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Functional validation of projection topography in the nigrostriatal dopamine system
0306-4522/82/040885-10~3.00/0 Pergamon Press Ltd 0 1982IBRO
Nanowience Vol. 7. No. 4, pp. 885 to 894. 1982 Printed in Great Britain.
FUNCTIONAL VALIDATION OF PROJECTION TOPOGRAPHY IN THE NIGROSTRIATAL DOPAMINE SYSTEM Department
P. REDGRAVEand I. MITCHELL of Psychology, University of Sheffield, Sheffield, SlO 2TN, U.K.
Abstract-Anatomical investigations have revealed that the nigrostriatal pathway is topographically organkd. In two experiments, nigrostriatal topography was investigated with catecholamine specific procedures, using paradigms which reflect the functional activity of dopaminergic neurones. Data were analysed with the intention of discovering possible relationships between the mesencephalic location of stimulating electrodes or injection cannulae, the extent and location of dopamine histofluorescence depletion within the striatum, and the effects of amphetamine and apomorphine on rotational behaviour. In animals pretreated with 250 mg/kg a-methyl-p-tyrosine it was found that unilateral stimulation with medially-placed nigral electrodes produced maximal depletion of dopamine histofluorescence in anterior dorso-medial regions of the striatum, while laterally-located electrodes principally depleted posterior, ventro-lateral areas. In the second experiment, 2pg of 6-hydroxydopamine in a volume of 0.5 pl was injected unilaterally at varying loci within the ventral midbrain of animals pre-treated with desmethylimipramine (25 mg/kg). It was discovered that the lateral injection coordinate was significantly associated with both the extent and location of the depletion of dopamine-related fluorescence from the ipsilateral striatum. Rotational behaviour, induced by dopamine-agonists was related firstly, to the overall extent of dopamine depletion from the striatum, and secondly, the contraversive turning induced by apomorphine in particular was related to the dorsoventral coordinate of the mesencephalic 6-hydroxydopamine injection. The results provide functional vahdation for the.pattern of topographical projection within the nigrostriatal dopaminergic system proposed on the basis of intracellular tracing techniques.
dopaminergic projections from the mesencephalon traditionally have been divided into three major groups, a nigrostriatal system,‘*25*36 a mesolimbic system and a mesocortical system.2,26*3s From those investigations, the nigrostriatal pathway was generally thought to arise from dopamine (DA) cells located in the pars compacta region of substantia nigra (SN) (A9 group of Dahlstrom & Fuxe’“) while the mesolimbic and mesocortical projections were considered to originate from the medial A10 cell group of the ventral tegmental area (VTA). Recent reports, however, suggest that the concept of SN and VTA cells giving rise to the striatal and limbic/cortical DA projections, respectively, may be overly simple. 3~12~14*1 s These investigations show that mesencephalic DA cell groups form a continuum, with both the VTA and SN providing DA projections to the majority of telencephalic terminal areas. Differential patterns of projection within the SN-VTA system are accounted for by the discovery of a clearly organ&d topography with medial-lateral, anteriorposterior and dorsoventral components. On the basis of this topography, it seems that the striatal projection derives from the ventral layer of DA cells in the SN and VTA within which there are clear medialAscending
Abbreviations: CN, caudate nucleus; DA, dopamine; SN, substantia nigra; VTA, ventral tegmental area; 6-OHDA, 6-hydroxydopamine.
lateral and anterior-posterior patterns of organisation. On the other hand, the majority of cortical and limbic projections arises from the dorsal layer of DA cells in the SN and VTA; again, there is some evidence for a medial-lateral topography within this subsystem. The functional properties of the ascending DA systems are not yet well understood. However, studies involving the manipulation of transmission in forebrain DA systems;9~13*16~22*30 indicate that locomotor activity responses seem to be more closely associated with limbic DA function while the striatal system is thought to mediate DA-stereotyped behaviour and postural control. With some exceptions,8*‘g*20~34 much of the work investigating the functions of the different DA systems at a behavioural level has manipulated DA neuronal activity within regions of the telencephalon. While these studies support the notion of distinct functional properties of striatal and limbic/ cortical DA systems, they do not distinguish between the traditional and more recent concepts concerning the arrangement of DA cells within the mesencephalon. There is, therefore, a particular need for experiments which seek to clarify the specific functions of DA cells at different locations within the ventral mesencephalon. In the preceding articleJ2 we reported experiments which were designed to test the validity of new procedures for determining the regional content of DA within the caudate nucleus (CN) by measuring the
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intensity of DA-induced histochemical flourescence. While conducting these experiments, it became clear that both unilateral electrical stimulation in animals pre-treated with a-methyl-p-tyrosine and injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra caused a regional depletion of DA fluorescence in the CN. In view of the need to clarify the functional properties of DA cells within different regions of the ventral mesencephalon, it seemed important to determine whether the precise location of the experimental manipulation within the ventral mesencephalon influenced the particular region of the CN maximally depleted of DA. In an extensive analysis of data obtained from experiments reported in the previous paper, we looked for specific relationships between (i) each of the stereotaxic co-ordinates of the mesencephalic electrode or injection cannula, (ii) the area of maximum DA fluorescence depletion within the CN, and (iii) in the 6-OHDA experiment, the effects of amphetamine and apormorphine on rotational behaviour. The purpose of the present paper is to report the results of these analyses.
EXPERIMENT 1: UNILATERAL ELECTRICAL STIMULATION OF SUBSTANTIA NIGRA IN ANIMALS PRE-TREATED WITH z-METHYL-p-TYROSINE
One paradigm used to test the validity of procedures for quantifying DA histofluorescence was to examine whether the relative depletion of fluorescence from the CN ipsilateral to a nigral electrode was related to the parameters of electrical stimulation applied to substantia nigra of animals pre-treated with a-methyl-p-tyrosine. Visual examination of CN tissue from animals used in Experiments 2A and 2B of the preceding paper3* revealed that the DA depletion on the stimulated side was largely confined to local areas of the CN. It was generally found that two or three of the six measurement positions (see Fig. 1B of the previous paper) were more severely depleted than the others. However, precisely which positions were affected was variable across animals. Clear differences in the degree of depletion were also observed in the anterior-posterior plane; this again was variable across animals. In view of the regional nature of the DA depletion, the present analysis was conducted in order to determine whether the area of maximum DA depletion within the striatum was related ition of the stimulating electrode in SN. EXPERIMENTAL
to the pos-
PROCEDURES
Following a period of nigral stimulation in rats pre-
treated with a-methyl-p-tyrosine (see previous paper) brain tissue was processed by a glyoxylic acid method for monoamine histofluorescence.” Fluoresanct intensity, integrated over a circular field (diameter 800pm). was measured photometrically. Measurements from six defined positions (see Fig. IB in the previous article) were taken
from the CN on each side of each coronal tissue wctlon. Thus. a grid of measurements was obtained from 1(1 (2islide) sections taken from different levels in the anteriorposterior plane of the CN. A complete dcscriptlon of the subJccts, surgtcal procedures. experimental conditions. histochemical processmg and fluorescence measurements can be found In I:xpcrlments 2A and 2B of the preceding article.” Duta
Each fluorescence from the side was from the derived from corresponding position the non-stimulated of the tissue section. gave a measure in the non-stimulated was acting an internal These individual scores for animal were over slides = 5) over measurement (!Y = Because animals different stimulating relative depletions slides were 1 to 5 (the sixth slide being omitted from animals in Experiment 2A) while depletions over positions were ranked 1 to 6; the highest depletion score attracted the highest rank. Thus, the slide ranks provided an indication of the relative extent of regional DA depletion within the anterior-posterior plane, while the position ranks were related to DA depletion within the dorsoventral and mediolateral dimensions of the CN. Subsequently. for each stimulated animal the value of five factors was determined. (1) A relative measure of the extent to which DA was depleted from anterior as opposed to posterior regions of the CN in each animal was obtained by subtracting the average rank of the posterior two slides from the average rank of the anterior two slides. (2) A relative measure of the extent lo which regions of the CN were differentially depleted of DA in the medial/lateral and dorsaliventral dimensions was achieved by allocating each animal to one of five groups presented in Table I on the
basis of which three measurement positions were most highly ranked for depletion. (3) The anterior-posterior electrode coordinate. (4) The lateral electrode coordinate. (5) The horizontal-vertical electrode coordinate. Finally, all the animals were ordered or grouped according lo their relative levels on each of the five factors. Having arranged the data in this manner it was possible to take individually each factor as the dependent variable and lo relate it to each of the other factors. Where a factor was continuously variable a trend analysis” was used to assess the relationship between it and the other factors. If, however. a factor was arranged in discon-
Table 1. A relative determination of fluorescence depletion in the medial-lateral and dorsal-ventral dimensions of the striatum was made by assigning animals to one of five groups depending on which measurement positions (see Fig. 1B of previous article) were maximally depleted Depletion group Medial Dorsomedial Dorsolateral Ventrolateral Ventromalial
Positions of maximal depktion VM. C, DM DM, C, D D, C. L L. c. VL VL, C. VM
Abbreviations: C. central; D, dorsal; DM, dorsomedial: L. lateral; VL, ventrolateral: VM. ventromedial.
