CHANGED EATING AND LOCOMOTOR BEHAVIOUR IN THE RAT AFTER 6-HYDROXYDOPAMINE LESIONS TO THE SUBSTANTIA NIGRA
CYNTHIA BROOK and SUSAN D. IVERSEN Department of Experimental Psychology.
University
of Cambridge,
Downing
Street, Cambridge
Summary -Bilateral 6-hydroxydopamine lesions to the substantia nigra of the rat result in SS-90~: loss of striatal dopamine. In such animals. amphetamine does not induce stereotyped behaviour but the running response to lower doses of amphetamine is unaffected. The lesion does, however. produce permanent changes in the spontaneous locomotor behaviour of the rat. Aphagia was observed only in animals with greater ditional forebrain noradrenaline loss.
striatal
dopamine
depletion
which was associated
with ad-
For many years it has been known that amphetamine interacts with endogenous amine transmitter systems in the brain and more recent neuropharmacological studies have strengthened this contention. In a variety of in vitro preparations (GLOWINSKI and BALDESSARINI, 1966) and during in uiuo perfusion of the ventricular system of the brain (MCKENZIE and SZERB, 1968; CARR and MOORE, 1969) it has been verified that amphetamine releases the endogenous catecholamines noradrenaline (NA) and dopamine (DA) from synaptic storage sites. In addition, fluorescence histochemical techniques have been developed for staining endogenous amines. These reveal that the amine transmitters NA and DA are localized to different neuronal systems having cell bodies in the hind and mid brain and fibres projecting to and innervating different forebrain structures (UNGERSTEDT, 1971a). For those seeking a correlation between endogenous amine transmitters and normal or drug-induced behaviour, this anatomical information opened up the prospect of investigating the role of amine transmitters at specific sites in the nervous system with different aspects of behavioural control. At low doses, amphetamine induces marked increases in spontaneous motor behaviour. Catecholamines are necessary for this behavioural effect of amphetamine because a-methyltyrosine (E-MT), which prevents the synthesis of NA and DA in amine neurones, has been shown to antagonize this stimulatory effect of amphetamine. However, z-MT, like many other biochemical agents of current interest, interferes with both DA and NA mechanisms and has therefore failed to provide definitive evidence of which of these amines is principally involved in amphetamine-induced stimulation of locomotor activity. Forebrain DA is almost entirely localized to a neuronal system arising from cells in the substantia nigra. Fibres of this system project to the corpus striatum, the so-called nigrostriatal tract, and selective lesions to this pathway provide a direct test of whether this part of the DA system is vital for the locomotor response to amphetamine. 6-Hydroxydopamine (6-OHDA), a chemical analogue of NA and DA, which, if injected into the brain. is taken up by and destroys amine-containing neurones and terminals and so provides an appropriate chemical lesion technique. If injected locally into the substantia nigra, severe damage of the nigro-striatal tract is produced in the absence of non-specific damage to nearby anatomical pathways (UNGERSTEDT, 1971 b). Using such a lesion technique, we have investigated in the adult rat, the locomotor responses to various doses of amphetamine after bilateral 6-OHDA lesions to the substantia nigra. 95
METHODS
Subjects Male albino Wistar rats of mean weight 240g were used for these experiments. were housed in groups of four under standard laboratory conditions with controlled ing and fed ad lihitum on dry pellet food. 6-Hydrosydopamine
They light-
lesiors to the suh.statltilr nigra
Rats were anaesthetized with Nembutal, used undiluted in a dose of 0.5 ml:lOOg body weight. They were then placed in a stereotaxic apparatus (Kopf Instruments). and the head angled according to the De Groot stereotaxic atlas, by placing the nose bar 5 mm above the intraaural line. Stereotaxic co-ordinates for the substantia nigra were modified after DE GR~OT (1959) for 240 g rats and the placements were A-P, 2.5 mm; L, 2 mm; H, 8 mm. A 30 gauge stainless steel cannula was lowered into the brain at this placement and after attaching a 5-~1 Hamilton syringe, a 6-OHDA or vehicle control injection was slowly infused. To prevent oxidation of the 6-OHDA, it was dissolved in Merlis solution (MERLIS, 1940) containing l.Omg/ml ascorbic acid. The concentration was 4pggjkd of 6-OHDA (as free base) and 2 ,~l (i.e. 8 pg 6-OHDA) was injected into the substantia nigra on each side. The vehicle solution lacked 6-OHDA but except in this respect, the lesion and control procedures were identical. Observation
qf immediatr
post-oprraticr
hrhaviour
The animals were weighed each day and their eating and drinking behaviour noted. All animals were offered highly palatable wet mash on the immediate post-operative days. The rats which failed to eat and were loosing weight rapidly, were tube fed until they were able to sustain themselves. Some animals did not survive at this stage in the experiment. Photocell
measureme~~ts
qf locomotor
actir!itJ
Locomotion was measured in a bank of 12 identical photocell cages. These were modified wire mesh rat cages (25.4 x 38.1 x 20.3 cm) with two photocell units equally placed on the long axis giving horizontal beams 2.54 cm above floor level. The two photocell beams reliably record ambulation involving the whole floor space of the cage. This occurs with spontaneous activity and with the increased locomotor activity seen after low doses of amphetamine. When high doses of amphetamine are used. a form of stereotyped motor behaviour emerges in which certain responses are repeated to the exclusion of all other behaviour. Sniffing, neck movements, rearing, and in the most intense stereotypy, licking and gnawing of the cage are seen. Photocell cages are unsuitable for quantifying stereotypy and caution is required when using this measure as an indication of the occurrence and intensity of stereotypy. With large doses of amphetamine intense stereotypy occurs which tends to produce low photocell counts because behaviour is generated at one position in the cage. If the animal happens to show stereotypy within the range of a beam, high scores on one photocell can be recorded. However, lower doses of amphetamine, which predominantly stimulate locomotor activity. induce mild stereotyped running during the second hour after administration. This is the case with 1.5 mgjkg and therefore counts during the 2nd hr reflect psychomotor stimulation exacerbated by the emergence of stereotypy. It can be appreciated that if, for some reason. an animal showed psychomotor stimulation to amphetamine but not stereotypy. low doses (1.5 mg! kg) of the drug would tend to produce in such animals, low photocell counts during the test hour. compared with controls. After high doses, the reverse would be true: with controls exhibiting stereotypy in one location, producing low counts and animals not showing stereotypy running about. Therefore when stereotypy is occurring. direct observation of the animal is required to evaluate photocell counts. Tc>stinyproceduw The amphetamine studies were performed gia and adipsia associated with the substantia
2- 3 weeks post-operatively, nigra lesion had recovered.
