The effect of surgical lesions to frontal cortex and substantia nigra on amphetamine responses in rats

BRAIN RESEARCH

295

THE EFFECT OF SURGICAL LESIONS TO F R O N T A L CORTEX AND SUBSTANTIA N I G R A ON A M P H E T A M I N E RESPONSES IN RATS

SUSAN D. 1VERSEN Department of Experimental Psychology, Cambridze University, Cambridge (Great Britain)

(Accepted March 5th, 1971)

INTRODUCTION If the synthesis of endogenous catecholamine transmitters in the CNS is inhibited, the locomotor response to amphetamine is blocked 2t,26. a-Methyltyrosine ((tMT) is one such metabolic inhibitor which prevents the synthesis of DOPA from its precursor and thereby the final synthesis of dopamine (DA) and noradrenaline (NA) both of which, despite their shared metabolic pathway, exist as autonomous neurotransmitter substances in discrete and different sites in the CNS 5. After (t-MT treatment, the blocked amphetamine response is asscciated with depletion of both DA and NA, and it could be inferred that either or both of these substances are vital for the normal locomotor responses to the drug. The solution of this problem demands selective depletion of one transmitter system rather than another but unfortunately, at effective doses, the toxicity of Disulphran, one such metabolic inhibitor of the dopamine to noradrenaline transformation, renders it an undesirable tool. The experiments to be reported represent a preliminary effort to pursue this problem with surgical techniques designed to selectively interfere with the endogenous store of the catecholamine transmitter DA. Using fluorescent histochemical techniques the distributions of endogenous DA, NA and 5-hydroxytryptamine (5-HT) have been plotted in the rat CNS1, a, and these studies show that a prominent dopaminergiccontaining system arises in the substantia nigra (zona compacta) and distributes terminals to the corpus striatum, which contains the highest endogenous DA level of any CNS structure. Considering the 'motor' nature of one class of amphetamine effects and their dependence on endogenous catecholamine levels, it seemed possible that this dopaminergic system might play a central role in these effectsI6, 2.5 - - a suggestion supported by observations that NA-containing neurones do not project strongly to the corpus striatum. The anatomical specificity of the dopaminergic pathway suggested that it might be possible to use surgical lesion techniques to interfere with this part of the extrapyramidal system and thereby its response to amphetamine. In particular we have attempted to assess the importance of frontostriatal and nigrostriatal projections to the amphetamine locomotor response in this way. Brain Research, 31 (1971) 295-311

In this report evidence is presented which suggests that the integrity of the dopaminergic pathway is necessary for amphetamine to produce its effects on locomotor activity. But this claim should not he interpreted as a parsimonious unitary theory to explain all the behavioural effects of amphetamine, some of which may be mediated by transmitters other than dopamine or a result of direct actions c,i the d r u g METHODS

Subjects Male Wistar rats weighing :> 150 g, housed in groups of 4, with tYee access to food and water, were used in these experiments.

Surgery Frontal lesions. The animals were anaesthetized with Nembutal (60 mg/kg) and the frontal cortex exposed through bilateral skull openings. Cortical tissue was removed by subpial aspiration using a small gauge sucker. Area 10 as delineated by Krieg 13 was removed using the bregma of the skull as a posterior landmark, Damage to the underlying striatum was avoided as far as possible and the connections between the olfactory bulb and the basal forebrain were spared. The lesion was packed with gel foam and the skin sutured. In the sham-operated controls the same surgical procedures were followed but no cortical tissue removed. Substantia nigra lesions. The animals were anaesthetized with Nembutal (60 mg/kg) and bilateral electrolytic lesions made to substantia nigra (SN) using coordinates from the atlas of K6nig and KlippetlL A current of 0.5 mA was passed for 10 sec. Sham-operated controls were also prepared using identical electrodes and co-ordinates but the needle was lowered only 5 mm into the cortex and current was not passed. About half the lesioned animals died within 10 days of the operation. Drugs DL-Amphetamine in doses of 3, 5 and t0 mg/ml was prepared in saline. L-amethyl-p-tyrosine (a-MT) was prepared as a suspension in saline at a concentration of 50 mg/ml. Injections were given intraperitoneatly and the animals received each dose or combination of doses studied on two separate occasions. In a given experiment all the animals received the doses of drugs in a random order and at least 3 days separated drug and placebo injections.

