Postnatal development of the dopaminergic system of the striatum in the rat

PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 5 7 5 - 9

Neuroscience Vol. 110, No. 2, pp. 245^256, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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POSTNATAL DEVELOPMENT OF THE DOPAMINERGIC SYSTEM OF THE STRIATUM IN THE RAT J. ANTONOPOULOS,a * I. DORI,a A. DINOPOULOS,a M. CHIOTELLIa and J. G. PARNAVELASb a

Department of Anatomy, School of Veterinary Medicine, University of Thessaloniki, 54006 Thessaloniki, Greece b

Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK

AbstractöThe dopaminergic innervation of the developing caudate^putamen (patches and matrix) and nucleus accumbens (shell and core) of the rat was examined with light and electron microscope immunocytochemistry, using antibodies against dopamine. Light microscopic analysis showed, in accordance with previous studies, that early in life, dopaminergic ¢bers were relatively thick and present throughout the striatum. Their distribution was heterogeneous, showing dense aggregations, the so-called dopamine islands. The pattern of innervation became more uniform during the third postnatal week with most of the dopamine islands no longer detectable. For electron microscopic analysis, parts of the caudate^putamen containing dopamine islands or matrix, and of the nucleus accumbens, from the shell and the core of the nucleus, were selected. This analysis revealed that symmetrical synapses between immunoreactive pro¢les and unlabeled dendritic shafts predominated throughout development but, at the late stages, symmetrical axospinous synapses also became a prominent feature. These ¢ndings indicate that: (1) although the caudate^putamen and the nucleus accumbens have di¡erent connections and functions, they exhibit similar types of dopaminergic synapses, and (2) the relatively late detection of dopaminergic axospinous synapses suggests that the development of the dopaminergic system in the striatum is an active process, which parallels the morphological changes of striatal neurons and may contribute to their maturation. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dopamine, caudate^putamen, nucleus accumbens, basal ganglia, ultrastructure, immunocytochemistry.

(Herkenham and Pert, 1981; Gerfen, 1984; Gerfen and Wilson, 1996). Dopamine (DA)-containing a¡erents show aggregations in the early postnatal striatum, the so-called DA islands, that correspond to patches in the adult (Graybiel, 1984). The dopaminergic innervation of patches is derived from a subset of neurons located in the ventral portion of the compact part of the substantia nigra, whereas the matrix is innervated by the ventral tegmental and retrorubral areas and by the dorsal lamina of the compact part of the substantia nigra (Gerfen et al., 1987; Jimenez-Castellanos and Graybiel, 1987). However, electron microscopic studies in the adult rat have revealed that tyrosine hydroxylase-containing axon terminals in both patches and matrix, despite their di¡erent origins, establish similar types of synapses, i.e., the symmetrical type preferably with dendritic spines and shafts of the medium spiny neurons (Arluison et al., 1984; Bouyer et al., 1984; Freund et al., 1984; Voorn et al., 1986; Kubota et al., 1987; Descarries et al., 1996; Hanley and Bolam, 1997). The ¢rst goal of the present study was to examine when and how this scheme of ultrastructural organization is established during development. This is particularly interesting in view of the fact that the compartmental organization of the striatum is involved in the regulation of the DA input (Gerfen, 1992). The NAc and CP have many common cytological features as well as similar a¡erent and e¡erent connections

The striatum, the major nucleus of the basal ganglia, is comprised of the caudate^putamen (CP) and the nucleus accumbens (NAc), as proposed by Heimer (1978). It is a homogeneous cell mass and consists mainly of medium spiny projection neurons and interneurons (DiFiglia et al., 1976; Bishop et al., 1982). The striatum receives inputs from most areas of the cerebral cortex (Calabresi et al., 1996; Sharpe and Tepper, 1998), the thalamus (Sharpe and Tepper, 1998), the brainstem (Bjo«rklund and Lindvall, 1984; Soghomonian et al., 1989; AstonJone et al., 1995) and the globus pallidus (Beckstead, 1983), and projects to the latter, the entopeduncular nucleus and the substantia nigra (for details see Gerfen and Wilson, 1996). Through these connections with other areas of the brain, the striatum plays an essential role in a wide variety of functions including motivation, attention, reward, and locomotor activity (LeMoal, 1995; Calabresi et al., 1997; Hauber, 1998). The CP is organized in compartments called patches (striosomes) and matrix, identi¢ed on the basis of neurochemical criteria (Graybiel and Ragsdale, 1978; Herkenham and Pert, 1981; Herkenham et al., 1984; Gerfen et al., 1985) and di¡erences in connectivity

