66
Brain Research, 563 (1991 ) 60 -7~ © 1991 Elsevier Science Publishers B.V, All rights reserved. 0006-8993/91/$03.51) ADONIS 0006899391171031 ~:
BRES 17103
Autoradiographic distribution of TRH binding sites in the human hypothalamus M. Najimi, F. Chigr, J. Champier, A. Tabib, N. Kopp and D. J o d a n i Laboratoire d'Anatornie Pathologique, Facultd de Mddecine Alexis Carrel, Lyon (France)
(Accepted 11 June 1991) Key words: Autoradiography; Hypothalamus; Human; Thyrotropin-releasing hormone binding sites
Using in vitro quantitative autoradiography and [3H]3MeTRH, a selective high affinity radioligand, we examined the rostrocaudal distribution of TRH binding sites in both the infant and the adult human hypothalamus. The saturation curve shows that the [3H]3MeTRH binds with high affinity to a single class of TRH binding sites and is saturable, the apparent constant of dissociation is in the namomolar range. TRH binding sites showed a wide distribution, principally in the anterior and mediobasal levels of the hypothalamus. TRH binding site concentration was highest within the diagonal band of Broca, the lateral preoptic area, the infundibular and the tuberal nuclei. TRH binding site concentration was moderate in the ventromedial nucleus and the medial preoptic area, whereas we observed low densities in the periventricular, paraventricular and mammillary nuclei. The distribution in the infant and the adult is generally similar. However, it is noteworthy that the infant tuberal nuclei displayed a lower binding site density when compared to the adult. On the other hand, the diagonal band of Broca is relatively more labeled in infant. The analysis of the whole hypothalamus allows us to ascertain the absence of lateral asymmetric distribution both in the infant and the adult. No significant difference is noticed when considering as paremeters of variation age, sex or post mortem delay.
INTRODUCTION Thyrotropin-releasing h o r m o n e ( T R H ) was initially isolated from bovine and porcine hypothalamus 2'6 and identified as the tripeptide amine: p G l u - H i s - P r o - N H 2. Using biochemical and immunohistochemical techniques, many studies have r e p o r t e d that the hypothalamus is the brain region containing the highest concentrations of this tripeptide in m a m m a l i a n species 5'8-1°'12' 16,26,27,43 including h u m a n 13'3°. Its biological actions have been r e p o r t e d to be m e d i a t e d by specific m e m b r a n e receptors TM. The location of these r e c e p t o r sites has been well d o c u m e n t e d in the central nervous system of many l a b o r a t o r y animals 18'35'37'38'41'42. In the rat, the T R H binding sites are widely distributed in m a n y brain regions 18'21'37'38. M o r e precisely, numerous T R H binding sites have been d o c u m e n t e d in several hypothalamic structures 18,37. In contrast to the murine brain, much less is known about the presence and the characteristics of T R H binding sites in the h u m a n brain. A few studies, p e r f o r m e d both by binding on tissue h o m o g e n a t e s and autoradiographic studies, have d e m o n s t r a t e d the presence of T R H
binding sites in the whole central nervous system 2°'3~. H o w e v e r , while the regional distribution of these binding sites has been studied in the h u m a n spinal cord 19 and hippocampus 11, no study had investigated the detailed regional distribution of such binding sites in the human hypothalamus. A recent r e p o r t from our l a b o r a t o r y indicates that the T R H levels, as m e a s u r e d by radioimmunoassay ( R I A ) , was higher in 3 structures, namely the ventromedial, the paraventricular and the dorsal nuclei, on the left side of the human h y p o t h a l a m u s 3. The aim of the present study is to investigate the biochemical characteristics and the detailed anatomical localization of T R H binding sites in the h u m a n hypothalamus using the well characterized analog [3H]MeTRH, by in vitro quantitative autoradiography. F u r t h e r , to investigate the d e v e l o p m e n t a l aspect of these binding sites, both infant and adult hypothalami have been processed. Finally, in o r d e r to confirm an eventual lateralization of T R H binding sites, the distribution and densities of labeling have been d e t e r m i n e d for the 2 hypothalamic sides.
Correspondence: M. Najimi, Laboratoire d'Anatomie Pathologique, Facult6 de Mrdecine Alexis Carrel, Lyon Cedex 08, France.