when any aphaBehavioural tcst-
6-OHDA lesions in rat substantia
nigra
97
ing was carried out in the mornings. On test days the animals were placed in the photocell cages and their activity recorded every 10 min for 40 min. Intraperitoneal injections of control vehicle or (+)-amphetamine were then given and activity recorded for a further two hours. (+ )-Amphetamine sulphate was dissolved in 0.9% saline, which was used alone as the control vehicle injection. Amphetamine doses of 1.5, 3.0 and 5.0 mg/kg were studied in Experiment 1 and 1.5 mg/kg in Experiment 3. In both experiments the lesion and control animals received the various injections of drugs and vehicle in a balanced order. Biochemical
USSUJ~ ofamine
levels
At the end of the experiments the animals were killed with a blow on the head, the brain removed and dissected on ice. A series of shallow cuts was made from the ventral surface, to isolate the hypothalamus and olfactory turbercle from surrounding tissue, and the brain was bisected in the saggital plane. Four brain regions (preoptic region, thalamus and tegmentum of midbrain. cerebral cortex and corpus striatum) were dissected with small blunt forceps and iris scissors, care being taken to avoid the inclusion of tissue which did not form part of the region being dissected. The assay used was developed by EVE-IX, FI~ZSIMONS and SETLEK( 1972). Tissues were blotted, weighed and homogenized in a mixture consisting of 1.5 ml 0.4 M perchloric acid and 0.1 ml EDTA (40 mg/ml) containing ascorbic acid (4 mg/ml). After addition of an internal indicator made up from three parts Chlordphenol Red to one part Bromocresol Green, the homogenate was brought to pH 6.5 by the addition of 4 M-potassium hydroxide and 1.0 M K,CO,. Precipitated material was removed by cooling and centrifugation and the supernatant was passed through a 2.0 x 0.25 cm micro-column filled with treated Amberlite CG. 120 N+ form resin. The homogenate was washed with 3 ml distilled water, and noradrenaline and dopamine were eluted with 1.5-ml portions of 1 Mand 2 M hydrochloric acid respectively. Noradrenaline was estimated by the fluorimetric method of EULER and LISHAJKO(1961) and dopamine by the method of CARLSSONand WALDECK (1958). Recovery of catecholamines was S&90%. Values obtained from experimental animals are expressed as a percentage of sham-operated or untreated controls which were included in all assays. Expet-imental
procedures
ExprtG~ettt
1. Six substantia nigra lesioned and six sham operated controls which were minimally aphasic and adipsic post-operatively were given I .5, 3.0 and 5.0 mg/kg (+ )amphetamine (two injections of each dose and two injections of saline in a random order) starting three weeks post-operatively. At least two rest days elapsed between drug or saline treatments. Locomotor behaviour was recorded in the photocell cages. At the end of the experiment dopamine levels in the striatum were assayed. E.uperitnetzt 2. Twelve substantia nigra lesioned animals with appropriate controls were prepared. Their spontaneous and drug induced (1.5 mg/kg amphetamine) behaviour was recorded 48 and 76 hr post-operatively. It was then apparent that they were severely aphagic and adipsic. They were maintained by tube feeding but because no recovery of spontaneous feeding occurred they were sacrificed and DA and NA levels of various segments of the forebrain assayed. E.uperittwttt 3. Twelve substantia nigra lesioned and 12 sham operated controls were prepared. Seven substantia nigra rats were minimally aphagic. They maintained their weight after post-operative day 3 and were subsequently used for drug studies. The drug testing was organised as in Experiment 1. Striatal DA levels were determined at the end of the experiments. The remaining five severely aphagic substantia nigra animals did not recover. They were tube-fed, their weight monitored and changes in their spontaneous activity recorded daily until death. Statistical
ttlethods
Analysis of variance was used for all data analysis.
98
BRWKLHI~
CYNTHIA
SUSAN
D.
IVERS~\
RESULTS
Experiment
1
Immediatr post-operutiw hrhaviour. When the 6-OHDA animals recovered from the anaesthetic they were very active in the cages when compared with animals receiving vehicle injection under identical conditions. This activity lasted for several hours and probably reflects the immediate effects of disruption of the normal activity of the DA pathway. In some cases circling activity was observed. which may indicate asymmetric damage of the nigro-striatal pathway. similar to that which can be produced permanently if the substantia nigra on only one side of the midbrain is injected with 6-OHDA. 1.5
30
50
h k I\
I
,‘I\\
1’
1 \
I
’
::
\
:
I I :
\\ \
1
’\I 4 \ .
\
i’
‘\
b”\l
I
‘1\ -Control - -
6-OHDA
\/’
I
I
I
I
,
2
I
2
hr
I
I 2
after Amphetamine
Fig. I. Mean responses in photocell cages of control (II = 6) and substantia nigra lesioned animals 1.5. 3.0 and 5.0 mglkg (+)-amphetamine.The responses of these animals to placebo saline responses have been subtracted from the drug response. The experiment was performed I4 days post-operatively.
(n = 6) after
Responses to amphetamine. The 6 substantia nigra lesioned animals used in this experiment were minimally aphagic and adipsic and drug testing began 2 weeks post-operatively. The mean responses of these animals compared with that of the control animals, to 1.5.3.0 and 5.0 mg/kg of (+ )-amphetamine are shown in Figure 1. In controls and substantia nigra lesioned animals, the lower doses (1.5 and 3 mg/kg) produced a different response pattern over time than that elicited by 5 mg/kg. At 1.5 mg/kg, despite an apparent lower response during the second test hour, the substantia nigra animals gave a response of the same magnitude (d.f. 1, 10; F = 1.1, P > 0.05) and time course (d.f. 1. 11 ; 110; F = 0.38. P > 0.05) as the controls. This was also the case with 3 mg/kg. However, at 5 mg/kg, although the time course of the responding was strikingly similar in both groups. the 6OHDA animals showed an enhanced response. This difference did not reach significance but probably reflects reduction in locomotor scores in the control animals because of the intense stereotypy induced in them by 5 mg/kg (+ )-amphetamine. Stereotypy was blocked in the 6-OHDA animals and therefore they did not become immobile but continued to show psychomotor stimulation to amphetamine and thus recorded high photocell counts. Although there was no significant treatment effect in the 5 mg/kg experiment, inspection of the locomotor scores 30 and 60 min after the drug injection revealed that some substantia nigra animals, like controls. showed low locomotor scores and presumably stereotypy. This indicates that heterogeneity of variance exists and in these circumstances the analysis of variance would not be expected to prove significant. The range of DA depletion in the striatum of the substantia nigra animals is presented in Table I. If the degree of block of stereotypy as reported above is plotted against DA depletion, the animals which sustained severe damage to the nigrostriatal system (907; dcpletion) show blocked stereotypy and those with about 50”,:, of the system intact continue to show stereotypy responses to 5 mg/kg (+ )-amphetamine and hence low locomotor counts (Fig. 2). When calculated over the 2 hr session or on the 30 min or 60 min values, the amount of activity is positively correlated with the severity of DA depletion (Spearman’s rank correlation coefficient, I’, = 0.886, II = 6, P < 0.05). If animals happen
6-OHDA
Table
I. Regional
nigra
I 2 3 4 5
I 2 3 4 5 6 7
x 9 IO II 12 Mean
nigra lesions. The values are presented animals
NA Cortex
30
I.6 X.0 I.0 5.0 0.0 4.3”’1” -+ 1.6 4.0 17.0 4X.0 20.0 6.0 15.5 21.0 13.0 13.0 30.0 15.0 22.0 190”, * 3.4
Mean
6-OHDA Suhstantia nigra Non-aphagic
NA Hypothalamus
DA Striatum
99
nigra
NA and DA levels after bilateral 6-OHDA substantia as a 7; k S.E.M. of those levels in control
Rat 6-OH DA Substantia Aphagic
lesions in rat substantia
Thalamus
80 90
60 14 15 15 27:i f 9
NA and midbrain
84 67 34 28 25 48x& 12
33 38 60:/” k 14
to remain in the path of a photobeam while stereotyping, the behaviour is measured as a high count on one beam and none on the other. It is interesting that the one control animal which gave a locomotor count of 275 produced 274 of those counts in one photobeam. By contrast, equivalent scores in the substantia nigra animals were equally distributed between the two beams, an indication that movement around the cage was occurring. Experiment
2
Immediately after recovering from the anaesthetic these animals showed high levels of spontaneous motor behaviour which subsided within 24 hr to leave them hypokinetic (d.f. 1, 8; F = 6.620; P < 0.05). At this stage they showed a normal response to 1.5 mg/kg (+)amphetamine. However, within the next 24 hr, evidence of behavioural supersensitivity emerged, for at 76 hr the response to amphetamine was greatly enhanced in the substantia nigra lesioned animals (d.f. 1, 8; F = 14.926; P < 0.01) (Fig. 3). Drug studies on these animals were then terminated because of the severe aphagia and when it was clear that recovery was not likely to occur, the animals were sacrificed for regioned brain NA and DA assays. These results are presented in Table 1. For comparative purposes this Table also includes the striatal DA levels in twelve 6-OHDA substantia nigra lesioned rats which were not aphagic and adipsic [Experiment l(6) and 3(6)]. 500
+ I’
t 30-40mln
Y
r
2
5 g b ;; E 8
300
0 60-70
.
0
0
200
mfn
.
;f0
s
+
100
I
IO
I 20
%
DA
I 30
I 40
0
.
+I 50
a
Control
remaining
Fig. 2. The locomotor counts of individual substantia nigra rats at 3&40 and 6&70min after 5 mg/kg (+)-amphetamine plotted against the percentage of remaining striatal DA. Comparable measures on the control animals are presented to the right of the figure.
loo
CYNTHIA
BROOKand SC~SAND.
IWRS~~
800 .*-:
48hr
76hr
.“’
‘,
0--t
1’
‘*6-OHM
: I
l\.
/’
l \.,._
l
.,,,
lCon+ro’
./
10
I
I
I
30
50
70
J i
90 mm
30
50
70
90
after Amphetamine
.