*Corresponding author. Tel : +30-31-999874; fax: +30-31-999842. E-mail address: [email protected] (J. Antonopoulos). Abbreviations : CP, caudate^putamen ; DA, dopamine; NAc, nucleus accumbens; P, postnatal day; TBS, Tris-bu¡ered saline. 245

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(Swanson and Cowan, 1975). However, there are also di¡erences in their connectivity. One major di¡erence is that the dopaminergic innervation of the NAc is derived primarily from the A10 group in the ventral tegmental area and that of the CP from the A9 group in the substantia nigra (Beckstead et al., 1979). Another di¡erence is that the NAc receives excitatory innervation from limbic structures such as the prefrontal cortex, hippocampus and amygdala (Swanson and Cowan, 1975; Domesick, 1981; Parent, 1990), whereas the CP receives excitation mainly from other areas of the cortex and the thalamus (Parent, 1990). Given these di¡erences, we sought to compare the development of the dopaminergic system in the CP with that in the NAc. The NAc is subdivided into two parts, the shell and the core (Groenewegen et al., 1989). Behavioral studies have shown that the NAc functions as an interface between the limbic system and the extrapyramidal motor system, with the shell representing the limbic- and the core the motor-associated compartment (Mogenson and Yim, 1981; Heimer et al., 1982). In addition, tyrosine hydroxylase immunoreactivity in the core of the NAc shows more morphological similarities with the CP than with the shell (Zahm, 1992). The third goal of the present study was to compare the DA innervation of the shell with that of the core of the NAc. Our aim then was to examine in detail the development of the DA system in the striatum of the rat and make comparisons between divisions and compartments of this complex area of the brain. More speci¢cally we used DA immunocytochemistry at the light and electron microscopic levels to: (i) describe the ultrastructural features of the dopaminergic synaptic contacts and how these features change with age; (ii) reveal possible variations between patches and matrix of the CP and shell and core in the NAc; (iii) compare the development of the dopaminergic system in the CP with that in the NAc. Resolving these issues is likely to enhance our understanding of the function of the striatum in normal and pathological behavior. A preliminary account of this work has been presented in abstract form (Dinopoulos et al., 2000).

EXPERIMENTAL PROCEDURES

Tissue preparation A total of 27 Wistar albino rats (bred in-house) of the following ages were used in this study: postnatal day 0 (P0; day of birth; n = 5), P4 (n = 2), P7 (n = 5), P14 (n = 5), P21 (n = 5) and P90 (adult; n = 5). All animal experiments were carried out in accordance with the European Communities Directive of 24 November 1986 (86/609/EEC) for the care and use of laboratory animals. All e¡orts were made to minimize animal su¡ering and to reduce the number of animals used. The rats were perfused, under ether anesthesia, through the heart (ages P0^P21) or through a cannula tied into the ascending aorta (adults), ¢rst with a small amount of saline and then with approximately 100^ 500 ml, depending on the age, of a ¢xative solution containing 5% glutaraldehyde in 0.05 M cacodylate bu¡er, pH 7.2. The brains were removed shortly after perfusion, post¢xed in the