Apparatus Locomotor activity and the stereotypy responses to amphetamine were measured in wire mesh cages (14 in. × l0 in.) with 2 photocells on the long axis. Activity cages of this kind accurately reflect locomotor activity levels but will also record stereotyped

Brain Research, 31 (1971) 295-3ll

LESIONS AND AMPHETAMINE RESPONSES IN RATS

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Brain Research, 31 (1971) 295 311

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behaviour although less reliably, as the animal may not be in the vicinity ol ~mc of the photobeams when showing repetitive stereotyped movements. All activity measures were made postoperatively and in each experiment the rats were habituated to the activity boxes for I h for 4 days before the drug experiments were begun. Biochemical methods

The animals were killed and the brains quickly removed. In the frontal lesion experiments the brains were divided into two samples, forebrain and mid/hind brain, by a cut between the mid-brain and the diencephalon. In the substantia nigra lesion experiments the brain was again divided into forebrain and mid/hind parts and the latter were fixed for subsequent histological processing. The forebrain was dissected into two samples for biochemical assay, the corpus striatum and the remaining forebrain. These brain samples were assayed to determine the endogeneous levels of DA and NA 23. The animals from Experiment 6 which survived for 120 days were prepared similarly but the brain samples were assayed for tyrosine hydroxylase activity as an indicator of the integrity of the dopaminergic neurones in the tissue 9. Histological procedures

The animals with substantia nigra lesions used in Experiments 4 and 5 were killed when behavioural testing was completed and the brains removed. Frozen sections were prepared from the fixed mid/hind brain portion and stained by the Nissl method to study the extent of the mid-brain damage. The extent of the SN damage in the 8 subjects of Experiment 6 is illustrated in Fig. 1. Except for No. 1, there was bilateral involvement of the zona compacta of the SN. There is varying involvement of more posterior structures such as the red nucleus and the field of Forel. The lesions in Experiments 4 and 5 have also been examined histologically and show similar extent s . Some animals with frontal lesions from Experiment 3 were used to verit~I histologically the extent of the lesion in these experiments. The animals were killed and when the brain was removed any damage to the olfactory bulb connections was noted. After fixation, frozen sections were prepared and stained by the Nissl method. The extent of the damage to Area 10 and any concomitant damage to the corpus striatum was noted. Procedures Experiment 1. The effect of intraperitoneal injections of 3.0, 5.0 and 10.0 mg/kg DL-amphetamine and saline was studied in 4 animals with bilateral frontal lesions and 4 sham-operated controls. The animals were placed in the photocell cages immediately after injection and the locomotor responses measured every 10 rain for 2 h. At the end of this experiment, which occupied 2 months, the animals were Brain Research, 31 (1971) 295-311