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Fig. 1. The six graphs show the relationship between the relative degree of depletion along the anteriorposterior axis (predominant depletion in the anterior caudate nucleus is indicated by positive numbers, while negative numbers indicate increasing relative depletion in the posterior caudate nucleus) and the mean depletion for each of the six measurement positions. The linear correlation and probability associated with each relationship is presented.
tinuous groups it was tested against the other factors with a Krusk~-Wa~Iis non-p~ametric analysis of variance.33
RESULTS Regional dopamine depletion within the caudate nuclars
Before an attempt was made to discover any possible relationships between specific areas of the DA depletion within the CN and the location of the nigral stimulating electrodes, it was first necessary to provide a three-dimensional description of the regional nature of DA depletion within the CN. This was accomplished by relating the factor indicating relative depletion within anterior and posterior regions of the CN with the factor representing relative depletion in the medial-lateral and dorsal-ventral aspects of the CN. The form of this relationship was characterised by an association between depletion in anterior and medial areas of the CN; depletion in posterior and lateral regions was also related. This relationship is illustrated in Fig. 1 where the mean ranks for depletion are shown for each of the six measurement positions when the animals were ordered in terms of relative depletion in the anterior-posterior dimension. Thus, it can be seen that for the dorso medial m~surement position, animals with rn~~~ depletion within the anterior CN (f 3) tended to have this position ranked highly for depletion, whereas animals principally depleted in the posterior regions of the CN (-3) generally had a low rank assigned to this position. A gradual reversal of this relationship
was observed as the measurement position assumed a more lateral location within the CN. These data indicate a clear association between DA depletion in anterior and medial and between posterior and lateral areas of the CN. An attempt was then made to relate specific areas of DA depletion within the CN to the location of stimulating electrodes in the substantia nigra. Nigral stimulation locus and regional depletion of sttiatal dopamine The first question to be considered was whether animals assigned to different groups on the basis of relative DA depletion among the six m~surement positions (see Table 1) would have consistently different positioning of the nigral stimulating electrode. Table 2 contains the mean electrode coordinates for each of the five depletion groups described in Table 1.
Table 2. Position depletion x electrode co-ordinates
Depletion group
N
Mean AP
Electrode Lat.*
Media) Dorsomediat Dorsolateraf Ventrolateral Ventral
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Co-ordinate HV -8.6 - 8.6 -8.3 -8.6 -8.5
The electrode coordinates were derived from the stereotaxic atlas of Pellegrino & Cushman.29 * Significant difference between means P < 0.001 (Kruskal-Wallis analysis of variance).
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Fig. 2. The six graphs show the relationship between the lateral coordinate of the nigral electrode placement and the mean depletion rank for each of the six measurement positions. The linear correlation and probability associated with each relationship is presented.
From these data, it is apparent that when proceeding in a clockwise direction around the CN from the medial depktion group the me-an lateral electrode coordinate for each group increased progressively. No signilkant relationship was found between relative depletion among the measurement positions and the other two electrode coordinates. Despite the manifest association between DA depletion in anterior and medial and in posterior and
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lateral regions of the CN a sign&ant relationship between relative depletion in the anterior-posterior dimension and the nigral ekctrode coordinates could not be demonstrated. However, a con&m@ trend towards anteriorly depleted animals having medially placed ekctrodes and posteriorly depleted animals having laterally placed ekctrodes Was noted. These analyses indicate that the specitlc area of the CN, which is maximally depkted of DA iluaranxnct
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Fig. 3. The five graphs show the rdnthmbip betweenthe lateral ~OO&UUCof the nigrpl ekclrodc placement and the man dqktioa rrdr for oath of the five slides. !%i& 1 cotttainui the two most anterior sections of CN tissue while the two most posterior sections were on slide 5. Tote linear correlation and probability associated with each relationship is presented.
Ni~ost~atal topography following electrical stimulation of substantia nigra, is significantly influenced by the lateral position of the stimulating electrode. Cotirmation of this relationship was sought by ordering the animals in terms of the lateral&y of their nigral electrode and plotting the average depletion rank for each of the six CN measurement positions. The resulting graphs (see Fig. 2) indicate that medial measurement positions attracted progressively lower depletion ranks as the lateral coordinate of the nigral electrode increased. This relationship gradually reversed as the measurement position itself became more lateral. The effects of laterality of nigral stimulation on DA depletion within the anterior-posterior dimension of the CN is shown in the graphs of Fig. 3. From this Figure it can be seen that animals with medially located electrodes tended to have anterior slides (1 and 2) ranked highly for deletion while posterior slides (4 and 5) were ranked lowly. Again this relationship seemed to reverse gradually as the eleo trode location became more lateral. In summary, it is concluded from these analyses that mediaRy placed nigral electrodes produced maximal depletion of DA ~stofluores~~ in anterior, dorsomedial areas of the CN whiie laterally located electrodes produced greater depletion in the posterior, ventrolateral regions of this structure.
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0.2 mgml ascorbate-saline solution) (N = 24) or an equivalent volume of the ascorbate vehicle (N = 6). The location of the intracranial injections was systematically varied in the medial-lateral dimension between L 0&3.0mm; the other coordinates were AP-2.5 and HV-7.8.29 The remaining six animals served as unoperated controls. Behavioural testing procedures
On days 11, 12 and 13 after surgery, behavioural measurement of rotation was obtained following the intraperitoneal injection of apomorphine (2 mg/kg) (MacFarlan Smith), ~phet~ine (1 m&kg) (Smith, Kline 8z French) or an equiv~ent volume of the 0.9% iialine vehicle. Each animal received each of the three drug treatments (1 treatment per day) in a counterbalanced order. The number and direction of 360 degree turns in a flat-bottomed bin (diameter 21 cm), were recorded in two 2 mitt periods, 25 and 40 min after injection. The scores for the two periods were combined, after which the number of turns ipsilateral to the intracranial 6-OHDA injection was subtracted from the number of contralateral turns to give a single measure of rotational behaviour. Histochemical processing and jluorescence measurement @I day 14 after surgery, all animals were killed and CN tissue was processed for ~t~hoI~ne histofluor~~n~ as described in the previous paper. In addition, the location of each intracranial injection was verified according to normal histological procedures.‘O Data analysis
EXPERIMENT 2: UNILATERAL INJE~IONS OF 6-HYDROXYDOPAMI~ AT DIFFERENT LOCATIONS WITHIN THE VENTRAL MESENCEPHALON
An alternative strategy used to validate procedures for the photometric assessment of DA histofluorescence was to determine the effects of 6-OHDA destruction of mesencephalic DA cell bodies on CN fluorescence. Again, preliminary visual examination of CN tissue from animals treated with 6-OHDA revealed regional patterns of DA depletion. Thus, on the basis of these informal observations, we decided to examine whether the location of the 6-OHDA injection site in the ventral me~ncephalon would predict the region of the CN maximally depleted of DA fluorescence. In the three days prior to death the effects of amphetamine, apomorphine and vehicle control injections on the rotational behaviour of each animal was determined. As a result of these me~uremen~ we were able to relate the behavioural performance, firstly to the location of the 6-OHDA injection and secondly, to the area of maximum DA fluorescence depletion within the CN. EXPERIME~AL
PROCEDURES
Subjects and surgical procedures
Thirty-six female hooded rats were used, of which 30 animals were treated with 25 mg/kg desmethyl-imipramine (Geigy) prior to a stereotaxically-positioned intracranial injection of either 6-OHDA (Sigma) (2pg free base in
For each animal injected with 6-OHDA, the value of nine factors was ‘determ~~; these have been item&l in Table 3. The coordinates for each injection site were derived from the stereotaxic system of De Groot.29 A relative measure of the extent to which each animal was depleted of DA, in anterior as opposed to posterior regions of the CN, was determined by subtracting the mean depletion score for tissue on the anterior two slides from the mean depletion score for tissue on the posterior two slides, This provided a continuous variable along which animals could be ordered. Indication of maximal depletion in the medial-lateral and dorsoventral planes of the CN was achieved by assigning animals to different groups, depending upon which of the six measurement positions (see Fig. 1B of previous paper) was most severely depleted. A total of four groups (medial, interm~ate/ medial, intermediate/lateral and lateral) was created to indicate relative depletion in the medial-lateral plane of the CN. Alternatively, animals were assigned to three groups (dorsal, intermediate and ventral) which were reflective of the relative depletion of dorsal us ventral measurement positions. As the rotational behaviour of each animal was expressed on a single continuously varying scale, two separate listings of the animals were created to indicate the relative amounts of rotational behaviour induced first by apomorphine and second, by amphetamine. Relationships between the factors were investigated either by cor~lational analyses or by the use of non-parametric analyses of variance (see above). RESULTS
The results will be discussed in relation to the following three issues: (i) the relationship between the
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P. Redgrave and I. MItchelI Table 3. The value for each animal treated with 6-hydroxydopamine was calculated on each of 9 dimensions -(i) 6-OHDA injection coordinates anterior-posterior lateral horizontal-vertical (ii) Striatal depletion of DA fluorescence
(d) (e) (f) (gj
(iii) Index of rotational behaviour after administration of:
location of the 6-OHDA injection in the ventral mesencephalon and the extent and location of DAdepletion from the CN, (ii) the relationships between the extent and location of striatal DA-depletion and rotational behaviour, and (iii) the relationships between the location of the 6-OHDA injection and rotational behaviour. GHydroxydopamine dopamine-depletion
injection
location
and
striatal
The major finding was that the overall extent of DA-depletion from the ipsilateral striatum was significantly related to the lateral coordinate of the 6-OHDA injection in the ventral midbrain. This relationship is illustrated in Fig. 4A and is best described as an inverted U-shaped function with maximal striatal DA-depletion achieved by injections placed approximately 1.5 mm lateral to the midline. The use of orthogonal components in tests for trends within these data3q revealed a best-fitting curve with a significant (P < 0.025) quadratic component (see Fig. 4A). A second finding was that when the animals were grouped according to striatal DA-depletion in the medial/lateral dimension it was found that maximum depletion in medial CN was associated with medial
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overall mean depletion anterior-posterior depletion medial-lateral depletion dorsal-ventral depletion apomorphine amphetamine
&OHDA injection coordinates while latmal depktion was associated with lateral coordinates. This relationship is illustrated in Fig. 4B; the di%mnces in the mean lateral coordinate between the groups was statistically significant @ < 0.001; Kruskal-Wallis analysis of variance). No other significant relationships between the ractors describing striatal DA depktion and the 5-OHDA injection coordinates were discovered. The reason for this may have been that the range of AP ,and HV measurements over which the animals varied was comparatively small. In summary, it was discovered that the lateral zooordinate of the 6-OHDA injection significantly affected both the overall extent and location of the DAdepletion from the ipsilateral striatum. Striatal dopamine-depletion
and rotational behaoiour
Significant linear relationships were found between the overall extent of DA-depletion from the ipsihteral striatum and the rotational behaviour induced by both amphetamine and apomorphine. Graphs A and B of Fig. 5 indicate that animals which showed most ipsilateral rotation after amphetamine and most xntralateral rotation after apomorphine tended to be the animals with the greatest overall depletion of DA
. .
