0306-4522/90 $3.00+0.00 Pergamon Press plc

Neurosdence Vol. 31,No. 2,pp. 387-398,1990 Printed in Great Britain

Q 1990IBRO

MICROTOPOGRAPHY OF D, DOPAMINERGIC BINDING SITES IN THE HUMAN SUBSTANTIA NIGRA: AN AUTORADIOGRAPHIC STUDY F. THIBAUT,* E. C. HIRSCH, R. RAISMAN, F. JAVOY-AGID and Y. AGID INSERM U289, HBpital de la SalpCtri&e, 47 Bd de I’hdpital, 75013 Paris, France Abstract-The autoradiographic distribution of 0, dopaminer~c binding sites was studied in the human ventrat mesencephalon using the D, antagonist rH]SCH 23390. [3H]SCH 23390 binding was characterized by a single class of sites with a K,, of 2.5 nM and a B,,,,, of 31 fmol/mg of tissue. The density of [‘H]SCH 23390 binding sites was high in the substantia nigra, moderate in the ventral tegmental area and low in the peri- and retrorubral field (catecholaminergic region A8). Binding densities were similar in pars compacta and pars reticulata of the substantia nigra, except for a peak value of high [3H]SCH 23390 in the pars reticulata, at a level just ventral to a zone of hyperdensity of melanized dopaminergic neurons in the pars compacta. The anatomical organization of the human ventral mesencephalon was analysed on adjacent sections stained for acetylcholinesterase histochemistry and tyrosine hydroxylase, substance P, dynorphin B, somatostatin and methionine-enkephalin immunohistochemistry, respectively. The similarity in distribution of 13H]SCH 23390 binding sites and substance P or dynorphyn B immunoreactivity suggests that D, binding sites are mainly located on the striatonigral projections. In accordance with these results: (I) the density of [3HJSCH 23390 binding sites was reduced in the substantia nigra of a patient with Huntington’s chorea, a disease associated with a degeneration of striatonigal neurons; (2) the density of [‘HJSCH 23390 binding sites was unaffected in the substantia nigra of a patient with Parkinson’s disease, a disorder characterized by a marked loss in nigral tyrosine hydroxylase-~sitive neurons. [-‘H]SCH 23390 binding sites showed a characteristic, heterogeneous distribution within the human ventral mesencephalon, confirming data obtained in other species. The preferential localization of D, dopamine receptors on striatonigral projections in human brain suggests that pharmacological manipulation of these receptors modulates the activity of striatonigral pathways, thereby affecting the various outputs of the nigral complex.

It is well accepted that the action of dopamine and dopaminergic drugs in the basal ganglia is mediated through at least two subtypes of dopamine receptors,48 the D, and the D, receptors, positively and negatively coupled, respectively, to a dopaminesensitive adenylate cycIase,3’ which might mediate different physiological responses.13 Autoradiographic localization of these receptors has shown that high densities of both subtypes are found in striatum and substantia nigra in rodents~9~‘o~‘7-Jq~*9~3~,~ non-human primates,5 and in human5,14,‘5brain. In the substantia nigra, the effect of stimulation of D, receptors by dopamine released from dendrites of the mesostriatal dopaminer~c neurons36 remains unknown, although studies in animals suggest that these receptors modulate the activity of the striatonigral projections, whereas D2 receptors seem to regulate nigrostriatal neurons.* In humans, the intranigrai djs~bution of D, receptors and their relation to catechoiaminergic and non-catecholaminergic neuronal systems have not yet been established. In the present study, we used the selective D, antagonist (R)-( + )-&-chIoro*To whom correspondence should be addressed. AChE, acetylcholinesterase; .5-HT, 5-hydroxytryptamine; SCH 23390, (R)-( + )-Gchloro2,3,4,5-tetrahydro-3-methyl-5-phenyl-IH-3-benzazepin7-01; TH, tyrosine hydroxylase.

