Autoradiography of antidepressant binding sites in the human brain: localization using [3h]imipramine and [3h]paroxetine

0306-4522/88 $3.00 + 0.00

Neuroscience Vol. 27, No. 2, pp. 473-496, 1988 Printed in Great Britain

Pergamon Press plc 0 1988 IBRO

AUTORADIOGRAPHY OF ANTIDEPRESSANT BINDING SITES IN THE HUMAN BRAIN: LOCALIZATION USING [”HIIMIPRAMINE AND [3H]PAROXETINE R. CORT&,*~ E.S~RIANO,~ A.P~zos,$A. PROBST*and J.M.PALACIOS$[~ *Department of Pathology, Division of Neuropathology, University of Basle, Schiinbeinstrasse 40, CH-4003-Basle, Switzerland and fPreclinica1 Research, Sandoz Ltd, CH-4002-Basle, Switzerland Ahatract--[3H]Imipramine and [‘Hlparoxetine were used to label sites associated with serotonin uptake mechanisms in post-mortem brain tissue from control subjects. The anatomical localization of these sites was examined by autoradiography and densities measured by microdensitometry. We found (‘Hlimipramine binding to increase with age in the cortex and amygdala, but to be independent of gender and post-mortem delay. Preliminary results indicate that the binding of both [3H]imipramine and [)H]paroxetine is diminished in the brain of patients treated with imipramine. The distribution of [3H]imipramine and [3H]paroxetine high-affinity binding sites was very similar, and correlated well with the distribution of serotonergic presynaptic markers in the brain. The highest densities of binding sites were found in the raphe nuclei and the midline thalamic nuclei. Other structures presenting high levels of binding were the substantia nigra, nucleus interpeduncularis, locus coeruleus, nucleus nervi hypoglossi, nucleus nervi facialis, mammillary bodies and other parts of the hypothalamus. In contrast, regions such as the neocortex, hippocampus, amygdala and cerebellum showed low densities of [3H]imipramine and [‘Hlparoxetine binding sites. This distribution seems to indicate that the ascending serotonergic pathways are the main site of action of antidepressants.

potently displace [‘Hlparoxetine binding, and that their potency to inhibit [3H]5-HT uptake correlates well with their potency to inhibit [‘Hlparoxetine action is presumed to be the blockade of the neuronal re-uptake of serotonin (5-HT), which results in an binding.B In addition, the destruction of serotonergic lesioning results increase in serotonergic neurotransmission.‘4*37*40*48 neurons by 5,7_hydroxytryptamine in almost total disappearance of specific binding of [3H]Imipramine binding to nerve terminal prepara[3H]paroxetine to rat cortical membranes.B These tions from mammalian brain has been described These results indicate that the specific [3H]paroxetine bindand extensively characterized. 7.8,10,22,31.35.57.58,~,62 ing sites on rat cortical membranes are associated studies have shown that the pharmacological charwith the 5-HT transporter. acteristics of the high-affinity [3H]imipramine binding site correlate well with those expected for a drugComparison of high-affinity [’ Hlparoxetine and recognition site associated with the S-HT uptake [3H]imipramine binding has shown that in platelet system, although some discrepancies have arisen.36 It membranes both ligands label the same number of sites,‘* suggesting that both interact with the same is known that, in addition, imipramine can bind with appreciable affinity to receptors and/or uptake sites 5-HT uptake site. In contrast, in neuronal memfor acetylcho1ine,69 histamine,*’ noradrenaline and branes the B,,,,, for [)H]imipramine is somewhat 5-HT.5’ higher than the B,, for [3H]paroxetine.4’ This sugThe non-tricyclic antidepressant paroxetine is a gests that, although a major part of the high-affinity more specific and potent inhibitor of 5-HT uptake rH]imipramine binding is localized on the 5-HT than imipramine. *2,33The binding of [‘Hlparoxetine transport mechanism, part of the [3H]imipramine binding occurs to other sites in brain tissue.4’ to rat and human neuronal and platelet membranes has been recently characterized.29.4’*42.54 It has been [‘H]Imipramine binding sites have been visualized demonstrated that only 5-HT uptake inhibitors in the rat brain using receptor autoradiography and found to correlate with the distribution of 5-HT nerve terminals.‘8,23*25,55 Axonal transport of imipramine tPresent address: Department of Histology, Karolinska binding sites has been demonstrated in the rat CNS Institute, P.O. Box 60400, S-10401 Stockholm, Sweden. §Present address: Dept de Farmacologia y Terapeutica, using the same technique.‘9 In the human brain Facultad de Medicina, Universidad de Santander, [3H]imipramine binding has been analysed in postE-39071Santander, Spain. mortem tissue by conventional membrane-binding ~(To whom correspondence should be addressed. procedures. 39,6’ The properties of these sites have been Abbreviations: ADBS, antidepressant binding sites; S-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin. found to be similar to those reported for the rat brain.

Imipramine is a dibenzazepine derivative widely used clinically as an antidepressant.24v30 Its mechanism of

473

474

R.

C'ORT~S

included in our study. The characteristics of these cases referring to age, gender, posr-mortem delay and cause trl’ death are given in Table 1. At autopsy brains were promptly removed from the skull and dissected. One hemisphere wa\ fixed on formahn for routine neuropathological examlnation. Blocks 4 5 mm thick were cut from the other hemisphere and brainstem, frozen at -80’C and later broughr to -20 C for storage. Sections 10pm thick were cut out from the blocks using a microtome-cryostat (Leltr 1710: Leitz, Wetzlar, F.R.G.) and mounted onto gelatin-coated 20 (‘ microscope glass slides. Tissue sections were kept ;It until use. Labelling of tissue sections with [‘Hjimipramine u:ts carried out as described by Crabowsky et tri.?’ Slidemounted tissues were first preincubated for 15 min in 50 mM Tris+HCl (pH 7.4) at room temperature and then Incubated for 1 h in the same buffer containing 150 mM NaCl. 5 mM KCI and 10 nM [‘Hlimipramine. After incubation sections were rinsed in cold fresh buffer for IO and 20 min consecutively and quickly dried under a stream of cold air. Blanks were obtained by the addition of 100 /rM desipramine 10 the incubation medium.

Since imipramine exerts important therapeutic effects and [3H]imipramine binding sites are present in the human brain it was interesting to analyse their distribution at a higher level of anatomical resolution. Therefore, we have applied quantitative autoradiographic techniques to visualize and measure the concentration of these sites in several brain nuclei in 16 neurologically control subjects. In addition we have compared the distribution of [3H]imipramine high-affinity binding sites with that of [3H]paroxetine. EXPERIMENTAL

PROCEDURES

Human brains were obtained at autopsy from 16 adult subjects (9 male and 7 female, mean age 67.9 _t 12 years, mean post-mortem delay 6.6 + 3.3 h) free of neurological or psychiatric disorders, who had not received antidepressant treatment prior to death. The brains of 3 adult patients treated with imipramine and from 3 infants were also Table

(years)

Post -mortem delay (h)

Age Case

Sex

Adult controls A M

1. Sources

Immediate

et ul.

of brain

cause of death

B C D

M F F

61 60 49 88

3 6.25 6.5 11.5

E F G

F M F

80 51 65

3 5.5 6

H I J K L M

F M F M M M

80 53 71 79 70 83

5 12 2 6.75 6.5 5.5

N

M

55

10

0 P

M F

68 68

12.5 3

Infants Q

M

5.5

8

Cardiac

R

M

7

4

S

F

6

9.5

Cardiac failure Respiratory failure Cardiac failure

Patients T

treated M

with imipramine 26 1.5

U

F

48

5

V

M

80

34

tissue

Pulmonary embolism Cardiac failure Pulmonary embolism Pulmonary embolism Bronchopneumonia Cardiac failure Cardiac failure Cardiac failure Cardiac failure Cardiac failure Bronchopneumonia Cardiac failure Cardiac failure Bleeding from dissecting of the aorta Acute peritonitis Pulmonary embolism Kidney transplantation Cardiac failure Pulmonary embolism Cardiac failure Pneumonia failure