Fig. 4. (A) The relationship between the lateral coordinate of the mesencephalic dhydroxydoplunine injection and the mean dopktion of rtriatal dopamine fluorsscsnce. The sign&ant quadratic treed (Q) is represented by the line of best fit. (9) The mean lateral injection coordinates of animaJa grouped according to differential depletion of DA fluorescence in the medial-lateral plane of the stinturn. Abbreviations: Med. medial; IM, intermediate-medial; IL, intermediate-lateral; Lat, lateral.
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Nigrostriatal topography
Fig. 5. (A) The linear relationship between turning induced by amphetamine and the mean depletion measure of striatal dopamine fluorescence.. (B) The linear relationship between turning induced by apomorphine and the mean depletion measure of striatal DA fluorescence.
from the CN. There was no consistent evidence to suggest that DA-depletion within any particular sub-
region of the CN was particularly rotation scores.
related to high
6-Hydroxydopamine injection location and rotation
It was found that the rotation induced by apomorphine was significantly related to the dorsoventral coordinate of the 6-OHDA injection into the ventral midbrain. Thus. Fig. 6 shows that high rates of contralateral rotation following the administration of apomorphine were associated with ventrally located injections of 6-OHDA. Apart from this result no other significant relationships between the location of the 6-OHDA injection and rotational behaviour were discovered. DISCUSSION Two experiments were performed in order to examine whether manipulation of the DA pathway afferent to the CN influences the intensity of DA-fluorescence within this structure. The extent to which the results of these experiments validated procedures for the quantitative measurement of DA-fluorescence was the concern of the preceding paper. The purpose of the present study was to analyse data from the two experiments with the intention of investigating the possible relationships between the location of treatments within the ventral mesencephalon, the extent and location of striatal DA depletion and the levels of rotational behaviour following the administration of amphetamine and apomorphine. Topographical relationships dopaminergic projection One important
in
the
the more caudal aspects of the CN. A much clearer description of nigrostriatal topography has been provided by anatomical studies using either silver impregnation techniques or the intraneuronal transport of horseradish peroxidase and [3H]1eucine/pro1ine.4*6*7*14*37 In general, it has been found that the pars compacta region of substantia nigra is organised so that the anteromedial portion of the CN receives efferents from the media1 substantia nigra, while cells in the lateral nigra project to the posterolateral CN. However, despite the generally convincing reasons given for considering that these patterns of projection relate principally to DA cells, it should be noted that the transport of horseradish peroxidase and [3H]amino acids is not specific to DA neurones. Thus it is important that the pattern of nigrostriatal topography described on the basis of intraneuronal transport techniques was, for the most part, confirmed in the present experiments in which histochemical procedures specific to catecholamines were used.” In addition, the protection of noradrenaline neurones by injections of desmethylimipramine, together with the finding that noradrenaline is present in the CN only in small amounts,1s*z3.3* probably means that the changes in the fluorescence intensity measurements observed in the present experiments were specifically related to changes in DA levels. In the first experiment of the present paper, a clear association between DA depletion in anterior and medial regions and between posterior and lateral areas of the CN was established. This is entirely consistent with the anteromedial and posterolateral topography proposed by investigators using tracing techniques. Further evidence for this topographical pattern was provided by the finding that mediallylocated electrodes caused maxima1 depletion of DA fluorescence in anterior dorsomedial areas of the CN, while laterally-placed electrodes produced most depletion in posterior ventro-lateral regions. Additional evidence for medial-lateral topography was found in the second experiment when it was observed that animals depleted of DA fluorescence principally in media1 regions of the CN had medially placed
nigrostriatal
aspect of the analyses presented in this paper is that direct functional evidence was provided for a topographical pattern of projection within the nigrostriatal DA-system. In the past, UngerstedP demonstrated that rostromedial substantia nigra lesions denervated the entire CN of DA whereas caudolateral nigral lesions only depleted DA levels in
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Fig. 6. The linear relationship between turning induced by apomorphine (2 mg/kg) and the dorsoventral coordinate of the mesencephalic 6-hydroxydopamine injection.
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6-OHDA injections while lateral CN depletion was associated with lateral placements of 6-OHDA. One way in which the current findings depart from observations made with intracellular tracers14 is that no differential patterns of projection were found which related to the anterior-posterior and dorsoventral placement of stimulating electrodes of 6-OHDA injections in the mesencephaion. This difference may be explained by the fact that most of the present stimulating electrodes and 6-OHDA injections were located towards the rostra1 pole of SN. In this position, it is unlikely they could affect differentially separate populations of anteriorly projecting DA-fibres. Of potential importance to the present investigation are some studies of Glowinski and his coworkers,24*2s where it is reported that in a variety of situations, there is an interdependence of the nigrostriatal dopaminergic systems on each side of the brain in the cat. Briefly, they found that when a particular unilateral manipulation of substantia nigra induced an ipsilateral change in striatal DA release it was often associated with an opposite change in the release of DA in the contralateral CN. If this bilateral interdependence of activity in the nigrostriatal DA pathways is also present in the rat, the difference measures in the present experiments will have been influenced. For example, unilateral electrical stimulation of substantia nigra may have caused not only an ipsilateral increase in DA release, but a corresponding decrease in release from the contralateral CN. Unfortunately, the calculation of simple subtraction or ratio measures of the difference between the fluorescence intensity on each side of the brain provides no clear indication of the relative importance of these two factors. While this limitation may restrict the use of the present photometric procedures in some instances, the estimation of the relative difference in the activity of DA neurons on each side of the brain seems to be unaffected. For example, the frequencyresponse function described in Fig. 6B of the previous paper is directly comparable with the data from experiments using alternative methods for measuring the activity of DA neurons. One final point in this section concerns the precise location of a nigral injection of 6-OHDA and its effect on the extent of DA-depletion in the ipsilateral striaturn. In the second experiment a standard 2pg of 6-OHDA was injected in a volume of 0.5 ~1 throughout. This is approximately one quarter the volume and dose that is commonly used to lesion mesencephalic DA cell bodies. 5*21Systematic variation of the lateral placement of this injection permits the conclusion that the most effective depletion of DA-fluorescence from the ipsilateral striatum can be achieved when the 6-OHDA injection is placed approximately 1.5 mm from the midline (see Fig. 4A). Behavioural correlates of striatal dopamine depletion
In the second experiment, measures of rotational behaviour following the administration of apomor-
phine and amphetamine were obtained. The first general finding was that rotational behaviour induced by both amphetamine and apomorphine was related to the overall extent of DA fluorescence depletion from the CN. Thus, it was observed that a high rate of both ipsilateral turning induced by amphetamine, and contralateral turning induced by apomorphine. tended to occur in association with large differences in the intensity of DA fluorescence between the CN on each side of the brain. These observations are in general agreement with previous findings which indicate that, in the present experimental paradigm, rotational behaviour reflects a relative imbalance between striatal DA activity on either side of the brain. (for reviews see Glick et a/.” and Pycock” ). The present results on turning are not, however. entirely consistent with the recent findings of Hefti er a/.,19 who reported that significantly greater depletions of striatal DA were required to produce rotation in response to apomorphine compared with that induced by amphetamine. Reference to Fig. 5 indicates that the amount of rotation induced by amphetamine and apomorphine over the present range of striatal DA depletions was comparable. This difference in results may have been caused by the use of different doses of the DA agonists, e.g. a higher dose of apomorphine (2 mg/kg cf. 1 mg/kg) and a lower dose of amphetamine (1 mg/kg cf. 5 mg/kg) was used in the present experiments. This explanation is. however, unlikely since Hefti et al., found that raising the dose to 5 mg/kg of apomorphine still failed to elicit contralateral turning in rats which clearly turned ipsilaterally in response to amphetamine. A more plausible account of the discrepant results may be found by considering differences in the procedures of 6-OHDA administration. In the present experiments. a small volume (0.5 ~1) of a solution of 6-OHDA whose concentration (4&$) has been demonstrated to be toxic to catecholaminergic neurons2’ was injected into different regions of the ventral tegmentum. Hefti et a1.,19 however, used a range of doses of 6-OHDA which were injected in the rather larger than normal volume of 4 pl. Given the topographical nature of the ascending DA systems it seems reasonable to suppose that the pattern of striatal DA depletion induced by these procedures may well be different, and that such differences may account for the discrepant results. The discovery of a relationship between the dorsoventral placement of 6-OHDA and turning is relevant to the understanding of the functional properties of different populations of mesencephahc DA cells. It was found that high rates of contralateral rotation in response to apomorphine were associated with the more ventral placements of QOHDA within the ventral tegmentum. In the intraneuronal tracing studies of Fallon & Moorei it was found that ventrallylocated cells within the SN-VTA complex projected mainly to the CN. whereas the cells which project to structures of the limbic forebrain were located more
Nigrostriatal topography dorsally. The present finding that a behaviour thought to be of striatal origin, i.e. turning, was particularly associated with ventrally-located injections of 6-OHDA provides functional evidence which is consistent with the arrangement of cells within the ventral tegmentum proposed by Fallon & Moore.14 Finally, in the present experiments no attempt was made to measure possible changes in the intensity of DA-related fluorescence in structures other than in
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the striatum. Thus, in future investigations of the functional properties of DA cells at different locations in the ventral mesencephalon, assessment of relative DA depletion in as many forebrain DA terminal areas as possible should be included. This may be achieved by slight modification of the present procedures for the photometric assessment of glyoxylic acid-induced fluorescence from the brain of a single subject (see preceding paper).
