
*
10
11
days of infusion
implantation
Of pumps
infusion
*
12
i
13 t Pumps removed
331
1Y---i%86 days after infusion
Fig. 5. Photocell measure (individual cages for a 60-min period, shown in countsj60min. measured between 09.00 and 12.00 am. each day) of changes in spontaneous locomotor activity of rats caused by the slow, discrete infusion of dopamine (50 ,xg delivered over a 24-hr period at a rate of 0.48 .nl:hr) into the mesolimbic tissue of rats selected as e--O low and m--m high activity responders. The spontaneous locomotion of low and high activity animals receiving infusion of dopamine solvent (0.48 &hr) is shown as -0 and ii&--U respectively. Responses are shown for the 13 days of infusion and 38 days post-infusion. Day 1 shows responses obtained in the morning before pump implantation in the afternoon. Postoperative-anaesthetic effect is shown in the reduced readings from the morning of day 2. n = 7. SEMs 2.5145”;. ,”
Readings taken over 24hr periods using the Automex activity meters and grouped rats also showed dopamine that infused into the accumbens, but not solvent, raised the level of spontaneous locomotor activity responding in both the low and high
‘ 0
1
*
1
3 1 2 4 implantation of pumps
*
4
*
‘
*
? 8 days of infusion
g
5
*
6
(
groups of rats (P < 0.01-P < 0.001, Fig. 6). Although there was a tendency for the development of peaks of hyperactivity on days 3 and 6. particularly in the rats classified as high activity responders. the clear biphasic development of hyperactivity observed with
activity
L ‘0
* ”
‘
t
i2 i3 Pumps removed
-*,b-J 7 14 26 38 ’ days after infusion
Fig. 6. Automex measure (from groups of five rats; readings taken at 60-mitt intervals were plotted for a 24-hr period and the area under the curve determined, values given here in arbitrary units) of changes in spontaneous locomotor activity of rats caused by the slow, discrete infusion of dopamine (S0/1g delivered over a 24-hr period at a rate of 0.48 &hr) into the mesolimbic tissue of rats selected as C---m low and m---m high activity responders. The spontaneous locomotion of low and high activity animals receiving infusion of dopamine solvent (0.48 &hr) is shown as O---O and U--U respectively. Responses are shown for the 13 days of infusion and 38 days post-infusion. 0 indicates the responses of animals during the 24 hr prior to implantation of pumps. Postoperativeeanaesthetic motor depression is shown clearly for the first day following pump impl~tation~ the depression of locomotion was also clear during the morning of day 2 (as detected using the photocell technique, see Fig. 4) but was somewhat masked as activity increased during the night period.
332
B.
et ul,
COSTALL
Duringinfusion
After
Infusion
O.?r
OL III
c2
I
I
I
I
4
6
8
10
a,
12 +
im~antatjon
Pumps
of pumps
removed
I
*
I
7
14
21
I
26
1
35
I
42days
Fig. 7. Locomotor hyperactivity and stereotypy responses to (-)NPA.
(0.05 mg/kg SC.), both during the 13 days of infusion with dopamine or its solvent and for 42 days after discontinuation of infusion. O---O indicates the response to (-)NPA of low activity animals receiving dopamine, O-O low activity animals receiving solvent, B--M high activity animals receiving dopamine and U---O high activity animals receiving solvent. Hyperactivity responses to (-)NPA were measured in counts;5 min. stereotypy responses were scored; this data was plotted graphically from onset to termination and the overall responses measured as areas under the hyperactivity and stereotypy curves; areas representing the hyperactivity or stereotypy response are expressed in arbitrary units. tz = IO. SEMs 4.5 I I.Yo
single rats between 9 a.m. and 12 noon was not apparent when measurements were taken over 24-hr periods from grouped rats (Fig. 6).
