D1- and D2 dopaminergic receptors in the developing cerebral cortex of macaque monkey: A film autoradiographic study

~)

Pergamon

0306-4522(94)0047 5-7

Neuroscience Vol. 65, No. 2, pp. 439-452, 1995 Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

D1- A N D D2 DOPAMINERGIC RECEPTORS IN THE DEVELOPING CEREBRAL CORTEX OF MACAQUE MONKEY: A FILM AUTORADIOGRAPHIC STUDY M. S. L I D O W Yale University School of Medicine, Section of Neurobiology, New Haven, CT 06510, U.S.A. Abstract--Film autoradiography was used to study the distribution of D t- and D2-dopaminergic receptors in the prefrontal association, somatosensory, primary motor and visual regions in the developing cerebral cortex of macaque monkeys. D~ receptors were labeled with [~25I]SCH23982, while D 2 sites were visualized with [t251]epidepride. D~- and D2-dopaminergic sites are already present in all cortical areas at embryonic day 73, the earliest age observed in this study. In contrast to the adult cortex, where D~ and D 2 receptors have different distributions, during development there are substantial similarities in the laminar patterns of these sites. In particular, both D~ and D 2 receptors tend to concentrate in the marginal zone and layer V of the developing cortical plate. The autoradiograms also show a high density of D~-dopaminergic sites in the transient ventricular and subventricular zones, where cortical neurons are generated. Although there is a significant rearrangement of the early laminar patterns, the adult distribution of both dopaminergic receptors in most cortical areas is achieved prenatally, soon after all cortical neurons assume their final positions. An early presence in the cerebral wall, a high density in the proliferative zones and fast maturation of the laminar distribution suggests that dopaminergic receptors may be involved in the regulation of cortical development.

T h e last decade has witnessed significant progress in o u r u n d e r s t a n d i n g of the cortical d o p a m i n e r g i c system. It has been s h o w n t h a t d o p a m i n e r g i c i n n e r v a t i o n a n d receptors are widely, distributed, in a laminar-specific m a n n e r , t h r o u g h o u t multiple sensory, motor and association cortical a r e a s . 4'5'16'18'26'3°'31'33'34'64 The presence of b o t h D~ a n d D2 classes o f d o p a m i n e r g i c receptors in the cerebral cortex has been establishedfl °,34,43 Behavioral a n d electrophysiological studies have indicated i m p o r t a n t roles played by d o p a m i n e in cognitive a n d m o t o r regulatory functions o f the cortex. 1°'41 It has also become accepted t h a t disturbances of the cortical d o p a m i n e r g i c system are involved in m a j o r psychiatric a n d neurological disorders, including schizop h r e n i a a n d P a r k i n s o n ' s disease.12'51'53'54'62Recently, it has been s h o w n t h a t cortical d o p a m i n e r g i c sites m a y be a m o n g the m a j o r targets o f antipsychotic agents. 38 F u r t h e r m o r e , the a c c u m u l a t e d d a t a suggest t h a t certain disorders, such as schizophrenia, T o u r e t t e ' s s y n d r o m e a n d hyperactivity in children, m a y be directly related to defects in the d e v e l o p m e n t o f cortical d o p a m i n e r g i c circuitryfl 1'62'63 All o f this m a k e s the knowledge of the o n t o g e n y o f the cortical d o p a m i n e r g i c system essential for u n d e r s t a n d i n g the d e v e l o p m e n t o f functional cortical circuitry a n d the etiology o f psychiatric a n d neurological disorders. In spite of wide interest, however, very little inform a t i o n is available o n the d e v e l o p m e n t of the cortical Abbreviations: E, embryonic day; P, postnatal day. 439

d o p a m i n e r g i c system in primates, 3,6,17,27 particularly o f its postsynaptic part. 36 This p a p e r describes the pre- a n d p o s t n a t a l changes in the l a m i n a r distribution o f DI- a n d D2-dopaminergic receptors in functionally diverse areas of the developing m o n k e y cerebral cortex.