67 TABLE I
Source of brain tissues Cases
Sex
Age
Post mortem
Cause of death
delay Adults A B C D E F G H
M F M F F M M F
22 y 27 y 34 y 42 y 45 y 47 y 67 y 82 y
30 h 25 h 28.5 h 12 h 20 h 12 h 7h 2.5 h
Cardiac failure Cardiac failure Sudden death Sudden death Coronary thrombosis Cardiac failure Infarctus Cardiac failure
Neonates I
F
J
M
2h 35 h
14 h 34 h
K
M
1 day
I0 h
L M N
M M F
1 month 1 month 1 year
20 h 5h 7h
Pulmonary hypoplasia Amniotic inhalation with gastric regurgitation Oedematic alveolitis with refractory hypoplasia Liver lesions Enterocolitis Hepatic necrosis
For saturation studies, sections from the anterior and mediobasal hypothalamus from 4 different human hypothalami (2 adults and 2 infants) were incubated with ligand concentrations ranging from 1 to 30 nM. For displacement experiments, serial sections from the preoptic area and infundibular nucleus were incubated in the same incubation medium containing graded concentrations of unlabeled TRH and 3Me-TRH (10-12-10-6 M). Kinetic analysis were computed by the method of Parker and Waud29, in which the computerized process is based on the conventional method of the least squares, including the calculation of the variance-covariance matrix. Standard errors on the best estimates of the Imax and IC50 values were calculated from the coefficients of the variance-covafiance matrix.
Chemicals [3H]MeTRH was purchased from NEN. All peptides were obtained from Peninsula; bovine serum albumin, gelatin, Tris and bacitracin from Sigma; MgCI2 from Merck.
RESULTS
Scatchard analysis of [3H]MeTRH binding sites The experiments have focused on 2 hypothalamic structures: the preoptic area in the anterior hypothalamus and the infundibular nucleus in the mediobasal hypothalamus. The saturation curve showed that specific [3H]MeTRH binding to the 2 structures analyzed is sat-
MATERIALS AND METHODS Fourteen brains (8 males and 6 females, 8 adults and 6 neonates), obtained from Edouard Herriot and Lyon Sud Hospitals, Lyon, France, were used in this study (Table I). None of them presented clinical, neurological or neuroendocrinological signs. Furthermore, no pathological lesions were observed after macroscopic and microscopic examination of the brains. After removing the brain at autopsy, the hypothalamus was dissected at 4 °C and stored at -80 °C. The hypothalamus sample was dissected, taking the parallel plane joining the optic chiasm and anterior commissure as frontal plane and the caudal plane just behind the mammillary complex. The sample remained 1-6 days at -20 °C before cutting into 20 pm-thick sections on a Micron cryostat at -20 °C. The sections were mounted on gelatine chromalun coated glass slides and stored at -20 °C until they were processed for autoradiography. For anatomical localization of the hypothalamic nuclei and areas, adjacent sections to those used for autoradiography were stained with Cresyl violet. Identification of the hypothalamic nuclei and areas was made according to an atlas of the human hypothalamus developed in our laboratory. In the autoradiography procedure we used, the sections were preincubated for 20 rain at room temperature in 0.05 M Tris HCI buffer (pH 7.4) containing 0.2 % bovine serum albumin (BSA). After drying, they were incubated at 4 °C in the same medium to which [3H]3MeTRH (10 nM) (specific activity: 87 Ci/mmol), MgC12 (5 x 10-6 M) and bacitracin (2 × 10-s M) had been added. Nonspecific binding was determined by incubation of adjacent sections in the same medium containing 10-6 M non-radioactive TRH. After a 2 h incubation period, the sections were washed 4 times in ice cold buffer (30 s/bath) and once in distilled water (15 s) and dried immediately at room temperature. The labeled sections, together with standards (Amersham), were apposed to 3H ultrofilm (Amersham) for 2 months at 4 °C. The films were processed in Dektol (Kodak) developer and the optical density of the autoradiographic images quantitated in terms of fmol/mg tissue using an Imstar-Starwise computer and image analysis system.
urable (Fig. 1). Scatchard analysis revealed that the apparent k D a values and maximal binding capacity are equivalent both in infant and adult (Table II).