Abbreviations:

2,3,4,S-tetrahydro-3-methyl-5-phenyI-lH-3-benzazepin-7-01 ([3H]SCH 2339Q6 to map D, dopaminergic receptors in relation to the striatonigral projections and dopaminergic neurons in the ventral mesencephalon of control human brain. In addition, to confirm location of the receptors, we examined brains from a patient with Huntington’s disease where there is extensive loss of striatonigral neurons and a patient with Parkinson’s disease where nigrostriatal neurons degenerate. EXPERIMENTALPROCEDURES Patients The study was performed on brain tissue from five patients (Table 1). Specimens were obtained from three control subjects without known history of psychiatric or neurologic disorders, who had received no dopaminergic drugs prior to death (cases 665, 694, 696; mean age at deathf S.E.M. = 83 f 4 years; post mortem delay before freezing f S.E.M. = 17 + 3 h). One patient had idiopathic Parkinson’s disease (case 716, age at death = 78 years; post mortem delay before freezing = 3 h). Duration of the disease was five years. Levodopa (mean daily dose = 500 mg; fast intake less than 24 h prior to death) was administers for four years, with no additional treatment. The clinical diagnosis of idiopathic Parkinson’s disease was confirmed by the existence of severe neuronal loss and the presence of Lewy bodies in the substantia nigra and locus coeruleus on neuropathologi~l examination. One patient had Huntington’s disease (case 644, age at death = 75 years; post mortem

387

38X

F. THIBAUTet ul. Table 1. Characteristics of the patients Diagnosis Control* Control* Control* Parkinson’s disease Huntington’s chorea

Brain number

Age (years)

Post mortem

Sex

696 694 665 716 644

F M M M F

92 80 71 78 75

18 II 24 3 9

delay (h)

Immediate cause of death Bronchopneumonia Bronchopneumonia Pulmonary embolism Bronchopneumonia Bronchopneumonia

*No known neurologic or psychiatric disorders. delay before freezing = 9 h). The disease had evolved for 10 years. The diagnosis was confirmed by neuropathological evidence of atrophy of the head of the caudate nucleus (with preservation of the convexity of its ventricular surface) as well as neuronal loss and astrocytic gliosis in the striatum. The patient was considered to be grade II according to the neuropathological classification of Huntington’s Disease defined by Von Sattel et al.” Tissue preparation

After autopsy, the brains were quickly removed and hemisected. The brainstem was cut in 2-cm-thick blocks, fresh frozen on dry ice reduced to powder and stored at -7O’C until sectioned. Consecutive transverse sections (15pm) of the mesencephalon, containing the substantia nigra, were cut at - 12°C to - 14°C in a cryostat. Sections were thaw-mounted onto gelatin-coated glass slides (double subbing) and dehydrated under vacuum for 1 h at - 10°C in the presence of desiccant. After dehydration, the sections were stored at -80°C in a box containing desiccant for at least two weeks before the experiments were performed. Series of 12 adjacent sections were taken at 10 regularly spaced intervals along the rostocaudal axis of the ventral mesencephalon. The sections were thawed before processing for [‘HISCH 23390 binding assays or immunohistochemistry.

ketanserin, a serotonin 2 and 1C ligand, and sulpiride, a D, antagonist, at five concentrations from 10m4 to 10m9M; atropine, clonidine and propanolol cholinergic and adrenergic ligands, respectively at 10el M. Following the binding assay, the tissue sections were dried and apposed to tritium-sensitive films (Amersham hyperfilm ‘H) in a Kodak x-ray cassette for one month at 4°C. An Amersham ‘H-microscale standard from 0.1 to 16 nCi/mg of tissue equivalent and from 3 to 1IO nCi/mg of tissue equivalent was included with every film. Films were developed with Kodak AL4 developer (1.5 min at 18”C), rinsed and fixed with Kodak film fixative (5 min). Image analysis

The density of [‘HISCH 23390 binding sites in the various regions of the ventral mesencephalon was determined with an image analyser (RAG 200 BIOCOM). Optical densities measured on the autoradiograms of specific and non-specific binding were first converted to nCi/mg tissue equivalent with the standard curve generated by the calibrated tritium microscales, then to fmol/mg of tissue equivalent. Background film staining was subtracted from values obtained for each section. Specific binding in each region was determined by subtracting the non-specific binding from total binding. The B,,,,, and Kd of [‘H]SCH 23390 binding were calculated by linear regression analysis of the specific binding (EBDA, Elsevier-Biosoft, Cambridge, U.K.).