Septicaemia

Pyelonephritis

Cardiac failure Bronchopneumonia

Pre-morfem condition Carcinoma of the lung Malignant tumor of oesophagus Chronic renal failure Diabetes mellitus

aneurism

lschemic heart disease Ischemic cardiopathy Chronic bronchitis, Emphysema Cor pulmonale Carcinoma of the stomach Coronary artery disease Rhabdomyosarcoma Carcinoma of the gall bladder Coronary artery disease Arterial hypertension Carcinoma of the rectum Chronic interstriatal nephritis Carcinoma Carcinoma

of the ureter of the breast

Cardiac malformation (truncus arteriosus communis) Acute lymphocytic leucemia Donohne syndrome Lennox epilepsy Multiple malformation Rhabdomyosarcoma of the leg (75 mg imipramine/day during last month before death) Carcinoma of the breast (75--l 50 mg imipramine/day for 14 weeks. Stop 1 I days before death) Arterial hypertension Ischemic heart disease Carcinoma of urinary bladder (25mg imipramine/day during 3 last days before death)

Antidepressant

binding sites in the human brain

In order to label tissue sections with [‘Hlparoxetine incubation buffers and the concentration of the radiolabelled ligand were selected taking in account the results from binding studies on rat brain membranesB Preliminary biochemical studies carried out with rat and human postmortem slide-mounted tissue sections showed that the characteristics of the binding of [‘Hlparoxetine to tissue sections are comparable to those reported from membrane binding assays. The best ratio of total/non-specific binding was obtained after two consecutive washes of 20min each. Therefore, the incubation conditions for [3H]paroxetine were as follows. Tissue sections were preincubated for 15 min at room temperature in 50 mM Tri-HCl (PH 7.4), as described for [3H]imipramine. The incubation was performed for I h at room- temperature in 50 mM T&-ICI containing 150 mM NaCl. 5 mM KC1 (oH 7.4) and 0.5 uM [‘Hlparoxetme. Tissue sections were then washed twice for 20min in the same buffer at 4°C and dried. To determine the non-specific binding, consecutive sections were incubated in the presence of 10 PM of the S-HT uptake inhibitor fluoxetine. To obtain autoradiograms, the labelled sections together with separate sets of brain gray and white matter standards were exposed to [‘H]Ultrofilm (LKB, Sweden) for 7-10 days in the case of [‘Hlimipramine and for 15 days in the case of [3Hlparoxetine. Autoradiograms were quantified by microdensitometry using a computerized image-analysis system as described previous1y.‘6 Brain areas and nuclei were identified using several atlases of the human brain.m.2’.” The raphe nuclei were named according to Braak6 and hippocampal fields according to Stephan.“’ [3H]Imipramine (75 Ci/mmol) and [’ Hlparoxetine (22.3 Ci/mmol) were obtained from New England Nuclear (Dreieich, F.R.G.). ”

,

-

RESULTS [’ H]Imipramine and [‘Hlparoxetine binding sites were heterogeneously

high-affinity

distributed throughout the human brain. The concentrations of binding sites of both [3H]ligands as determined by microdensitometry in several brain areas and nuclei are given in Table 2. As shown in this table and Figs 3-13, the anatomical localization of [3H]imipramine and [‘Hlparoxetine high-affinity binding sites in the human brain were almost identical. A quantitative correlation between [‘Hlimipramine and [3Hlparoxetine binding densities is illustrated in Fig. 1. This figure was obtained from specific binding values of [3Hlimipramine and [‘Hlparoxetine measured in single areas from three individual cases. The correlation between the binding of both ligands was r* = 0.737 (P < 0.0001). However, the levels of [‘Hlimipramine binding appeared to be proportionally higher than those of [3H]paroxetine. Nevertheless, there was a good correlation between the densities of [3Hlimipramine and [3Hlparoxetine binding and therefore in the following we will refer to these sites as antidepressant binding sites (ADBS). Although the densities of ADBS varied from case to case, their distribution was similar among the different control patients. We examined the possible influence of parameters such as age, gender and post-mortem delay on [‘Hlimipramine binding. In our series there were no significant differences in binding between male and female (Fig. 2A) or as a function

475

of the interval between death and freezing of the brain (Fig. 2B and C). In contrast, we found an increase in [3Hlimipramine binding with increasing age in several brain areas, including the frontal cortex (linear regression: correlation coefficient, r2 = 0.627; P -C0.0037; Fig. 2D), occipital cortex (r2 = 0.627; P < 0.0126; Fig. 2E), globus pallidus lateralis (r2 = 0.462; P < 0.0306), nucleus granularis (r2 = 0.677; P < 0.0121) and basalis amygdalae (r2 = 0.587; P < 0.0266), nucleus lateralis thalami (r2 = 0.552; P < 0.0219) and griseum centrale mesencephali (r2 = 0.556; P < 0.0211). The influence of age was, however, not observed in the other brain areas examined (Fig. 2F-I). The influence of these parameters on [3H]paroxetine binding was not analysed because of the small size of the population studied. The effects of treatment with imipramine on [3Hlimipramine and [‘Hlparoxetine binding were examined in three (cases T, U, V) and two (cases T, V) subjects respectively (see Table 1 for treatment details). In these patients the binding of both [3Hlligands was strongly diminished throughout the brain (Fig. 3, Table 3). In general [3Hlimipramine and [3H]paroxetine binding was reduced below 50% of adult control values. In most brain structures, no specific binding could be detected in such patients. In all cases analysed the highest densities of ADBS were detected in the raphe nuclei of the midbrain and upper pons and in the thalamus, while the lowest corresponded to white matter tracts, with one exception (see below). The non-specific binding of both ligands was generally homogeneous and ranged from 100 to 500 fmol/mg protein in the case of [3H]imipramine, and from 200 to 600 fmol/mg protein in the case of [)H]paroxetine. In the following description we will refer to the average content of [‘Hlimipramine and [3Hlparoxetine specific binding observed in adult control subjects. The reader is referred to Table 2 for quantitative data and to photographs in Figs 4-15. Brainstem and cervical cord (Figs 3-9)

In the brainstem ADBS were highly localized to specific nuclei. Very low levels of ADBS were found in the cervical cord (Fig. 4). The highest labelling was observed over a white matter tract located ventrolaterally, probably corresponding to the spino-olivary and olivospinal tracts, which showed densities of 1257 + 132 fmol/mg protein (as measured using white matter standards). Among gray matter nuclei, the highest densities of [3Hlimipramine binding sites were observed in the substantia gelatinosa and motor cells, which showed low levels of binding. In the medulla oblongata (Figs 4 and 5) the highest densities of ADBS were observed over the nuclei olivaris accessorius medialis and dorsalis and the nucleus nervi hypoglossi, which corresponded to high levels of binding. The nucleus raphe obscurus, nucleus solitarius, nucleus ambiguus, nucleus dorsalis nervi vagi, nucleus cuneatus lateralis and nucleus

R. CoRThset 01.

476

Table 2. [3H]Imipramine and [jH]paroxetine binding in the adult control human brain

n Medulla oblongata Nucleus raphe obscurus Nucleus tractus solitarius Nucleus nervi hypoglossi Nucleus olivaris inferior Caudal pons Nucleus raphe magnus Nucleus nervi facialis Nucleus rapht pontis Griseum pontis Nucleus reticularis tegmenti pontis Rostra1 pons Nucleus raphe linearis Nucleus raphe centralis Nucleus raphe dorsalis Nucleus raphe dorsalis, lateral part Griseum centrale metencephali Locus coeruleus Griseum pontis Nucleus parabrachialis lateralis Midbrain Nucleus rapht linearis Nucleus raphe centralis Nucleus raphe centralis, pars annularis Nucleus raphe dorsalis, pars supratrochearis Central gray Substantia nigra Nucleus interpeduncularis Red nucleus Nucleus tegmentalis pedundulo-pontinus Thalamus Nucleus anterior Nucleus medialis Nucleus lateralis anterior Nucleus paramedianus rotundocellularis Nucleus reuniens Zona incerta Hypothalamus Nucleus posterior Area lateralis Nucleus ventromedialis Nucleus tuberales Mammillary bodies Hippocampus Dentate gyrus CA3 (pyramidal layer) CA 1 (pyramidal layer) Subiculum Amygdala Nucleus lateralis Nucleus basalis Nucleus granularis Nucleus basalis accessorius Basal ganglia Nucleus caudatus Putamen Globus pallidus, pars lateralis Substantia innominata Cortex Frontal Occipital Parietal Cingular Cerebellum Molecular layer Granule cell layer Dentate nucleus