Inhibition analysis of [3H]MeTRH binding sites In these experiments, we have tested the ability of many T R H analogs to inhibit the binding of [3H]MeTRH. A m o n g the analogs used, only M e T R H and T R H inhibited competitively the specific binding in both preoptic area and infundibular nucleus by 50% with n a n o m o l a r potency. M e T R H was more potent than T R H (Fig. 2, Table III). By contrast, T R H - O H , DKP, HisPro-Glu, as well as L H R H , VIP, neurotensin, somatostatin and substance P, did not displace [3H]MeTRH binding in any of the regions studied (data not shown).
Autoradiographic distribution The data obtained from the quantitative autoradiographic study of the binding of [3H]MeTRH in the different hypothalamic structures are summarized in Table IV and Figs. 3-5. The resulting detailed mapping in the adult and infant hypothalamus appeared as follows: Adult hypothalamus. A. A n t e r i o r hypothalamus. This hypothalamic region (Figs. 3 A - C ; 4 A - C ) is very rich in T R H binding sites. The highest concentrations were located principally in the diagonal b a n d of Broca and the preoptic area. The T R H binding sites are distributed in the horizontal and the vertical limbs of the diagonal band of Broca with the same density and without sharp
68
PREOPTIC AREA
"~" 120
-
1oo"
60
"N
40
[3H] MeTRH (nM) INFUNDIBULAR NUCLEUS
_g g
,~.
[3H] Me TRN (nM)
Fig. I. Kinetics of [3H]3MeTRH binding to slide-mounted human hypothalamic sections as a function of [3H]3MeTRH concentration. Hypothalamic sections representing lateral preoptic area and infundibular nucleus were incubated for 2 h at 4 °C with increasing concentrations of tritiated 3MeTRH. Sections were apposed under ultrofilm and the binding was quantitated after autoradiography. Specific binding is defined by subtracting non-specific from total binding. Points are average of triplicate separate determinations. Average data from scatchard analysis are reported in Table II.
delimitation. Anteriorly, the labeling covers homogeneously the whole anatomical extent of the structure. This labeling does not change from the rostral to the caudal extent. Very high concentrations were also found
TABLE II Kinetics at equilibrium of [3H]3MeTRH binding in adult and infant hypothalamus Adult Preoptic area
kDa (nM) Bmax
Infant
4.8 ± 0.6 147 ± 14
5.3 -+ 0.5 135 -+ 12
7.6 ± 0.5 114 ± 5
6.5 ± 0.5 120 ± 16
(fmol/mg tissue) Infundibular nucleus
kDa (nM) Bran x
(fmol/mg tissue)
in the lateral preoptic area. This labeling is continuous with that present in the horizontal limb of the diagonal band of Broca. It decreases caudally. Thc medial part ot the preoptic area, in contrast to the lateral part, displays low densities. The paraventricular nucleus shows low but significant binding site densities. The labeling is distributed homogeneously in both the parvocellular and magnocellular parts of the nucleus. Further, similar densities were measured in the anterior and mediobasal levels of the nucleus. The suprachiasmatic and supraoptic nuclei do not display any significant labeling. Finally, the lateral hypothalamic area displays low densities. B. Mediobasal hypothalamus. Most parts of the mediobasal hypothalamus exhibit high to intermediate densities (Figs. 3D,E; 4D,E). The infundibular nucleus is one of the mediobasal structures displaying high densities. However, the labeling does not cover whole the nucleus, it is mainly concentrated in the ventral part. Along the anteroposterior extent of the nucleus, the autoradiographic grain density displays a heterogeneous distribution. While the T R H binding sites are very dense in the anterior part, they decrease in the posterior level and become rare. Caudally, the medial and lateral tuberal nuclei display very high densities. Labeling is homogeneous within these 2 structures. As for the infundibular nucleus, T R H binding sites were localized only in the ventral part. In contrast, no heterogeneous distribution was observed. More dorsally in the mediobasal hypothalamus, the ventromedial nucleus displayed intermediate T R H binding site densities. Labeling is distributed throughout the nucleus. As in the anterior region, low densities of T R H binding sites are observed in the lateral hypothalamic area. The median eminence as well as the fornix column do not display any significant labeling. C. Posterior hypothalamus. This hypothalamic level displays generally low T R H binding site concentrations (Figs, 3F; 4F). Low levels of T R H binding sites could be found in the medial mammillary nucleus. However, the lateral part of the mammillary nucleus displays intermediate T R H binding site densities. Autoradiographic labeling was absent further caudally. Dorsally, the subthalamic nucleus displays no autoradiographic labeling. Infant hypothalamus. The same general autoradiographic distribution of T R H binding sites (Fig. 5 A - F ) is observed in both the infant and adult hypothalamus. However, in the mediobasal hypothalamic level, while the adult tuberal nuclei display high densities, only low T R H binding site concentrations were detected in these nuclei in infants. Another discrepancy between the T R H binding site density distribution in the infant and the adult was observed in the diagonal band of Broca: this
69
INFUNDIBULAR NUCLEUS
PREOPTIC AREA 100
1°°t
80
t
6O
6O 3MeTRH
40-
40
20
20 0
I
- 3
-,2
"
I
"
•
-11
-; LOG
-'s
-'7
-6
0 -
-12
-11
-10
-9
-8
-7
-6
LOG M
M
Fig. 2. Competitive displacement of [3H]3MeTRH binding by TRH and 3MeTRH in lateral preoptic area and infundibular nucleus of the adult hypothalamus. The sections were incubated with 10 nM [3H]3MeTRH in the presence of increasing concentrations of displacing agohist. ICs0s were computed by the method of Parker and Waud.