Autoradiographic analysis of [‘H]SCH 23390 binding

Immunohistochemistry and hisiochemistry

Autoradiograms were obtained from sections incubated with a solution of [jH]SCH 23390 at room temperature, according to the procedure described by Savasta ef al.“’ Briefly, after a 15-min preincubation at room temperature in 50 mM Tris-HCl buffer pH 7.4, slide-mounted sections were covered with 500~1 of the radioactive solution in a humid atmosphere. Solutions of [3H]SCH 23390 (Amersham, specific activity 83 Ci/mmol) were prepared in a Tris-HCl buffer 50 mM, pH 7.4, containing (in mM): NaCl 120, KC1 5, CaCI, 2, MgCl, 1 and 1pM ketanserin in order to eliminate the serotonin receptor 5-hydroxytryptamine (S-HT,) and 5-HT,, binding component.’ The optimal experimental concentrations of radioligand were determined by Scatchard analysis. Duplicate sections were incubated for I h at room temperature, rinsed twice with cold buffer (4°C) from 10 min, quickly dipped into cold distilled water and dried under a current of cool air. Non-specific 13H]SCH 23390 binding was determined on alternate sections by adding IOpM of the specific D, antagonist cis-Z-piflutixol (Lundbeck Industries Chimiques, Paris, France) to the incubation medium. Scatchard analysis was performed on sections from two control brains (case 665 and 696), using 10 concentrations (from 20 to 0.5 nM) of [3H]SCH 23390. Consecutive transverse sections of the mesencephalon were examined at the level of the third cranial nerve fibers, and included the pars reticulata and the pars compacta of the substantia nigra, the peri- and retrorubral field (catecholaminergic region A8) and the ventral tegmental area (catecholaminergic region AIO). Competition experiments with [3H]SCH 23390 (4 nM) were performed in the presence of: piflutixol, a D, antagonist, at 10 concentrations ranging from IO-“ to lo-” M;

Tissue sections adjacent to those used for the binding were postfixed for 1.5 h in ice-cold 4% (w/v) parafonnaldehyde pH 7.4 in 0.1 M Na phosphate buffer pH 7.4 and rinsed twice in Tris-HCI buffer 0.25 M containing 0.9% NaCl. Immunohistochemistry was performed according to a protocol described elsewherez3 using primary antisera against tyrosine hydroxylase (TH) (lot 2013, dilution I : 120), dynorphin B (lot R832906 B2, dilution 1:350), methionineenkephalin (lot 864802, dilution 1:SOOO),somatostatin (lot S309, dilution 1:500). The sections were incubated for four days at 4°C with all the primary antisera, except one against substance P, where sections were incubated for 3 h at room temperature. The rabbit antiserum against substance P, kindly provided by Dr Feuerstein, was proven specific for substance P (personnal communication). The antiserum against somatostatin4 was kindly provided by Dr Benoit. The other antisera were purchased from Eugene Tech for the anti-TH antiserum, Milab for the anti-dynorphin B antiserum, Incstar for the anti-methionine-enkephalin, and characterized elsewhere.*,23,49Immunoreactivities were revealed by the peroxidase-antiperoxidase method with double bridge steps. 51 In all experiments, no staining was observed when the primary antiserum was omitted. Sections adjacent to those treated for immunohistochemistry were stained for acetylcholinesterase (AChE) activity according to a slightly modified version of the Geneser-Jensen and Blackstad method, as described elsewhere.22 Analysis ofthe anatomical distribution of the [3H]SCH 23390 binding sires

The rostrocaudal distribution of [3H]SCH 23390 binding sites was analysed in four regions of the ventral

389

D, receptors in the human substantia nigra

the binding studies, using an overhead slide projecto? as illustrated in Fig. 1. For more detailed analysis of the distribution of [3H]SCH 23390 binding sites in the substantia nigra, the density of the binding sites was determined at 11positions, mediolaterally, on each section. The rostrocaudal and the mediolateral distributions of the marker were used to construct a threedimensional representation of the intranigral distribution of [3H]SCH 23390 binding sites using the Statgraphics statistics program (STSC Inc.; Rockville, MD, U.S.A.). RESULTS Characterization of [3H]SCH 23390 binding The Scatchard plot of the saturation curve of [3H]SCH 23390 binding was linear (r = 0.96) indicating the existence of an homogeneous class of noninteracting binding sites with a K,fS.E.M. of 2.5 f 0.4 nM and a B,,,,, f S.E.M. of 31.5 f 6 fmol/ Fig. 1. Schematic representation of the various regions studied in the ventral mesencephalon. The landmarks of the regions were delineated on AChE-stained sections (see Fig. 6e) with the help of an overhead slide projector. The distribution of TH-immunostained neurons, i.e. dopamine cells (black dots), is from a representative transverse section through the mesencephalon of a control brain (case 694). This figure illustrates the procedure used to analyse the distribution of [‘H]SCH 23390 binding sites and immunoreactivity. The substantia nigra pars compacta (SN,) was delimited dorsally by the medial lemniscus and ventrally by the zone lacking TH-positive cell bodies. The substantia nigra pars reticulata (SN,,) was delineated by the AChErich zone containing fibers below the substantia nigra pars compacta. The catecholaminergic region A8 (A8) was taken to be the zone above the medial lemniscus and the red nucleus. No reliable anatomical landmarks are available to determine the boundaries of the ventral tegmental area, thus this area was arbitrary divided in three as indicated: medial ventral tegmental area (M), the zone containing the exiting fibers of the third cranial nerve in the pars compacta (III,), and the pars reticulata (III,) of the substantia nigra. CP, cerebral peduncle; RN, red nucleus. mesencephalon: the substantia nigra pars compacta and pars reticulata, the ventral tegmental area (corresponding to region M of Fig. 1) and the catecholaminergic region A8.z4 The boundaries of these brain areas were delimited on the AChE- and TH-stained sections, adjacent to those used for