EXPERIMENTAP LROCEDURES Tissue preparation Data for the present report were obtained from 26 Rhesus monkeys (Macaca mulatta) of both sexes ranging in age from embryonic day (E) 73 to five years of age (Figs 9, 10). The animals were obtained from Yale University Medical School Primate Breeding Colony (New Haven, CT) and New England Primate Regional Center (Farmington, MA). The monkeys were anesthetized with sodium pentobarbital (40 mg/kg) and perfused with ice-cold phosphate-buffered saline (pH 7.4; 1.25 1) followed by 0.1% paraformaldehyde containing increasing concentrations of sucrose in buffered saline: 500 ml 0% sucrose; 500ml 5% sucrose; 11 10% sucrose; 500ml 15% sucrose; and I1 20% sucrose. This fixation protocol enhances tissue preservation without measurably decreasing binding or altering kinetic constants. 5° The brains were rapidly removed, blocked and immersed in isopentane at - 3 0 ° C for 5 min before storing at -80°C. Tissue was cut into 20-pm-thick sections on a Bright Cryostat. Sections were mounted on acid-cleaned chrome alum-subbed slides and stored at - 8 0 ° C until the time of assay, conducted no more than two weeks after the tissue had been sectioned. The cortical regions examined in this study included prefrontal association (cytoarchitectonic area 4661), primary motor (area 49), somatosensory (areas 1 and 29) and primary visual (area 179) cortex.

440

M . S . Lidow

Binding assays The D~ class of dopaminergic sites was labeled with the antagonist [~25I]SCH2398272 Tissue sections were first preincubated for 20min at room temperature in 50mM Tris-HC1 buffer (pH 7.4). They were then incubated with 0.1-8 nM [~25I]SCH23982 for 90 min at room temperature in 50mM Tris HCI buffer (pH 7.4) containing 120mM NaC1, 5 m M KC1, 2raM CaClz, l m M MgC12 and 1/~M mianserin. The latter was added to block binding to 5-hydroxytryptamine 2, 5-hydroxytryptominelc and e2 adrenalin sites. After incubation, the tissue was rinsed twice (10 min each) in ice-cold 50 mM Tris-HCl buffer (pH 7.4). Nonspecific binding was determined in the presence of 1 # M

cis-flupentixol. The D 2 class of dopaminergic receptors was labeled with the antagonist [mSI]epidepride}8 Sections were preincubated as described for [125I]SCH23982 binding, and then incubated for 45 min at room temperature with 0.1-3.0 nM [125I]epidepride in 5 0 m M Tris HC1 buffer (pH 7.4) containing 120raM NaC1, 5 m M KC1, 2 m M CaCI 2, l mM MgC12, 0.1% ascorbic acid and 0.2 # M idazoxan to prevent labeling of c~2 sites. After incubation, the sections were rinsed as described for [msI]SCH23982 binding. Non-specific labeling was determined in the presence of 10 p M (+)-butaclamol. At the end of the labeling assay, all tissue sections were dipped in distilled water and apposed to 3H-sensitive Ultrofilm (Amersham Co. Arlington Heights, IL) for three days ([msI]SCH23982) or seven days ([125I]epidepride). After development for autoradiography, the tissue sections were stained with Cresyl Violet to allow analysis of the cytoarchitecture. At least five different concentrations of each radioligand were used in every assay. Our previous studies showed that five data points are sufficient for saturation analysis assuming a one-site receptor model. 18,3~32,34"49 At least five tissue sections were processed for every concentration of ligand, three for total and two for non-specific binding. All assays were repeated twice for each animal studied.

Analysis of autoradiograms Due to the difficulty in obtaining monkey fetuses, only a single animal was examined at most prenatal ages (Figs 9, 10). This precluded a rigorous quantitative evaluation of receptor development. Instead, this study concentrated on a detailed qualitative description of the laminar binding of dopamine receptor-specific radioligands in the developing monkey cerebral cortex. There are strong indications that the laminar patterns of radioligand binding autoradiographically visualized in this

study accurately reflect the distributions of binding sites characteristic for developmental ages examined. First, cortices of close ages had similar distributions of dopamine receptor-specific labeling, and there was a steady and systematic progression of developmental changes in binding distributions. Second, in all specimens, there were no significant differences between the equilibrium dissociation constants (Kd) of dopamine receptor-specific radioligand binding obtained in various cortical lamina (Table 1). This suggests that autoradiographic patterns generated in this study cannot be attributed to fluctuations in receptor affinity and reflect laminar variations the density of binding sites. The autoradiograms were examined with a BDS computer system (Biological Detection Systems, Inc., Pittsburgh, PA), which allows the overlay of digitized images of the Cresyl Violet-stained sections and the corresponding autoradiograms on the computer screen in order to facilitate histological identification of specific layers on the autoradiographic images. This computer system was also used for comparison of the optical densities of the film images with those of the mSI-standards (Amersham Corp., Arlington Heights, IL) that were apposed to the film along with the tissue sections. The system converts the optical densities of the autoradiograms into concentrations of labeled compounds per tissue volume. On all autoradiograms used in this study, the diffuse optical densities were between 0.08 and 0.80. In this range they are linearly related to tissue radioactivity on 3H-sensitive Ultrofilm? 9 When needed, the statistical analysis o f saturation binding was performed utilizing the non-linear curve fitting computer programs KINETIC/EDBA/LIGAND/LOWRY from ElsevierBIOSOFT Co. (Cambridge, U.K.).