same ¢xative for 1^2 h and processed accordingly for light or electron microscope immunocytochemistry. Light microscope immunocytochemistry Two brains from each age group were placed in 30% sucrose in phosphate bu¡er (pH 7.2) overnight for cryoprotection. Frozen sections were cut in the coronal plane and collected in 0.05 M Tris-bu¡ered saline (TBS; pH 7.2). Section thickness varied from 80 Wm in younger animals to 40 Wm in P21 and adult rats. After several washes in TBS, sections were processed for DA immunocytochemistry with the avidin^biotin complex method. The DA antiserum was used at a dilution of 1:1500 in TBS containing 1% sodium metabisul¢te and 0.5% Triton X-100. The DA antiserum, generously provided by Dr. R.M. Buijs (Brain Research Institute, Amsterdam, The Netherlands), was produced in rabbits. The procedures for raising the antiserum and all speci¢city tests have been described by Buijs et al. (1984) and Ge¡ard et al. (1984). In the present study, preincubation of the antiserum with 1035 M puri¢ed synthetic DA blocked all positive staining. However, the use of noradrenaline-adsorbed antiserum revealed no qualitative di¡erence in staining compared with the non-adsorbed DA antiserum. Details of the light and electron microscope immunocytochemical procedures used have been described previously (Papadopoulos et al., 1989; Dinopoulos et al., 1993). Electron microscope immunocytochemistry For electron microscope immunocytochemistry, three brains from each age group, except P4, were cut with a Vibroslice at 40^80 Wm and processed in the same way as the sections for light microscopy, with the exception that Triton X-100 was either omitted from the incubation solution or used at a concentration of 0.1%. Following immunocytochemical staining, sections were placed in 1% OsO4 , stained in 1% aqueous uranyl acetate, dehydrated through an ascending series of ethanols, passed through propylene oxide, and £at-embedded in Araldite. The £at-embedded specimens were ¢rst examined with the light microscope, and then selected parts of the CP containing patches (DA islands) or matrix, as well as selected parts of the NAc from the shell and the core of the nucleus, were removed and remounted on Araldite stubs for thin sectioning in the coronal plane. Here, we use the terms `patches' and `islands' interchangeably. Ultrathin sections were collected on 200-mesh formvar-coated grids, stained with uranyl acetate followed by lead citrate, and examined with a Zeiss electron microscope. Quantitative analysis Counts of synapses involving labeled immunoreactive varicosities were made in three animals of each of the ages examined, namely P0, P7, P14 and P21. Counts were made in single ultrathin sections of the patches or matrix in the CP and of the shell and the core in the NAc. In each subdivision of these areas, 1 mm2 , approximately equivalent to 100 grid squares of a 200-mesh grid, was examined. Immunoreactive varicosities were identi¢ed as round or elongated axon dilations ( s 0.4 Wm in diameter), containing numerous synaptic vesicles and, usually, one or two mitochondria. Synapses formed by labeled immunoreactive varicosities were identi¢ed by the presence of pre- and postsynaptic membrane specializations, a visible synaptic cleft, and the accumulation of synaptic vesicles in the presynaptic pro¢le. This de¢nition of varicosities and synapses was applied through all ages examined and is consistent with our earlier and other investigators' de¢nitions (Freund et al., 1984; Gerfen et al., 1987; Groves et al., 1994; Hanley and Bolam, 1997). In some cases, accumulation of vesicles and synapses was seen along small ( 6 0.2 Wm in diameter) axonal segments. These synapses were excluded from our quantitative analysis. Our aim was to determine, in single ultrathin sections, the percentages of labeled immunoreactive varicosities involved in synaptic contacts at di¡erent stages of development. In order to

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ensure that any £uctuations in the proportions of synapses during development were not due to changes in the sizes of DAimmunoreactive varicosities or of the synaptic appositions, we measured the lengths of a number of randomly selected synaptic junctions from those used in the synaptic counts and also the diameters (long axes) of the DA-immunoreactive varicosities in the same samples. Data are presented as mean þ S.E.M. Furthermore, we converted synaptic frequency from single sections to the whole volume of varicosities by using the stereological formula of Beaudet and Sotelo (1981), which takes into account the mean diameter of the varicosities (D), the length of the synaptic junctions (ls ), and the thickness of the section (t; about 90 nm in this study). The long axis of varicosities was used as the mean diameter D, as this has been suggested to give a reliable estimate of synaptic incidence (Umbriaco et al., 1994).

using Levene's test. The Kruskal^Wallis non-parametric test was applied to evaluate mean di¡erences in case of heterogeneity of variances, instead of Duncan's new multiple range test, used in case of homogeneity. Di¡erences of the synaptic frequency between compartments for a speci¢c area and age or di¡erences of density between areas for a speci¢c age were evaluated using both Mann^Whitney non-parametric test and t-test (Tables 2 and 3). The M2 procedure was also applied to test associations of age and type of synapses (Fig. 5). All analyses were conducted using the statistical software program SPSS for Windows (v. 6.1). Signi¢cance was declared at P90.05, unless otherwise noted.

RESULTS

Extrapolated synaptic frequency …%† ˆ Observed synaptic frequency…%† ls 2 t W ‡ D Z D In order to have a more reliable estimation, the synaptic density was also evaluated. This was determined by dividing the mean number of DA synapses by the total area of tissue examined. DA synapses were classi¢ed as groups of axosomatic, axodendritic, axospinous or total synapses and the density was expressed as number of synapses/1 mm2 . Finally, we recorded the type(s) of all the synapses counted by determining whether they were symmetrical or asymmetrical and identifying the postsynaptic neuronal elements. The aim of the ultrastructural analysis was to provide a description of the ¢ne features of the DA-immunoreactive pro¢les in the CP and the NAc, and a quantitative analysis of the frequency and type(s) of synapses formed by labeled pro¢les during development. Statistical analysis For the statistical evaluation of the results, one-way analysis of variance was performed, with or without transformed data, to determine possible signi¢cant e¡ects of age on the synaptic frequency or density. Variances were tested for homogeneity

247

Light microscopy Examination of the CP and the NAc showed that the pattern of development of the DA innervation and the morphology of ¢bers resembled closely the description given by Voorn et al. (1988). Therefore, only a brief account will be given here. At birth, and during the ¢rst postnatal week, DA ¢bers were present throughout the nuclei, with the highest density in the dorsolateral and ventrolateral CP, along the anterior commissure, and in the shell of the NAc. DA ¢bers in the CP appeared thicker than at older ages and were distributed unevenly, showing aggregations in places, the so-called DA islands (Fig. 1). During the subsequent 2 weeks labeled axons in the CP became more uniformly distributed, whilst most of the DA islands were no longer detectable, with the exception of the dorsolateral portion of the nucleus where some could still be discerned. At the same period, the pattern of DA innervation of the NAc was not altered substantially. In adult animals, the pat-

Fig. 1. Bright-¢eld photomicrograph illustrating DA islands in the CP of the rat at the time of birth. Scale bar = 83 Wm.