Fig. 3. Locomotor response to I.5 mgikg (+)-amphetamine tested at 48 and 76 hr after 6.OHDA (dashed line) or sham lesions (solid line) to the substantia nigra. The appropriate saline scores were subtracted from the drug responses.
Exprrimrnt
3
Twelve substantia nigra and 12 control animals were prepared for this experiment. Five were severely aphagic and adipsic and their spontaneous motor behaviour was studied as their weight fell. Six of the remaining recovered substantia nigra animals and six controls were tested in the photocell cages with I.5 mg/kg ( + )-amphetamine and placebo at 14 and 21 days and 2 months after operation. The responses at 2 months are shown in Figure 4. There was a significant difference between the responses of the two groups (d.f. 1. 11; F = 5.6; P < 0.05) characterised by a steeper fall off of response during the second test hour in the substantia nigra animals (d.f. 1. 11; 242; F = 2.82; P < 0.01) indicating that stereotyped running was not present in these animals as it was in the controls. The responses to 1.5 mg/kg (+)-amphetamine had been similar at 14 and 21 days post-operatively in these animals and on none of these occasions had there been any evidence in the substantia nigra animals of blocked locomotor response during the first hour. Spontaneous locomotor activity was also assessed in these animals immediately postoperatively and during the drug testing sessions at 14 and 21 days and 2 months postoperatively. At 2448 hr after the operation. the substantia nigra animals were markedly hypokinetic when placed in the photocell cages (d.f. 1, 10; F = 41.2; P < O.OOl), (Fig. 5). 300-
i \
e Cl 'a
/'
\ \ \,e -0,
l \
*Control
Nlgral
I 0
1 20
1 40
I 60
1 80
1 100
1 120
min after Amphetamw
Fig. 4. Mean responses in photocell cages of control (II = 6) and substantia nigra (II = 6) lesioned animals after I.5 mg/kg (+ )-amphetamine. The saline responses have been subtracted from the drug response. The experiment was performed 2 months post-operatively.
6-OHDA
lesions in rat substantia
101
nigra
3CO-
0
E
Control
200-
: =
\
.
8 P a
,oo
6-OH? \
6 f
0
\
\
\
\ \
\
\
‘o,_
I
IO
.-•
--G_ I
20
\ --C--l--_-(_
30
40
50
60
rnl"l" cage
Fig, 5. Spontaneous
locomotor
activity during the second post-operative stantia nigra lesioned animals.
day of control
and sub-
When placed in such a novel environment, normal animals exhibit declining activity levels, or so-called habitation, over time. Despite initial hypokinesia, the substantia nigra animals also show this behaviour and over the hour test their activity habituated to the same low level as that of the controls. Similar measures were taken during the three subsequent drug experiments, when 40 min habituation runs were given before the amphetamine injections. The results obtained 2 months post-operatively are shown in Figure 6 and the responses ofthe substantia nigra animals were very similar to those obtained immediately post-operatively. The hypokinesia is thus a persistent effect of this 6-OHDA lesion and furthermore is not attenuated when animals experience the same situation at short intervals. The habituation results shown in the left and right of Figure 6 were obtained at a 2-day interval. At these times the substantia nigra animals, like controls, again showed habituation over the test period but further analysis revealed that the time course of the habituation change was different in the two groups (d.f. 5, 100; F = 11.5; P < 0.001). The striatal DA levels in the animals from Experiment 3 are also presented in Table 1. There was again variation in the degree of DA depletion which tended to correlate with the block of stereotypy as measured indirectly by photocell activity (Fig. 7). However the correlation was in the opposite direction to that reported with 5 mg/kg (Fig. 2); with 15 mg/kg (+)-amphetamine, a block of stereotypy (i.e. massive DA depletion) resulted in lower photocell counts, whereas with 5 mg/kg the opposite was true.
300-
m E g 200 -
.
-Control
= -
-6-OHDA
8 g a 6
IOO-
i
Fig. 6. Spontaneous
locomotor activity in control and substantia nigra lesioned animals post-operatively. The measures were made at a 2 day interval.
2 months
102
CYNTHIA
and SUSAN D. IVEKS~~
BK~OK
30m1n .
.
100 ml”
0
800 L
0
700 ~ 0 600 ~ 2 s 500 8 b z 400E 8 cl 300 J 200 -
. .
. ‘.
q
B
.
q
.
i
. .
.
E .
.
30
40
L
Control %
DA
B
. .
. .
100 1I11 10 20
i
I
!
40 Control
109+---
remo~nmg
Fig. 7. The locomotor counts 01”individual substantia nigra rats recorded at 30 and 100 min alter the two doses of I.5 mg/kg (+)-amphetamine. plotted against the percentage of remaining striatal DA. The comparable responses of control animals are presented to the right of the figures. The calculations were made on drug responses measured Z months post-operatively.
The mean weight of the aphagic animals dropped from 250 to 190 g over the first postoperative week. Their spontaneous motor behaviour in photocell cages over 1 hr test periods on days 5, 6 and 7 are shown in Figure 8, and compared with that of sham operated and non-aphagic substantia nigra lesioned animals. The non-aphagic animals remained hypokinetic but the aphagic animals showed escalating spontaneous activity over the days (d.f. 1, 8; F = 5.9; P < 0.05).
DISCUSSION
6-Hydroxydopamine lesions to the substantia nigra change spontaneous and drug-induced motor behaviours. The profile of these effects varies with the post-operative interval. presumably reflecting the changing neuropharmacological status of the nigrostriatal tract and its postsynaptic receptors, consequent upon 6-OHDA injection. During the first postoperative week, biochemical and morphological studies indicate that presynaptic terminals are in a state of change. FAULL and LAVERTY (1969) report that within hours of
Postoperative
doy
6
‘6-OHDA
30
60
30
6-OHDA
Aphaglc
Non-
Aphaglc
60
Fig. 8. Spontaneous locomotor activity over a I-hr period in sham operated control, non-aphagic and aphagic substantia nigra lesioned animals. The tests were made on post-operatwe days 5. h and 7.
6-OHDAlesions
in rat substantia
nigra
103.
the lesion, DA levels rise in the striatum possibly due to the immediate post-operative disruption of neurotransmission on the nigrostriatal pathway and reduction in the normal release of synaptically stored DA. After 1 day, DA levels begin to fall consequent upon 6-OHDA-induced degeneration of the terminals and loss of the stored amine. Dopamine levels then presumably rise in the synaptic cleft and this change could be expected to have effects on spontaneous motor behaviour. Gradually the released DA is degraded and less synaptically stored DA is available for normal or drug-induced release. The labile state of striatal synapses would be expected to result, over the first few post-operative days, in a changing and complex pattern of motor behaviour and variable drug responses as indicated by the amphetamine results reported in Experiment 2. However, by 14 days post-operatively, the nigrostriatal system had stabilised. At this time the changes in spontaneous and drug-induced motor behaviour due to loss of striatal DA can be assessed. These experiments, in agreement with the related ones of CREESEand IVERSEN(1972), show that depletion of striatal DA abolishes the stereotyped behaviour seen with various doses of (+)-amphetamine. This result merely confirms further that the nigrostriatal system is essential for stereotyped behaviour (RANDRUPand MUNKVAD,1970; FOG. 1972). The question of whether the same DA system or independent neurochemical pathways containing NA mediate the running response to amphetamine remains unsolved. All we can state is that on average, DA depletion of 85-9051:)in the Creese and Iversen experiments and of 8 1% in the present study, while sufficiently severe to abolish stereotypy, does not impair amphetamine-induced locomotor behaviour. In view of this result. it could still be maintained that the DA system is involved in this response. The remaining DA system may be capable of sustaining one motor behaviour and not another, i.e. fewer intact DA-containing neurones are required to sustain amphetamine stimulation than stereotypy. In this case a procedure which produced total DA depletion would abolish the amphetamine locomotor response and this has not yet been achieved. At present, there is no selective biochemical manipulation of brain amines that has been studied which does block the locomotor response to amphetamine. Three neurochemical procedures have been described which do block the response (1) z-MT (WEISSMANand KOE, 1966; STOLKand RECH, 1970) (2) intracerebral injections of 6-OHDA in the neonate (CREESEadd IVERSEN,1973) and (3) intraventricular injection of 6-OHDA in the adult rat, (IVERSEN,unpublished observations). These procedures have two consequences in common; virtually total depletion of both forebrain DA and NA. Their success could, therefore, be due to the completeness of the DA loss or the involvement of the NA systems. The weight ofevidence at present favours the latter interpretation (IVERSEN,1973; FIBIGER. FIBIGERand ZIS, 1973). One could hope that substantia nigra lesion techniques will be developed which will result in selective and total DA loss; but to date such complete lesions produce very sick animals which are totally aphagic and adipsic and, therefore. poor behavioural subjects. Furthermore, in our experiments these complete substantia nigra lesions result in involvement of NA pathways and the amine loss is not selective to the DA system. Noradrenaline fibres are known to traverse the mid brain in the close vicinity of the substantia nigra and presumably a 6-OHDA injection which damages the whole of the substantia nigra is likely to spread to the adjacent fibre systems. Any pursuit of this problem to achieve more selective lesions should be guided by another neglected fact; namely that although we refer to the nigro-striatal system it is clear that the DA system is not a simple unitary projection. Fibres arising from neurones slightly medial to the substantia nigra innervate the nucleus accumbens and the amygdala. Furthermore, fibres innervating the different elements of the basal ganglia arise from different parts of the substantia nigra. The brain dissection techniques in current use take no account of this complexity and the “striatal” segment includes, in one assay sample, most of this heterogenous DA terminal arrangement. It is conceivable that one part of this system is involved in stereotypy and another in the locomotor response and that our lesion procedures damage the former much more completely than the latter. In this case a figure of 81”/;,striatal DA depletion may not be very informative.