[‘HlImipramine binding (fmol/mg protein) mean + S.E.M. min-max

3 3 5

611.3 + 68.2 604.9 _+37.0 785.5 & 56.7 415.5 + 54.4

3888898 556678 672-850 3055568

2 2 8 7 5

736.6 + 145.6 + 521.4 + 323.5 k 688.2 +

559-914 548-943 3217732 109-568 442-991

6

177.6 197.6 43.9 64.5 96.5

[ZHlParoxetine binding (fmol/mg protein) ?I mean + S.E.M. 3

3 3 3

I 1

529.5 + 244. I 697.0 & 122. I 880.0 + 68.8 368.0 _+94.X

3 3 2

782.6 1154.9 578.1 + 56.6 339.1 * 47.3 340.4 k 15.2

7 7 4 2 9 7 9 5

1590.3 + 152.9 1560.8 _+254.8 2400.2 k 302.5 2152.5 & 611.5 845.8 + 135.8 857.4 _+57.3 410.9 + 41.0 507.6 f 54.9

104-2163 821-2509 1779-3127 208 l-3424 399-l 720 726-l 177 2255653 365625

2 2 1

1162.3 k 306.2 1600.6 _+26.5 1990.9

2 2 3

1558.3 + 64.8 1375.3 _+203.8 473.4 + 58.3

8 7 7 I 14 13 9 10 9

1508.3 f 256.3 1710.3 + 263.5 1317.4 + 294.7 1981.6 f 322.2 564.7 * 53.5 800.1 + 67.6 805.3 k 126.0 424.3 -+ 47.3 404.9 + 57.8

713-2890 9643080 8443065 963-2999 213-964 483-1361 200-l 197 144610 276816

3

1015.5 C 142.5

2 3 3 3

1459.6 If 11.2 1714.6k97.3 921.2 + 122.3 123.4 & 90.1 1083.8 44.9

6 7

456.9 + 61.6 547.2 & 59.0 373.7 + 39.8 1346.9 + 222.3 1192.3 3 105.4 748.2 + 65.9

271l-611 269-700 259-559 523-2033 10091374 624 994

817.8 + 107.1 573.6 _+65.1 584.7 + 94.8 1006.0 f 99.0 1196.4+401.3

5441055 361-785 278-843 884-l 202 58881954

1

7 3 5 4 7

5 3 3

1 i 1 2 2

I 1 I

579.6 501.2 + 367.2 246.0 + 32.3 1941.6 541.7

1

908.2 927.8 + 124.2 517.3 1005.7 651.0

2

1 I

7 7 7 6

426.4 + 538.3 + 366.6 k 480.5 +

16.9 89.3 12.6 96.4

220-141 283-932 203-672 263-817

9 7 7 7

411.6 + 32.3 534.1 _+49.6 495.3 k 42.8 511.7 + 51.9

2717563 418-781 384696 382-799

8 9 8 7

522.9 & 44.9 633.9 + 49.0 502.1 * 55.5 714.7 rt: 77.8

323-747 371-838 2377701 420-904

3 3 1

354.9 + 108.4 513.9 + 99.5 234.5

8 7 I 5

371.0 + 401.1 * 307.1 + 336.2 +

39.0 37.0 41.2 31.1

156-513 25&510 86-466 289-458

2

307.1 + 5.4

5 5 5

180.7 + 17.3 358.8 + 30.3 401.6 + 43.9

142-234 29&438 264-541

Antidepressant

binding sites in the human brain

praepositus hypoglossi showed moderate levels of binding. Low densities of ADBS were associated with the substantia gelatinosa of the trigeminal nucleus, nucleus supraspinalis, and the nucleus olivaris inferior. The latter nucleus presented unusually high levels of non-specific binding, while in the nuclei olivaris accessorii the non-specific binding was within the normal range. Very low densities of ADBS were associated with the medullary reticular formation, although somewhat higher densities were observed in the ventromedial region of the nucleus reticularis gigantocellularis, laterally to the medial longitudinal fasciculus. Negligible binding was noted over other areas such as the pars interpolaris of the spinal trigeminal nucleus and vestibular nuclei.

471

In the lower part of the pons (Fig. 6) the highest levels of binding were associated with the nucleus nervi facialis. The distribution of binding sites in this nucleus was heterogeneous, with spots of high levels of binding being localized in the periphery of the nucleus, and lower levels in its center. Moderate densities of ADBS were seen over the nucleus rapht magnus and the rostra1 extension of the nucleus rapht obscurus, as observed in the medulla oblongata. Intermediate densities of ADBS were also measured in the nucleus reticularis gigantocellularis, over an area extending laterally from the raphe magnus and dorsally to the medial lemniscus. Other areas such as the griseum pontis had very low densities of binding sites. More rostrally, at the level of the motor

Abbreviations used in figures A ABa ABAc :Co AGr ALa Aq BST CA1 CA2 CA3 CG ChP Cn CU De DG E fx GCMi Gi GP GPl GPm Gr HDB Hi1 HDM HLa HPo HPV HVM IC ICL ICM IO IP IPC LC LP M MB MC MO Mo5

nucleus anterior thalami nucleus basalis amygdalae nucleus basalis accessorius amygdalae anterior white commissure nucleus corticalis amygdalae nucleus granularis amygdalae nucleus lateralis amygdalae aqueductus cerebri bed nucleus of the stria terminalis CA1 hippocampal subfield CA2 hippocampal subfield CA3 hiunocamual subfield central Gay (ghseum centrale mesencephali) choroid plexus nucleus cuneiformis nucleus cuneatus nucleus dentatus cerebelli dentate gyrus (molecular layer) entorhinal cortex fornix griseum centrale metencephali nucleus reticularis gigantocellularis griseum pontis globus pallidus, pars lateralis globus pallidus, pars medialis granule cell layer (cerebellar cortex) nucleus of the horizontal limb of the diagonal band of Broca hilus nucleus dorsomedialis hypothalami lateral hypothalamic area nucleus posterior hypothalami nucleus periventricularis hypothalami nucleus ventromedialis hypothalami inferior colliculus nucleus intralamellaris thalami, pars centralis lateralis nucleus intralamellaris thalami, pars centralis medialis inferior olivary nucleus nucleus interpeduncularis nucleus intralamellaris thalami, pars paracentralis locus coeruleus nucleus lateralis posterior (formatio lateralis thalami) nucleus medialis thalami mammillary bodies motor cells molecular layer (cerebellar cortex) nucleus motorius nervi trigemini

NRTP OAD OAM PBL Pdp Pf Pk PrH Pt Put F RC RCpa RDRDst

ReS RLi RMg Ro

ROb RP s SC SGe SI SN

SNC SNR so Sol Sth

Tub VA

vv VTA ZI

3 3A 4 10 12 VI

nucleus reticularis tegmenti pontis nucleus olivaris accessorius dorsalis nucleus olivaris accessorius medialis nucleus parabrachialis lateralis nucleus peripeduncularis nucleus parafascicularis (formatio intralamellaris thalami) Purkinje cell layer (cerebellar cortex) nucleus praepositus hypoglossi nucleus paratenialis (formatio paraventricularis thalami) putamen pyramidal cell layer of the hippocampus red nucleus nucleus raphe centralis nucleus rapht centralis, pars annularis nucleus raphe dorsalis nucleus raphd dorsalis, pars supratrochealis regio stellata nucleus rapht linearis nucleus raphe magnus nucleus paraventricularis rotundocellularis (= nucleus paramedianus) (formatio paraventricularis thalami) nucleus raphi obscurus nucleus raphe pontis subiculum superior colliculus substantia gelatinosa substantia innominata substantia nigra substantia nigra, para compacta substantia nigra, para reticulata spino-olivary/olivospinal tract nucleus tractus solitarius nucleus subthalamicus nucleus tuberales nucleus ventralis anterior (formatio lateralis thalami) nucleus ventralis ventralis (formatio lateralis thalami) ventral tegmental area zona incerta nucleus nervi occulomotorii nucleus accessorius nervi occulomotorii nucleus nervi trochlearis nucleus nervi facialis nucleus dorsalis nervi vagii nucleus nervi hypoglossi occipital cortex, lamina I occipital cortex, lamina VI

47x

R.