RESULTS

Dl-dopaminergic receptors in the developing cerebral cortex T h e earliest p r e n a t a l age o b s e r v e d in this study was E73. A t this age, the cortical plate c o n t a i n e d very few D l - d o p a m i n e r g i c r e c e p t o r s labeled with [12sI]SCH23982 (Figs 1-4, 9). M u c h higher r e c e p t o r densities were d e t e c t e d in several t r a n s i e n t e m b r y o n i c cortical zones. T h e s e include the m a r g i n a l z o n e overlying the f o r m i n g cortical plate a n d the v e n t r i c u l a r a n d s u b v e n t r i c u l a r proliferative z o n e s situated n e a r

Table l. Apparent affinity values for binding of dopaminergic radioligands in the prefrontal cortex (area 46) at several developmental ages [125I]SCH239892 Layers I II-III IV V VI Subplate Intermediate zone Subventricular zone (residual) One-way ANOVA

El07

E143

P60

8.35_+0.21 8.41+0.37 8.28+0.30 8.36+0.36 8.45_+0.23 8.44_+0.34

8.25+0.17 8.33+0.42 8.36+0.19 8.45_+0.24 8.26_+0.38 8.42_+0.39

8.40+_0.23 8.36+0.28 8.44_+0.31 8.29+0.17 8.34_+0.51 --

8.50+0.13

8.45-+0.35

--

8.27-+ 0.40

--

P > 0.05

P > 0.05

P > 0.05

[125I]Epidepride One-way ANOVA P>0.05 P>0.05 P>0.05 P>0.05 P>0.05

El07

E143

P60

One-way ANOVA

10.42_+0.12 10.10+0.21 10.11_+0.31 10.17-+0.37 10.19__+0.24 10.10_+0.16

10.20+0.17 10.09+0.34 10.17_+0.27 10.14-+0.31 10.11_+0.42 10.19_+0.22

10.11+0.22 10.21+0.30 10.15+0.19 10.18_+0.25 10.11_+0.18

P>0.05 P>0.05 P>0.05 P>0.05 P>0.05

10.18+0.29

10.21 +0.38

10.13 _+ 0.23

--

--

P > 0.05

P > 0.05

P > 0.05

Since the apparent affinity of the radioligands have a log-normal distribution, the data is presented as --log Kd. The K a values obtained in animals of other ages are similar to those presented in this table.

441

Development of dopaminergic receptors

Cresyl Violet

A

Binding

B

C

MZ

CP SP

IZ

SV VZ Fig. 1. Laminar distribution of [125I]SCH23982 binding (D:dopaminergic receptors) in the developing prefrontal cortex (area 46). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E 115. This laminar pattern is representative for specimens killed at E93, E 107, E115 and E 120. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at birth. This laminar pattern is representative for specimens killed at E128, E143, birth, one, two, four and eight months as well as one, three and five years of age. MZ, marginal zone future layer I; CP, cortical plate; SP, subplate zone; IZ, intermediate zone; WM, white matter (former subplate and intermediate zones); SV, subventricular germinal zone; VZ, ventricular germinal zone; VE, ventricle. To make evaluation and comparison of the binding patterns easier, the magnification of each image is adjusted to fit uniform dimensions. The images in this and other figures were digitized with the BDS computer system (Biological Detection Systems Inc. Pittsburgh, PA). The brightness, contrast and size were adjusted in Colorlt (MicroFrontier, Des Moines, IA). Then, the pictures were combined in MacDraw (Claris Corp., Santa Clara, CA) and printed on the CorrectPrint 300i high resolution color printer (RosterOps Co., Santa Clara, CA). the lateral ventricles (Figs 1-4, 9). In the latter two zones, the receptor density gradient corresponded to cell density (Figs 1, 3). The major difference between the distribution of D 1

Cresyl Violet

A

Binding

B

sites at E93 compared to the younger specimens was an addition of a new band of high receptor density corresponding to layer V of the cortical plate (Figs 3, 4, 9). Also, D l sites formed two bands

C

MZ CP

SP

IZ

Fig. 2. Laminar distribution of [1:5I]SCH23982 binding (D~ dopaminergic receptors) in the developing somatosensory cortex (area 1). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E115. This laminar pattern is representative for specimens killed at E93, El07, El 15 and El20. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at birth. This laminar pattern is representative for specimens killed at E128, E143, birth, one, two, four and eight months as well as one, three and five years of age. Abbreviations as in Fig. 1.