CYNTHIA BROOK and S~JSAND. IVERSEN
104
It is perhaps not surprising that it proves difficult to identify the neurological system mediating such a ubiquitous behaviour as locomotion. We assume that whenever it occurs locomotor behaviour is the same but behavioural analysis indicates that locomotion is a consequence of very many behavioural states. The present results emphasise the importance of describing the conditions under which locomotion is occurring. The substantia nigra lesions in these studies depressed the locomotion generated when the rat experienced a change of environment and this hypokinesia and abnormal habituation in a new environment is a permanent consequent of the substantia nigra lesion. However, once such an animal has habituated to the environment, the running induced by amphetamine is normal. Regarding the effects of nigral lesions on eating behaviour: these results indicate that. in general, the more severe the DA depletion, the more likely the animal is to show aphagia and adipsia. In view of the damage to the nigrostriated tract in these animals, one must conclude that the motor arousal associated with starvation is not mediated by a dopaminergic system. UNGERSTEDT (1971~) on the basis of histochemical analysis of striatal DA levels in substantia nigra lesioned animals, has also correlated aphagia and adipsia with the degree of DA loss. There are clearly exceptions, such as the animals in Experiment 1 with depletion of 96 and 94% striatal DA who were not severely aphagic and adipsic. Again total striatal DA may not prove the most reliable correlate of the eating disorder. However, in addition to this correlation with DA depletion. it is also clear that substantia nigra lesions which produce aphagia, result in substantial forebrain NA depletion as well. Possibly this is because a well placed cannulation of the substantia nigra which distributes 6-OHDA to virtually the whole of the substantia nigra, also involves some crucial part of the NA fibre systems which course through the midbrain in close vicinity to the substantia nigra. Classically, aphagia and adipsia have been observed after lateral hypothalamic lesions (TEITELBAUMand EPSTEIN, 1962). Recent histofluorescent findings indicate that the DA fibres of the nigrostriatal tract as well as ascending NA fibres pass through this region of the hypothalamus. UNGERSTEDT (1971~) therefore suggested that the classical lateral hypothalamic deficit described by TEITELBAUMand EPSTEIN(1962) after electrolytic lesions to the lateral hypothalamus was in fact due to severance of the DA fibres at this brain level. Several recent studies have demonstrated the behavioural similarity of the aphagia and adipsia associated with 6-OHDA lesions to the classical lateral hypothalamic syndrome (MARSHALL and TEITELRAUM.1973; FIBIGER, Zrs and MCGEER, 1973; ZIGMOND and STRICKER, 1973). However. in these investigations, in a study of electrolytic substantia nigra lesions (IVERSEN, 1971) and in the present study, both noradrenaline and dopamine were depleted. Selective lesions to the ventral and dorsal NA bundles (UNCXRSTEDT. 197 I b; CREESE,unpublished observations), which result in greater forebrain NA loss than substantia nigra lesions, do not produce aphagia (UNCXRSTWT, 1971b; CREESE.unpublished obscrvations). We are not claiming, therefore, an involvement of NA rather than DA in these feeding deficits. It does appear, however, that the data do not at present justify a simple shift of emphasis from NA to DA and an exclusive role for the nigrostriatal tract in feeding behaviour. Such a conclusion would ignore much evidence of a role of NA systems in certain aspects of the control of food intake (HOEBEL, 197 1) and reinforcement (STEIN. 1964). Not least important, it also totally neglects recent theoretical (BINDRA, 1968) and experimental work (CAMPBELL and BAEZ, 1974; CROW. 1973) which supports the view that the behaviour associated with need and its satisfaction are complex and involve the integration of several functionally distinct neural systems of the forebrain and we suggest. of their different neurotransmitters. Acknowledgements-Thiswork was supported by MRC grant C 97lj242 B to S.D.I. The authors thank Professor 0. L. ZANGWILLfor laboratory facilities and encouragement for this work. KLITH Evl.rrs performed
the biochemical
assays. REFERENCES
BINIIRA, D. (1968).Neuropsychologicalinterpretationof the effects of drive and incentive-motivation activity and instrumental behaviour. P.s~chol. Rev. 75: I-22.
on general
6-OHDA
lesions in rat suhstantia
nigra
105
CAMPIXI.L. B. A. and BA~,z, L. A. (1974). Dissociation of arousal and regulatory behaviours following lesions of the lateral hypothalamus. J. conzp. phq~siol. PsJx+~/. (In press). CARLSSON.A. and WALDUX. B. (1958). Fluorimetric determination of dopamine. Acta physiol. stand. 44: 293-299. CAKR. L. A. and MOORE, K. E. (1969). Norepinephrine: release from brain by o-amphetamine in rice. Science, N.Y. 164: 322---37x CRLIX. I. and IVI:KSFN.S. D. (1972). Amphetamine response in rat after dopamine neurone destruction. Nnturr ?jl+t Biol. 238: 247-248. C~ttst. 1. and IVERSEN.S. D. (1973). Blockage of amphetamine induced motor stimulation and stereotypy in the adult rat following neonatal treatment with 6-hydroxydopamine, Brain Res. 55: 369-382. CKOW, T. J. (1973). Catecholamine-containing neurones and electrical self stimulation: 2. A theoretical interpretation and some psychiatric implications. Psycho/. Med. 3: 6&73. DE G~oor. J. (1959). The rat forebrain in stereotaxic coordinates. V’rh. X. Ned. Akad. Wt. 52: l-40. EVETTS. K. D.. Frrzsrhno~s, J. T. and SETLER, P. E. (1972). Eating caused by 6-hydroxydopamine-induced release of nor~~dre~~linc in the diencephalon of the rat. J. P~~.~~~~.223: 35.-47. EULVR. U. S. VONand IXHAJICO, F. (1961). Improved techniques for the fluorimetric estimation of catecholamines. ,lctcl phyml. scrrrld. 51: 348-356. FAL’LL. R. L. M. and LAV~KTY. R. (1969). Changes in dopamine levels in the corpus striatum following lesions in the substantia nigra. Ezpl. Neural. 23: 332-340. FIBI~;~R. H. C.. FIHIC~ER,H. P. and ZIS. A. P. (1973). Attenuation of amphetamine-induced motor stimulation and stcrcotype by h-hydroxydopamine in the rat. By. .I. Phctrmac. 47: 683-693. FIIXEK. H. C., ZIS. A. P. and MCGEF.K, E. G. (1973). Feeding and drinking deficits after 6-hydroxydopamine admillist~tion in the rat: similarities to the lateral hypot~dlan~ic syndrome. Br& Rrs. 55: 135-148. Foe;. R. (1972). On stereotypy and catalepsy: studies on the effect of amphetamine and neuroleptics in rats. iicta WWW/. .scur~d.48: Suppl. 50. GLOWIXSKI. J. and BALD~SSAKINLR. J. (1966). Metabolism of norepinephrine in the central nervous system. Pharmuc. Rr~rl.lx: I ?Ol- 1238. Ho~H~L. B. G. (1971). Feeding: neural control of intake. AWL Rev. Physiol. 33: 533-568. IVI:RS~N, S. D. (1971). The effect of surgical lesions to frontal cortex and suhstantia nigra on amphetamine responses in rats. Brcti/z Res. 31: 295-3 l I. IV~KS~N. S. D. (1973). h-Hydroxydopamine; a chemical lesion technique for studying the role of amine neurotransmitters in hchaviour. T/XI Neuroscirnces 3rd Study Proyram (SC~IMITT.F. 0. and WORDEN, F. G., Eds.) pp. 705-71 I. M.I.T. Press, Cambridge. Mass. McK~~zrc. G. M. and SZERH, J. C. (1968). The effect of dihydroxyphenylalanine, preniprazine and dextroamphetamine on the i/r rive release of dopamine from the caudate nucleus. J. Pharmac. exp. Ther. 162: 302-308. MAKSHALL. J. F. and T~ITELBAUM,P. (1973). A comparison of the eating in response to hypothermic and glucoprivie challenges after nigrai 6-hydroxydopamine and lateral hypothalamic electrolytic lesions in rats. Brain Rrs. 55 229-233. MERLIS. J. K. (1940). The effect ofchanges in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Alt1. J. Ph~xioi. 131: 67-72. RANIXX,P, A. and MUNKVAD. I. (1970). Biochemical, anatomical and psychological investigations of stereotyped behaviour induced by amphetamines. In: Amphetumines urzd Related Compounds: Proceedings of‘ the Merio R’qri lrwtitzrtrs fbr Pharnxxologicul Research. (E. COSTA and S. GARATTINI Eds.) pp. 69s-713. Raven Press, New York. STrilu, L. (1964). ~IfstimLllation of the brain and the central stimuIant action ofamphetam~nc. F&. Proc. Fedn. Am. Sots. rsp. Bioi. u: 836-850. STQLK. J. M. and Rr:c~, R. H. (1970).Antagonism of D-amphetamine by r-methyl-L-tyrosine. Behavioural evidence for the participation of catecholamine stores and synthesis in the amphetamine stimulant response. Neurophurmuc~o/o~~~ 9: 149-263. TEITELBACM,P. and EPSTEIN. A. N. (1962). The lateral hypothalamic syndrome: recovery of feeding and drinking after lateral hypothalamic lesions. Psyrhot. Rev. 69: 74-90. UNGEKSTI:DT. U. (I97Iaf. Stereotoxic mapping of the monoamine pathway in the rat brain. Acfa p~~~~i~~~. stand. Suppl. 367: I -4x. UNGI:RST~:IX. U. (1971h). Histochemical studies on the effects of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurones in the rat brain. In: 6-Npdroxgdopumine and Catrchoiamine ~Veworws (MAL~ORS. T. and 'IHo~.NI:N, H., Eds.) pp. 101-127. North-Holland. Amsterdam. Uuc;r~STI:ur, LJ. (1971~). Adipsia and aphagia after &hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta.physiol. stand. Suppl. 367: 95-122. WIXSMAU. A. and Kot-1, B. K. (1966). Antiamphetamine effects following inhibition of tyrosine hydroxylase. J. Piiccrmtrt. rrp. Thu. IS 339--3X?. ZIC;.%%~NIX M. J. and STRITKER, E. H. (1973). Recovery of feeding and drinking by rats after intraventricu~r 6hydroxydopaminc or lateral hypothalamic lesions. Science. N.Y. 182: 717-720.