LESIONS A N D A M P H E T A M I N E RESPONSES IN RATS

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tested with various doses of D-amphetamine 1° and about 5 months after operation the same animals were retested with 5 mg/kg DL-amphetamine. Experiment 2. Groups of 6 frontal and 6 sham-operated rats were prepared and in these animals the effect of ct-MT pretreatment on the amphetamine responses was studied. The animals received injections of 1.5 and 2.5 mg/kg D-amphetamine, 50 mg/kg ~z-MT 2 h before 1.5 and 2.5 mg/kg D-amphetamine and control injections of ~z-MT and saline. Locomotor activity recording started 1 h after the amphetamine or saline injections and records were taken every l0 rain for I h. The order of drug injections was randomized and at least two days separated injections. Experiment 3. The effect of bilateral frontal lesions on the endogenous levels of NA and DA in the forebrain and mid-brain/hind-brain was studied using 10 lesioned and 10 sham-operated controls. These animals were killed 3 weeks postoperatively and the brains assayed biochemically. Experiment 4. The behaviour of 6 animals with substantia nigra lesions and 6 controls was studied on various occasions during the first l0 days immediately after the operation. Spontaneous locomotor activity, neck movements and gnawing behaviour were recorded each day by observing: (i) The photocell counts during the first 10 rain period after introduction into the activity box. (ii) The number of discrete neck movements in the following 3 rain observation period. (iii) The percentage of 100 sec spent gnawing a glass rod pushed into the activity box. The gnawing was studied after the activity counts and neck movements had been recorded. Experiment 5. Testing was begun 2 weeks postoperatively and the effect on locomotor activity of 0.5, 3, 5, and 10 mg/kg DL-amphetamine and saline was studied in 6 animals with bilateral substantia nigra lesions and 6 sham-operated controls. Experiment 6. Experiment 5 was repeated with new groups of animals (8 with substantia nigra lesions, 8 controls) using 3 and 5 mg/kg DL-amphetamine. Behavioural testing was begun 2 weeks postoperatively with habituation trials. The response to 3 and 5 mg/kg DL-amphetamine was tested on 3 separate occasions during the following 105 days. Experiment 7. Groups of substantia nigra lesioned animals and sham-operated controls were prepared for biochemical assay of the endogenous striatal DA and NA levels. Twelve animals (6 lesioned and 6 control) were killed at 27 and 72 h postoperatively. The 12 animals from Experiment 4 were killed 17 days postoperatively and the 16 animals of Experiment 6 at 120 days. RESULTS

Analysis and stat&tics In the drug response time courses the values presented are the total counts in response to the drug after subtraction of the appropriate saline placebo scores (e.g.,

Brain Research, 31 (1971) 295-311

300

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Fig. 3. Response of frontal lesioned (squares) and control (circles) rats to 3, 5 and 10 mg/kg DLamphetamine. Each score is the mean of 2 drug treatments with the appropriate mean saline score subtracted. After 10 mg/kg only the values at 10 and 120 rain are significantly different.

Fig. 5). The differences in responses between the lesioned and the control animals have been compared with Students's t test and significance values refer to this procedure. Experiment l. Bilateral frontal lesions significantly increased the spontaneous locomotor activity of rats during the first 50 rain in the photocell cage but during the second hour the frontals were no more active than the controls (Fig. 2). In Fig. 3 the characteristic time course of the responses to 3, 5 and 10 mg/kg oL-amphetamine is presented. In control animals 3 mg/kg resulted in a significant stimulation of locomotor activity during the 2 h of the test session, whereas 5 mg/kg stimulated activity for only the first 10 rain after injection. This dose has a tendency to suppress spontaneous locomotor activity for about 1 h, followed by a significant increase in recorded locomotor counts which are observed mainly to reflect the emerBrain Research, 31 (1971) 295-311

LESIONS AND AMPHETAMINE RESPONSES IN RATS

301

RESPONSE of FRONTALS to AMPHI~TAMINE

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TABLE T L A C K OF E F F E C T OF F R O N T A L LESIONS O N F O R E B R A I N

NA

AND DA

LEVELS 1N T H E R A T

Values are m e a n s {; S.E.M. for groups of 10 animals.

Noradrenaline ( itg/g)

Dopamine (,ug/g)