. &

mg of tissue equivalent (mean of two experiments) (Fig. 2). Non-specific binding (at the &), determined in the presence of 1OpM cis-Z-piflutixol corresponded to 20% and 36% of the total binding in the substantia nigra pars reticulata and pars compacta, respectively. Kd and B,,, values (Table 2) calculated from Scatchard plots were similar in pars compacta and pars reticulata of the substantia nigra. Higher Kd and lower B,,,,, values were found in the ventral tegmental area and in the catecholaminergic region A8 as compared with the substantia nigra (Table 2). cis-Z-Piflutixol displaced [3H]SCH 23390 binding with high affinity (1~~~:8 x 1O-7 M). rcs,, values were similar in the substantia nigra pars compacta and pars reticulata (Table 3). The preference of [3H]SCH 23390 for the D, rather than the D, receptor was demonstrated by the lcso of sulpiride (> 10e3 M). The S-HT antagonist ketanserin also displaced [‘H]SCH 23390 binding with very low affinity (rcsO:10m4M). None of the competition curves for [3H]SCH 23390 could be fitted to models of two binding sites (data not shown). Micromolar concentrations of atropine, propanolol and clonidine showed little or no effect on [3H]SCH 23390 binding levels (Table 3).

60 60 40

.

3H-SCH 23390

.

A

(nM)

0

5

10

15

20

25

30

35

Bound (fmoles/mg tissue sq.)

Fig. 2. Characterization of [3H]SCH 23390 binding sites in the human substantia nigra. (A) Saturation analysis of [3H]SCH 23390 specific binding to slide-mounted tissue sections from human substantia nigra. Data from a single representative experiment are reported in which [‘HISCH 23390 concentrations ranged from 0.5 to 20 nM and non-specific binding was defined as the amount of binding in the presence of 10 PM cis-Z-piflutixol, as described in Experimental Procedures. (B) Scatchard analysis of the saturation data presented in A indicated a Kd of 2.5 + 0.4 nM and a B,, of 31 k 6 fmol/mg of tissue equivalent. Data are the average of two separate experiments on tissue sections from two control brains (in each experiment [‘H]SCH 23390 binding levels were determined on triplicate sections).

390

F.

THIBAUT et al.

Ac~ordingIy, the distribution of [3H]SCM 23390 binding sites in the ventral mesencephalon was investigated using the labeled ligand at a concentration of 4 nM.

Table 3. Inhibition of 13H]SCH 23390 binding by various compounds in the human substantia nigra