CYNTHIA BROOK and SUSAN D. IVERSEN Department of Experimental Psychology.
University
of Cambridge,
Downing
Street, Cambridge
Summary -Bilateral 6-hydroxydopamine lesions to the substantia nigra of the rat result in SS-90~: loss of striatal dopamine. In such animals. amphetamine does not induce stereotyped behaviour but the running response to lower doses of amphetamine is unaffected. The lesion does, however. produce permanent changes in the spontaneous locomotor behaviour of the rat. Aphagia was observed only in animals with greater ditional forebrain noradrenaline loss.
striatal
dopamine
depletion
which was associated
with ad-
For many years it has been known that amphetamine interacts with endogenous amine transmitter systems in the brain and more recent neuropharmacological studies have strengthened this contention. In a variety of in vitro preparations (GLOWINSKI and BALDESSARINI, 1966) and during in uiuo perfusion of the ventricular system of the brain (MCKENZIE and SZERB, 1968; CARR and MOORE, 1969) it has been verified that amphetamine releases the endogenous catecholamines noradrenaline (NA) and dopamine (DA) from synaptic storage sites. In addition, fluorescence histochemical techniques have been developed for staining endogenous amines. These reveal that the amine transmitters NA and DA are localized to different neuronal systems having cell bodies in the hind and mid brain and fibres projecting to and innervating different forebrain structures (UNGERSTEDT, 1971a). For those seeking a correlation between endogenous amine transmitters and normal or drug-induced behaviour, this anatomical information opened up the prospect of investigating the role of amine transmitters at specific sites in the nervous system with different aspects of behavioural control. At low doses, amphetamine induces marked increases in spontaneous motor behaviour. Catecholamines are necessary for this behavioural effect of amphetamine because a-methyltyrosine (E-MT), which prevents the synthesis of NA and DA in amine neurones, has been shown to antagonize this stimulatory effect of amphetamine. However, z-MT, like many other biochemical agents of current interest, interferes with both DA and NA mechanisms and has therefore failed to provide definitive evidence of which of these amines is principally involved in amphetamine-induced stimulation of locomotor activity. Forebrain DA is almost entirely localized to a neuronal system arising from cells in the substantia nigra. Fibres of this system project to the corpus striatum, the so-called nigrostriatal tract, and selective lesions to this pathway provide a direct test of whether this part of the DA system is vital for the locomotor response to amphetamine. 6-Hydroxydopamine (6-OHDA), a chemical analogue of NA and DA, which, if injected into the brain. is taken up by and destroys amine-containing neurones and terminals and so provides an appropriate chemical lesion technique. If injected locally into the substantia nigra, severe damage of the nigro-striatal tract is produced in the absence of non-specific damage to nearby anatomical pathways (UNGERSTEDT, 1971 b). Using such a lesion technique, we have investigated in the adult rat, the locomotor responses to various doses of amphetamine after bilateral 6-OHDA lesions to the substantia nigra. 95
METHODS
Subjects Male albino Wistar rats of mean weight 240g were used for these experiments. were housed in groups of four under standard laboratory conditions with controlled ing and fed ad lihitum on dry pellet food. 6-Hydrosydopamine
They light-
lesiors to the suh.statltilr nigra
Rats were anaesthetized with Nembutal, used undiluted in a dose of 0.5 ml:lOOg body weight. They were then placed in a stereotaxic apparatus (Kopf Instruments). and the head angled according to the De Groot stereotaxic atlas, by placing the nose bar 5 mm above the intraaural line. Stereotaxic co-ordinates for the substantia nigra were modified after DE GR~OT (1959) for 240 g rats and the placements were A-P, 2.5 mm; L, 2 mm; H, 8 mm. A 30 gauge stainless steel cannula was lowered into the brain at this placement and after attaching a 5-~1 Hamilton syringe, a 6-OHDA or vehicle control injection was slowly infused. To prevent oxidation of the 6-OHDA, it was dissolved in Merlis solution (MERLIS, 1940) containing l.Omg/ml ascorbic acid. The concentration was 4pggjkd of 6-OHDA (as free base) and 2 ,~l (i.e. 8 pg 6-OHDA) was injected into the substantia nigra on each side. The vehicle solution lacked 6-OHDA but except in this respect, the lesion and control procedures were identical. Observation
qf immediatr
post-oprraticr
hrhaviour
The animals were weighed each day and their eating and drinking behaviour noted. All animals were offered highly palatable wet mash on the immediate post-operative days. The rats which failed to eat and were loosing weight rapidly, were tube fed until they were able to sustain themselves. Some animals did not survive at this stage in the experiment. Photocell
measureme~~ts
qf locomotor
actir!itJ
Locomotion was measured in a bank of 12 identical photocell cages. These were modified wire mesh rat cages (25.4 x 38.1 x 20.3 cm) with two photocell units equally placed on the long axis giving horizontal beams 2.54 cm above floor level. The two photocell beams reliably record ambulation involving the whole floor space of the cage. This occurs with spontaneous activity and with the increased locomotor activity seen after low doses of amphetamine. When high doses of amphetamine are used. a form of stereotyped motor behaviour emerges in which certain responses are repeated to the exclusion of all other behaviour. Sniffing, neck movements, rearing, and in the most intense stereotypy, licking and gnawing of the cage are seen. Photocell cages are unsuitable for quantifying stereotypy and caution is required when using this measure as an indication of the occurrence and intensity of stereotypy. With large doses of amphetamine intense stereotypy occurs which tends to produce low photocell counts because behaviour is generated at one position in the cage. If the animal happens to show stereotypy within the range of a beam, high scores on one photocell can be recorded. However, lower doses of amphetamine, which predominantly stimulate locomotor activity. induce mild stereotyped running during the second hour after administration. This is the case with 1.5 mgjkg and therefore counts during the 2nd hr reflect psychomotor stimulation exacerbated by the emergence of stereotypy. It can be appreciated that if, for some reason. an animal showed psychomotor stimulation to amphetamine but not stereotypy. low doses (1.5 mg! kg) of the drug would tend to produce in such animals, low photocell counts during the test hour. compared with controls. After high doses, the reverse would be true: with controls exhibiting stereotypy in one location, producing low counts and animals not showing stereotypy running about. Therefore when stereotypy is occurring. direct observation of the animal is required to evaluate photocell counts. Tc>stinyproceduw The amphetamine studies were performed gia and adipsia associated with the substantia
2- 3 weeks post-operatively, nigra lesion had recovered.