Control

Frontal lesion

Control

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0.34 4~ 0.02

0.33 ± 0.03

0.69 ~ 0.04

0.68 ~ 0.02

Brain Research, 31 (1971) 295-311

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gence of stereotyped behaviour. The depression followed by stereotypy is seen even more clearly at the largest dose used, 10 mg/kg. The responses of the frontals to these doses of DL-amphetamine had similar time courses but were significantly enhanced. When animals with frontal lesions were retested 5 months postoperatively the response to 5 mg/kg DE-amphetamine was not significantly different from that previously recorded (Fig. 4). Experiment 2. The response of frontal lesioned animals and controls to 1.5 o-amphetamine is shown in Fig. 5 together with the effect of pretreatment with 50 mg/kg a-MT. The frontals in this study again showed an enhanced response to Damphetamine, a-MT significantly blocks this response in both frontals and controls and indeed under these conditions the activity patterns of the two groups of animals were similar. Experiment 3. Bilateral frontal lesions did not significantly alter the endogenous levels of NA or DA in any brain area (Table I). Experiment 4. Immediately postoperatively marked changes were observed in the substantia nigra lesioned animals. The animals did not eat or drink and those who survived were kept alive by force feeding and drinking. Gradually over several days, eating and drinking was reinstated and seemed to be aided by the use of wet palatable food (e.g., high protein baby food). Other behavioural changes became apparent a few hours after the lesion. The animals were highly active, irritable and many showed persistent stereotyped neck movements and gnawing of any objects encountered. It proved necessary to restrain them in small covered plastic boxes to prevent them from dying of exhaustion. Systematic observation was made for 10 days, starting 9 h postoperatively and spontaneous locomotor activity, neck movements and gnawing were found to be greatly enhanced in the substantia nigra animals (Fig. 6). Gradually over the 10 days these spontaneous behaviours decreased and after a week there were no significant differences between the lesion and control animals on any of these measures. Experiment 5. The substantia nigra animals in this experiment showed similar behaviour patterns immediately postoperatively. The testing was begun two weeks 180--

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Brain Research, 31 (1971) 295-311

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postoperatively by which time the acute behavioural changes had subsided and this is substantiated by the identical responses of the substantia nigra and control animals to the saline injections given during the course of the amphetamine experiment (Fig. 7)~ However, the average responses to DL-ampbetamine were different in the two groups. 0.5 mg/kg produced an insignificant stimulation of locomotor activity in both lesioned and control animals but to doses of 3, 5 and I 0 mg/kg the controls gave the characteristic response patterns (Fig. 8). The substantia nigra animals showed on the average significantly smaller locomotor responses to the most effective doses (3 and 5 mg/kg)~ The responses of the substantia nigra animals were also notable because of the more prolonged time course of the effects, and because of the similarity of the responses produced by different doses of amphetamine in these animals (Fig. 9). The responses of the 6 nigral animals were averaged for Figs. 8 and 9, but from the results obtained with individual animals, it was apparent that the magnitude of the response varied considerably amongst the group of nigrat animals. Nevertheless, the time course of the amphetamine was the same in all lesioned animals ........a graduallY rising response rate over the 2 h period. Two animals out of these 6 actualty showed Brain Research, 31 (1971) 295-3tl

305

LESIONS AND AMPHETAMINE RESPONSES IN RATS RESPONSE of NIGRAL LESIONED RATS to INCREASING DOSES of d, AMPHETAMINE

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higher response rates to amphetamine than the controls and the remaining 4 showed only very low responses to the drug 2°. Experiment 6. The mean results of the second experiment on animals with SN lesions are presented in Fig. 10. As in Experiment 5 the SN subjects could be divided into two groups - - high and low responders. However, while in Experiment 5 the majority of the lesioned animals were low responders the reverse was true in Experiment 6. The results from the two experiments could therefore be subdivided further to illustrate this point. This has been done for Experiment 5 and the low responders show on the average an almost total lack of locomotor responses to amphetamine ~0 while the high responders, at least after operation, show a significantly enhanced response. Brain Research, 31 (1971) 295-31 l