COKTiS

r'fu/

3000T

z ._ w 5

2000

t

rk0.787,

P<0.0001

0 00 3H-lmlpramlne

binding

Fig. 1. Correlation between [3H]imipramine and [‘Hlparoxetine binding. The densities (fmol/mg protein) of [3H]imipramine- and [3H]paroxetine-specific binding were obtained from consecutive sections from three adult control subjects. Linear regression analysis showed a good correlation between the specific binding densities of the two ligands. 3 H-lmipramine

A

Gender

Ei

1500-

binding

C

nigra

Substantla

1500

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3000

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5 $ I 4

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m

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f

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. ____--

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10

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15 8

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cortex

SOOT

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cortex

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1000, T

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----ef

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r2=0.62i PCO.0037

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Fig. 2. Effects of gender (A), posr-morrem delay (B, C) and age (II-I) of the patients on [‘Hlimipramine binding. (A) Distribution of [’ Hhmipramine binding in males (m) and females (f) in the substantia nigra and central gray. In both structures the two groups show comparable concentrations of binding sites. (II, C) The delay between death and freezing of the brain does not have significant effects on the binding of [3H]imipramine. (D-I) At older ages [‘Hlimipramine binding increases in the frontal and occipital cortices. No such effect is observed in the other regions illustrated. See text for details.

lb0

as*

Fig. 3. Total binding of [3H~m~pm~a~ (A, C) and ~3Hjparoxetine(B, E3)TVm~braia sections From a control case (A, B) and a representativepatient who received antidepressant tre&ment before death {C, D). Note that the binding of both iigands is markedly reduced over all strwtures and nuclei in the case treated with imipramine. Bar i-j3 mm.

~~e~ing~ nucleus (Fig. 7A), the d~~h~tj~ of ADBS was rather homo~~eous, low densities of

ADBS being found over most nuclei. In contrast to other raphi nuclei, the nucleus raphi pcmtis presented only intermediate to law levels of binding. At this level, the highest densities of ADBS were noted over

an area iucaked rn~i~l~~ in the ventral tegme~tnm~ which probably corresponds to the nucleus reticularis tegmenti pontis. In the upper pans (Fig. 713)and midbrain (Figs 3, g and 9) very high densities of ADBS were aksociated with the different raphe nuclei, including the nucleus

480

R. CORT~S (‘I ul. Table

3. Binding

of [JH]imipramine

and [‘Hlparoxetine

in adult

patients

treated

[‘H]Imipramine binding Mean &-S.E.M. Medulla oblongata Nucleus raphe obscurus Nucleus tractus solitarius Nucleus nervi hypoglossi Nucleus olivaris inferior Caudal pons Nucleus raphC magnus

different

from adult

n

(fmolimg

%

n

1 I I

81.1 161.8 341.4

13 21 82

I

1

55.5

8

1

I

Nucleus nervi facialis Nucleus rapht pontis Griseum pontis Rostra1 pons Nucleus raphk linearis Nucleus raphi centralis Nucleus rapht dorsalis Locus coeruleus Midbrain Nucleus raphC linearis Nucleus raphi centralis, pars annularis Nucleus rapht dorsalis, pars supratrochearis Central gray Substantia nigra Nucleus interpeduncularis Red nucleus Nucleus reticularis tegmenti pontis Thalamus Nucleus anterior Nucleus medialis Nucleus lateralis anterior Nucleus paramedianus rotundocellularis Hypothalamus Nucleus posterior Area lateralis Mammillary bodies Hippocampus Dentate gyrus CA3 (pyramidal layer) CA I (pyramidal layer) Subiculum Amygdala Nucleus lateralis Nucleus basalis Nucleus granularis Nucleus basalis accessorius Basal ganglia Nucleus caudatus Putamen Globus pallidus, pars lateralis Substantia innominata Cortex Frontal Occipital Parietal Cingular Cerebellum Granular cell layer Dentate nucleus Significantly

protein)

control

values:

with imipramine [‘HlParoxetine

1

1

Mean i S.E.M. (fmollmg protein)

115.8 k 83.2*** 149.4 k 45.6

22 46

20 37 27 74

0.0 168.1 105.3 28.0

15 18 4

1

I

114.7 229.9 333.4

1 I 1

1

136.2

2 2 2 2 3 1 1 2

251.2 730.0 757.5 161.5 412.1 609.0 136.1 115.0

2

240.2 + 19.8 211.4 189.9 + 30.1* 229.0

53 39

I

334.0 250.0 199.0

41 44 17

1 1 1 I

210.0 211.0 186.0 172.0

49 39 51 36

1 1 1 1

237.0 312.0 313.0 227.0

58 58 63 44

2 2 2 1

216.0 k 54.0** 323.6 f 50.4** 255.8 + 41.2* 162.0

41 51 23

2 1 1

188.3 + 53.7* 254.0 285.0 524.0

51 63 93 156

1 1

132.1 284.5

I 2

I 1 1

1

‘Y”

105.9 254.8 241.1 272.3

I 2 2

binding

I0 14 17

16 + + + f +

207.8** 46.0 205.5 43.S** 38.6**

+ 35.0*

17 53 38 29 52 76 32 28

1 2 1

I

1233.0 790.8 + 527.7 592.8

84 46 64

1

558.9 52.3

117

52

I

335.9

67

1

216.9

11

I 1

15

1

54.2 0.0 99.0

42

1

337.7

110

51 17

51

0

37 71

*2P < 0.07; **2P < 0.05; ***2P < 0.005 (t-test).

raphk linearis, centralis and dorsalis. Both in the pons and midbrain, binding to the dorsal raphi nucleus extended laterally beyond the limits of the nucleus into the central gray. These lateral extensions were the regions of the dorsal raphk nucleus and of the entire human brain the most enriched in ADBS. Structures such as the locus coeruleus, substantia

nucleus interpeduncularis, nigra pars compacta, area tegmentalis ventralis, nucleus paranigralis, nucleus accessorius nervi occulomotorii and griseum centrale metencephali contained high concentrations of ADBS. Moderate concentrations were found in the superior colliculus, dorsomedial aspect of the inferior colliculus, griseum centrale mesencephali (central

Antidepressant

binding sites in the human brain

481

Fig. 4. Figs 4-15. Autoradiographic mapping of the distribution of rH]imipramine and [‘Hlparoxetine highaffinity binding sites in the control human brain. Bright-field photomicro~p~ are shown. Dark regions correspond to nuclei enriched in binding sites. Observe that the localization of r3Hlparoxetine binding sites closely parallels that of [‘Hlimipramine binding sites. Fig. 4. Autoradiograms showing the distribution of [3H]imipramine binding sites in the cervical cord (A) and medulla oblongata (C). The non-specific binding obtained with 100 pM desipramine is illustrated in Band D, respectively. Note the high labelling over the spino-olivary/olivospinal tract, which is specifically inhibited in B. In contrast, the nucleus olivaris inferior shows a high non-specific binding (D). Note the presence of moderate densities of binding sites in the nucleus raphe obscurus and nucleus praepositus hypoglossi. Bars = 3 mm.

gray), nuclei parabrachialis lateralis and medialis, substantia nigra pars reticulata and in an area lying ventrally to the medial longitudinal fasciculus which probably corresponds to the regio stellata as described by Braak.6 Other areas such as the nucleus cuneifo~is, nucleus tegmentalis ~d~culo~ntinus, nucleus ruber, nucleus net-vi occulomotorii, nucleus nervi trochlearis and nucleus reticularis pontis oralis had low densities of ADBS.

Diencephalon (Figs 10 and 11)

Most nuclei of the thalamus had intermediate or low densities of ADBS. However, some nuclei of the paraventricular and intralaminar formations located near the encephalon midline presented very high densities of specific binding. Among these nuclei the highest concentrations of sites were associated with the nucleus paraventricularis rotund~eliula~s (also

Fig 5. Localization of [‘Hlimipramine (A) and L’H]paroxetine (B) binding sites in the medulla oblongata Observe the similarity between the pattern of labelling of both ligands. Areas enriched in binding are the nucleus olivaris accessorius medialis (OAM) and dorsalis, nucleus nervi hypoglossi, nucleus dorsalis nervi vagi, nucleus tractus solitarius. nucleus cuneatus lateralis, as well as the choroid plexus of the fourth ventricle. Bar = 3 mm

Antidepressant

binding sites in the human brain

Fig. 6. Binding of [‘H]imipramine (A) and [3H]paroxetine (B) in the lower pons. The densest labelhng occurs in the nucleus raphe magnus and in the nucleus nervi facialis. In the latter nucleus note that binding sites present an irregular distribution, with patches very rich in binding sites being located along the nucleus. Bar = 3 mm.