REFERENCES 1. And&n N. E., Carlsson A., DahlstSm A., Fuxe K., Hillarp N. A.& Larsson K. (1964) Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 3, 523-530. 2. Andtn N. E., Dahlstriim A., Fuxe K., Larsson L., Olson L. & Ungerstedt U. (1966) Ascending monoamine neurons to the telencephalon and diencephalon. Acta. physiol. stand. 67, 313-326. 3. Avendand C., Reinoso-Suarez F. & Llamas A. (1976) Projections to gyrus sigmoideus from the substantia nigra in the cat, as revealed by the horseradish peroxidase retrograde transport technique. Neuroscience Letters 2, 61-65. 4. Bedard P., Larochelle L., Parent A. & Poirier L. J. (1969) The nigrostriatal pathway: a correlative study based on neuroanatomical criteria in the cat and the monkey. Expl Neural. 25, 365-377. 5. Butcher L. L., Eastgate S. M. & Hodge G. K. (1974) Evidence that punctuate intracerebral administration of 6-hydroxydopamine fails to produce selective neuronal degeneration. Naunyn-Schmeidebergs Arch. Pharmac. 285, 31-70. 6. Carpenter M. D. & Peter D. (1972) Nigrostriatal and nigrothalamic fibres in the cat. J. camp. Neural. 144, 93-116. 7. Carter D. A. & Fibiger H. C. (1977) Ascending projections of presumed dopamine-containing neurons in the ventral tegmentum of the rat as demonstrated by horseradish peroxidase. Neuroscience 2, 569-576. 8. Costall B., Marsden C. D., Naylor R. J. & Pycock C. J. (1976) The relationship between striatal and mesolimbic dopamine dysfunction and the nature of circling responses following 6-hydroxydopamine and electrolytic lesions of the ascending dopamine systems. Brain Res. 118, 87-l 13. 9. Costall B. & Naylor R. J. (1973) The role of telencephalic dopaminergic systems in the mediation of apomorphine stereotyped bchaviour. Eur. J. Pharmac. 24, 8-24. 10. Dahlstriim A. & Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system-I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta. physiol. stand. 62, Suppl. 232, l-55. 11. De la Torre J. C. & Surgeon J. W. (1976) A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method. Histochemistry 49, 81-93. 12. Domesick V. B., Beckstead R. M. JL Nauta W. J. H. (1976) Some ascending and descending projections of the substantia nigra and ventral tegmentum area in the rat. Neuroscience Abs. 2, 61. 13. Ernst A. M. & Smelik P. G. (1966) Site of action of dopamine and apomorphine on compulsive gnawing behaviour. Experientia 22, 837-838. 14. Fallon J. H. & Moore R. Y. (1978) Catecholamine innervation of the basal forebrain-IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. camp. neural. 180, 545-580. 15. Fallon J. H., Riley J. N. & Moore R. Y. (1978) Substantia nigra dopamine neurons: separate populations project to neostriatum and allocortex. Neuroscience Letters 7, 157-162. 16. Fog. R., Randrup A. & Pakkenberg H. (1970) Lesions in corpus striatum and cortex of rat brains and the effect on pharmacologically-induced stereotyped, aggressive and cataleptic behaviour. Psychophormacologia (Berl.) l&346-356. 17. Glick S. D., Jerussi T. P. & Fleisher L. N. (1976) Turning in circles: the neuropharmacology of rotation. Life Sci. 1% 889-896. 18. Grzanna R., Morrison J. H., Coyle J. T. & Molliver M. E. (1977) The immunohistochemical demonstration of noradrenergic neurons in the rat brain: the use of homologous anti-serum to dopamine-B-hydroxylase. Neuroscience Letters 4, 127-134. 19. Hefti F., Melamed E., Sahakian B. J. & Wurtman R. J. (1980) Circling behaviour in rats with partial, unilatera1, nigro-striatal lesions. Effects of amphetamine, apomorphine and DOPA. Pharmac. Biochem. Behau. 12, 185-188. 20. Hodge G. K. & Butcher L. L. (1979) Role of pars compacta of the substantia nigra in circling behaviour. Pharmac. Biochem. Behao. 10, 695-709. 21. Javoy F., Sotelo C., Herbert A. & Agid Y. (1976) Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system. Brain Res. 102, 201-215. 22. Kelly P. H., Seviour P. W. & Iversen S. D. (1975) Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Bruin Res. 94, 507-522. 23. Koslow S. H., Racagni G. & Costa E. (1974) Mass fragmentographic measurement of norepinephrine, dopamine, serotonin and acetylcholine in seven discrete nuclei of the rat tel-diencephalon. Neuropharmacology 13, 1123-l 130. 24. Leviel V., Cheramy A., Nieoullon A. & Glowinski J. (1979) Symmetric bilateral changes in dopamine release from the caudate nuclei of the cat induced by unilateral nigral application of glycine and GABA-related compounds. Brain Res. 175, 259-270.
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25. Lindvali 0. & Bjorklund A. (1974) The organisation of the ascending catecholamine neuron systems in the rat brarn as revealed by the glyoxylic acid fluorescence method. Acta physiol. stand. Suppl. 412, 1-48. 26. Lindvall O., Bjorklund A., Moore R. Y. & Stenevi U. (1974a) mesencephalic dopamine neurons projecting to nencortex. Brain Res. 81, 325-331. 27. Lindvall O., Bjiirkhmd A. & Svensson L. A. (19746) Fluorophore formation from catccholamines and related compounds in the glyoxylic acid fluorescence histochemical method. Hisrochemisrrj~ 39, 197 227. 28. Nieoullon A., Cheramy A. & Glowinski J. (1977) Interdependence of the nigrostriatal dopaminergic systems on the two sides of the brain in the cat. Science N. y 198, 416481. 29. Pelligrino L. J. & Cushman A. J. (1967) A Stereotaxic Atlas of the Rat Brain Appleton-Century-Crofts, New York. 30. Pijnenburg A. J. J., Honig W. M. M., Heyden J. A. M. & van Rossum J. M. (1976) Effects of chemical stimulation of the mesolimbic dopamine system upon locomotor activity. Eur. .I. Pharmac. 35, 4.5-58. 31. Pycock C. J. (1980) Turning behaviour in animals. Neuroscience 5.461-514. 32. Redgrave P. & Mitchell I. (1982) Photometric assessment of glyoxylic acid-induced fluorescence of dopamine in the caudate nucleus. Neuroscience 7, 871-883. 33. Siegel S. Non-parametric Statisticsfor the Behauioural Sciences pp. 184193. McGraw-Hill, New York. 34. Thal L., Mishra R. K., Gardner E. L., Horowitz S. G.. Varmuza S. & Makman M. H. (1979) Dopamine antagonist binding increases in two behaviourally distinct striatal denervation syndromes. Brain Res. 170, 381-386. 35. Thierry A. M., Blanc G., Sobel A., Stinus L. & Glowinski J. (1973) Dopaminergic terminals in rat cortex. Science N. ‘i: 18&499-501.
36. Ungerstedt U. (1971) Sterotaxic mapping of the monoamine pathways in the rat brain. Acra ph.vsioi. stand. SSQL 367, l-48. 37. Wsunoff K. G., Hassler R., Romansky
K., Usunova R. P. & Wagner A. (1976) The nigrostriatal projection in the cat-Part 1. Silver impregnated study. J. neural. Sci. 28, 265-288. 38. Versteeg D. H. G., van der Gugten J., de Jong W. & Palkovits M. (1976) Regional concentrations of noradrenaline and dopamine in rat brain. Bruin Res. 113, 563-574. 39. Winer B. J. (1971) Statistical principles in experimental design. McGraw-Hill Book Company, New York. 40. Wolf G. (1971) Elementary histology for neurophysiologists. In Methods in Psychobiology (ed. Myers R. D.) Vol. I. pp. 281-300. Academic Press, London. (Acceprd
19 September 1981)
Nanowience Vol. 7. No. 4, pp. 885 to 894. 1982 Printed in Great Britain.