structure displays higher densities in infants than in
which afford many advantages, such as a high degree of
adults.
population homogeneity, the control of sex, age and post m o r t e m delay, the use of h u m a n samples might be influenced by these parameters 39. As far as the cases used
DISCUSSION The present study represents, to our knowledge, the first detailed mapping of the distribution of T R H binding sites in the h u m a n hypothalamus. The in vitro quantitative autoradiographic technique was chosen to the
TABLE IV
m e m b r a n e h o m o g e n a t e binding method, which did not afford a high anatomical resolution. We used the [3H]MeTRH as radioligand, which has b e e n reported to be more appropriate than [3H]TRH37. In addition, this
Values are presented as the mean and standard error of the mean (S.E.M.), measured in fmol/mg tissue for 6 infants and 8 adults. Both in infant and adult, the values are given for the fight and left (values in italics) hypothalamic sides. The specific binding in each structure analyzed is obtained by subtracting non-specific binding from total binding.
T R H radioanalog, which is biologically more potent than T R H , has b e e n well tested in previous h u m a n brain studies, both in the m e m b r a n e binding preparations and in in vitro quantitative autoradiography, where it has b e e n shown to exhibit a high binding affinity 19"31. Unlike experiments performed in laboratory animals
TRH binding site densities in the human
Hypothalamic structures
Diagonal band of Broca
[3H]MeTRH binding sites (fmol/mg tissue) Infant
Adult
183 +- 3
160 - 7* 156 +- 8* 144 +-6 142 +-6 51 -+2 55 -+3 40 +-2 42 -+3 22 -+1 23 +-1 127 +-3 121 +-2 73 -+4 72 +-2 36 -+2 34 -+3 126 -+ 3** 120 -+ 5"* 19 +-1 21 +-1
182 +- 3
Lateral preoptic area
152 -+ 4 155 +- 7
TABLE III
Medial preoptic area
Displacement kinetics o f [3H]MeTRH binding in adult and infant hypothalamus
Paraventricular nucleus
47 +- 8 49 -+3
Pefiventricular nucleus Adult
Infant
Infundibular nucleus
TRH IC50 (nM) MeTRH IC5o (nM)
17.4 +- 2.1
16.5 -+ 3.2
1.4 --- 0.2
1.1 +- 0.4
21 -+ 1 21 -+2
Infundibular nucleus Preoptic area
38 +- 1 37++-1
124 -+ 2 116 +- 3
Ventromedial nucleus
79 +- 3 84++-4
Lateral hypothalamic area
32 +- 4 35 +-3
TRH IC5o (nM) MeTRH IC5o (nM)
26.3 -+ 2.6 1.9 -+ 0.2
Tuberal nuclei
76 +- 2
Mammillary nuclei
23 -+ 1
24.2 -+ 3.4 1.7 -+ 0.3
75 +- 1 25+-1
*P < 0.05 and **P < 0.01 by Student's t-test.