483

Antidepressant

binding sites in the human brain

485

Fig. 8. Locahxation of [3~Jimipmmine (A) and 13~~roxetine (B) binding sites in the caudal midbrain. At this level, tbe highest densities are found over the nuclei rapht dorsalis pars supratrocblearis and linearis. The substantia nigra pars compacta, nucleus interpeduncularis, and central gray contain also high concentrations of binding sites. Lower densities are associated with the inferior colliculus, nucleus cuneiformis and substantia nigra pars reticulata. Bar = 3 mm.

paramedianus) as described by Krieg (in Ref. 21). Other nuclei such as the reuniens, paratenialis, parafascicularis and nucleus intralamellark pars centralis medialis were also enriched in ADBS. Somewhat lower levels of binding were found in the pars medialis lateralis and paracentralis of the nucleus intralamellaris, as well as in the zona incerta and nucleus peripeduncularis. Only very low levels of binding were observed in the nucleus subthalamicus. In the hypothalamus very high densities of ADBS were measured in the mammillary bodies and tuberal nuclei. The nuclei posterior, arcuate (infundibularis), periventricular and dorsomedial showed high levels of binding. Somewhat lower levels were associated with the ventromedial nucleus and the lateral hypothalamic area. However, very high densities were observed in perifornical parts of the lateral area.

called nucleus

Basal ganglia and basal forebrain (Fig. 12)

Moderate levels of ADBS were associated with the nucleus caudatus, putamen and nucleus accumbens.

Lower densities were measured in the globus pallidus, both in the lateral and medial segments, while high densities were found in the island magna of Calleja. In the basal forebrain, intermediate densities were observed in the substantia innominata, including the nucleus basalis of Meynert and nuclei of the diagonal band of Broca. Limbic system (Fig. 13) [3H]Imipramine binding sites were homogeneously distributed throughout the amygdala. Low densities of sites were observed over most nuclei, with somewhat higher densities being present in the nucleus corticalis, nucleus medialis and lateral part of the nucleus basalis. [3H]Paroxetine binding was not studied in the amygdala. The hippocampal formation contained low concentrations of ADBS. The highest densities were found in the stratum pyramidalis of the CA3 subfield. The densities of sites over this layer decreased in the CA1 sector and slightly increased again in the subicular region.

Fig. 7. Binding of ]3H]imipramine in the caudal (A) and rostra1 (B) pans. (A) AH nuclei at the level of the motor nucleus of the trigeminal nerve contain low densities of [SH]imipramine binding sites, including the nucleus raphb pontis. (B) In contrast, in the upper pons very high densities are localized in the nuclei raphe linearis, centralis and dorsalis. Lower levels of binding can be seen in the locus coeruieus, griseum oentrale metencephali, nucleus parabrachialis lateralis and regio stellata. Bars = 3 mm.

Cerebral cortex, cerebellum and chornidplexus (Figs 5, 14 and IS) Low lev&. OF binding were found in the frontat, cingulate, parieta& entorhinal and occipital cortices (Fig. 14). ADBS densities were similar from region to

---.~-_..~--

--

region. In general, deeper cortical laminae contained higher levels of ADBS than the more superftcial layers. In the cerebellum (Fig. 15), law to very low levels of specific ~~H~~rn~~rarni~eand f3~~~ar~~e~~~e binding were found. The highest densities were detected over the nucleus dentatus and the lowest

;___l_“____l-_l”-. __..~,. l___“^“.--~-ll-..--.-“.

“_.-

10. Corcmal sections of th5 ~~~~~~~1~~ lab&d with ~3~~im~~r~~n~(A) and ~~~~~arox~ti~~{IQ, Very high de&t& of sites are locaked over the nucleus ~ara~entr~cularj~ rotundoce~~~a~s and mammillary bodies, Lower Ievels of binding can be seen over the nucleus ~arafascic~r~s, posterior nucleus of the hypothalamus and nucleus peripeduncularis, among others. Bar = 3 mm. Fig.

Antidepressant

binding sites in the human brain

481

R. CORT~S et al.

Fig. 11. Autoradiogram of the anterior hypothalamus and thalamus obtained with [’ Hjimipramine. High densities of binding are seen in the tuberal, periventricular and dorsomedial hypothalamic nuclei, as well as in the nucleus rotundocellularis and nucleus paratenialis of the thalamic paraventricular formation. Lower levels of binding are associated with the lateral and ventromedial nuclei of the hypothalamus, zona incerta and anterior and ventral anterior nuclei of the thalamus. Bar = 3 mm.

Fig. 12. Autoradiograms showing [3H]imipramine (A) and [)H}paroxetine (B) binding to sections through the basal ganglia and basal forebrain. The highest, although only moderate, labelling is found in the substantia innominata and nucleus of the horizontal limb of the diagonal band. Slightly lower concentrations of binding sites are associated with the putamen, while only very low binding occurs in the globus pallidus pars lateralis and pars medialis, and in the bed nucleus of the stria terminalis. Bar = 3 mm.

Antidepressant

binding sites in the human brain

Fig. 12.

489

R. CORT~

et al.

Antidepressant binding sites in the human brain

491

Fig. 14. [3H]Imipramine binding in the occipital cortex. Note that the binding is only low, and mainly concentrated

in the deeper

layers

(VI) as opposed

over the molecular layer of the cerebellar cortex. In the cortex the Purkinje cell layer could be seen as a narrow band slightly more enriched in binding sites. High densities of ADBS were observed in the choroid plexus (Fig. 5). DISCUSSION

The present study provides a detailed mapping of the distribution of ADBS in the human brain. These sites were labelled using two different [‘Hlligands in parallel: [‘Hlimipramine and [3H]paroxetine. To our knowledge, this is the first report on the binding of [jH]paroxetine to human brain tissue. Distribution of antidepressant binding sites in human brain: correlation with serotonergic markers

There is a considerable amount of evidence suggesting a presynaptic localization of [3Hlimipramine and [3H]paroxetine high-affinity binding sites on 5HT-containing nerve terminals. For example, the distribution of [3Hlimipramine binding sites has been shown to overlap with the presence of 5-HT terminals

to outer

layers (I). Bar = 3 mm.

in the rat’8.23*25,47*62,65 and also in the human brain39 (present report, see below). Furthermore, axonal transport of [3Hlimipramine binding sitesI and losses of [‘Hlimipramine and [‘Hlparoxetine binding following destruction of serotonergic neurons by 5,7-dihydroxytryptamine lesioning’838~29~32~65~M have been shown in the rat. On the other hand, there is a good correlation between the potency of various drugs to inhibit the uptake of [3H]5-HT and their potency to displace [3Hlimipramine and [3Hlparoxetine binding. These findings have been interpreted as indicating that imipramine and paroxetine bind to the 5-HT transporter complex, although it is controversial for imipramine. 36Since imipramine and 5-HT interact in a non-competitive manner, it is generally accepted that [‘Hlimipramine does not bind directly to the 5-HT uptake site, but rather to an allosteric site which modulates the activity of 5-HT uptake mechanisms.’ The distributions of [‘Hlimipramine and [3H]paroxetine reported here are highly comparable. The similarities observed support the concept that both ligands label the same protein or protein complex.

Fig. 13. Autoradiographic images of the amygdala (A) and hippocampus (B) labelled with [3Hlimipramine. Both structures are characterized by low levels of binding. In the amygdala the highest binding is localized in the nucleus corticalis, and in the hippocampal formation in the pyramidal layer in the CA3 and CA2 subfields. Bars = 3 mm.

492

R. CORtiS

Pt

d.