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tern of distribution and the density of labeled axons were similar to those observed at P21, with a complete absence of DA islands. Electron microscopy For electron microscopic analysis, sections were selectively taken through the dorsolateral portion of the CP and through the shell and the core of the NAc (Fig. 2). These regions could be clearly distinguished in our DA immunohistochemical preparations (see also Zahm, 1991, 1992). Labeled DA axonal segments and varicosities were observed in ultrathin sections in all parts of the CP and the NAc. The immunoreactive varicosities, as well as the synapses formed by them, showed fairly uniform morphological features in both the CP and the NAc and they will be described together. Their size varied between 0.76 þ 0.12 Wm and 0.93 þ 0.23 Wm in CP and between 0.84 þ 0.29 Wm and 0.90 þ 0.21 Wm in NAc, at all ages examined. Newborn (P0)

Fig. 2. Camera lucida drawing of a coronal section of the rat brain showing the areas of the CP or NAc from which selected parts were taken for the electron microscopic analysis. Abbreviations: ac, anterior commissure ; FStr, fundus striati ; gcc, genu corpus callosum; LS, lateral septum; MS, medial septum; NAcC, nucleus accumbens core ; NAcS, nucleus accumbens shell; Tu, olfactory tubercle; VDB, nucleus of the vertical limb of the diagonal band of Broca. Scale bar = 1 mm.

A substantial number of immunoreactive varicosities (diameter: 0.91 þ 0.23 Wm in CP; 0.86 þ 0.22 Wm in NAc) were found throughout the striatum at the time of birth. They were closely apposed to dendritic shafts and less often to perikarya without any glial processes intervening between them. At times, they formed symmetrical synapses with dendritic shafts (Fig. 3a, b), whilst few axosomatic symmetrical synapses were also encountered. The latter were more common at this age than in older animals (Fig. 5a, b). Synapses with symmetrical membrane specializations were also found along thin labeled axonal segments and presumably are synapses en passant (Freund et al., 1984; Groves et al., 1994). The majority

Table 1. Synaptic frequency of DA varicosities in the developing striatum of the rat Age

P0 P7 P14 P21 P0 P7 P14 P21

Number of varicosities

CP Patch Matrix Patch Matrix Patch Matrix Patch Matrix NAc Shell Core Shell Core Shell Core Shell Core

Number and percent of synapses

1

2

3

T

1

2

3

T

m ´ (%)

e (%)

229 202 123 95 125 106 229 226

175 179 139 100 130 155 198 272

190 226 218 100 116 96 195 160

594 607 480 295 371 357 622 658

57 54 41 19 39 17 43 24

45 43 29 28 34 37 48 40

50 51 67 25 32 25 42 11

152 148 137 72 105 79 133 75

25.64 24.44 28.31 24.33 28.31 21.98 22.24 11.17

77.51 73.87 98.41 84.13 91.29 71.37 62.88 35.50

244 81 207 150 170 180 111 137

186 135 218 176 190 200 220 212

202 152 199 128 200 135 193 171

632 368 624 454 560 515 524 520

73 13 68 35 50 46 13 19

39 24 78 51 60 54 55 35

45 29 57 30 80 34 48 22

157 66 203 116 190 134 116 76

24.38 17.63 32.42 25.24 33.66 25.91 20.52 14.41

73.05 52.73 95.67 75.14 99.76 76.05 71.38 47.12

Counts were made in an area of 1 mm2 of patches or matrix of the CP and shell or core of the NAc in each of three animals at all ages examined. T = total number of varicosities or synapses; m ´ = mean synaptic frequency in single sections ; e = mean synaptic frequency extrapolated from single sections to whole volume of varicosities.