70 A
¢--'-
v
m
M,
!,
A
r
/
.2
Fig. 3. Schematic coronal sections of the human hypothalamus drawn from adult, representing the distribution and densities of TRH binding sites at different hypothalamic levels (A-C: anterior level; D,E: mediobasal level; F: posterior level). 1~-~1,>140 fmol/mg tissue; [ ] 101-140 fmol/mg tissue; [K-I, 45-100 fmol/mg tissue; [7],
Brain Research, 563 (1991 ) 60 -7~ © 1991 Elsevier Science Publishers B.V, All rights reserved. 0006-8993/91/$03.51) ADONIS 0006899391171031 ~:
BRES 17103
Autoradiographic distribution of TRH binding sites in the human hypothalamus M. Najimi, F. Chigr, J. Champier, A. Tabib, N. Kopp and D. J o d a n i Laboratoire d'Anatornie Pathologique, Facultd de Mddecine Alexis Carrel, Lyon (France)
(Accepted 11 June 1991) Key words: Autoradiography; Hypothalamus; Human; Thyrotropin-releasing hormone binding sites
Using in vitro quantitative autoradiography and [3H]3MeTRH, a selective high affinity radioligand, we examined the rostrocaudal distribution of TRH binding sites in both the infant and the adult human hypothalamus. The saturation curve shows that the [3H]3MeTRH binds with high affinity to a single class of TRH binding sites and is saturable, the apparent constant of dissociation is in the namomolar range. TRH binding sites showed a wide distribution, principally in the anterior and mediobasal levels of the hypothalamus. TRH binding site concentration was highest within the diagonal band of Broca, the lateral preoptic area, the infundibular and the tuberal nuclei. TRH binding site concentration was moderate in the ventromedial nucleus and the medial preoptic area, whereas we observed low densities in the periventricular, paraventricular and mammillary nuclei. The distribution in the infant and the adult is generally similar. However, it is noteworthy that the infant tuberal nuclei displayed a lower binding site density when compared to the adult. On the other hand, the diagonal band of Broca is relatively more labeled in infant. The analysis of the whole hypothalamus allows us to ascertain the absence of lateral asymmetric distribution both in the infant and the adult. No significant difference is noticed when considering as paremeters of variation age, sex or post mortem delay.
INTRODUCTION Thyrotropin-releasing h o r m o n e ( T R H ) was initially isolated from bovine and porcine hypothalamus 2'6 and identified as the tripeptide amine: p G l u - H i s - P r o - N H 2. Using biochemical and immunohistochemical techniques, many studies have r e p o r t e d that the hypothalamus is the brain region containing the highest concentrations of this tripeptide in m a m m a l i a n species 5'8-1°'12' 16,26,27,43 including h u m a n 13'3°. Its biological actions have been r e p o r t e d to be m e d i a t e d by specific m e m b r a n e receptors TM. The location of these r e c e p t o r sites has been well d o c u m e n t e d in the central nervous system of many l a b o r a t o r y animals 18'35'37'38'41'42. In the rat, the T R H binding sites are widely distributed in m a n y brain regions 18'21'37'38. M o r e precisely, numerous T R H binding sites have been d o c u m e n t e d in several hypothalamic structures 18,37. In contrast to the murine brain, much less is known about the presence and the characteristics of T R H binding sites in the h u m a n brain. A few studies, p e r f o r m e d both by binding on tissue h o m o g e n a t e s and autoradiographic studies, have d e m o n s t r a t e d the presence of T R H
binding sites in the whole central nervous system 2°'3~. H o w e v e r , while the regional distribution of these binding sites has been studied in the h u m a n spinal cord 19 and hippocampus 11, no study had investigated the detailed regional distribution of such binding sites in the human hypothalamus. A recent r e p o r t from our l a b o r a t o r y indicates that the T R H levels, as m e a s u r e d by radioimmunoassay ( R I A ) , was higher in 3 structures, namely the ventromedial, the paraventricular and the dorsal nuclei, on the left side of the human h y p o t h a l a m u s 3. The aim of the present study is to investigate the biochemical characteristics and the detailed anatomical localization of T R H binding sites in the h u m a n hypothalamus using the well characterized analog [3H]MeTRH, by in vitro quantitative autoradiography. F u r t h e r , to investigate the d e v e l o p m e n t a l aspect of these binding sites, both infant and adult hypothalami have been processed. Finally, in o r d e r to confirm an eventual lateralization of T R H binding sites, the distribution and densities of labeling have been d e t e r m i n e d for the 2 hypothalamic sides.
Correspondence: M. Najimi, Laboratoire d'Anatomie Pathologique, Facult6 de Mrdecine Alexis Carrel, Lyon Cedex 08, France.