Fig. 15. Distribution of [)H]imipramine (A) and pH]paroxetine (B) binding sites in the cerebellum. Both in the dentate nucleus and cortex, the concentrations of binding sites for both ligands are low. Bar = 3 mm. in regions very enriched in ADBS [3H]imipramine-specific binding was proportionally higher than [‘Hlparoxetine-specific binding. These differences were not due to film over-exposure and saturation of silver grain density, but rather represent binding to other sites, probably different from the 5-HT uptake complex, as suggested by Mellerup and Plenge.4’ These authors have observed that, while in platelets the number of [3H]imipramine and However,

[‘Hfparoxetine binding sites is the same,“’ in rat neuronal membranes the B,,, for [‘Hlimipramine for [3H]paroxetine. is slightly higher than the B,,,,, Nevertheless, in agreement with these authors, our results indicate that the majority of [“H]imipramine binding sites appear to be associated with the 13H]paroxetine binding site. There was a widespread distribution of ADBS throughout the human brain, with the highest density

Antidepressant binding sites in the human brain being localized over the rapht nuclei in the midbrain. High densities were also found in some midline thalamic nuclei, parts of the hypothalamus and substantia nigra, among others, while lower levels of binding were observed in the neocortex, basal ganglia, amygdala, hippocampus and in the remainder of the thalamus. This distribution agrees closely with previous data on the localization of [3H]imipramine binding sites in the human brain as determined by classical binding assays.39*6’However, the densities we measured by microdensitometry are higher than those determined by membrane binding assays. For instance, Langer et a1.39have reported BnlBX values of about 500 fmol/mg protein in the hypothalamus, 300 fmol/mg protein in the frontal cortex, 260 fmol/mg protein the hippocampus and 220 fmol/mg protein in the nucleus caudatus, while in our study the average [3H]imipramine specific binding levels in these regions were about 700, 400, 450 and 500 fmol/mg protein respectively. In addition, it has to be taken in account that our density values were obtained using 10 nM [3H]imipramine, a concentration which leads to a 55-70% occupancy (Kd = 4.2-7.6 nM, Ref. 39). Such increased densities measured in intact tissue as opposed to membrane homogenates have been also observed for several drug and neurotransmitter receptors56 and could be due to a depletion in the number of binding sites during the homogenization process. The localization of [3Hlimipramine binding sites in the human brain was similar to that found in the rat brain.‘8*23.25,55 Differences were noted, however, in the lateral amygdaloid nucleus and locus coeruleus. In the latter no [‘Hlimipramine-specific binding has been reported in the rat. As observed in the rat brain, the distribution of ADBS we report for the human brain is in good agreement with the endogenous levels of 5-HT and its metabolites in human4*“.76 and monkeyU brain. High concentrations of 5-HT and 5-hydroxyindoleacetic acid (5-H&4) are found in the midbrain, particularly in the substantia nigra, raphe nuclei, hypothalamus and some thalamic regions, which are structures rich in ADBS, whereas the cerebral and cerebellar cortices that contain low concentrations of 5-HT and 5-HIAA are poor in ADBS. However, while Birkmayer et a1.4 have detected very high concentrations of 5-HT in the nucleus ruber and nucleus accumbens, as well as in the globus pallidus, we have found only intermediate or low densities of ADBS in these areas. The lack of information of 5-HT distribution at the light microscopic level precludes further detailed comparison. The anatomical distribution of ADBS compares well with that of 5-HT, receptors,49 particularly the 5-HT,, subtype. For example, very high densities of both ADBS and 5-HT,, receptors are associated with the nuclei raphe centralis, dorsalis and linearis&

493

and with some midline thalamic nuclei. In contrast, there are pronounced differences between ADBS and 5-HT,, densities and distribution in the neocortex and hippocampus. In these regions the distribution and density levels of ADBS are closer to 5-HT,, receptors. The localization of 5-HT,, receptors and ADBS also compares well in the choroid plexus and substantia nigra, but is remarkably different in the globus pallidus. In general there is a good parallelism between ADBS and 5-HT, receptor distribution in the amygdala, thalamus, hypothalamus, midbrain and brainstem. On the other hand, there is a bad correlation between the distribution of ADBS and 5-HT, receptors in the human brain.” Our results in the human brain indicate that, as in the rat brain, the ascending serotonergic fibre systems may be the main target of imipramine action, since we found a good topographical overlap of ADBS distribution and the localization of serotonergic perikarya and terminals of these pathways. The ventral and dorsal ascending serotonergic pathways arise respectively from the cell groups B6, B7 and B8 and B7, B5 and B3 according to Dahlstrom and Fuxe,” which correspond to the raphe centralis (B6 + B8), rapht dorsalis (B7), raphe pontis (B5), and raphe magnus (B3) of Braak6 (see also Refs 5, 15, 44, 71 and 72). These nuclei contain very high densities of ADBS, with exception of the raphe magnus, which has intermediate densities, and the raphe pontis, which is very poor in ADBS. Structures such as the hypothalamus, substantia nigra, midline thalamic nuclei, as well as the griseum centrale mesencephali, all receive afferents from the two ascending pathways5.‘5.4’,” and are heavily labelled by [‘HIimipramine and [‘Hlparoxetine. However, the hippocampus, striatum, cerebral cortex, amygdala and septal nuclei, which also receive terminals from these ascending projections,5*43v74contain relatively low levels of ADBS. In addition to the ascending serotonergic systems, the descending propriobulbar pathway also Seems to contain ADBS. Some of these fibres project to the locus coeruleus and reticular formation,5J5~63~71 which we found to be enriched in ADBS. On the contrary, the cerebellar serotonergic pathway appears to be devoid of ADBS. It has also been reported that some parts of the hypothalamus, including the nuclei ventromedialis and dorsomedialis, and the mammillary bodies, rich in ADBS, also contain serotonergic neuronsI Of particular interest is the presence of high densities of ADBS in the substantia nigra and locus coeruleus, since the largest dopaminergic and noradrenergic pathways emerge from these areas. This localization suggests a possible role of ADBS in the regulation of dopamine and noradrenaline neurotransmission. The presence of serotonergic perikarya in the locus coeruleus in monkey@ should also be mentioned in this context.

494 Fuctors sites

it. CORTi:S afecting

the density

of antidepressant binding

ff

d

Other authors, however, found no modifications“ (11 only regional alterations34 in the B,,, of [‘H]imtpramine following such treatments in rats. On the other hand, Graham et al.*” have reported a lack of modification of [‘Hlparoxetine binding after armdepressant administration in rats. These authors have argued that previously reported decreases in [‘Hlimipramine binding might be due to the presence of the drug in tissue. Although we routinely preincubated all tissue samples to avoid possible interferences, we cannot rule out the possibility that imipramine was incompletely washed out from the brain in our patients. We have not analysed whether the decreases in [‘Hlimipramine and [‘H]paroxetine binding observed after antidepressant administration are due to a reduction in the B,,, or to an increase in the Kn of the ligands for their binding sites. Patients T and V had received imipraminc until the last day before death. In these cases the loss of antidepressant binding could be due to imipramine remaining in tissue, especially in case V who was treated only for 3 days. In contrast, a downregulation of ADBS might have occurred in patient U, because this case had received imipramine for 14 weeks, and stopped the treatment 11 days before death. However, depression also might be the cause of the decrease in antidepressant binding observed, since reduced [3H]imipramine binding has been reported in brain structures of depressive patients.” The effects of antidepressant treatment on the density of ADBS in the human brain clearly deserve further studies.

In the present study we found high variability in ADBS densities among the different cases studied. We therefore examined the possible influences of age, sex and post-mortem delay of the patients on the density and distribution of ADBS. In our patient cohort we did not observe significant differences in [‘Hlimipramine binding in relation to post-mortem delay or gender, which is in contradiction with data from animal studies.75 In most brain areas, the levels of [‘Hlimipramine-specific binding were also independent of the age of the patients. However, in a few regions such as the globus pallidus, frontal and occipital cortices and amygdala, [3H]imipramine binding increased as a function of age. These results are in agreement with data from membrane binding studies in human brain45.67 and platelets.3B Other authors, however, have reported that [3Hlimipramine binding in the human cortex, as well as other presynaptic serotonergic markers, is independent of age. ’ An age-related increase in ADBS might explain the clinical observations indicating that antidepressant treatment is more effective in older patients.59 Although we were unable to find significant differences between male and female patients, clinical data suggest that gender might play a role in the efficiency of antidepressant therapy effectiveness.5y In cases treated with imipramine before death [‘Hlimipramine and [‘Hlparoxetine binding was markedly decreased in all brain structures. Our results are in agreement with previous studies showing reductions in [3H]imipramine binding in rat2.3.5K Acknowledgement-We wish to thank Dr C. Richard Jones and in cat” brain after antidepressant treatment. for critical reading of the manuscript.