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Fig. 3. DA-immunoreactive axonal varicosities in the patches (a, d) and matrix (b, e) of the CP or in the core (c) of the NAc forming symmetrical (a^d) or asymmetrical (e) synapses (arrows) with dendritic shafts (D) (a^d) and spines (Sp) (e) at birth (a, b) or at P7 (c^e). Scale bar = 0.3 Wm.

of the postsynaptic neurons displayed morphological characteristics, such as soma size (diameter of perikarya 10^20 Wm) and rounded nuclei, typical of medium spiny neurons (DiFiglia et al., 1976; Bishop et al., 1982). Occasionally, DA terminals formed symmetrical synapses on presumptive cholinergic neurons as judged by their size ( s 20 Wm in diameter), abundant granular endoplasmic reticulum, and deeply indented nuclei. However, doublelabeling experiments are needed to establish unequivocally the identity of the DA-receptive neurons. Pro¢les immunopositive for DA were also found in close apposition with blood vessels in both the CP and the NAc. These pro¢les were commonly in direct contact with the basal lamina of the capillaries, but in some cases a thin glial process was interposed between them. Postnatal days 7^21 Abundant immunoreactive varicosities (diameter: 0.90 þ 0.31 Wm in CP and 0.87 þ 0.24 Wm in NAc at

P7; 0.93 þ 0.23 Wm in CP and 0.90 þ 0.21 Wm in NAc at P14; 0.76 þ 0.18 Wm in CP and 0.84 þ 0.29 Wm in NAc at P21) were seen during the second and third postnatal weeks. At the beginning of the second postnatal week the morphological features of the DA synapses formed by these varicosities and the postsynaptic elements involved in these synaptic contacts were similar to those observed in newborn animals (Fig. 3c, d). A few axospinous synapses were seen for the ¢rst time in the CP and the NAc (Fig. 3e). The percentage of immunoreactive pro¢les involved in synaptic contacts was higher in the patches of the CP and in the shell of the NAc than in the matrix and the core of these areas, respectively (Table 1). At P14 the majority of synapses, which in general displayed more mature features than at earlier stages, were axodendritic with symmetrical membrane specializations (Fig. 4a, b, e). The number of axospinous synapses (Figs. 4c and 5a, b) appeared slightly increased. Similar synaptic relationships were observed at the end of the third postnatal week (Fig. 4f), except for a

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Fig. 4. DA-immunoreactive axonal varicosities in the patches (a, b) and matrix (f) of the CP or in the shell (d, e) and core (c) of the NAc forming symmetrical synapses (arrows) with perikarya (P) (f), dendritic shafts (D) (a, b, e) and spines (Sp) (c) at P14 (a^e) or P21 (f). In d, a DA varicosity is in close apposition to a dendritic shaft (arrowhead) arising from the cell body of a large neuron. The nucleus (N) of this neuron is also included in the photomicrograph. This varicosity is shown to form a synapse in a subsequent section (e). Postsynaptic elements also receive asymmetrical synapses (open arrows) from unlabeled axon terminals in c and f. Scale bar = 0.3 Wm (a^c, e, f), 0.5Wm (d).

marked increase of axospinous synapses in both nuclei (Fig. 5a, b). These synapses were even more frequent in adult animals. Immunopositive pro¢les in close apposition to blood vessels were a constant feature throughout development. Quantitative analysis The results of the quantitative analysis of synaptic frequency and density in the developing striatum are

summarized in Tables 1^3. As shown in Table 1, the proportion of labeled varicosities forming synapses extrapolated from single sections to whole volume of varicosities (e) in DA islands increased from 77.51% in newborn animals to 98.41% at P7, then declined slightly to 91.29% at P14 and markedly to 62.88% at P21. In the matrix of the CP, this proportion increased from 73.87% in newborn animals to 84.13% at P7, then declined to 71.37% at P14 and substantially to 35.50% at P21. The corresponding values of the mean synaptic frequency in

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Fig. 5. Histograms illustrating the percentages of DA varicosities that form synapses with somata, dendritic shafts and spines in the CP (a) and NAc (b) of the rat at various postnatal ages. The values represent the means þ S.D. of counts made in three animals at each developmental age. The total number of synapses counted appeared in Table 1.

single sections (m ´ ) were not signi¢cantly di¡erent in the patches of the CP at the ages examined; in the matrix, the same values were not signi¢cantly di¡erent at P0, P7 and P14, but all di¡ered signi¢cantly from the value at P21 (P90.05). Comparing the same values between patches and matrix, at the same postnatal ages, we found that only at P21 was there a signi¢cant di¡erence (P90.05) between them (Table 2). In the shell of the NAc, the proportion (e) increased from 73.05% in newborn animals to 95.67% at P7 and to 99.76% at P14, then declined to 71.38% at P21. In the core of the NAc, the proportion (e) increased from 52.73% in newborn animals to 75.14% at P7 and to 76.05% at P14 and declined to 47.12% by P21 (Table 1). The corresponding values (m ´ ) in the shell of the NAc were not signi¢cantly di¡erent at P0, P7 and P14; the value at P21 was signi¢cantly di¡erent (P90.05) from those at P7 and P14. In the core of the NAc the values at P7 and P14 di¡ered signi¢cantly