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This mean enhanced response fades with time (Fig. 10), but even after I00 days is much greater than in the low responders. The significance of these enhanced responses and their relationship to the blocked effect in animals with similar lesions remains obscure. However, it may be relevant that irrespective of the magnitude of the response other features of the normal amphetamine response is changed in all the animals, notably the time course and dose-dependency. Irrespective of whether the nigral lesioned animals were high or low responders the pattern of responding over the 2 h period was the same - - gradually increasing to a peak at 2 h. The early locomotor activity response to low doses (3 and 5 mg/kg) in controls is not seen and the magnitude of the response is the same for both doses. In this respect the results of Experiments 5 and 6 are in agreement. Experiment 7. Twenty-four hours, 72 h, 17 and 120 days postoperatively groups of nigral animals were killed and the brains assayed for either striatal dopamine or tyrosine hydroxylase (Fig. 11). There was a progressive decline in striatum DA and at 17 days a significant depletion ( 4 0 ~ ) was achieved, similar to the depletion of tyrosine hydroxylase measured at 120 days. DISCUSSION

The effect of frontal lesion on spontaneous locomotor activity Bilateral lesions to the frontal cortex of the rat increased spontaneous locomotor activity. The increase was small but significant, was seen immediately postoperatively,

Brain Research, 31 (1971) 295-311

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307

and was still present at least 5 months later. However, in our experimental procedure this increased activity was seen only during the first hour of the testing sessions. The literature contains conflicting reports of increased motor activity after frontal lesions. Spontaneous locomotor activity has been measured in a variety of apparatus and it is erroneous to assume that in the different environments locomotor scores reflect the same behaviour. It seems possible for example that when animals are placed in photocell cages, such as those used in the presentexperiments, exploratory activity occurs initially followed, when habituation is complete, by a lower sustained baseline activity. The former behaviour is enhanced by frontal lesions, the latter is not. Such an interpretation is consonant with findings that frontal lesions impair habituation to novel stimuli s,ll, but fail to increase activity on long test sessions in stabilimeters a4. The effect o f frontal lesions on the locomotor and stereotypy responses to amphetamine

The present results show that frontal lesions result in a marked enhancement of both the locomotor and stereotypy responses to various doses of DL-amphetamine. Lynch et al. 14 have also reported an enhanced locomotor activity in frontals after doses up to 4.5 mg/kg of the racemate but in contrast in the present study no decrement in the enhanced responses was seen 5 months postoperatively. The frontal cortex projects widely subcortically 4,26 and it is generally supposed that the prevailing influence from the frontal cortex is inhibitory. Amphetamine may act at various sites in the CNS which receive such inhibitory projections and Lynch et al. I4 have chosen to relate their findings to the release, by frontal lesions, of a site of amphetamine action in the reticular formationL Neither the site nor the frontal projection can be denied but it seems unlikely that the non-specific activating centres are the final common pathway for the highly specific behavioural effects of amphetamine. It is possible that the corpus striatum represents a more important focus for both descending frontal projections and the pharmacological effect of amphetamine. It has been shown anatomically 4,26 and behaviourally 7,s that the frontal cortex and the corpus striatum are functionally related and it is reasonable to suppose that the output from the striatum to other motor centres would change if the telencephalic circuitry was disrupted. This hypothesis could explain both the changed normal activity levels and the enhanced effect of amphetamine in frontal animals. There is evidence that amphetamine acts by releasing endogenous amine transmitters from the forebrain 3,15. The principal ascending dopamine-containing system has terminals in the striatum and this system has been implicated in pathological conditions which result in motor disorderslT, TM and in the amphetamine effects on motor behaviour16, za. Contrary to reports of Nielson 17, frontal lesions in our experience did not significantly increase endogenous levels of striatal DA and therefore a greater availability of neurotransmitter for release cannot be the explanation for the present results. However, the fact that a-MT blocks both the control and frontal response to amphetamine supports the contention that catecholamines in the striatum are necessary for the locomotor response. Furthermore the similarity of the response Brain Research, 31 (1971) 295-311

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in frontals and controls after c~-MT suggests that catecholamines play a ~cnOal roJt: in both the normal and the enhanced response to amphetamine, tn this scn:se the enhanced amphetamine response would not seem to be a non-specific release phenon-~enon although the widespread depletion achieved by (z-MT prevents an3' more precise identification of the site of the catecholamines vital for the nor~T:at and enhanced amphetamine response.