~~stri~utj~n of E3H]SCH 23390 banding sites in the

cis-Z-Piflutixol Sulpiride Ketanserin Atropine Clonidine Propanolol

Compounds ventral mesencephalon

qf control subjects

Specific [3H]SCH 23390 binding on autoradiograms was mainly confined to the substantia nigra pars compacta and pars reticulata (Fig. 3A), whereas non-specific binding was more uniformly distributed. In the pars compacta, however, small spots of nonspecific binding were observed (Fig. 3B). Superimposition of the autoradiograms and the counterstained sections showed a close correspondence between dots of non-specific binding and neuromelanin accumulation. Specific ligand binding was highest in the substantia nigra (mean densitykS.E.M.: 16.5 +_ 1.8 and 18.2 + 1.7 fmol/mg of tissue equivalent in the pars reticulata and pars compacta, respectively), with maximal levels in the medioventral region (Fig. 3). Mean binding density was significantly lower in cell group A8 (9 i 0.8 fmol/mg of tissue equivalent) and in the ventral tegmental area (8.1 + 0.35 fmoljmg of tissue equivalent). Binding density varied along the rostrocaudal axis of the mesencephalon. Within the substantia nigra, the density of [3H]SCH 23390 binding sites was higher at caudal than at rostra1 levels (Fig. 4). The pattern of distribution of [3H]SCH 23390 binding sites was similar in both parts of the substantia nigra, except at the rostra1 pole where the density of binding was strikingly higher in the pars reticulata than in the pars compacta. In areas corresponding to the dopaminer~i~ ceil group A8 and the ventral tegmental area, a peak density of [3H]SCH 23390 binding was observed but more caudal than that in the substantia nigra (pars compacta or pars reticulata). The mediolateral pattern of distribution of [‘H]SCH 23390 binding sites was analysed only in the pars reticulata of the substantia nigra. The density increased gradually from the lateral part to the level at which fibers of the third carnial nerve exit, then

Table 2. Characteristics of [‘H]SCH 23390 binding in the human ventral mesencenhalon

&I (nM) Substantia nigra (total) oars reticulata pars compacta Peri- and retrorubral field (A8) Ventral tegmental area (A-10) The

2.53 +I 0.40 2.42+0.15 2.71 z 0.30 8.55 If. 0.50 5.05 * 0.70

B lnar

(fmolimg tissue) 31.5+6 33 + 3 31 I5 5 + 0.8 8.2& 1

values were determined on autoradiograms by Scatchard analysis as described in Ex~rimentai Procedures. Values are the means&S.E.M. of triplicate sections from two control brains.

K, (nM) 2080 f 200 > 100,000 > 100,000 > I 00,M)o > 100,000 > 100,000

Each K, value, obtained from competition studies, represents the mean+S.E.M. of data from one experiment with two control brains. K, values were determined using computer analysis (EBDA, Efsevier-Biosoft, Cambridge, U.K.) from ICKY values with the equation rc, = 1cm (I + IL]/&).

decreased medially to values similar to those observed laterally. A three-dimensional distribution of 13H]SCH 23390 binding sites within the substantia nigra pars reticulata was constructed (Fig. 5) by combining the lateromedial and rostrocaudal patterns on the series of sections. A peak value of [3H]SCH 23390 binding density (38 fmofjmg of equivalent tissue), two- to three-fold higher than the mean estimation for the whole substantia nigra, was detected just ventral to a zone of the substantia nigra pars compacta containing a high density of melanized neurons. Distribution of tyrosine hydroxylase und peptide imntunosta~n~ngin the human ventral mesence~ha~on

TH immunoreactivity was distributed heterogeneously within the human ventral mesencephalon. The pattern of distribution was strikingly different from the organization of specific [‘H]SCH 23390 binding sites, in both parts of the substantia nigra (Fig. 6a). TH immunoreactivity was most intense in the substantia nigra pars compaeta, mainly confined to neural cells and faint in neuronal processes within the pars reticulata (Fig. 6~). Immunostaining was also detectable but less intense in cell group A8, the ventral tegmental area and the central gray substance. An intense piexus of substance P-immunoreactive fibers was observed throughout the substantia nigra. The intensity of substance P staining did not differ markedly between the pars compacta and the pars reticulata of the substantia nigra (Fig. 6b). Substance P immunoreactivity predominated in the medial part of the substantia nigra. This pattern of distribution was strikingly similar to that of t3H]SCH 23390 binding sites (Fig. 6a,b). Dense fields of dynorphin B-immunoreactive terminals were observed within the substantia nigra. Immunoreactivity was most intense in the pars reticulata but was also strong in the pars compacta of the

D, receptors in the human substantia nigra

391

Fig. 3. Autoradiograms of pH]SCH 23390 binding sites in the human ventral me~ncephalon. Total binding {A) was determined in the presence of 4nM of [3H]SCH 23390. Non-specific binding (B) was estimated at the same ligand concentration in the presence of 10 PM cir-Z-piflutixol. Note that the most intense non-specific binding appears as black dots (indicated by arrows) shown to correspond to neuromelanin on the tissue sections. SNpr, substantia nigra pars reticulata; RN, red nucleus; ML, medial lemniscus. Scale bar = 2 mm.