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from those at P0 and P21 (P90.05). Comparing the same values between shell and core, at the same postnatal ages, we found that they were signi¢cantly di¡erent (P90.1) at P0, P7 and P14 (Table 2). Thus, the proportion of immunoreactive pro¢les forming synapses was higher in DA islands of the CP and in the shell of the NAc than in the matrix and the core of these nuclei, respectively. Densitometric measurements (see Table 3) of DA synapses from the developing CP showed that their total density (axosomatic, axodendritic and axospinous synapses pooled together) decreased signi¢cantly (P90.05) during the ¢rst two postnatal weeks and increased slightly during the third. The density of axospinous synapses showed a signi¢cant increase (P90.05), whereas the densities of axosomatic and axodendritic synapses decreased signi¢cantly (P90.05). In the NAc, the densities of axospinous and axosomatic DA synapses showed the same trends as in the CP, while the total density and the density of axodendritic synapses increased during the ¢rst two postnatal weeks and declined during the third. These alterations were signi¢cant (P90.05) only for the axodendritic synapses. Comparing the mean values at the same age and for the same groups of synapses between CP and NAc, we found signi¢cant di¡erences (P90.05) between axodendritic synapses at P0, total synapses at P7, and total, axosomatic, and axodendritic synapses at P14 (Table 3). Finally, the M2 procedure revealed that the incidence of the three types of synapses (axosomatic, axodendritic and axospinous) depends signi¢cantly (P90.05) on age in both CP and NAc (Fig. 5). The mean length of DA synaptic junctions was estimated by measuring the lengths of the synaptic active zones in both CP and NAc and was found to be 0.32 þ 0.01 Wm at P0, 0.26 þ 0.01 Wm at P7, 0.30 þ 0.01 Wm at P14 and 0.26 þ 0.01 Wm at P21 in CP and 0.30 þ 0.01 Wm at P0, 0.32 þ 0.01 Wm at P7, 0.33 þ 0.01 Wm at P14 and 0.26 þ 0.01 Wm at P21 in the NAc. These measurements showed that the mean length of DA synaptic junctions remained virtually unchanged during development.

DISCUSSION

The role of the dopaminergic innervation in the developing striatum Neurogenesis in the striatum of the rat begins at embryonic day 12 and continues throughout embryonic life (Phelps et al., 1989; Bhide, 1996; Sheth and Bhide, 1997). Medium spiny neurons found in patches begin to di¡erentiate at embryonic day 12, followed by those in the matrix (Bayer, 1984; Van der Kooy and Fishell, 1987). They all undergo a prolonged postnatal maturation (Tepper et al., 1998). As these cells are the main targets of DA a¡erents, their development and maturation may be closely related to alterations of their DA innervation. Here, we found that during the ¢rst two postnatal weeks, the postsynaptic targets of DA inputs in both the CP and the NAc are mainly dendrites

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Table 2. Comparative analysis of synaptic frequency of DA varicosities in single sections through the CP (patches and matrix) and the NAc (shell and core) of the rat at di¡erent postnatal ages Age

P0 P7 P14 P21

CP

NAc

Patches

Matrix

Shell

Core

25.64 þ 0.41aA 28.31 þ 3.79aA 28.31 þ 1.50aA 22.24 þ 1.01aA

24.44 þ 1.22aA 24.33 þ 2.33aA 21.98 þ 3.03aA 11.17 þ 2.29bB

24.38 þ 2.79abA 32.42 þ 2.07bA 33.66 þ 3.22bA 20.52 þ 4.40aA

17.63 þ 0.87aB * 25.24 þ 1.86bB * 25.91 þ 0.55bB * 14.41 þ 1.08aA

Values are expressed as means þ S.E.M. Mean values in the same column without a superscript in common (a b) are signi¢cantly di¡erent (P90.05). Mean values in the same row and for the same area without a superscript in common (A B) are signi¢cantly di¡erent (P90.05); *P90.10.