REFERENCES 1. Allen S. J., Benton J. S., Goodhardt M. J., Haan E. A., Sims N. R., Smith C. C. T., Spillane J. A., Bowen D. M. and Davison A. N. (1983) Biochemical evidence of selective nerve cell changes in the normal ageing human and rat brain. J. Neurochem. 41, 25&265. 2. Arora R. C. and Meltzer H. Y. (1986) Effect of subchronic treatment with imipramine, chlorpromazine and the combination on [‘Hlimipramine binding in rat blood platelets and frontal cortex. Life Sci. 39, 22892296. 3. Barbaccia M. L., Bnmello N., Chuang D. M. and Costa E. (1983) On the mode of action of imipramiw: Relationship between serotonergic axon terminal function and down-regulation of @-adrenergic receptors. Neuropharmuco/og.v 22, 3733383. 4. Birkmayer W., Danielczyk W., Neumayer E. and Riederer P. (1974) Nucleus ruber and L-DOPA psychosis: Biochemical postmortem findings. J. neural Transm. 35, 93-l 16. 5. Bobillier P., Seguin S., Petitjean F., Salvert D., Touret M. and Jouvet M. (1976) The rapht nuclei of the cat brainstem: A topographic atlas of their efferent projections as revealed by autoradiography. Brain Res. 113, 449486. 6. Braak H. (1970) Ueber die Kerngebiete des menschlichen Hisnstammes: II. Die Raphekerne. 2. Zel~or.reh. 107, 1233141. I. Briley M., Langer S. Z. and Raisman R. (1979) Specific [‘Hlimipramine binding in rat brain. Br. J. Pharmac. 67, 434435. 8. Briley M. S., Fillion G., Beaudoin D., Fillion M. P. and Langer S. Z. (1980) [‘HjImipramine binding in neuronal and glial fractions of horse striatum. Eur. J. Pharmac. 64, 191-194. 9. Briley M., Langer S. Z. and Sette M. (1981) Allosteric interaction between [‘Hlimipramine binding site and the serotonin uptake mechanism. Br. J. Pharmac. 74, 817P-818P. 10. Briley M., Raisman R., Arbilla S., Casadamont M. and Langer S. Z. (1982) Concomitant decrease in L3H]imipramine binding in cat brain and platelets after chronic treatment with imipramine. Eur. J. Pharmac. 81, 309314.

11. Bucht G., Adolfsson R., Gottfries C. G., Roos B. E. and Winblad B. (1981) Distribution of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in human brain in relation to age, drug influence, agonal status and circadian variation. J. neural Transm. 51,185-203. effects in mice by three 12. Buus-Lassen J. (1978) Potent and long-lasting potentiation of two %hydroxytryptophan-induced selective 5-HT uptake inhibitors. Eur. J. Pharmac. 47, 351-355.

Antidepressant

binding sites in the human brain

495

13. Chan-Palay V. (1977) Indoleamine neurons and their processes in the normal rat brain and in chronic diet-induced thiamine deficiency demonstrated by uptake of rH]serotonin. J. camp. Neural. 176, 467494. 14. Charney D. S., Menkes D. B. and Heninger G. R. (1981) Receptor sensitivity and the mechanism of action of antidepressant treatment. Arch. gen. Psych&. 38, 1160-t 180. 15. Conrad L. C. A., Leonard C. M. and Pfaff D. W. (1974) Connections of the median and dorsal rapht nuclei in the rat: An autoradiographic and degeneration study. J. camp. Nemo/. 156, 179-206. 16. Cartes R., Probst A. and Palacios J. M. (1984) Quantitative light autoradiographic localization of cholinergic muscarinic receptors in human brain: Brainstem. Neuroscience 12, 1003-1026. 17. Dahlstrijm A. and Fuxe K. (1965) Evidence for the existence of monoamine-neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta physiol. stand. 62 (Suppl. 232), l-55. 18. Dawson T. M. and Wamsley J. K. (1983) Autoradiographic localization of [‘Hlimipramine binding sites: Association with serotonergic neurons. Brain Res. 11, 325-334. 19. Dawson T. M., Gehlert D. R., Snowhill E. W. and Wamsley J. K. (1985) Quantitative autoradiographic evidence for axonal transport of imipramine receptors in the central nervous system of the rat. Neurosci. Len. 55, 261-266. 20. DeArmond S. J., Fusco M. M. and Dewey M. M. (1976) Structure of the Human Brain. A Photographic Atlas, 2nd edn. Oxford University Press, New York. 21. Dewulf A. (1971) Anatomy of the Normal Human Thalamus. Topometry and Standardized Nomenclature. Elsevier, Amsterdam. 22. Dumbrille-Ross A., Tang S. W. and Coscina D. V. (1981) Differential binding of rH]imipramine and [‘Hlmianserine in rat cerebral cortex. Life Sci. 29, 2049-2058. 23. Fuxe K., Calza L., Benfenati E., Zini I. and Agnati L. F. (1983) Quantitative autoradiographic localization of [‘Hlimipramine binding sites in the brain of the rat: Relationship to ascending 5-hydroxytryptamine neuron systems. Proc. natn. Acad. Sci. U.S.A. 80, 38363840.

24. Glassman A. H. and Perel J. M. (1973) The clinical pharmacology

of imipramine. Implications for therapeutics.

Arch. gen. Psychiat. 28, 649653.

25. Grabowsky K. L., McCabe R. T. and Wamsley J. K. (1983) Localization of [3H]imipramine binding sites in rat brain by light microscopic autoradiography. Life Sci. 32, 23552361. 26. Graham D., Tahraqui L. and Langer S. Z. (1987) Effect of chronic treatment with selective monoamino oxidase inhibitors and specific 5-hydroxytryptamine uptake inhibitors on [3H]paroxetine binding to cerebral cortical membranes of the rat. Neuropharmacology 26, 1087-1092. 27. Green J. P. and Maayami S. (1977) Tricyclic antidepressant drugs block histamine H, receptor in brain. Nature 269, 1633165.

28. Gross G., Gothert M., Ender H. P. and Schumann H. J. (1981) [3H]Imipramine binding sites in the rat brain. Selective localization on serotonergic neurons. Naunyn-Schmiedeberg’s Arch. Pharmac. 317, 3lCk314. 29. Harbert E., Graham D., Tahraqui L., Claustre Y. and Langer S. Z. (1985) Characterization of [‘Hlparoxetine binding to rat cortical membranes. Eur. J. Pharmac. 118, 1077114. 30. Hollister L. E. (1978) Tricyclic antidepressants. New Engl. J. Med. 229, 11061109, 1168-l 172. 31. Hrdina P. D. (1984) Differentiation of two components of specific [‘Hlimipramine binding in rat brain. Eur. J. Pharmac. 102, 481488.