substantia nigra, particularly in the dorsal band corresponding to the zone fi described by Olszewski and Baxter.38 Although, the intensity of staining of dynorphin B immunoreactivity was not as intense as that of substance P, its pattern of distribution was superimposable on that of [3H]SCH 23390 binding sites at the macroscopic level (Fig. 6b,d). The pattern of somatostatin immunoreactivity contrasted with that of the other peptides. Intense staining was observed in a restricted zone of the nigral complex, located in the laterodorsal part of the

substantia nigra, and extending through the caudal third of the structure (Fig. 6e). Beside this zone of high somatostatin immunoreactivity, faint immunostaining was present throughout the rest of the structure. The distributions of somatostatin immunoreactivity and [3H]SCH 23390 binding sites were clearly different. Methionine-enkephaiin immunoreactivity was sought for, but could not be analysed in these tissue preparations because of the high non-specific background.

392

F.

THIBAUT

PI ul.

30 20 10 0 20 10 0

Lateral

2 Rastral

4

6

{~)

10 ca4iilrl

8

Fig. 4. Rostrocaudal distribution of [‘HISCH 23390 binding in the ventral mesencephalon of a control brain. A series of 10 regulariy spaced sections from the mesencephalon of a control brain (case 694) were incubated in the presence of t3H]SCH 23390 (4 nM), as described in Experimental Procedures. Non-specific binding was defined by IO PM cis-Zpiflutixol. Data are the average of two experiments performed on triplicate sections. Similar data were obtained with the other control brains. Values are presented separately for the pars reticulata (SNpr) and the pars compacta (SNpc) of the substantia nigra (SN). A8, peri- and retrorubra1 catecholaminergic region; AIO. ventral tegmental area, also described as M in Fig. I.

Fig. 5. Three-dimensional representation of the distribution of the [‘H]SCH 23390 binding sites in the substantia nigra pars reticulata from a control brain. Transverse sections were taken at 0%mm intervals along the rostrocaudal axis of the substantia nigra (case 694). Measurements along the mediolateral axis were made at a different positions on a transverse section, perpendicular to the midline of the brainstem. Similar data were obtained with the other control brains. Data are the average of two experiments performed on triplicate sections.

tissue equivalent): substantia nigra pars compacta (18.2 + OS), pars reticulata (18.9 f l.S), catecholaminergic region A8 (5.6 + 1); data for A10 were not available. Interestingly, non-specific binding in the substantia nigra pars compacta was 32% lower than in control subjects, in parallel with the loss of pigment. Indeed, the small dots of unspecific binding associated with neuromelanin in the pars compacta of control subjects (Fig. 3A) could no longer be detected in the brain of the parkinsonian patient (Fig. 7B). Distribution oj’ [sH]SCH 23390 binding sites and This may explain why the total binding (Fig. 7A) is tyrosine hydroxyluse and peptide immunoreuctioity in higher in the control than in parkinsonian substantia the ventral me.~enceph~~onqf’putients with Purki~s~~~‘.s nigra (Fig. 7B) although specific binding values are disease und Hunt~ll~t[)n,s disease similar. As expected, a massive loss of TH-immunostained The distribution of [-‘H]SCH 23390 binding sites neurons was observed in the substantia nigra of the in the ventral mesencephalon of the parkinsonian ~~rkinsonian patient (Fig. 7H) compared with conpatient was similar to that observed in control subtrols (Fig. 7G). TH immunostaining was almost jects: (I) the same heterogeneous pattern was obcompletely absent from the pars reticulata of the served throughout the rostrocaudal and mediolateral extent of the nigral complex; (2) the density of specific substantia nigra (Fig. 7H). The pattern of distribu[jH]SCH 23390 binding was within the range of tion and the intensity of staining for substance P (Fig. 7E), dynorphin B and somatostatin (data not controls values (mean+S.E.M. in fmoi/mg of

Fig. 6. Distribution of [‘HJSCH 23390 binding sites and peptides and TH immunoreactivity in the substantia nigra of a control brain. Nearly serial sections through the ventral mesencep~lon of control brain 694 at the caudal third of the substantia nigra representing (a) an autorddiogram of f3H]SCH 23390 binding, and sections treated by immunohistochemistry with antibodies against substance P (b), TH (c), dynorphin B (d). somatostatin (e) or AChE histochemistry (f). Asterisk indicates a zone labeled on [jH]SCH 23390 autoradiography and stained for substance P or dynorphin B immunohistochemistry but with low TH and somatostatin immunost~ining. ML, medial lemniscus; SNpc, substantia nigra pars compacta. Scale bar = 2 mm.