The maturation of the DA system in the striatum parallels morphological changes of striatal neurons, as was discussed above, and other developmental events that occur in this area during postnatal life. At birth, a substantial number of DA axons are present throughout the striatum and a substantial number of synapses have already been formed. DA ¢bers exhibit substantial alterations in their morphology and density before they establish a pattern of distribution similar to that seen in adults. Early in postnatal life, the proportion of DA varicosities forming synapses increases gradually to reach a peak at the end of the ¢rst postnatal week in CP and the second postnatal week in NAc. This increase coincides with high glial cell line-derived neurotrophic factor immunoreactivity, which has been suggested to regulate the DA innervation of the striatum (LopezMartin et al., 1999). At the end of the third postnatal week, the proportion of DA varicosities forming synapses showed a marked decline. Immunoblot analysis in this period of development showed an abrupt increase of the striatal-enriched protein tyrosine phosphatase, which is thought to play a regulatory role in striatal cell maturation and synaptogenesis (Okamura et al., 1997). It should be mentioned that the total density of DA and other types of synapses peak around the beginning (CP) or the end (NAc) of the ¢rst postnatal week. This shows a di¡erent trend for the DA synapses when

(over 80%) and cell somata. In this period, the spines of the medium spiny neurons have not yet been formed (Chronister et al., 1976; Trent and Tepper, 1993; Tepper et al., 1998). During the second postnatal week, few axospinous DA synapses were found but, in the third week, they increased to as much as 12% of the total number of DA synapses. Medium spiny neurons show the highest density of spines at this time (Trent and Tepper, 1993; Tepper et al., 1998). These ¢ndings suggest that the development of the DA system in the striatum is an active process that parallels morphological changes of striatal neurons. In addition, the fact that DA axons contact di¡erent parts of the postsynaptic neurons, depending on the age, appears to be closely related to the state of maturation of the DA neuron itself. This notion is based on evidence showing that the majority of newly formed synapses by grafted DA neurons in the striatum of the rat and the monkey are with dendritic shafts and somata of striatal neurons (Freund et al., 1985; Leranth et al., 1998). These ¢ndings suggest that developing and transplanted animals exhibit the same type of synapses in contrast to adult animals. It seems likely that the DA inhibition (Siggins, 1978; Herrling and Hull, 1980) acts on cell somata or proximal dendrites and, thus, can more e¡ectively block the excitatory signals arising from the cortex and the thalamus (Sharpe and Tepper, 1998).

Table 3. Density of DA synapses in the developing striatum of the rat Age

CP Total density of DA synapses Density of axosomatic synapses Density of axodendritic synapses Density of axospinous synapses NAc Total density of DA synapses Density of axosomatic synapses Density of axodendritic synapses Density of axospinous synapses

P0

P7

P14

P21

50.00 þ 2.16aA 5.83 þ 1.07aA 44.16 þ 2.12aA ^

34.83 þ 7.07abA 2.66 þ 0.55bA 32.00 þ 7.63abA 0.16 þ 0.15a

30.66 þ 3.37bA 0.66 þ 0.33bcA 28.50 þ 2.99bA 1.50 þ 0.34bA

36.00 þ 5.90abA 2.00 þ 0.51cA 26.16 þ 4.88bA 7.83 þ 1.62cA

37.16 þ 8.51aA 8.33 þ 3.20aA 28.83 þ 5.59aB ^

53.16 þ 7.57aB 4.83 þ 1.49abA 48.33 þ 6.40bA ^

54.00 þ 6.30aB 2.00 þ 0.36bB 48.33 þ 5.56bB 3.66 þ 1.25aA

32.00 þ 6.88aA 1.00 þ 0.44bA 25.66 þ 6.43aA 5.33 þ 0.95aA

Values are mean þ S.E.M. numbers of DA synapses/1 mm2 counted in each of three animals at all ages examined. Mean values in the same row without a superscript in common (a b c) are signi¢cantly di¡erent (P90.05). Mean values at the same age and for the same group of synapses without a superscript in common (A B) are signi¢cantly di¡erent (P90.05).

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Dopaminergic innervation of the developing striatum

compared to that of the general population of synapses. Indeed, Hattori and McGeer (1973) found that the greatest increase in the density of synapses in the striatum occurred between P13 and P17, whereas the density of symmetrical synapses reached a peak by P17. These data indicate that the process of synapse formation by DA axons in the striatum shows exuberance during development, as was shown previously for the lateral septum of the rat (Antonopoulos et al., 1997). The formation of such synapses suggests that DA may play a role in developmental events in brain areas it innervates. It has been suggested that DA, apart from its function as a neurotransmitter, plays an important role in growth cone motility, target cell selection and speci¢city of synapse formation (Spencer et al., 1998). In addition, DA neurons have been shown to contain various neurotrophins, including brain-derived neurotrophic factor and neurotrophin 3 (Seroogy et al., 1994), which may play an important role in spine survival (Ingham et al., 1993). In conclusion, the establishment of dopaminergic synapses with neurons in the CP (mainly the medium spiny neurons) and in the NAc corresponds to morphological and functional changes of these neurons and may contribute to their maturation. These ¢ndings add to the accumulating information about the developmental and functional role of these inhibitory a¡erents in both the neonatal and adult striatum. Finally, as Tepper et al. (1998) stated, ``characterizing the di¡erences in the physiology and morphology of neostriatal neurons among neonates and adults, and the time course of the maturation of these neurons is important not only for determining how the functional organization of the adult neostriatum comes about, but may also have relevance to interpreting the results of physiological and morphological studies of neostriatal grafts and how these di¡er from the normal intact striatum''. Dopaminergic innervation of the striatum: changes during development and comparisons between CP and NAc and their subdivisions We showed here that although there are substantial changes in the density of axodendritic and axospinous synapses, the total density of DA synapses in the CP and NAc remained fairly constant during the developmental period examined. Comparison of these results with previous studies shows that DA synapses undergo similar changes as those of the general population (Sharpe and Tepper, 1995; Tepper et al., 1998) of symmetrical synapses. Thus, the presumably inhibitory DA inputs develop early in accordance with the early maturation of the total inhibitory circuits of the striatum, as was suggested by electrophysiological studies (Sharpe and Tepper, 1995). We demonstrated that the proportion of DA varicosities making synapses decreases after P7 in the CP and P14 in the NAc. This change may be attributed to alterations in the size of varicosities or the length of the synaptic junctions. Our data show that, while there was a decrease in the mean size of DA varicosities (diameter) between P14 and P21 in CP and NAc, the mean length of