The ability of (-)NPA to stimulate locomotor activity was virtually abolished (P < 0.001) in both low and high activity responders from the second day of dopamine infusion to its termination. this being most marked in the group of rats which normally gave a marked response to (-)NPA (Fig, 7). Thirteen days of solvent infusion did not at any time significantly alter the responses to (-)NPA of either high or low activity responders (P > 0.05, Fig. 7). Stereotyped responding to f-)NPA was markedly reduced by the dopamine infusion, but not by the solvent (P < 0.001, Fig. 7). After discontinuation of infusion of solvent all animals maintained levels of hyperactivity and stereotypy responses to (-)NPA which were indistingLlishable from control values obtained before infusion (Fig. 7). The reduced stereotypy responses observed in both groups of animals receiving chronic dopamine returned towards normal over the 5-week period after discontinuing the infusion, although a reduction below solvent values was still apparent by the fifth
week (P < 0.01). In contrast, animals initially selected as low hyperactivity responders to (-)NPA showed markedly enhanced hyperactivity responses (P < 0.001) which developed between 2 and 3 weeks after dopamine withdrawal and then persisted at the level of normal high activity responders to the termination of the experiment. In further contrast, the markedly reduced responses to (-)NPA of animals which initially gave high responses remained below or approximating to the normal responsiveness of rats initially classified as low activity responders (reduction in responding highly significant, P < 0.001). Effects qf‘ potential antagonists 011 the incrrusrd sponlocomotor uctirity caused by the intro-orrumhens in~usiurlof (~opurnin~, taneous
Of the potential antagonists tested, only pretreatment with haloperidol and a-Hupenthixol was shown to antagonize the increased locomotion caused by the dopamine infusion, both during days 3-5 and 10 and 11; propranolol, piperoxan and methysergide were without effect (Table 1). @%cts C$ chronic ~l~.~~~jn~hi~ dopnmine irtfision WI the number of‘ [31-I)NPA hinding sites in mesolimhic tissue Three clear differences could be identified between groups of animals from the binding studies. First, that there were significantly more [‘H]NPA binding sites
Chronic Table
I. Selective locomotion
intra-accumbens
dopamine
antagonism by neuroleptics of the enhanced spontaneous caused by dopamine infusion into the accumbens Hyperactivity Dose (mg/kg i.p.)
Antagonist
tissue low
than
795 and that
normal 190 fmol/mg
chronic
of normal activity
protein,
infusion
of
high
activity
(B,,,
animals respectively),
dopamine
134 129 110 149 141
ani-
values
secondly,
increased
the
in animals initially classified as low activity responders but showing a markedly enhanced response at the time of sacrifice (from B,,, 190 to 415 fmol/mg protein) and, thirdly, that the chronic dopamine infusion can, conversely, decrease the C3H]NPA binding sites in mesolimbic tissue of rats classified initially as high activity responders but exhibiting a markedly reduced response to (-)NPA at the time of sacrifice (from B,,, 795 to 485 fmol/mg protein) (Fig. 8). Whilst Scatchard analysis revealed clear changes in the number of binding sites, the similarity in K, values 0.760.96 nM indicated no change in receptor affinity.