M. S. Lidow

442

Cresyl Violet

A

Binding

B

MZ ~ CP

SP

IZ

SV VZ

C

D

I

II

III IVa IVb IVcct lVc[3 V VI SP

IZ SV VE Fig. 3. Laminar distribution of ['25I]SCH23982 binding (D, dopaminergic receptors) in the developing primary visual cortex (area 17). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E93. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E 120. This laminar pattern is representative for specimenskilled at E 107, E115, El20 and E128. (D) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E143. This laminar pattern is representative for specimens killed at E143, birth, one, two, four and eight months as well as one, three and five years of age. Abbreviations as in Fig. 1.

matching the two strips of high cell density observed in the ventricular and subventricular zones (Fig. 3). In El07 and older specimens, the laminar distribution of D~ receptors in the cortical plate showed regional differences. Also, after El07, D, receptors gradually disappear in the ventricular and subventricular zones as these zones become exhausted. The prefrontal and somatosensory cortex of specimens killed at El07, E115 and El20 displayed receptor distributions similar to that observed at E93. Layers I (former marginal zone) and V contained the highest receptor densities (Figs 1, 2, 9). The adult distribution of D~ sites in the prefrontal and somatosensory regions was already achieved in

the specimen obtained at E128. At this and all following ages [E143, birth (postnatal day 1, P1) and one month (P30), two months (P60), four months (PI20), either months (P240), one year (P365), three years (P1095) and five years of age (P1825)], the highest densities of D~ sites were observed in superficial layers I and II, the upper half of layer III and deep layers V and VI (Figs 1, 2, 9). In the primary visual cortex of El07, ELI5, El20 and E128 specimens, the distribution of D~ sites was different from that of earlier ages in that in addition to high densities in layers I and V, the receptors were also concentrated in sublayer IVa (Figs 3, 9). The adult pattern of the D~ receptor distribution in this

Development of dopaminergic receptors area was first observed at E143. In this and older specimens, the highest densities of D~ sites were observed in layers I, II, IVa, V and VI (Figs 3, 9). In contrast to the laminar patterns in other cortical areas, the distribution of D~ receptors in the primary motor cortex did not become adult-like before birth. In the El07, ELI5, El20, E128, E143, newborn and one-month-old specimens, the primary motor cortex displayed the highest density of D 1 receptors in layer V (Figs 4, 9). In the second postnatal month, this laminar pattern was replaced by a high receptor density in layer llI (Figs 4, 9). The adult distribution of D~ receptors was observed in the animals at eight

Cresyl Violet

A

Binding

443

months of age. In these and older specimens, the highest densities of D~ receptors in the primary motor cortex were detected in layers I, II and the upper half of layer III (Figs 4, 9).

D2 dopaminergic receptors in the developing cerebral cortex In the prefrontal and somatosensory cortex of E73 specimens, the marginal zone contained the highest density of D 2 dopaminergic receptors visualized with [t2SI]epidepride (Figs 5, 6, 10). In E93, El07, El 15 and El20 embryos, this zone (and its descendent, layer I) was joined by layer V as the laminae with the highest

B

C

MZ cP

sP IZ

D

E

Fig. 4. Laminar distribution of [J25I]SCH23982 binding (191 dopaminergic receptors) in the developing primary motor cortex (area 4). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E93. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E143. This laminar pattern is representative for specimens killed at El07, E115, El20, E128, E143, birth and one month of age. (D) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at four months of age. This laminar pattern is representative for specimens killed at two and four months of age. (E) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at one year of age. This laminar pattern is representative for specimens killed at eight months and one, three and five years of age. Abbreviations as in Fig. 1.