when any aphaBehavioural tcst-
6-OHDA lesions in rat substantia
nigra
97
ing was carried out in the mornings. On test days the animals were placed in the photocell cages and their activity recorded every 10 min for 40 min. Intraperitoneal injections of control vehicle or (+)-amphetamine were then given and activity recorded for a further two hours. (+ )-Amphetamine sulphate was dissolved in 0.9% saline, which was used alone as the control vehicle injection. Amphetamine doses of 1.5, 3.0 and 5.0 mg/kg were studied in Experiment 1 and 1.5 mg/kg in Experiment 3. In both experiments the lesion and control animals received the various injections of drugs and vehicle in a balanced order. Biochemical
USSUJ~ ofamine
levels
At the end of the experiments the animals were killed with a blow on the head, the brain removed and dissected on ice. A series of shallow cuts was made from the ventral surface, to isolate the hypothalamus and olfactory turbercle from surrounding tissue, and the brain was bisected in the saggital plane. Four brain regions (preoptic region, thalamus and tegmentum of midbrain. cerebral cortex and corpus striatum) were dissected with small blunt forceps and iris scissors, care being taken to avoid the inclusion of tissue which did not form part of the region being dissected. The assay used was developed by EVE-IX, FI~ZSIMONS and SETLEK( 1972). Tissues were blotted, weighed and homogenized in a mixture consisting of 1.5 ml 0.4 M perchloric acid and 0.1 ml EDTA (40 mg/ml) containing ascorbic acid (4 mg/ml). After addition of an internal indicator made up from three parts Chlordphenol Red to one part Bromocresol Green, the homogenate was brought to pH 6.5 by the addition of 4 M-potassium hydroxide and 1.0 M K,CO,. Precipitated material was removed by cooling and centrifugation and the supernatant was passed through a 2.0 x 0.25 cm micro-column filled with treated Amberlite CG. 120 N+ form resin. The homogenate was washed with 3 ml distilled water, and noradrenaline and dopamine were eluted with 1.5-ml portions of 1 Mand 2 M hydrochloric acid respectively. Noradrenaline was estimated by the fluorimetric method of EULER and LISHAJKO(1961) and dopamine by the method of CARLSSONand WALDECK (1958). Recovery of catecholamines was S&90%. Values obtained from experimental animals are expressed as a percentage of sham-operated or untreated controls which were included in all assays. Expet-imental
procedures
ExprtG~ettt
1. Six substantia nigra lesioned and six sham operated controls which were minimally aphasic and adipsic post-operatively were given I .5, 3.0 and 5.0 mg/kg (+ )amphetamine (two injections of each dose and two injections of saline in a random order) starting three weeks post-operatively. At least two rest days elapsed between drug or saline treatments. Locomotor behaviour was recorded in the photocell cages. At the end of the experiment dopamine levels in the striatum were assayed. E.uperitnetzt 2. Twelve substantia nigra lesioned animals with appropriate controls were prepared. Their spontaneous and drug induced (1.5 mg/kg amphetamine) behaviour was recorded 48 and 76 hr post-operatively. It was then apparent that they were severely aphagic and adipsic. They were maintained by tube feeding but because no recovery of spontaneous feeding occurred they were sacrificed and DA and NA levels of various segments of the forebrain assayed. E.uperittwttt 3. Twelve substantia nigra lesioned and 12 sham operated controls were prepared. Seven substantia nigra rats were minimally aphagic. They maintained their weight after post-operative day 3 and were subsequently used for drug studies. The drug testing was organised as in Experiment 1. Striatal DA levels were determined at the end of the experiments. The remaining five severely aphagic substantia nigra animals did not recover. They were tube-fed, their weight monitored and changes in their spontaneous activity recorded daily until death. Statistical
ttlethods
Analysis of variance was used for all data analysis.
98
BRWKLHI~
CYNTHIA
SUSAN
D.
IVERS~\
RESULTS
Experiment
1
Immediatr post-operutiw hrhaviour. When the 6-OHDA animals recovered from the anaesthetic they were very active in the cages when compared with animals receiving vehicle injection under identical conditions. This activity lasted for several hours and probably reflects the immediate effects of disruption of the normal activity of the DA pathway. In some cases circling activity was observed. which may indicate asymmetric damage of the nigro-striatal pathway. similar to that which can be produced permanently if the substantia nigra on only one side of the midbrain is injected with 6-OHDA. 1.5
30
50
h k I\
I
,‘I\\
1’
1 \
I
’
::
\
:
I I :
\\ \
1
’\I 4 \ .
\
i’
‘\
b”\l
I
‘1\ -Control - -
6-OHDA
\/’
I
I
I
I
,
2
I
2
hr
I
I 2
after Amphetamine
Fig. I. Mean responses in photocell cages of control (II = 6) and substantia nigra lesioned animals 1.5. 3.0 and 5.0 mglkg (+)-amphetamine.The responses of these animals to placebo saline responses have been subtracted from the drug response. The experiment was performed I4 days post-operatively.