number
of binding
min)
After antagonist
Before antagonist
n = 5. SEMs are given. *Reduction significant to P < 0.001 (Student’s
mals
(counts/5
-
0.1 0.05 5.0 10.0 2.5
Haloperidol a-Flupenthixol Propranolol Piperoxan Methysergide
in the mesolimbic
333
infusion
sites for C3H]NPA
f f jI & f
7 6 12 12 21
52 37 137 168 153
f 12’ + 5* * 9 f 11 17
r-test)
action in rats of differing sensitivities, It is emphasized that there was no difference in the hyperactivity response to dopamine between high and low activity groups. The prolonged infusion of dopamine into the nucleus accumbens led to deposition of oxidative products of dopamine in both the areas of the nucleus accumbens and tuberculum olfactorium. This possibly
DISCUSSION
The present study was initially directed to the development of a simple method to allow dopamine to be infused chronically into cerebral tissue. The technique described, of coupling intracerebral injection units to Alzet osmotic minipumps proved entirely satisfactory for the injection of dopamine into a mesolimbic area of the rat brain. Using this technique the effects of persistent dopamine stimulation from the area of the nucleus accumbens on (a) spontaneous motor behaviour and (b) dopamine agonist induced behavioural effects of hyperactivity and stereotypy were determined, with particular regard to the importance of basal locomotor activity levels for determining drug action. (-)N-n-propylnorapomorphine [( -)NPA] proved to be a most useful dopamine agonist for causing both dose-related hyperactivity and dose-related stereotypy which can be dissociated on a temporal basis (Costall et al., 1980). The present use of (-)NPA allowed a clear subdivision of rats into two groups, those that responded with a marked hyperactivity and those which did not. The stereotypy response was the same in all animals. This subdivision of rats into “high” and “low” activity responders was the essential basis of the attempt to distinguish differences in drug
0
100
200
300
400
500
600
f mol bound
700
/mg
800 protein
Fig. 8. Scatchard analyses of effects of dopamme infusion into the accumbens on the binding of C3H]NPA to mesolimbic tissue. Data is given for low activity animals subject to infusion of vehicle (0) or dopamine (0) and high activity animals subject to vehicle (0) or dopamine (m). The vehicle treatments did not significantly alter the r3H]NPA binding from normal, the normal control values for low and high activity animals being K, = 0.94 nM, B,,, = 205 fmol;mg protein and K, = 0.88 nM. E,,, = 730 fmoljmg protein respectively. At the time of sacrifice (6 weeks after discontinuing infusion) the dopamine-treated rats showed maximally changed locomotor hyperactivity responding to (-)NPA, low activity animals showing a markedly enhanced response. high activity animals a reduced response. n = 6 (the limited amount of mesolimbic tissue precluding replicate experiments). SEMs < IO”,,.
334
B. COSTALL et al.
reflects a diffusion of dopamine from the deposition site to the tuberculum olfactorium and, therefore, the discussion is confined to the effects of dopamine infusion into the “mesolimbic area” of the rat brain. During such an infusion the rats exhibited enhanced locomotor responsiveness, measured both as individual activity in the photocell cages and as grouped activity on the Automex activity meters. Using the photocell technique of measurement, the increased locomotion over the 13-day period of infusion was shown to assume a biphasic pattern, peak responses being obtained on days 3-5 and again on days 10 and 11 of infusion. This biphasic pattern was not apparent from the less precise measure of group “reactivity”, although this technique clearly showed a raised level of locomotor responding during the 13-day period of infusion. That discrete injections of dopamine into the mesolimbic area of the rat brain can increase locomotor activity has been shown in numerous studies using the acute intracerebral injection technique (Pijnenburg and Van Rossum. 1973; Costall and Naylor, 1975), although these have frequently assessed the dopamine response after monoamine oxidase inhibition; here it is demonstrated that the chronic infusion of dopamine into mesolimbic tissue could enhance locomotor activity in the absence of monoamine oxidase inhibition. During this stage of infusion when locomotor activity-reactivity was enhanced, the responsiveness of rats to both the hyperactivityand stereotypy-inducing actions of the dopamine agonist, ( -)NPA, was markedly reduced, which may be behavioural reflections of reduced receptor “sensitivity”, a term used here in its broadest sense, during the period of persistent dopamine stimulation. It is interesting that animals initially selected as low and high activity responders to challenge with (-)NPA were indistinguishable in tests for spontaneous locomotion before, during, and after the period of dopamine infusion, and were indistinguishable in their non-responsiveness to (-)NPA during infusion. The only differentiation between these two groups, other than in their initial selection. was in their changed responsiveness to the hyperactivity component of the action of (-)NPA after termination of dopamine infusion. As with observations made before and during infusion, there were no differences in the stereotypy patterns of the two groups of rats during the weeks following infusion; the ability of (-)NPA to induce stereotypy reappeared simultaneously and to the same extent in both groups, slowly returning towards control values (although, even 5 weeks after dopamine withdrawal, this behavioural component was still of reduced intensity). In complete contrast, the two groups of high and low activity rats did give differential hyperactivity responses to (-)NPA after withdrawal of dopamine, but the nature of the change in responsiveness of the two groups was virtually to reverse the initial classification Thus. animals initially classified as high ac-
tivity responders to (-)NPA maintained the low activity response, established during infusion, for the entire 5-week post-infusion period, whilst those animals initially classified as low activity responders demonstrated, within 2-3 weeks of ceasing the dopamine infusion, a persistent high activity response to (-)NPA which approximated in intensity to responses of control high activity animals. Observations made at the behavioural level were mirrored by the biochemical changes, although the use of [3H]NPA to label dopamine receptors (see Creese, Padgett, Fazzini and Lopez, 1979; Titeler and Seeman, 1979) only allowed correlations to be made with the activity component of behavioural responses to (-)NPA. Thus, normal or animals treated with control solvent which were shown to give a high hyperactivity response to (-)NPA challenge were found to possess more binding sites for (-)NPA in their mesolimbic areas than normal or control animals giving low activity responses to (-)NPA. After 13 days of continuous dopamine infusion, when high activity animals exhibited markedly reduced responses to (-)NPA, their normal number of mesolimbic C3H]NPA binding sites was shown to be markedly reduced whilst the numbers of [3H]NPA binding sites in the mesolimbic tissue of initially low activity responders were significantly increased above control values. It was of interest that the affinity of the receptors for [“H]NPA did not change. That the behavioural and biochemical consequences of chronic mesolimbic dopamine infusion can correlate with the basal locomotor responsiveness of rats to a dopamine agonist is the most important finding of the present study. In order to detect the differential changes caused by persistent overstimulation in the mesolimbic dopamine system, not only is careful preselection of rats essential, but this must be further selective in terms of motor response studied (in the present situation, hyperactivity and not stereotypy) and careful in terms of methodology of behavioural measure (for hyperactivity, individual or grouped responses). The changes which occur as a consequence of persistent mesolimbic dopamine stimulation would appear to involve an interaction with dopamine receptors since both the response to the dopamine itself and to the (-)NPA, which is used to characterize the behavioural change, were specifically antagonized by the neuroleptic agents haloperidol and r-flupenthixol. Whilst the present data are preliminary in that further doses of dopamine and other agonists are clearly required to establish the specificity of action, the concept is proposed that overstimulation of dopamine mechanisms may finally function to reduce an “active” motor state or increase the activity of an “underactive” state, and this would appear particularly pertinent to an understanding of behavioural change consequent on exaggerated mesolimbic dopamine function, or on peripheral administration of a dopamine agonist to modify motor function.
Chronic
intra-accumbens
Ackno~~lrdyements This work was supported by the Wellcome Trust. The authors are grateful for gifts of drugs from I.C.I., Janssen Pharmaceutics, Lundbeck, Research Biochemicals Inc.. Roche and Sandoz. REFERENCES Carlsson A. (1978) Antipsychotic drugs, neurotransmitters, and schizophrenia. Am. J. Psychiat. 135: 164173. Costall B.. Fortune D. H., Law S-J.. Naylor R. J., Neumeyer J. L. and Nohria V. (1980) (-)N-(Chloroethyl)norapomorphine inhibits striatal dopamine function via irreversible receptor binding. Nature, Lond. 285: 571-573. Costall B.. Hui S.-C. G. and Naylor R. J. (1980) Denervation in the dopaminergic mesolimbic system: funtional changes followed using (-)N-n-propylnorapomorphine depend on the basal activity levels of rats. Neuropharmacolog_r 19: 1039-1048. Costall B. and Naylor R. J. (1975) The behavioural effects of dopamine applied intracerebrally to areas of the mesolimbic system. Eur. J. Pharmac. 32: 87792.
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