32. Hrdina P. D., Pappas B., Biolik R. and Ryan C. (1982) Regulation of [‘Hlimipramine binding in rat brain regions: Effect of neonatal 5,7_dihydroxytryptamine treatment. Eur. J. Pharmac. 83, 343-344. 33. Hyttel J. (1982) Citalopram: pharmacological profile of a specific serotonin-uptake inhibitor with antidepressant activity. Prog. Neuropsychopharmac. Biol. Psychiat. 6, 277-295. 34. Kinnier W. J., Chuang D. M. and Costa E. (1980) Down regulation of dihydroalprenolol and imipramine binding sites in brain of rats repeatedly treated with imipramine. Eur. J. Pharmac. 67, 289-294. 35. Kinnier W. J., Chuang D. M., Gwynn G. and Costa E. (1981) Characteristics and regulation of high affinity [3H]imipramine binding to rat hippocampal membranes. Neuropharmacology. 20, 411419. 36. Laduron P. M., Robbyns M. and Schote A. (1982) [3H]Desipramine and [‘Hlimipramine binding are not associated with noradrenaline and serotonin uptake in the brain. Eur. J. Pharmac. 78, 491493. 37. Langer S. Z., Moret C., Raisman R., Dubocovich M. L. and Briley M. (1980) High affinity [‘HJimipramine binding in the rat hypothalamus is associated with the uptake of serotonin but not norepinephrine. Science, N.Y. 210, 113331135. 38. Langer S. Z., Briley M. S., Raisman R., Henry J.-F. and Morselli P. L. (1980) Specific [3H]imipramine binding in human platelets. Influence of age and sex. NaunynSchmiedeberg’s Arch. Pharmac. k3, 189-194. _ 39. Lanaer S. Z., Javov-Auid F.. Raisman R.. Brilev M. and Acid Y. (1981) Distribution of soecific hieh affinitv bindine sites-for [3H]imipramiie in human brain.‘J. Neurochem. 37; 2677271. ’ 40. Lidbrink P., Jonsson G. and Fuxe K. (1971) The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenaline and 5-hydroxytriptamine neurons. Neuropharmacology 10, 521-536. 41. Mellerup E. T. and Plenge P. (1986) High affinity binding of [‘Hlparoxetine and [3H]imipramine to rat neuronal membranes. Psychopharmacology 89, 436439. 42. Mellerup E. T., Plenge P. and Engelstoft M. (1983) High affinity binding of [3H]paroxetine and [‘Hlimipramine to human platelet membranes. Eur. J. Pharmac. %, 303-309. 43. Moore R. Y. and Halaris A. E. (1975) Hippocampal innervation by serotonin neurons of the midbrain rapht in the rat. J. camp. Neurol. 164, 171-183. 44. Nieuwenhuys R., Voogd J. and van Huijzen C. (1981) The Human Cenrral Nervous System. A Synopsis and Atlas, 2nd edn. Springer Verlag, Berlin. 45. Owen F., Chambers D. R., Cooper S. J., Crow T. J., Johnson J. A., Lofthouse R. and Poulter M. (1986) Serotonergic mechanisms in brains of suicide victims. Brain Res. 362, 185-188. 46. Palacios J. M., Probst A. and Cortis R. (1983) The distribution of serotonin receptors in the human brain: high density of [‘H]LSD binding sites in the raphb nuclei of the brainstem. Brain Res. 274, 15&155. 47. Palkovits M., Saavedra J. M., Jacobowitz D. M., Kiter J. S., Zaborszky L. and Brownstein M. J. (1977) Serotonergic innervation of the forebrain: Effect of lesions on serotonin and tryptophan hydroxylase levels. Brain Res. 130, 121-134.

496

R. (‘oHT~% L’I ui.

hmdrng iabel 48. Paul S. M., Rehavi M., Rice K. C., Yttah T. and Skolnick P. (1981) Does high affinity [‘Hlimipramme serotonin reuptake sites in brain and platelets? L$e Sci. 28, 2753-2760. Autoradiographic mappmg 49. Pazos A., Probst A. and Palacios J. M. (1987) Serotonin receptors in the human brain-Ill. of serotonin-I receptors. Neuroscience 21, 97- 122. Autoradiographic mapping 50. Pazos A., Probst A. and Palacios J. M. (1987) Serotonin receptors in the human brain--IV. of serotonin-2 receptors. Neuroscience 21, 123~ 139. treatment decreases spiroperidol-labeled serotonm 51. Peroutka S. J. and Snyder S. H. (1980) Long-term antidepressant receptor binding. Science, N. Y. 210, 88890. B. E. and Perry R. H. (1983) Decreased imipramine binding m 52. Perry E. K., Marshall E. F., Blessed G., Tomlinson the brains of patients with depressive illness. Er. J. Psychiat. 142, 188-192. high-affinity binding sites in rat brain. Effects of imipramine and 53. Plenge P. and Mellerup E. T. (1982) [)H]Imipramine lithium. Psychopharmacology 77, 9497. drugs can change the affinity of [‘Hlimipramine and [‘Hlparoxetine 54. Plenge P. and Mellerup E. T. (1985)Antidepressive binding to platelet and neuronal membranes. Eur. J. Pharmac. 119, l-8. localization of imipramine binding in rat brain. 55. Rainbow T. C., Biegon A. and McEwen B. S. (1982) Autoradiographic Eur. J. Pharmac. 77, 3633364. 56. Rainbow T. C., Biegon A. and Berck D. J. (1984) Quantitative receptor autoradiography with tritium-labeled ligands: Comparison of biochemical and densitometric measurements. J. Neurosci. Meth. 11, 231-241. binding sites in rat brain. Narure 281, 57. Raisman R., Briley M. and Langer S. 2. (1979) Specific tricyclic antidepressant 1488150. binding sites in rat brain characterized by 58. Raisman R., Briley M. S. and Langer S. Z. (1980) Specific antidepressant high-affinity [)H]imipramine binding. Eur. J. Pharmac. 61, 3733380. drugs. J. new. Menfal His. 159, 120-130. 59. Raskin A. (1974) Age-sex differences in response to antidepressant M. (1978) Multiple binding sites of tricyclic antidepressant drugs to mammalian brain 60. Rehavi M. and Sokolovsky receptors. Brain Res. 149, 5255529. 61. Rehavi M., Paul S. M., Skolnick P. and Goodwin F. K. (1980) Demonstration of specific high affinity binding sites for [‘Hlimipramine in human brain. Life Sci. 26, 2273-2279. 62. Rehavi M., Skolnick P. and Paul S. M. (1983) Subcellular distribution of high affinity [3H]imipramine binding and [3H]serotonin uptake in rat brain. Eur. J. Pharmac. 87, 3355339. 63. Sakai K., Touret M., Salvert P., Leger L. and Jouvet M. (1977) Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique. Brain Res. 119, 2141. 64. Schofield S. P. M. and Everitt B. J. (1984) The organization of indoleamine neurons in the brain of the Rhesus monkey (Macaca mulata). J. camp. Neurol. 197, 3699383. 65. Sette M., Raisman R., Briley M. and Langer S. Z. (1981) Localization of tricyclic antidepressant binding sites on serotonin nerve terminals. J. Neurochem. 37, 40-42. 66. Sette M., Ruberg M., Raisman R., Scatton B., Zivkovic B., Agid Y. and Langer S. Z. (1983) [3H]lmipramine binding in subcellular fractions of rat cerebral cortex after chemical lesions of serotonergic neurons. Eur. J. Pharmac. %,41--S I. 61. Severson J. A., Marcusson J. O., Osterburg H. H., Finch C. E. and Winblad B. (1985) Elevated density of [‘Hlimipramine binding in aged human brain. J. Neurochem. 45, 1382-1389. distribution in primate brain--IV. Indoleamine68. Sladek J. R., Garver D. L. and Cummings J. P. (1982) Monoamine containing perikarya in the brainstem of Macaca arctoides. Neuroscience 7, 477493. H. I. (1977) Antidepressants and the muscarinic acetylcholine receptor. Arch. gen. 69. Snyder S. H. and Yamamura Psychiaf. 34, 236269. 70. Stephan H. (1975) Handhuch der Mikroskopischen Anatomic des Menschen. 4. Band: Nervensystem. Springer Verlag, Berlin. 71. Taber-Pierce E., Foote W. E. and Hobson J. A. (1976) The efferent connection of the nucleus raphe dorsalis. Brain Res. 107, 1377144. U. (1971) Stereotaxic mapping of the monoamine pathways in the rat brain. Acta physiol. stand. (Suppl.) 72. Ungerstedt 367, 149. D. C., Greenberg D. A., Sheehan P. P. and Snyder S. H. (1978) Tricyclic antidepressants: therapeutic 73. UPrichard properties and affinity for noradrenergic receptor binding sites in the brain. Science, N.Y. 199, 197-198. G. K. (1977) Inhibition of neurons in the amygdala by dorsal rapht stimulation: Mediation 74. Wang R. Y. and Aghajanian through a direct serotonergic pathway. Brain Res. lu), 85-102. binding sites to chronic 75. Wilson M. A. and Roy E. J. (1985) Age alters the observed response of imipramine antidepressant treatment in female rats. Eur. J. Pharmac. 106, 381-391. and monoamine metabolites in brains from 76. Winblad B., Bucht G., Gottfries C. G. and Roos B. E. (1979) Monoamines demented schizophrenics. Acta psychiat. stand. 60, 17-28. (Accepted 26 April 1988)