Substantia nigra lesions (i) General observations All the lesioned animals showed severe adipsia and aphagia after the operation and were force-fed and watered until self-sustaining. Presumably this effect was the major reason for the high mortality rate observed after these lesions, although in addition to emaciation almost all of the animals who died several days postoperatively showed an engorged and bleeding penis. The significance of these symptoms is not apparent. In the experiments (4 and 7) where results are presented for the immediately postoperative days, data from obviously sick animals who subsequently died were not included. Ungerstedt z2 has reported similar effects on eating and drinking behaviour after lesions to the substantia nigra induced by local injections of 6-OH-DA. (ii) Immediately postoperative behaviour In addition to altered eating and drinking behaviour, the substantia nigra animals were observed to be hyperactive as soon as they recovered from the anaesthetic. Some aspects of the hyperactivity were quantified and spontaneous locomotor activity, neck movements, and elicited gnawing were found to be significantly greater than in the controls for the first 10 postoperative days. The time course of these enhanced responses is similar to that observed for the depletion of catecholamines from the striatum by Faull and Laverty 6, consequent upon degeneration of the nigrostriatal pathway. It is difficult to explain precisely why such degeneration and depletion should enhance motor output. It is possible that as the DA terminals in the striatum degenerate, there is a sudden release of endogenous DA. The effect on the postsynaptic membrane might be expected to mimic that of DA released by other means, for example by amphetamine, which is known to enhance the same aspects of motor behaviour. Alternatively, it could be suggested that the lesion also disrupts the complex circuitry of the extrapyramidal system and thus its output and that this general disruption is gradually compensated for. (iii) Spontaneous locomotor activity in photocell cages Two weeks postoperatively the tests with amphetamine were begun and during the course of the experiments the locomotor response to saline was tested on two separate occasions. There were no significant differences between the response of the lesioned and control animals over the 2 h test periods. This finding is in keeping with recovery at l0 days of spontaneous locomotor activity in the SN animals (Fig. 6) and does not suggest that the lesion irretrievably altered the normal output of the extrapyramidal system. Brain Research, 3t (1971) 295-311

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(iv) The response to amphetamine The SN animals could be clearly divided into two groups, those in which the normal hyperactivity response to amphetamine was greatly reduced, 'low responders', and those in which the response was considerably enhanced, 'high responders'. In the low responder group the peak locomotor activity normally seen about 1 h after small doses of amphetamine (1.5 mg/kg D-amphetamine or 3 mg/kg DL-amphetamine) was greatly reduced, but stereotyped behaviour, characteristically seen at long intervals (2 h) after larger doses (2.5 mg/kg D-amphetamine or 5 mg/kg DL-amphetamine), was unaffected. Both the time course and dose-dependence of the normal amphetamine response was thus altered in these animals. Similar changes in the time course of the amphetamine response were also observed in the 'high responder' group of SN lesioned animals, but in these there was also a significantly increased hyperactivity response, at least immediately after the operation. In the biochemical experiments it was found that bilateral substantia nigra lesions resulted in a significant reduction of DA and tyrosine hydroxylase activity from the corpus striatum without any change in forebrain NA levels. Two major issues arise from these findings: (1) Can the partially blocked amphetamine responses be related to the partial DA depletion in the corpus striatum which resulted from the SN lesions? (2) Why was there a depletion of striatal DA and tyrosine hydroxylase of only 40 % in view of the histologically verified bilateral involvement of the zona compacta of the SN in almost all of the animals? Unfortunately, we have so far obtained biochemical and behavioural data on only animalslof Experiment 6, so that we do not yet have sufficient results to determine whether the variable amphetamine responses observed after SN lesions can be correlated with the extent of DA or tyrosine hydroxylase loss from the striatum. In general histological examination of SN lesions indicated the lesions may have often failed to damage the most anterior part of the zona compacta and in several cases the lesions extended beyond the SN posteriorly, sometimes involving the red nucleus, which may explain the hind limb rigidity noted in these animals when they were held for injection. Indeed, the 'high responders' in Experiments 5 and 6 appeared to be animals who had sustained more extensive posterior damage. These results do not provide a definitive answer to the question of whether the integrity of the nigrostriatal pathway is essential for the normal locomotor responses to amphetamine. In pursuing the problem it is hoped to study amphetamine responses after local microinjections of 6-OH-DA to the anterior substantia nigra 23. This compound is specifically taken up by and destroys amine containing neurones 24 and in preliminary experiments we have confirmed that an almost total depletion of striatal DA can be achieved in this way. Such information is vital to be able to evaluate the present partial results. It may be of relevance that both Faull and Laverty 6 working on the rat and Poirieretal.lS, 19 on the cat also obtained only partial DA depletions with discrete nigral lesions and reported that more extensive lesions involving the tegmentum were necessary for total depletion. These findings suggest that there are substantial ascending DA neurones in the regions outside the substantia nigra, which are not damaged by local surgical Brain Research, 3[ (1971) 295-311