444

Cresyl Violet

M. S. Lidow

A

Binding

B

C

Fig. 5. Laminar distribution of [~251]epidepride binding (D 2 dopaminergic receptors) in the developing prefrontal cortex (area 46). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at EI 15. This laminar pattern is representative for specimens killed at E93, E107, E115 and El20. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed E143. This laminar pattern is representative for specimens killed at E128, E143, birth, one, two, four, and eight months as well as one, three and five years of age. Abbreviations as in Fig. 1.

receptor density (Figs 5, 6, 10). The adult distribution of D 2 receptors was already detected in the prefrontal and somatosensory areas of a specimen killed at E128. At this and all subsequent ages, the highest receptor density was observed in layer V (Figs 5, 6, 10). In the primary visual cortex of E73 embryos, layer V contained the highest density of D2 receptors (Figs 7, 10). At E93, in addition to layer V the marginal zone was also enriched in these receptor

Cresyl Violet

A

Binding

B

sites (Figs 7, 10). In El07, El 15, and E l 2 0 specimens, the third lamina of high receptor density, corresponding to sublayer IVc~, became visible (Figs 7, 10). In the visual cortex of E128 embryo, the highest densities of D: sites were observed in layers I, IVa, IVce and V (Figs 7, 10). The adult receptor distribution in this area was first detected in the E143 specimens. At this and all postnatal ages, layers IVa and V contained the highest densities of D2 receptors (Figs 7, 10).

C

Fig. 6. Laminar distribution of [125I]epidepride binding (D 2 dopaminergic receptors) in the developing somatosensory cortex (area 1). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E 115. This laminar pattern is representative for specimens killed at E93, E107, E115 and El20. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed E143. This laminar pattern is representative for specimens killed at E128, E143, birth, one, two, four and eight months as well as one, three and five years of age. Abbreviations as in Fig. I.

Development of dopaminergic receptors

Cresyl Violet

A

Binding

445

C

B

MZ

CP

SP IZ SV VZ

D

E

1 IV IV

Fig. 7. Laminar distribution of [l:5I]epidepride binding (D 2 dopaminergic receptors) in the developing primary visual cortex (area 17). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E93. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E115 (note that at this age, primary motor cortex displays layer IV that at older ages is no longer visible). This laminar pattern is representative for specimens killed at El07, El 15 and El20. (D) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E 128. (E) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at eight months of age. This laminar pattern is representative for specimens killed at E143, birth, one, two, four and eight months as well as one, three and five years of age. Abbreviations as in Fig. 1.

In the primary m o t o r cortex of E73 specimens, O 2 receptors concentrated in the marginal zone and layer V (Figs 8, 10). In addition to these laminae, the deepest one-third of layer III was also rich in D2 sites at El07, E l l 5 and E l 2 0 (Figs 8, 10). The m o t o r cortex of El28, E143 and newborn specimens had the highest receptor densities in layers I and II, the upper half of layer III, and layer V (Figs 8, 10). The adult pattern, with the highest receptor density exclusively in layer V, was first observed in this area in the one-month-old animals (Figs 8, 10). N o changes in this pattern of receptor distribution were observed in the specimens up to five years of age (Fig. 10). NSC 65/2

F

DISCUSSION

In most cortical areas, the adult distribution o f dopaminergic receptors is achieved prenatally Previous reports showed that in adult rhesus monkeys, D~ and D 2 dopaminergic receptors display specific laminar distributions in multiple areas of the primate cerebral cortex. 1s'28,31'33'34 The present results demonstrate that both D~ and D2 dopaminergic receptors are present in the developing monkey cerebral cortex at least as early as E73, which is the beginning of the second one-third of gestation (in rhesus monkeys, pregnancy lasts 165 days), just after

446

M.S. Lidow

infragranular layers of the cortical plate have been f o r m e d . 16'47'48 It was also found that, with exception of the primary m o t o r region, the mature laminar distribution of D1 and D 2 dopaminergic sites is achieved prenatally in all cortical regions studied. A particularly early maturation was observed in the prefrontal and somatosensory areas, where the adultlike laminar patterns of both DI and O 2 sites can be detected at E128, soon after all cortical neurons assume their final positions 16'47'4s and prior to the maturation of the laminar pattern of G A B A e r g i c and glutamatergic immunoreactivity 56 (Schwartz M. L., personal communication) and the establishment of the adult distribution of thalamocortical and cortico-

Cresyl Violet

A

cortical fibers. 16'49'55 Moreover, the formation of the adult laminar distribution of dopaminergic receptors in these areas precedes that of most other monoaminergic sites. 36'37 In addition, at E128, the adult-like receptor distributions may be observed in the parietal and temporal association and the extrastriate cortical areas (Lidow M. S., unpublished observation). The early achievement of adult-like distributions is also characteristic of dopaminergic innervation in most areas of the primate cerebral cortex. 5'6 Furthermore, the establishment of mature distributions of dopaminergic innervation in some cortical regions, most notably in area 46 of the prefrontal and areas 1, 2 and 3 of the somatosensory cortex, 3'6 closely corresponds