FUNCTIONAL VALIDATION OF PROJECTION TOPOGRAPHY IN THE NIGROSTRIATAL DOPAMINE SYSTEM Department
P. REDGRAVEand I. MITCHELL of Psychology, University of Sheffield, Sheffield, SlO 2TN, U.K.
Abstract-Anatomical investigations have revealed that the nigrostriatal pathway is topographically organkd. In two experiments, nigrostriatal topography was investigated with catecholamine specific procedures, using paradigms which reflect the functional activity of dopaminergic neurones. Data were analysed with the intention of discovering possible relationships between the mesencephalic location of stimulating electrodes or injection cannulae, the extent and location of dopamine histofluorescence depletion within the striatum, and the effects of amphetamine and apomorphine on rotational behaviour. In animals pretreated with 250 mg/kg a-methyl-p-tyrosine it was found that unilateral stimulation with medially-placed nigral electrodes produced maximal depletion of dopamine histofluorescence in anterior dorso-medial regions of the striatum, while laterally-located electrodes principally depleted posterior, ventro-lateral areas. In the second experiment, 2pg of 6-hydroxydopamine in a volume of 0.5 pl was injected unilaterally at varying loci within the ventral midbrain of animals pre-treated with desmethylimipramine (25 mg/kg). It was discovered that the lateral injection coordinate was significantly associated with both the extent and location of the depletion of dopamine-related fluorescence from the ipsilateral striatum. Rotational behaviour, induced by dopamine-agonists was related firstly, to the overall extent of dopamine depletion from the striatum, and secondly, the contraversive turning induced by apomorphine in particular was related to the dorsoventral coordinate of the mesencephalic 6-hydroxydopamine injection. The results provide functional vahdation for the.pattern of topographical projection within the nigrostriatal dopaminergic system proposed on the basis of intracellular tracing techniques.
dopaminergic projections from the mesencephalon traditionally have been divided into three major groups, a nigrostriatal system,‘*25*36 a mesolimbic system and a mesocortical system.2,26*3s From those investigations, the nigrostriatal pathway was generally thought to arise from dopamine (DA) cells located in the pars compacta region of substantia nigra (SN) (A9 group of Dahlstrom & Fuxe’“) while the mesolimbic and mesocortical projections were considered to originate from the medial A10 cell group of the ventral tegmental area (VTA). Recent reports, however, suggest that the concept of SN and VTA cells giving rise to the striatal and limbic/cortical DA projections, respectively, may be overly simple. 3~12~14*1 s These investigations show that mesencephalic DA cell groups form a continuum, with both the VTA and SN providing DA projections to the majority of telencephalic terminal areas. Differential patterns of projection within the SN-VTA system are accounted for by the discovery of a clearly organ&d topography with medial-lateral, anteriorposterior and dorsoventral components. On the basis of this topography, it seems that the striatal projection derives from the ventral layer of DA cells in the SN and VTA within which there are clear medialAscending
Abbreviations: CN, caudate nucleus; DA, dopamine; SN, substantia nigra; VTA, ventral tegmental area; 6-OHDA, 6-hydroxydopamine.
lateral and anterior-posterior patterns of organisation. On the other hand, the majority of cortical and limbic projections arises from the dorsal layer of DA cells in the SN and VTA; again, there is some evidence for a medial-lateral topography within this subsystem. The functional properties of the ascending DA systems are not yet well understood. However, studies involving the manipulation of transmission in forebrain DA systems;9~13*16~22*30 indicate that locomotor activity responses seem to be more closely associated with limbic DA function while the striatal system is thought to mediate DA-stereotyped behaviour and postural control. With some exceptions,8*‘g*20~34 much of the work investigating the functions of the different DA systems at a behavioural level has manipulated DA neuronal activity within regions of the telencephalon. While these studies support the notion of distinct functional properties of striatal and limbic/ cortical DA systems, they do not distinguish between the traditional and more recent concepts concerning the arrangement of DA cells within the mesencephalon. There is, therefore, a particular need for experiments which seek to clarify the specific functions of DA cells at different locations within the ventral mesencephalon. In the preceding articleJ2 we reported experiments which were designed to test the validity of new procedures for determining the regional content of DA within the caudate nucleus (CN) by measuring the
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intensity of DA-induced histochemical flourescence. While conducting these experiments, it became clear that both unilateral electrical stimulation in animals pre-treated with a-methyl-p-tyrosine and injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra caused a regional depletion of DA fluorescence in the CN. In view of the need to clarify the functional properties of DA cells within different regions of the ventral mesencephalon, it seemed important to determine whether the precise location of the experimental manipulation within the ventral mesencephalon influenced the particular region of the CN maximally depleted of DA. In an extensive analysis of data obtained from experiments reported in the previous paper, we looked for specific relationships between (i) each of the stereotaxic co-ordinates of the mesencephalic electrode or injection cannula, (ii) the area of maximum DA fluorescence depletion within the CN, and (iii) in the 6-OHDA experiment, the effects of amphetamine and apormorphine on rotational behaviour. The purpose of the present paper is to report the results of these analyses.
EXPERIMENT 1: UNILATERAL ELECTRICAL STIMULATION OF SUBSTANTIA NIGRA IN ANIMALS PRE-TREATED WITH z-METHYL-p-TYROSINE
One paradigm used to test the validity of procedures for quantifying DA histofluorescence was to examine whether the relative depletion of fluorescence from the CN ipsilateral to a nigral electrode was related to the parameters of electrical stimulation applied to substantia nigra of animals pre-treated with a-methyl-p-tyrosine. Visual examination of CN tissue from animals used in Experiments 2A and 2B of the preceding paper3* revealed that the DA depletion on the stimulated side was largely confined to local areas of the CN. It was generally found that two or three of the six measurement positions (see Fig. 1B of the previous paper) were more severely depleted than the others. However, precisely which positions were affected was variable across animals. Clear differences in the degree of depletion were also observed in the anterior-posterior plane; this again was variable across animals. In view of the regional nature of the DA depletion, the present analysis was conducted in order to determine whether the area of maximum DA depletion within the striatum was related ition of the stimulating electrode in SN. EXPERIMENTAL
to the pos-
PROCEDURES
Following a period of nigral stimulation in rats pre-
treated with a-methyl-p-tyrosine (see previous paper) brain tissue was processed by a glyoxylic acid method for monoamine histofluorescence.” Fluoresanct intensity, integrated over a circular field (diameter 800pm). was measured photometrically. Measurements from six defined positions (see Fig. IB in the previous article) were taken
from the CN on each side of each coronal tissue wctlon. Thus. a grid of measurements was obtained from 1(1 (2islide) sections taken from different levels in the anteriorposterior plane of the CN. A complete dcscriptlon of the subJccts, surgtcal procedures. experimental conditions. histochemical processmg and fluorescence measurements can be found In I:xpcrlments 2A and 2B of the preceding article.” Duta
Each fluorescence from the side was from the derived from corresponding position the non-stimulated of the tissue section. gave a measure in the non-stimulated was acting an internal These individual scores for animal were over slides = 5) over measurement (!Y = Because animals different stimulating relative depletions slides were 1 to 5 (the sixth slide being omitted from animals in Experiment 2A) while depletions over positions were ranked 1 to 6; the highest depletion score attracted the highest rank. Thus, the slide ranks provided an indication of the relative extent of regional DA depletion within the anterior-posterior plane, while the position ranks were related to DA depletion within the dorsoventral and mediolateral dimensions of the CN. Subsequently. for each stimulated animal the value of five factors was determined. (1) A relative measure of the extent to which DA was depleted from anterior as opposed to posterior regions of the CN in each animal was obtained by subtracting the average rank of the posterior two slides from the average rank of the anterior two slides. (2) A relative measure of the extent lo which regions of the CN were differentially depleted of DA in the medial/lateral and dorsaliventral dimensions was achieved by allocating each animal to one of five groups presented in Table I on the
basis of which three measurement positions were most highly ranked for depletion. (3) The anterior-posterior electrode coordinate. (4) The lateral electrode coordinate. (5) The horizontal-vertical electrode coordinate. Finally, all the animals were ordered or grouped according lo their relative levels on each of the five factors. Having arranged the data in this manner it was possible to take individually each factor as the dependent variable and lo relate it to each of the other factors. Where a factor was continuously variable a trend analysis” was used to assess the relationship between it and the other factors. If, however. a factor was arranged in discon-
Table 1. A relative determination of fluorescence depletion in the medial-lateral and dorsal-ventral dimensions of the striatum was made by assigning animals to one of five groups depending on which measurement positions (see Fig. 1B of previous article) were maximally depleted Depletion group Medial Dorsomedial Dorsolateral Ventrolateral Ventromalial
Positions of maximal depktion VM. C, DM DM, C, D D, C. L L. c. VL VL, C. VM
Abbreviations: C. central; D, dorsal; DM, dorsomedial: L. lateral; VL, ventrolateral: VM. ventromedial.