D, receptors in the human substantia nigra

393

Fig. 7. Distribution of fHfsew 23390 binding sites and peptide and TH immunoreactivity in the substantia nigra of a control subject, a patient with Parkinson’s disease and a patient with Huntington’s disease. Nearly serial sections through the substantia nigra at the level of the exiting fibers of the cranial nerve III of control subject 694 (A, D, G), a patient with Parkinson’s disease (B, E, H) and a patient with Huntington’s disease (C, F, I). For each case, atuoradiograms obtained after [3HJSCH 23390 binding (A, B, C) substance P immunoreactivity @, E, P) and TH immunoreactivity (G, H, I) are illustrated. Subnormal TH immunoreactivity is observed in the substantia nigra of the patient with Parkinson’s disease (H) as compared with the control subject (G). [3H)SCH 23390 binding (C) and substance P immunoreactivity (F) are decreased in the substantia nigra of the patient with Huntington’s chorea (A) compared with a control (G). Scale bar = 3 mm.

D,

receptors in the human substantia nigra

shown) immunoreactivity did not markedly differ from that of controls (Fig. 7D) throughout the rostrocaudal extent of the substantia nigra. As in control brains, methionine-enkephalin specific staining was not detectable in the parkinsonian substantia nigra. In the substantia nigra from the patient who died with Huntington’s disease, tissue shrinkage was observed macros~opically. The density of [lH]SCH 23390 binding sites decreased strikingly compared with controls (mean densitykS.E.M. in fmol/mg of tissue equivalent: 3.7 +0.2 and 5.2 + 0.5 in pars compacta and pars reticulata, respectively, Fig. 7C). However, in the catecholaminergic area A8 and in the ventral tegmental area, the density of [3H]SCH 23390 binding was the same as in controls (mean values&S.E.M.: 5.64 + 1 and 7.7 + 0.4 fmol/mg of tissue equivalent, respectively). Despite the marked decrease in binding density, the pattern of the distribution of [3H]SCH 23390 along the rostrocaudal axis was not altered (Fig. 7A, C). Little neuropeptide immunoreactivity remained in either part of the substantia nigra of this patient: substance P and dynorphin B staining was difficult to distinguish from non-specific staining (Fig. 7F). In contrast, the pattern of distribution and the intensity of TH immunoreactivity were not altered in this pathological condition (Fig. 71). The tissue section at the level of the zone of intense somatostatin immunoreactivity, observed in the control substantia nigra (Fig. 6E), was not available for study. DISCUSSION P~u~~ucQf~g~cu~ pro$le of 13H]SCH 23390 binding sites in the human ventral mesencephalon

395

al.3 in rat and cat brain. Discrepant data are, however, reported in the literature: in the human brain, higher levels have been reported in the pars compacta with lower densities in its dorsolateral regionI whereas in the rat, cat and monkey a higher density of D, receptors has been observed in the pars reticulata compared with the pars compacta.“‘*” The discrepancies reported in [3H]SCH 23390 binding levels between the two regions of the substantia nigra, among the different species investigated, could be related to species differences in organization of the nigral complex. It may only reflect the fact that the pars compacta is relatively more developed in the human substantia nigra than in other species.43 The low levels of [‘H]SCH 23390 binding the catecholaminergic cell group A8 and in the ventral tegmental area indicates that D, receptor density is low in these areas of the human brain as in rat, cat and monkey.‘-3,5.47 The density of [3H]SCH 23390 varied throughout the extent of the substantia nigra suggesting a heterogeneously organized distribution of D, receptors. The highest density of D, receptors was observed in the medial part of both subdivisions of the substantia nigra, in agreement with data in cat, monkey and rat brain.‘.ss45The prominent binding found in the medial part of the pars reticulata coincides with the presence in this area of ventrally extending dendrites of dopaminergic neurons of the pars compacta. In cat and monkey brain, this zone of high D, receptor density in the pars reticulata is located ventrally to a group of densely packed dopaminergic cell bodies in the pars compacta. In these species, the neurons located in this so-called “denso~ellular zone” have been shown to project towards the D, receptor enriched striosomal compartment of the striatum,28 and may do so as well in human brain.