253

DA synaptic junctions remained virtually unchanged during development. These data suggest that the di¡erence in the proportion of synapses between P14 and P21 may be even greater than that suggested in Table 1. This reduction in the proportion of DA varicosities making synapses might suggest a reduced in£uence of DA in the mature striatum. However, the total synaptic density remained relatively constant during development. In fact, since the volume of the striatum in rats and mice increases signi¢cantly during development (Fentress et al., 1981), the absolute number of DA synapses shows a proportional increase. The shell and core are clearly distinct subdivisions of the NAc as they give rise to di¡erent e¡erent connections (Heimer et al., 1991) and are also distinguished in DA (Voorn et al., 1986) and tyrosine hydroxylase (Zahm, 1991) immunohistochemical preparations (see also Introduction). In addition, earlier studies (Zahm, 1992) have shown that the core of the NAc resembles the neostriatum whereas the shell does not. However, our ¢ndings show that, at least during development, DA terminals form similar types of synapses and have similar postsynaptic targets in the shell and the core of the NAc as well as in the patch and matrix of the CP. These ¢ndings suggest that the DA innervation of the striatum is essentially the same in the CP and NAc as well as in their subdivisions in conjunction with their cytoarchitectonic similarities. However, the accumulation of DA ¢bers in patches, the higher synaptic incidence of DA varicosities in this subdivision of the CP than in the matrix at all ages examined, and the signi¢cant di¡erence between them at P21 (22.24% in patches; 11.17% in matrix; see Table 2) may suggest that DA exerts its action(s) mainly through the patches during development. This pattern of dopaminergic innervation is di¡erent from that of the lateral septum. We have described two di¡erent DA inputs in the lateral septum (Antonopoulos et al., 1997). The ¢rst develops earlier in life and, through symmetrical axodendritic synapses, a¡ects remote parts of neurons, whereas the second develops later and through asymmetrical axosomatic synapses a¡ects neuronal somata. These data and the present results indicate that the dopaminergic system shows area speci¢city in the developing brain. Dopaminergic regulation of blood £ow in the developing striatum DA axons have been shown to contact blood vessels in the retina (Favard et al., 1990), the dorsal root ganglia (Weil-Fugazza et al., 1993) and the cerebral cortex (Krimer et al., 1998). Our ¢ndings show that, in addition to those areas, DA-immunopositive axons are in close apposition to blood vessels, at all postnatal ages examined, in both CP and NAc, and suggest a possible vasomotor function of DA and a role in the regulation of local blood £ow from the early stages of postnatal development. Other substances that have been suggested to play a role in local cerebral blood £ow in the striatum include neuropeptide Y, calcitonin gene-related peptide, arginine-vasopressin, substance P and the cate-

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cholamines isoproterenol, epinephrine and norepinephrine (Sercombe et al., 1975; Michel et al., 1986; Van Zwieten et al., 1988; Suzuki et al., 1989). The interaction of all these chemical substances upon the wall of the striatal capillaries is of particular interest since regional changes in blood £ow may alter the functional state of this area of the brain and particularly the shell of the NAc, which is probably implicated in the etiology of

psychiatric disorders such as psychosis, addiction and major depression (Heimer et al., 1997). AcknowledgementsöWe wish to thank Dr. R.M. Buijs for the DA antiserum, Dr. Ch. Batzios for help with the statistical analysis and Mrs. A. Tsipinia for secretarial help. This work was supported by a grant from the Hellenic Ministry of Development (PENED, 1995).

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