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Fig. 1. The six graphs show the relationship between the relative degree of depletion along the anteriorposterior axis (predominant depletion in the anterior caudate nucleus is indicated by positive numbers, while negative numbers indicate increasing relative depletion in the posterior caudate nucleus) and the mean depletion for each of the six measurement positions. The linear correlation and probability associated with each relationship is presented.
tinuous groups it was tested against the other factors with a Krusk~-Wa~Iis non-p~ametric analysis of variance.33
RESULTS Regional dopamine depletion within the caudate nuclars
Before an attempt was made to discover any possible relationships between specific areas of the DA depletion within the CN and the location of the nigral stimulating electrodes, it was first necessary to provide a three-dimensional description of the regional nature of DA depletion within the CN. This was accomplished by relating the factor indicating relative depletion within anterior and posterior regions of the CN with the factor representing relative depletion in the medial-lateral and dorsal-ventral aspects of the CN. The form of this relationship was characterised by an association between depletion in anterior and medial areas of the CN; depletion in posterior and lateral regions was also related. This relationship is illustrated in Fig. 1 where the mean ranks for depletion are shown for each of the six measurement positions when the animals were ordered in terms of relative depletion in the anterior-posterior dimension. Thus, it can be seen that for the dorso medial m~surement position, animals with rn~~~ depletion within the anterior CN (f 3) tended to have this position ranked highly for depletion, whereas animals principally depleted in the posterior regions of the CN (-3) generally had a low rank assigned to this position. A gradual reversal of this relationship
was observed as the measurement position assumed a more lateral location within the CN. These data indicate a clear association between DA depletion in anterior and medial and between posterior and lateral areas of the CN. An attempt was then made to relate specific areas of DA depletion within the CN to the location of stimulating electrodes in the substantia nigra. Nigral stimulation locus and regional depletion of sttiatal dopamine The first question to be considered was whether animals assigned to different groups on the basis of relative DA depletion among the six m~surement positions (see Table 1) would have consistently different positioning of the nigral stimulating electrode. Table 2 contains the mean electrode coordinates for each of the five depletion groups described in Table 1.
Table 2. Position depletion x electrode co-ordinates
Depletion group
N
Mean AP
Electrode Lat.*
Media) Dorsomediat Dorsolateraf Ventrolateral Ventral
8 16 5 7 3
-2.0 -2.3 -2.2 -2.3 -2.4
1.2 1.6 1.6 1.8 1.9
Co-ordinate HV -8.6 - 8.6 -8.3 -8.6 -8.5
The electrode coordinates were derived from the stereotaxic atlas of Pellegrino & Cushman.29 * Significant difference between means P < 0.001 (Kruskal-Wallis analysis of variance).
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Fig. 2. The six graphs show the relationship between the lateral coordinate of the nigral electrode placement and the mean depletion rank for each of the six measurement positions. The linear correlation and probability associated with each relationship is presented.
From these data, it is apparent that when proceeding in a clockwise direction around the CN from the medial depktion group the me-an lateral electrode coordinate for each group increased progressively. No signilkant relationship was found between relative depletion among the measurement positions and the other two electrode coordinates. Despite the manifest association between DA depletion in anterior and medial and in posterior and
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lateral regions of the CN a sign&ant relationship between relative depletion in the anterior-posterior dimension and the nigral ekctrode coordinates could not be demonstrated. However, a con&m@ trend towards anteriorly depleted animals having medially placed ekctrodes and posteriorly depleted animals having laterally placed ekctrodes Was noted. These analyses indicate that the specitlc area of the CN, which is maximally depkted of DA iluaranxnct
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Fig. 3. The five graphs show the rdnthmbip betweenthe lateral ~OO&UUCof the nigrpl ekclrodc placement and the man dqktioa rrdr for oath of the five slides. !%i& 1 cotttainui the two most anterior sections of CN tissue while the two most posterior sections were on slide 5. Tote linear correlation and probability associated with each relationship is presented.
Ni~ost~atal topography following electrical stimulation of substantia nigra, is significantly influenced by the lateral position of the stimulating electrode. Cotirmation of this relationship was sought by ordering the animals in terms of the lateral&y of their nigral electrode and plotting the average depletion rank for each of the six CN measurement positions. The resulting graphs (see Fig. 2) indicate that medial measurement positions attracted progressively lower depletion ranks as the lateral coordinate of the nigral electrode increased. This relationship gradually reversed as the measurement position itself became more lateral. The effects of laterality of nigral stimulation on DA depletion within the anterior-posterior dimension of the CN is shown in the graphs of Fig. 3. From this Figure it can be seen that animals with medially located electrodes tended to have anterior slides (1 and 2) ranked highly for deletion while posterior slides (4 and 5) were ranked lowly. Again this relationship seemed to reverse gradually as the eleo trode location became more lateral. In summary, it is concluded from these analyses that mediaRy placed nigral electrodes produced maximal depletion of DA ~stofluores~~ in anterior, dorsomedial areas of the CN whiie laterally located electrodes produced greater depletion in the posterior, ventrolateral regions of this structure.
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0.2 mgml ascorbate-saline solution) (N = 24) or an equivalent volume of the ascorbate vehicle (N = 6). The location of the intracranial injections was systematically varied in the medial-lateral dimension between L 0&3.0mm; the other coordinates were AP-2.5 and HV-7.8.29 The remaining six animals served as unoperated controls. Behavioural testing procedures
On days 11, 12 and 13 after surgery, behavioural measurement of rotation was obtained following the intraperitoneal injection of apomorphine (2 mg/kg) (MacFarlan Smith), ~phet~ine (1 m&kg) (Smith, Kline 8z French) or an equiv~ent volume of the 0.9% iialine vehicle. Each animal received each of the three drug treatments (1 treatment per day) in a counterbalanced order. The number and direction of 360 degree turns in a flat-bottomed bin (diameter 21 cm), were recorded in two 2 mitt periods, 25 and 40 min after injection. The scores for the two periods were combined, after which the number of turns ipsilateral to the intracranial 6-OHDA injection was subtracted from the number of contralateral turns to give a single measure of rotational behaviour. Histochemical processing and jluorescence measurement @I day 14 after surgery, all animals were killed and CN tissue was processed for ~t~hoI~ne histofluor~~n~ as described in the previous paper. In addition, the location of each intracranial injection was verified according to normal histological procedures.‘O Data analysis
EXPERIMENT 2: UNILATERAL INJE~IONS OF 6-HYDROXYDOPAMI~ AT DIFFERENT LOCATIONS WITHIN THE VENTRAL MESENCEPHALON
An alternative strategy used to validate procedures for the photometric assessment of DA histofluorescence was to determine the effects of 6-OHDA destruction of mesencephalic DA cell bodies on CN fluorescence. Again, preliminary visual examination of CN tissue from animals treated with 6-OHDA revealed regional patterns of DA depletion. Thus, on the basis of these informal observations, we decided to examine whether the location of the 6-OHDA injection site in the ventral me~ncephalon would predict the region of the CN maximally depleted of DA fluorescence. In the three days prior to death the effects of amphetamine, apomorphine and vehicle control injections on the rotational behaviour of each animal was determined. As a result of these me~uremen~ we were able to relate the behavioural performance, firstly to the location of the 6-OHDA injection and secondly, to the area of maximum DA fluorescence depletion within the CN. EXPERIME~AL
PROCEDURES
Subjects and surgical procedures
Thirty-six female hooded rats were used, of which 30 animals were treated with 25 mg/kg desmethyl-imipramine (Geigy) prior to a stereotaxically-positioned intracranial injection of either 6-OHDA (Sigma) (2pg free base in
For each animal injected with 6-OHDA, the value of nine factors was ‘determ~~; these have been item&l in Table 3. The coordinates for each injection site were derived from the stereotaxic system of De Groot.29 A relative measure of the extent to which each animal was depleted of DA, in anterior as opposed to posterior regions of the CN, was determined by subtracting the mean depletion score for tissue on the anterior two slides from the mean depletion score for tissue on the posterior two slides, This provided a continuous variable along which animals could be ordered. Indication of maximal depletion in the medial-lateral and dorsoventral planes of the CN was achieved by assigning animals to different groups, depending upon which of the six measurement positions (see Fig. 1B of previous paper) was most severely depleted. A total of four groups (medial, interm~ate/ medial, intermediate/lateral and lateral) was created to indicate relative depletion in the medial-lateral plane of the CN. Alternatively, animals were assigned to three groups (dorsal, intermediate and ventral) which were reflective of the relative depletion of dorsal us ventral measurement positions. As the rotational behaviour of each animal was expressed on a single continuously varying scale, two separate listings of the animals were created to indicate the relative amounts of rotational behaviour induced first by apomorphine and second, by amphetamine. Relationships between the factors were investigated either by cor~lational analyses or by the use of non-parametric analyses of variance (see above). RESULTS
The results will be discussed in relation to the following three issues: (i) the relationship between the
890
P. Redgrave and I. MItchelI Table 3. The value for each animal treated with 6-hydroxydopamine was calculated on each of 9 dimensions -(i) 6-OHDA injection coordinates anterior-posterior lateral horizontal-vertical (ii) Striatal depletion of DA fluorescence
(d) (e) (f) (gj
(iii) Index of rotational behaviour after administration of:
location of the 6-OHDA injection in the ventral mesencephalon and the extent and location of DAdepletion from the CN, (ii) the relationships between the extent and location of striatal DA-depletion and rotational behaviour, and (iii) the relationships between the location of the 6-OHDA injection and rotational behaviour. GHydroxydopamine dopamine-depletion
injection
location
and
striatal
The major finding was that the overall extent of DA-depletion from the ipsilateral striatum was significantly related to the lateral coordinate of the 6-OHDA injection in the ventral midbrain. This relationship is illustrated in Fig. 4A and is best described as an inverted U-shaped function with maximal striatal DA-depletion achieved by injections placed approximately 1.5 mm lateral to the midline. The use of orthogonal components in tests for trends within these data3q revealed a best-fitting curve with a significant (P < 0.025) quadratic component (see Fig. 4A). A second finding was that when the animals were grouped according to striatal DA-depletion in the medial/lateral dimension it was found that maximum depletion in medial CN was associated with medial
s
I I
overall mean depletion anterior-posterior depletion medial-lateral depletion dorsal-ventral depletion apomorphine amphetamine
&OHDA injection coordinates while latmal depktion was associated with lateral coordinates. This relationship is illustrated in Fig. 4B; the di%mnces in the mean lateral coordinate between the groups was statistically significant @ < 0.001; Kruskal-Wallis analysis of variance). No other significant relationships between the ractors describing striatal DA depktion and the 5-OHDA injection coordinates were discovered. The reason for this may have been that the range of AP ,and HV measurements over which the animals varied was comparatively small. In summary, it was discovered that the lateral zooordinate of the 6-OHDA injection significantly affected both the overall extent and location of the DAdepletion from the ipsilateral striatum. Striatal dopamine-depletion
and rotational behaoiour
Significant linear relationships were found between the overall extent of DA-depletion from the ipsihteral striatum and the rotational behaviour induced by both amphetamine and apomorphine. Graphs A and B of Fig. 5 indicate that animals which showed most ipsilateral rotation after amphetamine and most xntralateral rotation after apomorphine tended to be the animals with the greatest overall depletion of DA
. .