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intervention but may be by 6-OH-DA. Ungerstedt 2z has reported that bilateral 6-OHo DA injections into the SN result in changes in motor behaviour similar ,o 0lose we have observed during the first 10 days after bilateral surgical lesions of tl>, structure. It is possible that the blocked amphetamine response, like the imm,:diate postoperative behaviour are unrelated to the states of the DA system and merely reflect surgical interference in the functioning of the extrapyramidal system. This seems unlikely on account of the enhanced output of the motor system immediately posloperatively and the subsequent normal spontaneous locomotor activit2~. However. this tenet is also on weak anatomical ground. The striatum does project to the SN but functionally the important outputs of the corpus striatum are via the pallidum and the thalamus. SUMMARY

Bilateral lesions to the frontal cortex of the rat resulted in enhancement of the characteristic hyperactive responses to amphetamine, By contrast, bilateral lesions to the substantia nigra (SN) reduced the locomotor response to amphetamine and changed its characteristics. These results suggest that the corpus striatum, and in particular the dopaminergic pathway from the SN to the caudate nucleus play a central role in some of the behavioural effects of amphetamine. ACKNOWLEDGEMENTS

B. A. Simpson, S. Wilkinson, J. A. Wilde and P. H. Bradney have made contributions to these studies. It is a pleasure also to thank Mrs. J. Baker who performed the biochemical assays.

REFERENCES 1 ANDI~N, N.-E., FUXE, K., HAMBERGER, B., AND HOKFELT, T., A quantitative study on the nigrostriatal dopamine neuron system in the rat, Acta physiol, scand., 67 (1958) 306-312. 2 BRADLEY, P. B., Synaptic transmission in the central nervous system and its relevance for drug action, Int. Rev. Neurobiol., 11 (1968) 1-56. 3 CARR, L. A., AND MOORE, K. A., Norepinephrine: Release from brain by D-amphetamine in vivo, Science, 164 (1969) 322-323. 4 COWAN, W. M., CARMAN, J. B., AND POWELL, T. P. S., The organisation of cortico-striate connex ions in the rabbit, Brain, 86 (1963) 525-562. 5 DAHLSTR6M, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurones in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, stand., Suppl. 232 (1964) 1-55. 6 FAULL, R. L. M., AND LAVERTY, R., Changes in dopamine levels in the corpus striatum following lesions in the substantia nigra, Exp. Neurol., 23 (1969) 332-340. 7 GROSS, C. G., CHOROVER, S. L., AND COHEN, S. M., Caudate, cortical, hippocampal and dorsal thalamic lesions in rats: Alternation and Hebb-Williams maze performance, Neuropyschologia. 13 (1965) 53-68. 8 HARRELL, N. W., AND ISAAC, W,, Frontal lesions and illumination effects upon the activity of the albino rat, Physiol. Behav., 4 (1969) 477-478.

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