67 TABLE I
Source of brain tissues Cases
Sex
Age
Post mortem
Cause of death
delay Adults A B C D E F G H
M F M F F M M F
22 y 27 y 34 y 42 y 45 y 47 y 67 y 82 y
30 h 25 h 28.5 h 12 h 20 h 12 h 7h 2.5 h
Cardiac failure Cardiac failure Sudden death Sudden death Coronary thrombosis Cardiac failure Infarctus Cardiac failure
Neonates I
F
J
M
2h 35 h
14 h 34 h
K
M
1 day
I0 h
L M N
M M F
1 month 1 month 1 year
20 h 5h 7h
Pulmonary hypoplasia Amniotic inhalation with gastric regurgitation Oedematic alveolitis with refractory hypoplasia Liver lesions Enterocolitis Hepatic necrosis
For saturation studies, sections from the anterior and mediobasal hypothalamus from 4 different human hypothalami (2 adults and 2 infants) were incubated with ligand concentrations ranging from 1 to 30 nM. For displacement experiments, serial sections from the preoptic area and infundibular nucleus were incubated in the same incubation medium containing graded concentrations of unlabeled TRH and 3Me-TRH (10-12-10-6 M). Kinetic analysis were computed by the method of Parker and Waud29, in which the computerized process is based on the conventional method of the least squares, including the calculation of the variance-covariance matrix. Standard errors on the best estimates of the Imax and IC50 values were calculated from the coefficients of the variance-covafiance matrix.
Chemicals [3H]MeTRH was purchased from NEN. All peptides were obtained from Peninsula; bovine serum albumin, gelatin, Tris and bacitracin from Sigma; MgCI2 from Merck.
RESULTS
Scatchard analysis of [3H]MeTRH binding sites The experiments have focused on 2 hypothalamic structures: the preoptic area in the anterior hypothalamus and the infundibular nucleus in the mediobasal hypothalamus. The saturation curve showed that specific [3H]MeTRH binding to the 2 structures analyzed is sat-
MATERIALS AND METHODS Fourteen brains (8 males and 6 females, 8 adults and 6 neonates), obtained from Edouard Herriot and Lyon Sud Hospitals, Lyon, France, were used in this study (Table I). None of them presented clinical, neurological or neuroendocrinological signs. Furthermore, no pathological lesions were observed after macroscopic and microscopic examination of the brains. After removing the brain at autopsy, the hypothalamus was dissected at 4 °C and stored at -80 °C. The hypothalamus sample was dissected, taking the parallel plane joining the optic chiasm and anterior commissure as frontal plane and the caudal plane just behind the mammillary complex. The sample remained 1-6 days at -20 °C before cutting into 20 pm-thick sections on a Micron cryostat at -20 °C. The sections were mounted on gelatine chromalun coated glass slides and stored at -20 °C until they were processed for autoradiography. For anatomical localization of the hypothalamic nuclei and areas, adjacent sections to those used for autoradiography were stained with Cresyl violet. Identification of the hypothalamic nuclei and areas was made according to an atlas of the human hypothalamus developed in our laboratory. In the autoradiography procedure we used, the sections were preincubated for 20 rain at room temperature in 0.05 M Tris HCI buffer (pH 7.4) containing 0.2 % bovine serum albumin (BSA). After drying, they were incubated at 4 °C in the same medium to which [3H]3MeTRH (10 nM) (specific activity: 87 Ci/mmol), MgC12 (5 x 10-6 M) and bacitracin (2 × 10-s M) had been added. Nonspecific binding was determined by incubation of adjacent sections in the same medium containing 10-6 M non-radioactive TRH. After a 2 h incubation period, the sections were washed 4 times in ice cold buffer (30 s/bath) and once in distilled water (15 s) and dried immediately at room temperature. The labeled sections, together with standards (Amersham), were apposed to 3H ultrofilm (Amersham) for 2 months at 4 °C. The films were processed in Dektol (Kodak) developer and the optical density of the autoradiographic images quantitated in terms of fmol/mg tissue using an Imstar-Starwise computer and image analysis system.
urable (Fig. 1). Scatchard analysis revealed that the apparent k D a values and maximal binding capacity are equivalent both in infant and adult (Table II).