Fig. 4. (A) The relationship between the lateral coordinate of the mesencephalic dhydroxydoplunine injection and the mean dopktion of rtriatal dopamine fluorsscsnce. The sign&ant quadratic treed (Q) is represented by the line of best fit. (9) The mean lateral injection coordinates of animaJa grouped according to differential depletion of DA fluorescence in the medial-lateral plane of the stinturn. Abbreviations: Med. medial; IM, intermediate-medial; IL, intermediate-lateral; Lat, lateral.
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Nigrostriatal topography
Fig. 5. (A) The linear relationship between turning induced by amphetamine and the mean depletion measure of striatal dopamine fluorescence.. (B) The linear relationship between turning induced by apomorphine and the mean depletion measure of striatal DA fluorescence.
from the CN. There was no consistent evidence to suggest that DA-depletion within any particular sub-
region of the CN was particularly rotation scores.
related to high
6-Hydroxydopamine injection location and rotation
It was found that the rotation induced by apomorphine was significantly related to the dorsoventral coordinate of the 6-OHDA injection into the ventral midbrain. Thus. Fig. 6 shows that high rates of contralateral rotation following the administration of apomorphine were associated with ventrally located injections of 6-OHDA. Apart from this result no other significant relationships between the location of the 6-OHDA injection and rotational behaviour were discovered. DISCUSSION Two experiments were performed in order to examine whether manipulation of the DA pathway afferent to the CN influences the intensity of DA-fluorescence within this structure. The extent to which the results of these experiments validated procedures for the quantitative measurement of DA-fluorescence was the concern of the preceding paper. The purpose of the present study was to analyse data from the two experiments with the intention of investigating the possible relationships between the location of treatments within the ventral mesencephalon, the extent and location of striatal DA depletion and the levels of rotational behaviour following the administration of amphetamine and apomorphine. Topographical relationships dopaminergic projection One important
in
the
the more caudal aspects of the CN. A much clearer description of nigrostriatal topography has been provided by anatomical studies using either silver impregnation techniques or the intraneuronal transport of horseradish peroxidase and [3H]1eucine/pro1ine.4*6*7*14*37 In general, it has been found that the pars compacta region of substantia nigra is organised so that the anteromedial portion of the CN receives efferents from the media1 substantia nigra, while cells in the lateral nigra project to the posterolateral CN. However, despite the generally convincing reasons given for considering that these patterns of projection relate principally to DA cells, it should be noted that the transport of horseradish peroxidase and [3H]amino acids is not specific to DA neurones. Thus it is important that the pattern of nigrostriatal topography described on the basis of intraneuronal transport techniques was, for the most part, confirmed in the present experiments in which histochemical procedures specific to catecholamines were used.” In addition, the protection of noradrenaline neurones by injections of desmethylimipramine, together with the finding that noradrenaline is present in the CN only in small amounts,1s*z3.3* probably means that the changes in the fluorescence intensity measurements observed in the present experiments were specifically related to changes in DA levels. In the first experiment of the present paper, a clear association between DA depletion in anterior and medial regions and between posterior and lateral areas of the CN was established. This is entirely consistent with the anteromedial and posterolateral topography proposed by investigators using tracing techniques. Further evidence for this topographical pattern was provided by the finding that mediallylocated electrodes caused maxima1 depletion of DA fluorescence in anterior dorsomedial areas of the CN, while laterally-placed electrodes produced most depletion in posterior ventro-lateral regions. Additional evidence for medial-lateral topography was found in the second experiment when it was observed that animals depleted of DA fluorescence principally in media1 regions of the CN had medially placed
nigrostriatal
aspect of the analyses presented in this paper is that direct functional evidence was provided for a topographical pattern of projection within the nigrostriatal DA-system. In the past, UngerstedP demonstrated that rostromedial substantia nigra lesions denervated the entire CN of DA whereas caudolateral nigral lesions only depleted DA levels in
- 0.0
8’1 78
-15
33 6teonlratal.15 - - @ailat)
Tming
Fig. 6. The linear relationship between turning induced by apomorphine (2 mg/kg) and the dorsoventral coordinate of the mesencephalic 6-hydroxydopamine injection.
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P. Redgrave and I. Mitchell
6-OHDA injections while lateral CN depletion was associated with lateral placements of 6-OHDA. One way in which the current findings depart from observations made with intracellular tracers14 is that no differential patterns of projection were found which related to the anterior-posterior and dorsoventral placement of stimulating electrodes of 6-OHDA injections in the mesencephaion. This difference may be explained by the fact that most of the present stimulating electrodes and 6-OHDA injections were located towards the rostra1 pole of SN. In this position, it is unlikely they could affect differentially separate populations of anteriorly projecting DA-fibres. Of potential importance to the present investigation are some studies of Glowinski and his coworkers,24*2s where it is reported that in a variety of situations, there is an interdependence of the nigrostriatal dopaminergic systems on each side of the brain in the cat. Briefly, they found that when a particular unilateral manipulation of substantia nigra induced an ipsilateral change in striatal DA release it was often associated with an opposite change in the release of DA in the contralateral CN. If this bilateral interdependence of activity in the nigrostriatal DA pathways is also present in the rat, the difference measures in the present experiments will have been influenced. For example, unilateral electrical stimulation of substantia nigra may have caused not only an ipsilateral increase in DA release, but a corresponding decrease in release from the contralateral CN. Unfortunately, the calculation of simple subtraction or ratio measures of the difference between the fluorescence intensity on each side of the brain provides no clear indication of the relative importance of these two factors. While this limitation may restrict the use of the present photometric procedures in some instances, the estimation of the relative difference in the activity of DA neurons on each side of the brain seems to be unaffected. For example, the frequencyresponse function described in Fig. 6B of the previous paper is directly comparable with the data from experiments using alternative methods for measuring the activity of DA neurons. One final point in this section concerns the precise location of a nigral injection of 6-OHDA and its effect on the extent of DA-depletion in the ipsilateral striaturn. In the second experiment a standard 2pg of 6-OHDA was injected in a volume of 0.5 ~1 throughout. This is approximately one quarter the volume and dose that is commonly used to lesion mesencephalic DA cell bodies. 5*21Systematic variation of the lateral placement of this injection permits the conclusion that the most effective depletion of DA-fluorescence from the ipsilateral striatum can be achieved when the 6-OHDA injection is placed approximately 1.5 mm from the midline (see Fig. 4A). Behavioural correlates of striatal dopamine depletion
In the second experiment, measures of rotational behaviour following the administration of apomor-
phine and amphetamine were obtained. The first general finding was that rotational behaviour induced by both amphetamine and apomorphine was related to the overall extent of DA fluorescence depletion from the CN. Thus, it was observed that a high rate of both ipsilateral turning induced by amphetamine, and contralateral turning induced by apomorphine. tended to occur in association with large differences in the intensity of DA fluorescence between the CN on each side of the brain. These observations are in general agreement with previous findings which indicate that, in the present experimental paradigm, rotational behaviour reflects a relative imbalance between striatal DA activity on either side of the brain. (for reviews see Glick et a/.” and Pycock” ). The present results on turning are not, however. entirely consistent with the recent findings of Hefti er a/.,19 who reported that significantly greater depletions of striatal DA were required to produce rotation in response to apomorphine compared with that induced by amphetamine. Reference to Fig. 5 indicates that the amount of rotation induced by amphetamine and apomorphine over the present range of striatal DA depletions was comparable. This difference in results may have been caused by the use of different doses of the DA agonists, e.g. a higher dose of apomorphine (2 mg/kg cf. 1 mg/kg) and a lower dose of amphetamine (1 mg/kg cf. 5 mg/kg) was used in the present experiments. This explanation is. however, unlikely since Hefti et al., found that raising the dose to 5 mg/kg of apomorphine still failed to elicit contralateral turning in rats which clearly turned ipsilaterally in response to amphetamine. A more plausible account of the discrepant results may be found by considering differences in the procedures of 6-OHDA administration. In the present experiments. a small volume (0.5 ~1) of a solution of 6-OHDA whose concentration (4&$) has been demonstrated to be toxic to catecholaminergic neurons2’ was injected into different regions of the ventral tegmentum. Hefti et a1.,19 however, used a range of doses of 6-OHDA which were injected in the rather larger than normal volume of 4 pl. Given the topographical nature of the ascending DA systems it seems reasonable to suppose that the pattern of striatal DA depletion induced by these procedures may well be different, and that such differences may account for the discrepant results. The discovery of a relationship between the dorsoventral placement of 6-OHDA and turning is relevant to the understanding of the functional properties of different populations of mesencephahc DA cells. It was found that high rates of contralateral rotation in response to apomorphine were associated with the more ventral placements of QOHDA within the ventral tegmentum. In the intraneuronal tracing studies of Fallon & Moorei it was found that ventrallylocated cells within the SN-VTA complex projected mainly to the CN. whereas the cells which project to structures of the limbic forebrain were located more
Nigrostriatal topography dorsally. The present finding that a behaviour thought to be of striatal origin, i.e. turning, was particularly associated with ventrally-located injections of 6-OHDA provides functional evidence which is consistent with the arrangement of cells within the ventral tegmentum proposed by Fallon & Moore.14 Finally, in the present experiments no attempt was made to measure possible changes in the intensity of DA-related fluorescence in structures other than in
893
the striatum. Thus, in future investigations of the functional properties of DA cells at different locations in the ventral mesencephalon, assessment of relative DA depletion in as many forebrain DA terminal areas as possible should be included. This may be achieved by slight modification of the present procedures for the photometric assessment of glyoxylic acid-induced fluorescence from the brain of a single subject (see preceding paper).