Binding

B

MZ CP

SP

IZ

C

D

I II

Ill

V VI SP IZ Fig. 8. Laminar distribution of [J25I]epidepride binding (D 2 dopaminergic receptors) in the developing primary motor cortex (area 4). (A) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E73. (B) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E 115. This laminar pattern is representative for specimens killed at E93, E 107, E115 and El20. (C) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at E143. This laminar pattern is representative for specimens killed at E128, E143 and birth. (D) The autoradiogram and matching Cresyl Violet section showing the laminar pattern observed at eight months of age. This laminar pattern is representative for specimens killed at one, two, four and eight months as well as one, three and five years of age. Abbreviations as in Fig. 1.

Development of dopaminergic receptors

447

Prefrontal cortex (2) UI

{2) (1X1X1](1)(1) r~moco m (3) III

ilnllllltll Ill

rt

(2)

(2)

(2)

(2)

(3)

et

a.

Ck

I~.

CI.

(1)

(3)

13.

IZ

$omatosensorv cortex

motor cortex

..

J

J

Fig. 9. Diagrammatic representation of the developmental changes in the distribution of [125I]SCH23982 binding (D~ dopaminergic receptors) in the prefrontal, somatosensory, primary motor and primary visual cortex of macaque monkey. Proliferative zones are not shown. Developmental ages studied are plotted on logarithmic scale in days. Above each age mark, the number of animals studied at this age is shown in parentheses. Lines connect binding patterns to the appropriate age marks. Abbreviations as in Fig. I.

to that for dopaminergic receptor sites observed in the present study. Such early and synchronized maturation of laminar distribution of both preand postsynaptic parts of the cortical dopaminergic system may suggest a role for dopaminergic contacts in the development of cortical circuitry. This may provide some clues to why many psychiatric and neurological disorders can be traced to abnormalities in the development of the cortical dopaminergic system. ~1,62,63 It should be mentioned, however, that although the adult distributions of dopaminergic innervation and receptors in the primate cerebral cortex seem to be achieved rather early, a full maturation of the cortical dopaminergic system is completed only near puberty (three years of age). Thus, previous quantitative study of neurotransmitter receptors during postnatal development revealed that the densities of both D] and D2 dopaminergic sites in the monkey cerebral cortex undergo a significant increase from birth to two months of age and then decrease steadily to adult levels for the following three years. 35'36 The concentration of cortical dopamine also undergoes significant changes during several years after birth. 17

Both D~ and D: dopaminergic receptors tend to concentrate in the marginal zone and layer V o f the developing cortical plate In the adult monkey cerebral cortex, D 1 and D 2 dopaminergic receptors have very different distributions. 18'34 On that basis it was speculated that in adult animals, D l and D 2 dopaminergic receptors may be largely associated with different cellular elements of the cortex and are involved in different cortical activities/8 Thus, it was surprising to find significant similarities in the development of both dopaminergic receptor sites. For example, in the prefrontal and somatosensory cortex of E73 specimens, both receptors were present at high density in the marginal zone. Also, in the cortical plate of the prefrontal, somatosensory and visual areas of E93 specimens, D~ and D2 receptors had identical laminar distributions. Finally, although distributions of D 1 and D2 receptors in the primary motor cortex of the E93 specimens did not correspond precisely, high densities of both receptor sites were detectable in layers I and V. These observations may indicate a higher level of interaction between dopaminergic receptor subtypes

448

M. S. Lidow

Prefrontal cort(~x (2) uI

(2) 141

(1X1X1](1)(1) P.Loo=o ¢~ (3) lalttltlll~ ttl

(2)

(2)

(2)

(3)

(1)

(3)

o

o~

el

~omatosensory ¢OrtQX

Primary motor cortex

(2)

w.

I

J

J

J

W=L___I

Primarv visual cortex

I I

I_.L_.__ l__

I. S

Fig. 10. Diagrammatic representation of the developmental changes in the distribution of [~25I]epidepride binding (D 2 dopaminergic receptors) in the prefrontal, somatosensory, primary motor and primary visual cortex of macaque monkey. Abbreviations as in Fig. I.