Inhibition analysis of [3H]MeTRH binding sites In these experiments, we have tested the ability of many T R H analogs to inhibit the binding of [3H]MeTRH. A m o n g the analogs used, only M e T R H and T R H inhibited competitively the specific binding in both preoptic area and infundibular nucleus by 50% with n a n o m o l a r potency. M e T R H was more potent than T R H (Fig. 2, Table III). By contrast, T R H - O H , DKP, HisPro-Glu, as well as L H R H , VIP, neurotensin, somatostatin and substance P, did not displace [3H]MeTRH binding in any of the regions studied (data not shown).
Autoradiographic distribution The data obtained from the quantitative autoradiographic study of the binding of [3H]MeTRH in the different hypothalamic structures are summarized in Table IV and Figs. 3-5. The resulting detailed mapping in the adult and infant hypothalamus appeared as follows: Adult hypothalamus. A. A n t e r i o r hypothalamus. This hypothalamic region (Figs. 3 A - C ; 4 A - C ) is very rich in T R H binding sites. The highest concentrations were located principally in the diagonal b a n d of Broca and the preoptic area. The T R H binding sites are distributed in the horizontal and the vertical limbs of the diagonal band of Broca with the same density and without sharp
68
PREOPTIC AREA
"~" 120
-
1oo"
60
"N
40
[3H] MeTRH (nM) INFUNDIBULAR NUCLEUS
_g g
,~.
[3H] Me TRN (nM)
Fig. I. Kinetics of [3H]3MeTRH binding to slide-mounted human hypothalamic sections as a function of [3H]3MeTRH concentration. Hypothalamic sections representing lateral preoptic area and infundibular nucleus were incubated for 2 h at 4 °C with increasing concentrations of tritiated 3MeTRH. Sections were apposed under ultrofilm and the binding was quantitated after autoradiography. Specific binding is defined by subtracting non-specific from total binding. Points are average of triplicate separate determinations. Average data from scatchard analysis are reported in Table II.
delimitation. Anteriorly, the labeling covers homogeneously the whole anatomical extent of the structure. This labeling does not change from the rostral to the caudal extent. Very high concentrations were also found
TABLE II Kinetics at equilibrium of [3H]3MeTRH binding in adult and infant hypothalamus Adult Preoptic area
kDa (nM) Bmax
Infant
4.8 ± 0.6 147 ± 14
5.3 -+ 0.5 135 -+ 12
7.6 ± 0.5 114 ± 5
6.5 ± 0.5 120 ± 16
(fmol/mg tissue) Infundibular nucleus
kDa (nM) Bran x
(fmol/mg tissue)
in the lateral preoptic area. This labeling is continuous with that present in the horizontal limb of the diagonal band of Broca. It decreases caudally. Thc medial part ot the preoptic area, in contrast to the lateral part, displays low densities. The paraventricular nucleus shows low but significant binding site densities. The labeling is distributed homogeneously in both the parvocellular and magnocellular parts of the nucleus. Further, similar densities were measured in the anterior and mediobasal levels of the nucleus. The suprachiasmatic and supraoptic nuclei do not display any significant labeling. Finally, the lateral hypothalamic area displays low densities. B. Mediobasal hypothalamus. Most parts of the mediobasal hypothalamus exhibit high to intermediate densities (Figs. 3D,E; 4D,E). The infundibular nucleus is one of the mediobasal structures displaying high densities. However, the labeling does not cover whole the nucleus, it is mainly concentrated in the ventral part. Along the anteroposterior extent of the nucleus, the autoradiographic grain density displays a heterogeneous distribution. While the T R H binding sites are very dense in the anterior part, they decrease in the posterior level and become rare. Caudally, the medial and lateral tuberal nuclei display very high densities. Labeling is homogeneous within these 2 structures. As for the infundibular nucleus, T R H binding sites were localized only in the ventral part. In contrast, no heterogeneous distribution was observed. More dorsally in the mediobasal hypothalamus, the ventromedial nucleus displayed intermediate T R H binding site densities. Labeling is distributed throughout the nucleus. As in the anterior region, low densities of T R H binding sites are observed in the lateral hypothalamic area. The median eminence as well as the fornix column do not display any significant labeling. C. Posterior hypothalamus. This hypothalamic level displays generally low T R H binding site concentrations (Figs, 3F; 4F). Low levels of T R H binding sites could be found in the medial mammillary nucleus. However, the lateral part of the mammillary nucleus displays intermediate T R H binding site densities. Autoradiographic labeling was absent further caudally. Dorsally, the subthalamic nucleus displays no autoradiographic labeling. Infant hypothalamus. The same general autoradiographic distribution of T R H binding sites (Fig. 5 A - F ) is observed in both the infant and adult hypothalamus. However, in the mediobasal hypothalamic level, while the adult tuberal nuclei display high densities, only low T R H binding site concentrations were detected in these nuclei in infants. Another discrepancy between the T R H binding site density distribution in the infant and the adult was observed in the diagonal band of Broca: this
69
INFUNDIBULAR NUCLEUS
PREOPTIC AREA 100
1°°t
80
t
6O
6O 3MeTRH
40-
40
20
20 0
I
- 3
-,2
"
I
"
•
-11
-; LOG
-'s
-'7
-6
0 -
-12
-11
-10
-9
-8
-7
-6
LOG M
M
Fig. 2. Competitive displacement of [3H]3MeTRH binding by TRH and 3MeTRH in lateral preoptic area and infundibular nucleus of the adult hypothalamus. The sections were incubated with 10 nM [3H]3MeTRH in the presence of increasing concentrations of displacing agohist. ICs0s were computed by the method of Parker and Waud.