The pharmacological profile of rH]SCH 23390 binding determined by autoradiographic analysis of sections of human substantia nigra (Table 2) is Distribution of D, receptors in the humun substunt~ consistent with data obtained in homogenates of nigra in relation to the nigrostriatal and striatonigral innervation nigral and striatal tissue from human brain.12~37~41~“,46 Competition studies (Table 3) suggest that, under the In an attempt to determine where nigral D, receppresent experimental conditions, rH]SCH 23390 pritors are located with respect to the striatonigral input marily labeled D, dopaminergic receptors in the or nigral dopaminergic neurons in human brain, the ventral mesencephalon. Most, if not all, unspecific autoradiographic distribution of D, receptors and the binding is associated with the presence of neuropattern of distribution of TH-immunostained neurmelanin (Fig. 3). This is further supported by the ons and peptidergic afferences were compared in the decrease in non-specific binding in the substantia substantia nigra from control subjects and in patients nigra pars compacta of the parkinsonian patient. with a characteristic neuronal deficiency. However, These data may account for the higher level of this approach has some limitations. (I) D, receptors non-sp~ific 13H]SCH 23390 binding in human than were assessed by film autoradiography (not at the in rat substantia nigra.” cellular level) on fresh frozen tissue, since [3H]SCH 23390 binding was not detectable on fixed material Distribution qf 13H]SCff 23390 binding sites in the (data not shown) whereas antibody immunostaining human uentrai mesence~haion of control subjects was not satisfactory on unfixed tissue (data not Ligand binding levels were similar in the pars shown). Therefore, immunohistochemical detection reticulata and pars compacta of the substantia nigra, was performed on postfixed sections adjacent to those with the exception of a restricted zone (discussed treated for autoradiography. Accordingly, the distribelow). The data are in agreement with observations bution of the immunostaining and of the D, receptors of Boyson et al.” and Beckstead* and Beckstead et could only be compared macroscopically. (2) Some

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F. THIBALJT et (11

antigens, methionine-enkephalin for example, which are well immunostained on immersion fixed tissue25 were not detectable on postfixed sections. Despite these technical limitations, the present data offer new insights into the distribution of D, receptors with respect to dopaminergic and peptidergic innervation of the human substantia nigra. In control brain, the distribution of D, receptors was closely superimposed on that of substance P and dynorphin B immunoreactivity, but differed from that of somatostatin and TH immunostaining. This observation, in accordance with data in experimental animals,“.27.32.33.42 suggests that in human substantia nigra D, receptors are localized on substance P- and dynorphin B-containing terminals originating in the striatum. This conclusion is further supported by the neuropathological cases examined in this study. (I) A concomitant decrease in D, receptors density, and substance P and dynorphin B immunoreactivity was observed in the substantia nigra of the patient with Huntington’s disease, in agreement with previous observations for and for substance P immunostainD, receptors” ing. ‘“J’J’J~,~~ (2) The levels of D, receptors were similar in the substantia nigra of controls and the patient with Parkinson’s disease, in accordance with previous biochemica140,44and autoradiographic findings. I5 Parkinson’s disease is associated with severe loss of nigral dopaminergic neurons24.2h whereas innervation by substance P- and dynorphin B-contain-

ing projections is essentially spared.2’~25These results confirm experiments in the rat, demonstrating a decreased number of D, receptors in the substantia pars reticulata following excitotoxic lesions of the striatum, whereas destruction dopaminergic neurons in the substantia nigra with 6-hydroxydopamine does not modify D, receptor content in this structure.‘8.‘9

CONCLUSIONS

Altogether the data support the view that D, dopaminergic receptors are heterogeneously distributed throughout the substantia nigra and that most if not all are located on striatonigral fibers. In animals, nigral D, receptors are known to be tonically stimulated by local release of dopamine from dopaminergic dendrites. 35In human as in animal brain, they may therefore control the activity of both the nigral dopaminergic neurons and the output of the nigral complex, via the release of neurotransmitter from striatonigral pathways. Acknowledgements-We

thank Drs Dubois

and

Scatton

(LERS, Bagneux) for helpful discussion, Drs Duyckaerts, Hauw (HBpital de la Salp&itre, Paris), Laurent, Piette and Sachet (H6pital Charles Foix, Ivry) for providing human material and Drs Benoit (HBpital general de Montreal. Montreal) and Feuerstein (Inserm U3 18, Grenoble) for their gift of antisera. It is a pleasure to acknowledge the valuable comments on the manuscript of Dr M. Ruberg.

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(Accepted 23 April 1990)