in the developing compared to the adult cerebral cortex. Indeed, it has been shown that in the developing cortex, D 1 and D 2 dopaminergic receptors may work together in ways lost with maturity. For example, both of them may assume the role of autoreceptors? 9 O~ autoreceptors have never been reported in the cortex of adult animals. ~'59 In general, during development, both D~ and D 2 dopaminergic receptors tend to concentrate in the marginal zone. One can only speculate on the roles played by dopaminergic sites in this lamina. For instance, during cortical formation, all new neurons first arrive at the border between the cortical plate and the marginal zone. 47'48 There, future pyramidal neurons form apical dendritic bouquets which penetrate and then grow within the marginal zone (for review see Ref. 42). Based on the early appearance and high density of monoaminergic, including dopaminergic, innervation in the marginal zone, 6'15 it has been proposed that the above-mentioned dendritic bouquets are formed in response to action of monoaminergic neurotransmitters. 746 As described here, concentration of dopaminergic receptors in the marginal zone allows further elaboration of this hypothesis. Thus, I am proposing that dopaminergic receptors in particular are involved in regulation of the initial growth and ramification of the apical dendritic processes of the cortical pyramidal cells.

That stimulation of dopaminergic receptors can influence outgrowth of neuronal processes has previously been shown in cultures of cortical and retinal neurons. 25'6° In these systems, excitation of the neuronal D2 receptors promotes, while activation of Dt sites inhibits, extension and branching of neurites. The other lamina of the embryonic cerebral cortex rich in dopaminergic receptors, layer V, contains bodies of large pyramidal neurons projecting to subcortical structures (for review see Ref. 13). It is possible, therefore, that both dopaminergic receptors are heavily involved in the regulation of the development of these cortical cells and, thus, are necessary for the establishment of the properly functional cortical output. In this respect, it may be indicative that the pyramidal neurons of layer V become the most severely affected in cerebral cortices developed in the absence of dopaminergic input. 23 D 1 dopaminergic receptors may regulate generation o f cortical cells The implication of dopamine as a neurotrophic factor stemmed from observations that development in the absence of dopaminergic innervation results in reduction of cortical thickness 22 and underdevelopment of cortical neurons. 23 The specific distributions of dopaminergic receptors, particularly of the Dr sites, observed in this study further support a possible

Development of dopaminergic receptors neurotrophic role for dopamine in cortical development. For example, a high concentration of D 1 receptors was detected in the transient ventricular and subventricular proliferative cortical zones. Moreover, the gradient of receptor densities in these zones clearly corresponds to the gradient of cell densities and, thus, the receptors have to be be associated with proliferating and/or newly-formed cortical cells occupying these laminae.8 We also observed that the number of receptors in the ventricular and subventricular zones declined in parallel with subsiding proliferative activity. The ability of dopamine to influence DNA synthesis is well documented. 14,39 However, all previous studies concentrated on the ability of dopamine to arrest DNA synthesis through D 2 receptors. Thus, it is not surprising that proliferative zones are poor in D 2 dopaminergic sites. The results of this study, on the other hand, suggest that dopamine may also stimulate DNA synthesis through D t receptors. The ability to both suppress and stimulate cell division through different receptor subtypes has been documented for other neurotransmitters, notably noradrenaline:5 In addition, it was shown recently that the cell division-stimulating ~ l-adrenergic receptors are present in the proliferative zones of the cerebral wall. 37 Therefore, it is possible that DNA synthesis in the cortical proliferative zones is stimulated by several neurotransmitters through specific receptor subtypes and second messenger cascades.

Factors that may influence distribution of dopaminergic receptor sites While the adult distributions of dopaminergic innervation and receptors correspond rather well34 and are achieved concurrently during development, the early developmental patterns of dopaminergic innervation and receptors do not correspond well. For example, prior to formation of the mature laminar distribution, a high density of both D l and D2 sites can be observed in layer V of most cortical areas. During the same developmental period, this layer is practically devoid of dopaminergic innervation throughout the cortex. 3'6 Layer VI, on the other hand, contains dopaminergic fibers,3'6 but is poor in receptors. This observation indicates that while neurotransmitter-specific innervation may influence the maturation of the laminar distribution of dopaminergic receptors, there is a significant degree of independence between development of pre- and postsynaptic parts of the cortical dopaminergic system. This independence is particularly evident when quantitative developmental changes in the dopamine concentration17 are compared with changes in dopaminergic receptor density. 36 No meaningful correlation between these two parameters can be found. 36 The spatial mismatch between monoamine neurotransmitter innervation and receptors in adult and developing cerebral cortex and other brain structures has been noted in multiple studies, eg 29,31.36The reason