structure displays higher densities in infants than in
which afford many advantages, such as a high degree of
adults.
population homogeneity, the control of sex, age and post m o r t e m delay, the use of h u m a n samples might be influenced by these parameters 39. As far as the cases used
DISCUSSION The present study represents, to our knowledge, the first detailed mapping of the distribution of T R H binding sites in the h u m a n hypothalamus. The in vitro quantitative autoradiographic technique was chosen to the
TABLE IV
m e m b r a n e h o m o g e n a t e binding method, which did not afford a high anatomical resolution. We used the [3H]MeTRH as radioligand, which has b e e n reported to be more appropriate than [3H]TRH37. In addition, this
Values are presented as the mean and standard error of the mean (S.E.M.), measured in fmol/mg tissue for 6 infants and 8 adults. Both in infant and adult, the values are given for the fight and left (values in italics) hypothalamic sides. The specific binding in each structure analyzed is obtained by subtracting non-specific binding from total binding.
T R H radioanalog, which is biologically more potent than T R H , has b e e n well tested in previous h u m a n brain studies, both in the m e m b r a n e binding preparations and in in vitro quantitative autoradiography, where it has b e e n shown to exhibit a high binding affinity 19"31. Unlike experiments performed in laboratory animals
TRH binding site densities in the human
Hypothalamic structures
Diagonal band of Broca
[3H]MeTRH binding sites (fmol/mg tissue) Infant
Adult
183 +- 3
160 - 7* 156 +- 8* 144 +-6 142 +-6 51 -+2 55 -+3 40 +-2 42 -+3 22 -+1 23 +-1 127 +-3 121 +-2 73 -+4 72 +-2 36 -+2 34 -+3 126 -+ 3** 120 -+ 5"* 19 +-1 21 +-1
182 +- 3
Lateral preoptic area
152 -+ 4 155 +- 7
TABLE III
Medial preoptic area
Displacement kinetics o f [3H]MeTRH binding in adult and infant hypothalamus
Paraventricular nucleus
47 +- 8 49 -+3
Pefiventricular nucleus Adult
Infant
Infundibular nucleus
TRH IC50 (nM) MeTRH IC5o (nM)
17.4 +- 2.1
16.5 -+ 3.2
1.4 --- 0.2
1.1 +- 0.4
21 -+ 1 21 -+2
Infundibular nucleus Preoptic area
38 +- 1 37++-1
124 -+ 2 116 +- 3
Ventromedial nucleus
79 +- 3 84++-4
Lateral hypothalamic area
32 +- 4 35 +-3
TRH IC5o (nM) MeTRH IC5o (nM)
26.3 -+ 2.6 1.9 -+ 0.2
Tuberal nuclei
76 +- 2
Mammillary nuclei
23 -+ 1
24.2 -+ 3.4 1.7 -+ 0.3
75 +- 1 25+-1
*P < 0.05 and **P < 0.01 by Student's t-test.
70 A
¢--'-
v
m
M,
!,
A
r
/
.2
Fig. 3. Schematic coronal sections of the human hypothalamus drawn from adult, representing the distribution and densities of TRH binding sites at different hypothalamic levels (A-C: anterior level; D,E: mediobasal level; F: posterior level). 1~-~1,>140 fmol/mg tissue; [ ] 101-140 fmol/mg tissue; [K-I, 45-100 fmol/mg tissue; [7],