(n = 6) after
Responses to amphetamine. The 6 substantia nigra lesioned animals used in this experiment were minimally aphagic and adipsic and drug testing began 2 weeks post-operatively. The mean responses of these animals compared with that of the control animals, to 1.5.3.0 and 5.0 mg/kg of (+ )-amphetamine are shown in Figure 1. In controls and substantia nigra lesioned animals, the lower doses (1.5 and 3 mg/kg) produced a different response pattern over time than that elicited by 5 mg/kg. At 1.5 mg/kg, despite an apparent lower response during the second test hour, the substantia nigra animals gave a response of the same magnitude (d.f. 1, 10; F = 1.1, P > 0.05) and time course (d.f. 1. 11 ; 110; F = 0.38. P > 0.05) as the controls. This was also the case with 3 mg/kg. However, at 5 mg/kg, although the time course of the responding was strikingly similar in both groups. the 6OHDA animals showed an enhanced response. This difference did not reach significance but probably reflects reduction in locomotor scores in the control animals because of the intense stereotypy induced in them by 5 mg/kg (+ )-amphetamine. Stereotypy was blocked in the 6-OHDA animals and therefore they did not become immobile but continued to show psychomotor stimulation to amphetamine and thus recorded high photocell counts. Although there was no significant treatment effect in the 5 mg/kg experiment, inspection of the locomotor scores 30 and 60 min after the drug injection revealed that some substantia nigra animals, like controls. showed low locomotor scores and presumably stereotypy. This indicates that heterogeneity of variance exists and in these circumstances the analysis of variance would not be expected to prove significant. The range of DA depletion in the striatum of the substantia nigra animals is presented in Table I. If the degree of block of stereotypy as reported above is plotted against DA depletion, the animals which sustained severe damage to the nigrostriatal system (907; dcpletion) show blocked stereotypy and those with about 50”,:, of the system intact continue to show stereotypy responses to 5 mg/kg (+ )-amphetamine and hence low locomotor counts (Fig. 2). When calculated over the 2 hr session or on the 30 min or 60 min values, the amount of activity is positively correlated with the severity of DA depletion (Spearman’s rank correlation coefficient, I’, = 0.886, II = 6, P < 0.05). If animals happen
6-OHDA
Table
I. Regional
nigra
I 2 3 4 5
I 2 3 4 5 6 7
x 9 IO II 12 Mean
nigra lesions. The values are presented animals
NA Cortex
30
I.6 X.0 I.0 5.0 0.0 4.3”’1” -+ 1.6 4.0 17.0 4X.0 20.0 6.0 15.5 21.0 13.0 13.0 30.0 15.0 22.0 190”, * 3.4
Mean
6-OHDA Suhstantia nigra Non-aphagic
NA Hypothalamus
DA Striatum
99
nigra
NA and DA levels after bilateral 6-OHDA substantia as a 7; k S.E.M. of those levels in control
Rat 6-OH DA Substantia Aphagic
lesions in rat substantia
Thalamus
80 90
60 14 15 15 27:i f 9
NA and midbrain
84 67 34 28 25 48x& 12
33 38 60:/” k 14
to remain in the path of a photobeam while stereotyping, the behaviour is measured as a high count on one beam and none on the other. It is interesting that the one control animal which gave a locomotor count of 275 produced 274 of those counts in one photobeam. By contrast, equivalent scores in the substantia nigra animals were equally distributed between the two beams, an indication that movement around the cage was occurring. Experiment
2
Immediately after recovering from the anaesthetic these animals showed high levels of spontaneous motor behaviour which subsided within 24 hr to leave them hypokinetic (d.f. 1, 8; F = 6.620; P < 0.05). At this stage they showed a normal response to 1.5 mg/kg (+)amphetamine. However, within the next 24 hr, evidence of behavioural supersensitivity emerged, for at 76 hr the response to amphetamine was greatly enhanced in the substantia nigra lesioned animals (d.f. 1, 8; F = 14.926; P < 0.01) (Fig. 3). Drug studies on these animals were then terminated because of the severe aphagia and when it was clear that recovery was not likely to occur, the animals were sacrificed for regioned brain NA and DA assays. These results are presented in Table 1. For comparative purposes this Table also includes the striatal DA levels in twelve 6-OHDA substantia nigra lesioned rats which were not aphagic and adipsic [Experiment l(6) and 3(6)]. 500
+ I’
t 30-40mln
Y
r
2
5 g b ;; E 8
300
0 60-70
.
0
0
200
mfn
.
;f0
s
+
100
I
IO
I 20
%
DA
I 30
I 40
0
.
+I 50
a
Control
remaining
Fig. 2. The locomotor counts of individual substantia nigra rats at 3&40 and 6&70min after 5 mg/kg (+)-amphetamine plotted against the percentage of remaining striatal DA. Comparable measures on the control animals are presented to the right of the figure.
loo
CYNTHIA
BROOKand SC~SAND.
IWRS~~
800 .*-:
48hr
76hr
.“’
‘,
0--t
1’
‘*6-OHM
: I
l\.
/’
l \.,._
l
.,,,
lCon+ro’
./
10
I
I
I
30
50
70
J i
90 mm
30
50
70
90
after Amphetamine
.
Fig. 3. Locomotor response to I.5 mgikg (+)-amphetamine tested at 48 and 76 hr after 6.OHDA (dashed line) or sham lesions (solid line) to the substantia nigra. The appropriate saline scores were subtracted from the drug responses.
Exprrimrnt
3
Twelve substantia nigra and 12 control animals were prepared for this experiment. Five were severely aphagic and adipsic and their spontaneous motor behaviour was studied as their weight fell. Six of the remaining recovered substantia nigra animals and six controls were tested in the photocell cages with I.5 mg/kg ( + )-amphetamine and placebo at 14 and 21 days and 2 months after operation. The responses at 2 months are shown in Figure 4. There was a significant difference between the responses of the two groups (d.f. 1. 11; F = 5.6; P < 0.05) characterised by a steeper fall off of response during the second test hour in the substantia nigra animals (d.f. 1. 11; 242; F = 2.82; P < 0.01) indicating that stereotyped running was not present in these animals as it was in the controls. The responses to 1.5 mg/kg (+)-amphetamine had been similar at 14 and 21 days post-operatively in these animals and on none of these occasions had there been any evidence in the substantia nigra animals of blocked locomotor response during the first hour. Spontaneous locomotor activity was also assessed in these animals immediately postoperatively and during the drug testing sessions at 14 and 21 days and 2 months postoperatively. At 2448 hr after the operation. the substantia nigra animals were markedly hypokinetic when placed in the photocell cages (d.f. 1, 10; F = 41.2; P < O.OOl), (Fig. 5). 300-
i \
e Cl 'a
/'
\ \ \,e -0,
l \
*Control
Nlgral
I 0
1 20
1 40
I 60
1 80
1 100
1 120
min after Amphetamw
Fig. 4. Mean responses in photocell cages of control (II = 6) and substantia nigra (II = 6) lesioned animals after I.5 mg/kg (+ )-amphetamine. The saline responses have been subtracted from the drug response. The experiment was performed 2 months post-operatively.
6-OHDA
lesions in rat substantia
101
nigra
3CO-
0
E
Control
200-
: =
\
.
8 P a
,oo
6-OH? \
6 f
0
\
\
\
\ \
\
\
‘o,_
I
IO
.-•
--G_ I
20
\ --C--l--_-(_
30
40
50
60
rnl"l" cage
Fig, 5. Spontaneous
locomotor
activity during the second post-operative stantia nigra lesioned animals.
day of control
and sub-
When placed in such a novel environment, normal animals exhibit declining activity levels, or so-called habitation, over time. Despite initial hypokinesia, the substantia nigra animals also show this behaviour and over the hour test their activity habituated to the same low level as that of the controls. Similar measures were taken during the three subsequent drug experiments, when 40 min habituation runs were given before the amphetamine injections. The results obtained 2 months post-operatively are shown in Figure 6 and the responses ofthe substantia nigra animals were very similar to those obtained immediately post-operatively. The hypokinesia is thus a persistent effect of this 6-OHDA lesion and furthermore is not attenuated when animals experience the same situation at short intervals. The habituation results shown in the left and right of Figure 6 were obtained at a 2-day interval. At these times the substantia nigra animals, like controls, again showed habituation over the test period but further analysis revealed that the time course of the habituation change was different in the two groups (d.f. 5, 100; F = 11.5; P < 0.001). The striatal DA levels in the animals from Experiment 3 are also presented in Table 1. There was again variation in the degree of DA depletion which tended to correlate with the block of stereotypy as measured indirectly by photocell activity (Fig. 7). However the correlation was in the opposite direction to that reported with 5 mg/kg (Fig. 2); with 15 mg/kg (+)-amphetamine, a block of stereotypy (i.e. massive DA depletion) resulted in lower photocell counts, whereas with 5 mg/kg the opposite was true.
300-
m E g 200 -
.
-Control
= -
-6-OHDA
8 g a 6
IOO-
i
Fig. 6. Spontaneous
locomotor activity in control and substantia nigra lesioned animals post-operatively. The measures were made at a 2 day interval.
2 months
102
CYNTHIA
and SUSAN D. IVEKS~~
BK~OK
30m1n .
.
100 ml”
0
800 L
0
700 ~ 0 600 ~ 2 s 500 8 b z 400E 8 cl 300 J 200 -
. .
. ‘.
q
B
.
q
.
i
. .
.
E .
.
30
40
L
Control %
DA
B
. .
. .
100 1I11 10 20
i
I
!
40 Control
109+---
remo~nmg
Fig. 7. The locomotor counts 01”individual substantia nigra rats recorded at 30 and 100 min alter the two doses of I.5 mg/kg (+)-amphetamine. plotted against the percentage of remaining striatal DA. The comparable responses of control animals are presented to the right of the figures. The calculations were made on drug responses measured Z months post-operatively.
The mean weight of the aphagic animals dropped from 250 to 190 g over the first postoperative week. Their spontaneous motor behaviour in photocell cages over 1 hr test periods on days 5, 6 and 7 are shown in Figure 8, and compared with that of sham operated and non-aphagic substantia nigra lesioned animals. The non-aphagic animals remained hypokinetic but the aphagic animals showed escalating spontaneous activity over the days (d.f. 1, 8; F = 5.9; P < 0.05).