449

for these findings is still unknown. However, the recent immunocytochemical studies of several modulatory neurotransmitter receptors 44'5s in the cerebral cortex showed that the majority of such receptors are not associated with synapses formed by their neurotransmitter-specific fibers. Rather, they are found in asymmetric synapses involved in excitatory amino acid-based transmission. The activation of these modulatory receptors most likely occurs by neurotransmitter release some distance away and diffuses through intracellular space. This may suggest that this "volume" neurotransmission may be widespread in the cortex and, thus, a precise matching of neurotransmitter innervation and receptors is not a requirement for a properly functional modulatory neurotransmitter system. Another cortical input that may conceivably influence the laminar distribution of dopaminergic receptors is thalamocortical and corticocortical innervation. However, distribution of such fibers in the developing and adult cerebral cortex 2'1921,40,49.57is very different from those of either D~ or D 2 receptors, and thus they cannot be directly responsible for the development of cortical dopaminergic sites. This conclusion is also supported by a recent study of monkey visual cortex which, due to an early prenatal binocular enucleation, never received specific thalamic input. The lack of such input produced no abnormalities in the density or distribution of dopaminergic receptors.5 It is interesting that such functionally diverse cortical regions as the prefrontal association and somatosensory cortex, which have very different connections ~9'2~ but similar cytoarchitecture,9 display identical developmental and adult laminar patterns of dopaminergic receptor sites. In contrast, adjacent cortical regions which have different cytoarchitectonic features, such as motor and somatosensory areas, 9 show differential patterns of receptor development, even though the adult receptor distributions in these regions may be very similar. It is possible, therefore, that the areal cytoarchitecture may play a significant role in the determination of the laminar patterns of developing dopaminergic receptors.

Technical note: differences between autoradiograms generated by [J25I]epidepride and [3H]raclopride In the present study, D 2 dopaminergic sites were labeled with the highly selective antagonist [125I]epidepride, which as an iodinated radioligand allowed easy visualization of low density cortical receptors. 24 This radioligand became available very recently, and in most of the previous studies, my colleagues and I used another D 2 antagonist, [3H]raclopride. 18'3°'34-36 Both of these radioligands belong to the same chemical class of substituted benzomidesfl4'28 However, our preliminary comparative study in the adult monkey cerebral cortex showed that they display different laminar patterns of labelingfl8This is particularly evident in the primary visual

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cortex, where [3H]raclopride concentrates predominantly in layer V, while [125I]epidepride, in addition to this layer, labels sublayer IVa. A comparison of postnatal development of D 2 sites previously visualized with [3H]raclopride36 with the data presented in this paper based on [125I]epidepride binding shows no significant difference in most cortical areas. However, in the primary visual cortex, these two radioligands produce very different developmental laminar patterns. Thus, at birth, [3H]raclopride concentrates in the lower one-third of layer III and sublayer IV of this area. Within the next two months, the binding begins to accumulate in layer V and, thereafter, this layer displays binding density far greater than any other cortical layer. In contrast, both sublayer IVa and layer V exhibit a high density of [~25I]epidepride binding from birth to adulthood. While the reason for dissimilarities in the distribution of [125I]epidepride and [3H]raclopride binding in the cerebral cortex is presently unknown, it is possible that this may be due to differences in the selectivity of these two radioligands for various subtypes constituting the D 2 dopaminergic receptor group. 28 Indeed, the preliminary binding studies in a battery of cell cultures expressing individual dopaminergic receptor subtypes (Lidow M. S., unpublished observation) showed that [~25I]epidepride has a high affinity (Ka < 1.0 nM) to all known subtypes of the D 2 recep-

tor class. In contrast, [3H]raclopride displays high affinity (Kd= 0.8-3.0 riM) only t o Dz~longand short) and D3 receptor subtypes. Its affinity for 0 4 receptor subtype is below the level needed for autoradiographic visualization of this site (K d = 300 riM). CONCLUSIONS This study showed that both D~ and D2 dopaminergic receptors are present in multiple areas of the primate cerebral cortex from early stages of its formation. Moreover, DL receptors display a high density in the transient embryonic proliferative zones where cortical neurons are generated. Finally, the adult-like distribution of dopaminergic sites in the primate cerebral plate is achieved prenatally, soon after all cortical neurons assume their final position and prior to the maturation of the laminar pattern of GABAergic and glutamatergic immunoreactivity and the establishment of the adult distribution of thalamocortical and corticocortical fibers. All this suggests that dopaminergic receptors may be involved in the regulation of cortical development. Acknowledgements--The author wishes to thank Dr Pasko

Rakic for encouragement and many helpful suggestions in the preparation of this manuscript. This work was supported by NIH grants PO1-NS22807-06 and P50-MH44866-03.

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