REFERENCES 1. And&n N. E., Carlsson A., DahlstSm A., Fuxe K., Hillarp N. A.& Larsson K. (1964) Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 3, 523-530. 2. Andtn N. E., Dahlstriim A., Fuxe K., Larsson L., Olson L. & Ungerstedt U. (1966) Ascending monoamine neurons to the telencephalon and diencephalon. Acta. physiol. stand. 67, 313-326. 3. Avendand C., Reinoso-Suarez F. & Llamas A. (1976) Projections to gyrus sigmoideus from the substantia nigra in the cat, as revealed by the horseradish peroxidase retrograde transport technique. Neuroscience Letters 2, 61-65. 4. Bedard P., Larochelle L., Parent A. & Poirier L. J. (1969) The nigrostriatal pathway: a correlative study based on neuroanatomical criteria in the cat and the monkey. Expl Neural. 25, 365-377. 5. Butcher L. L., Eastgate S. M. & Hodge G. K. (1974) Evidence that punctuate intracerebral administration of 6-hydroxydopamine fails to produce selective neuronal degeneration. Naunyn-Schmeidebergs Arch. Pharmac. 285, 31-70. 6. Carpenter M. D. & Peter D. (1972) Nigrostriatal and nigrothalamic fibres in the cat. J. camp. Neural. 144, 93-116. 7. Carter D. A. & Fibiger H. C. (1977) Ascending projections of presumed dopamine-containing neurons in the ventral tegmentum of the rat as demonstrated by horseradish peroxidase. Neuroscience 2, 569-576. 8. Costall B., Marsden C. D., Naylor R. J. & Pycock C. J. (1976) The relationship between striatal and mesolimbic dopamine dysfunction and the nature of circling responses following 6-hydroxydopamine and electrolytic lesions of the ascending dopamine systems. Brain Res. 118, 87-l 13. 9. Costall B. & Naylor R. J. (1973) The role of telencephalic dopaminergic systems in the mediation of apomorphine stereotyped bchaviour. Eur. J. Pharmac. 24, 8-24. 10. Dahlstriim A. & Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system-I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta. physiol. stand. 62, Suppl. 232, l-55. 11. De la Torre J. C. & Surgeon J. W. (1976) A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method. Histochemistry 49, 81-93. 12. Domesick V. B., Beckstead R. M. JL Nauta W. J. H. (1976) Some ascending and descending projections of the substantia nigra and ventral tegmentum area in the rat. Neuroscience Abs. 2, 61. 13. Ernst A. M. & Smelik P. G. (1966) Site of action of dopamine and apomorphine on compulsive gnawing behaviour. Experientia 22, 837-838. 14. Fallon J. H. & Moore R. Y. (1978) Catecholamine innervation of the basal forebrain-IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. camp. neural. 180, 545-580. 15. Fallon J. H., Riley J. N. & Moore R. Y. (1978) Substantia nigra dopamine neurons: separate populations project to neostriatum and allocortex. Neuroscience Letters 7, 157-162. 16. Fog. R., Randrup A. & Pakkenberg H. (1970) Lesions in corpus striatum and cortex of rat brains and the effect on pharmacologically-induced stereotyped, aggressive and cataleptic behaviour. Psychophormacologia (Berl.) l&346-356. 17. Glick S. D., Jerussi T. P. & Fleisher L. N. (1976) Turning in circles: the neuropharmacology of rotation. Life Sci. 1% 889-896. 18. Grzanna R., Morrison J. H., Coyle J. T. & Molliver M. E. (1977) The immunohistochemical demonstration of noradrenergic neurons in the rat brain: the use of homologous anti-serum to dopamine-B-hydroxylase. Neuroscience Letters 4, 127-134. 19. Hefti F., Melamed E., Sahakian B. J. & Wurtman R. J. (1980) Circling behaviour in rats with partial, unilatera1, nigro-striatal lesions. Effects of amphetamine, apomorphine and DOPA. Pharmac. Biochem. Behau. 12, 185-188. 20. Hodge G. K. & Butcher L. L. (1979) Role of pars compacta of the substantia nigra in circling behaviour. Pharmac. Biochem. Behao. 10, 695-709. 21. Javoy F., Sotelo C., Herbert A. & Agid Y. (1976) Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system. Brain Res. 102, 201-215. 22. Kelly P. H., Seviour P. W. & Iversen S. D. (1975) Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Bruin Res. 94, 507-522. 23. Koslow S. H., Racagni G. & Costa E. (1974) Mass fragmentographic measurement of norepinephrine, dopamine, serotonin and acetylcholine in seven discrete nuclei of the rat tel-diencephalon. Neuropharmacology 13, 1123-l 130. 24. Leviel V., Cheramy A., Nieoullon A. & Glowinski J. (1979) Symmetric bilateral changes in dopamine release from the caudate nuclei of the cat induced by unilateral nigral application of glycine and GABA-related compounds. Brain Res. 175, 259-270.
P. Redgrave and I. Mitchell
894
25. Lindvali 0. & Bjorklund A. (1974) The organisation of the ascending catecholamine neuron systems in the rat brarn as revealed by the glyoxylic acid fluorescence method. Acta physiol. stand. Suppl. 412, 1-48. 26. Lindvall O., Bjorklund A., Moore R. Y. & Stenevi U. (1974a) mesencephalic dopamine neurons projecting to nencortex. Brain Res. 81, 325-331. 27. Lindvall O., Bjiirkhmd A. & Svensson L. A. (19746) Fluorophore formation from catccholamines and related compounds in the glyoxylic acid fluorescence histochemical method. Hisrochemisrrj~ 39, 197 227. 28. Nieoullon A., Cheramy A. & Glowinski J. (1977) Interdependence of the nigrostriatal dopaminergic systems on the two sides of the brain in the cat. Science N. y 198, 416481. 29. Pelligrino L. J. & Cushman A. J. (1967) A Stereotaxic Atlas of the Rat Brain Appleton-Century-Crofts, New York. 30. Pijnenburg A. J. J., Honig W. M. M., Heyden J. A. M. & van Rossum J. M. (1976) Effects of chemical stimulation of the mesolimbic dopamine system upon locomotor activity. Eur. .I. Pharmac. 35, 4.5-58. 31. Pycock C. J. (1980) Turning behaviour in animals. Neuroscience 5.461-514. 32. Redgrave P. & Mitchell I. (1982) Photometric assessment of glyoxylic acid-induced fluorescence of dopamine in the caudate nucleus. Neuroscience 7, 871-883. 33. Siegel S. Non-parametric Statisticsfor the Behauioural Sciences pp. 184193. McGraw-Hill, New York. 34. Thal L., Mishra R. K., Gardner E. L., Horowitz S. G.. Varmuza S. & Makman M. H. (1979) Dopamine antagonist binding increases in two behaviourally distinct striatal denervation syndromes. Brain Res. 170, 381-386. 35. Thierry A. M., Blanc G., Sobel A., Stinus L. & Glowinski J. (1973) Dopaminergic terminals in rat cortex. Science N. ‘i: 18&499-501.
36. Ungerstedt U. (1971) Sterotaxic mapping of the monoamine pathways in the rat brain. Acra ph.vsioi. stand. SSQL 367, l-48. 37. Wsunoff K. G., Hassler R., Romansky
K., Usunova R. P. & Wagner A. (1976) The nigrostriatal projection in the cat-Part 1. Silver impregnated study. J. neural. Sci. 28, 265-288. 38. Versteeg D. H. G., van der Gugten J., de Jong W. & Palkovits M. (1976) Regional concentrations of noradrenaline and dopamine in rat brain. Bruin Res. 113, 563-574. 39. Winer B. J. (1971) Statistical principles in experimental design. McGraw-Hill Book Company, New York. 40. Wolf G. (1971) Elementary histology for neurophysiologists. In Methods in Psychobiology (ed. Myers R. D.) Vol. I. pp. 281-300. Academic Press, London. (Acceprd
19 September 1981)