DISCUSSION
6-Hydroxydopamine lesions to the substantia nigra change spontaneous and drug-induced motor behaviours. The profile of these effects varies with the post-operative interval. presumably reflecting the changing neuropharmacological status of the nigrostriatal tract and its postsynaptic receptors, consequent upon 6-OHDA injection. During the first postoperative week, biochemical and morphological studies indicate that presynaptic terminals are in a state of change. FAULL and LAVERTY (1969) report that within hours of
Postoperative
doy
6
‘6-OHDA
30
60
30
6-OHDA
Aphaglc
Non-
Aphaglc
60
Fig. 8. Spontaneous locomotor activity over a I-hr period in sham operated control, non-aphagic and aphagic substantia nigra lesioned animals. The tests were made on post-operatwe days 5. h and 7.
6-OHDAlesions
in rat substantia
nigra
103.
the lesion, DA levels rise in the striatum possibly due to the immediate post-operative disruption of neurotransmission on the nigrostriatal pathway and reduction in the normal release of synaptically stored DA. After 1 day, DA levels begin to fall consequent upon 6-OHDA-induced degeneration of the terminals and loss of the stored amine. Dopamine levels then presumably rise in the synaptic cleft and this change could be expected to have effects on spontaneous motor behaviour. Gradually the released DA is degraded and less synaptically stored DA is available for normal or drug-induced release. The labile state of striatal synapses would be expected to result, over the first few post-operative days, in a changing and complex pattern of motor behaviour and variable drug responses as indicated by the amphetamine results reported in Experiment 2. However, by 14 days post-operatively, the nigrostriatal system had stabilised. At this time the changes in spontaneous and drug-induced motor behaviour due to loss of striatal DA can be assessed. These experiments, in agreement with the related ones of CREESEand IVERSEN(1972), show that depletion of striatal DA abolishes the stereotyped behaviour seen with various doses of (+)-amphetamine. This result merely confirms further that the nigrostriatal system is essential for stereotyped behaviour (RANDRUPand MUNKVAD,1970; FOG. 1972). The question of whether the same DA system or independent neurochemical pathways containing NA mediate the running response to amphetamine remains unsolved. All we can state is that on average, DA depletion of 85-9051:)in the Creese and Iversen experiments and of 8 1% in the present study, while sufficiently severe to abolish stereotypy, does not impair amphetamine-induced locomotor behaviour. In view of this result. it could still be maintained that the DA system is involved in this response. The remaining DA system may be capable of sustaining one motor behaviour and not another, i.e. fewer intact DA-containing neurones are required to sustain amphetamine stimulation than stereotypy. In this case a procedure which produced total DA depletion would abolish the amphetamine locomotor response and this has not yet been achieved. At present, there is no selective biochemical manipulation of brain amines that has been studied which does block the locomotor response to amphetamine. Three neurochemical procedures have been described which do block the response (1) z-MT (WEISSMANand KOE, 1966; STOLKand RECH, 1970) (2) intracerebral injections of 6-OHDA in the neonate (CREESEadd IVERSEN,1973) and (3) intraventricular injection of 6-OHDA in the adult rat, (IVERSEN,unpublished observations). These procedures have two consequences in common; virtually total depletion of both forebrain DA and NA. Their success could, therefore, be due to the completeness of the DA loss or the involvement of the NA systems. The weight ofevidence at present favours the latter interpretation (IVERSEN,1973; FIBIGER. FIBIGERand ZIS, 1973). One could hope that substantia nigra lesion techniques will be developed which will result in selective and total DA loss; but to date such complete lesions produce very sick animals which are totally aphagic and adipsic and, therefore. poor behavioural subjects. Furthermore, in our experiments these complete substantia nigra lesions result in involvement of NA pathways and the amine loss is not selective to the DA system. Noradrenaline fibres are known to traverse the mid brain in the close vicinity of the substantia nigra and presumably a 6-OHDA injection which damages the whole of the substantia nigra is likely to spread to the adjacent fibre systems. Any pursuit of this problem to achieve more selective lesions should be guided by another neglected fact; namely that although we refer to the nigro-striatal system it is clear that the DA system is not a simple unitary projection. Fibres arising from neurones slightly medial to the substantia nigra innervate the nucleus accumbens and the amygdala. Furthermore, fibres innervating the different elements of the basal ganglia arise from different parts of the substantia nigra. The brain dissection techniques in current use take no account of this complexity and the “striatal” segment includes, in one assay sample, most of this heterogenous DA terminal arrangement. It is conceivable that one part of this system is involved in stereotypy and another in the locomotor response and that our lesion procedures damage the former much more completely than the latter. In this case a figure of 81”/;,striatal DA depletion may not be very informative.
CYNTHIA BROOK and S~JSAND. IVERSEN
104
It is perhaps not surprising that it proves difficult to identify the neurological system mediating such a ubiquitous behaviour as locomotion. We assume that whenever it occurs locomotor behaviour is the same but behavioural analysis indicates that locomotion is a consequence of very many behavioural states. The present results emphasise the importance of describing the conditions under which locomotion is occurring. The substantia nigra lesions in these studies depressed the locomotion generated when the rat experienced a change of environment and this hypokinesia and abnormal habituation in a new environment is a permanent consequent of the substantia nigra lesion. However, once such an animal has habituated to the environment, the running induced by amphetamine is normal. Regarding the effects of nigral lesions on eating behaviour: these results indicate that. in general, the more severe the DA depletion, the more likely the animal is to show aphagia and adipsia. In view of the damage to the nigrostriated tract in these animals, one must conclude that the motor arousal associated with starvation is not mediated by a dopaminergic system. UNGERSTEDT (1971~) on the basis of histochemical analysis of striatal DA levels in substantia nigra lesioned animals, has also correlated aphagia and adipsia with the degree of DA loss. There are clearly exceptions, such as the animals in Experiment 1 with depletion of 96 and 94% striatal DA who were not severely aphagic and adipsic. Again total striatal DA may not prove the most reliable correlate of the eating disorder. However, in addition to this correlation with DA depletion. it is also clear that substantia nigra lesions which produce aphagia, result in substantial forebrain NA depletion as well. Possibly this is because a well placed cannulation of the substantia nigra which distributes 6-OHDA to virtually the whole of the substantia nigra, also involves some crucial part of the NA fibre systems which course through the midbrain in close vicinity to the substantia nigra. Classically, aphagia and adipsia have been observed after lateral hypothalamic lesions (TEITELBAUMand EPSTEIN, 1962). Recent histofluorescent findings indicate that the DA fibres of the nigrostriatal tract as well as ascending NA fibres pass through this region of the hypothalamus. UNGERSTEDT (1971~) therefore suggested that the classical lateral hypothalamic deficit described by TEITELBAUMand EPSTEIN(1962) after electrolytic lesions to the lateral hypothalamus was in fact due to severance of the DA fibres at this brain level. Several recent studies have demonstrated the behavioural similarity of the aphagia and adipsia associated with 6-OHDA lesions to the classical lateral hypothalamic syndrome (MARSHALL and TEITELRAUM.1973; FIBIGER, Zrs and MCGEER, 1973; ZIGMOND and STRICKER, 1973). However. in these investigations, in a study of electrolytic substantia nigra lesions (IVERSEN, 1971) and in the present study, both noradrenaline and dopamine were depleted. Selective lesions to the ventral and dorsal NA bundles (UNCXRSTEDT. 197 I b; CREESE,unpublished observations), which result in greater forebrain NA loss than substantia nigra lesions, do not produce aphagia (UNCXRSTWT, 1971b; CREESE.unpublished obscrvations). We are not claiming, therefore, an involvement of NA rather than DA in these feeding deficits. It does appear, however, that the data do not at present justify a simple shift of emphasis from NA to DA and an exclusive role for the nigrostriatal tract in feeding behaviour. Such a conclusion would ignore much evidence of a role of NA systems in certain aspects of the control of food intake (HOEBEL, 197 1) and reinforcement (STEIN. 1964). Not least important, it also totally neglects recent theoretical (BINDRA, 1968) and experimental work (CAMPBELL and BAEZ, 1974; CROW. 1973) which supports the view that the behaviour associated with need and its satisfaction are complex and involve the integration of several functionally distinct neural systems of the forebrain and we suggest. of their different neurotransmitters. Acknowledgements-Thiswork was supported by MRC grant C 97lj242 B to S.D.I. The authors thank Professor 0. L. ZANGWILLfor laboratory facilities and encouragement for this work. KLITH Evl.rrs performed
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