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Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase
Developmental Brain Research, 57 (1990) 43-53 Elsevier
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Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase Carlos Ifiiguez 1, Joaquin De Juan 1, Ali A1-Majdalawi 2 and Manuel J. Gayoso 2 IDepartarnento de Histologia, Universidad de Alicante, Alicante (Spain) and 2Departamento de Biologia Celular, Universidad de Valladolid, Valladolid (Spain) (Accepted 17 July 1990) Key words: Basal ganglia; Caudate nucleus; Striatum; Wheat germ agglutinin-horseradish peroxidase transport; Postnatal development
This paper deals with the postnatal development of afferent and efferent connections of the rat striatum as revealed by the transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). Tracer was injected weekly from birth to the end of the first postnatal month in the head of the caudate nucleus. To control for transport from cortical areas contaminated by the micropipette, injections in newborn rats were made by either vertical or lateral penetrations. In addition some newborn and 14-day-old animals were injected only in the cortex. The results showed that at birth there was retrograde transport to the thalamus, substantia nigra and raphe nuclei. Labelling in the cortex was seen at birth but was probably due to cortical contamination. Transport from the striatum was clearly established on day 7, when a few labelled neurons were observed on both the ipsi and contralateral sides. These neurons increased in number and were distributed through layers III to VI by day 14. At this time labelled cell bodies were observed in the claustrum and lateral amygdaloid nucleus as well as in the globus pallidus and entopeduncular nucleus. On day 21 the contralateral labelling of the lateral amygdaloid nucleus was apparent. The anterograde transport from the striatum to globus paUidus, entopeduncular nucleus and substantia nigra was already visible at birth although its intensity increased during the first postnatal month. INTRODUCTION The rat striatum is an essential c o m p o n e n t of the basal ganglia. This area is sometimes referred to as the dorsal striatum since the nucleus accumbens septi and some areas of the olfactory tubercle are also considered to be striatal structures and constitute the ventral striatum 19. In this p a p e r we will refer to the so-called dorsal striatum as the striatum. O u r morphological knowledge of the connectivity of the basal ganglia has increased with i m p r o v e m e n t of tract-tracing techniques, together with identification of n e u r o t r a n s m i t t e r s by immunocytochemistry. This has also led to a b e t t e r understanding of the role p l a y e d by the striatum in the functioning of the brain 2'x6"19. T h r e e inputs (cortical, thalamic and nigrostriatal) have been classically considered as the main source of afferents to the striatum. The whole neocortex projects to the striatum in a topographically o r d e r e d m a n n e r 27"5z'53 and glutamate is the c o m m o n l y accepted n e u r o t r a n s m i t t e r for these afferents 14'43. Neurons from the amygdaloid complex that project to the striatum release somatostatin, cholecystokinin (CCK) and p e r h a p s vasoactive intestinal p e p t i d e (VIP) 32"39, whereas neurons from the claustrum and the a d j a c e n t piriform cortex release C C K 32. The
thalamic p r o j e c t i o n is also t o p o g r a p h i c a l l y organized and originates mainly in the i n t r a l a m i n a r nuclei, although some of its fibres arise from the ventral, posterior and midline neuronal groups 4'49'52. A n o t h e r group of diencephalic afferents comes from the globus pallidus and e n t o p e d u n c u l a r nucleus 24"36'44'48 but the neuroactive substances used by these two pathways have not been elucidated. The main afferents from the brain-stem originate in neuronal groups A9, A10 and A 8 of D a h l s t r 6 m and Fuxe 9 located in the pars c o m p a c t a of the substantia nigra, t e g m e n t a l ventral area and r e t r o r u b r a l field respectively. All are d o p a m i n e r g i c 3"47'51 although a small n o n - d o p a m i n e r g i c c o m p o n e n t has been found 46"5°. In addition, a serotoninergic p r o j e c t i o n reaches the striatum from the raphe nuclei, which also has a non-serotoninergic c o m p o n e n t 16'33'45. The a f o r e m e n t i o n e d afferents end mainly in the ipsilateral striatum, but bilateral projections from the cortex and the m e s e n c e p h a l o n have been described 6"1s'3°'54"55. The efferents from the striatum are directed towards the substantia nigra, globus pallidus and nucleus e n t o p e d u n c u l a r i s (globus pallidus lateralis and medialis in primates) 12'a7"41. All these pathways use y - a m i n o b u t y r i c acid ( G A B A ) as the neur o t r a n s m i t t e r but the coexistence of different peptides
Correspondence: C. Ifiiguez, Departamento de Histologia, Apdo. Correos 374, E-03080 Alicante, Spain. 0165-3806/90/$03.51) © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
44 has been d e t e c t e d in them. Thus, enkephalin is present in the pallidal projection 8"~° whereas substance P and dynorphin have been associated with the fibres that end in the nucleus entopeduncularis and the pars reticulata of the substantia nigra 37.
Giemsa to facilitate the identification of brain structures 2~'. Pairs of adjacent sections, one showing the HRP reaction and the other counterstained, were drawn using a camera lucida attached to a microscope under dark- and bright-field illumination.
In contrast with data from adults, knowledge about the d e v e l o p m e n t of these pathways is scarce. Most of these studies have been carried out in the cat, an animal which is born with fairly m a t u r e striatal connections and, according to both electrophysiological and morphological results, the afferents from the cortex, thalamus and midbrain as well as the efferents from the caudate are present and functional at birth 1"7"28"34'35. The results
RESULTS
o b t a i n e d in kittens with wheat germ a g g l u t i n i n - h o r s e radish peroxidase ( W G A - H R P ) transport showed that there is no postnatal addition of afferents to the striatum and the extent of the axonal arborizations is relatively similar in neonates and adults 13. With respect to this, the lack of d a t a in the rat is a gap to be filled in our k n o w l e d g e since this animal is very commonly used in neuroscience and its striatal d e v e l o p m e n t could be s o m e w h a t different to the cat as h a p p e n s in other areas of the brain. The aim of the present work was therefore to establish w h e t h e r all the striatal connections in the rat transport H R P at birth and to describe their postnatal d e v e l o p m e n t and maturation. The results are c o m p a r e d with d a t a from o t h e r animals.
MATERIALS AND METHODS Albino Wistar rats, ages 1, 7, 14, and 21 days, in groups of four plus two adults, were used for the experiments. The animals received WGA-HRP injections in the head of the caudate, chosen to diminish the risk of contamination on non-striatal structures. All the injections were made via vertical penetrations except for newborn animals in which both vertical and lateral penetrations were made because brain immaturity at this age allows WGA-HRP diffusion to adjacent cortical areas. Nevertheless, as some contamination along the trajectory of the micropipette could not be avoided, two additional rats received cortical injections on days 1 and 14 to compare the transport of the tracer with those injected in the striatum. The stereotaxic coordinates for adult animals were calculated from the atlas of KOnig and Klippe125, and corrected for the different ages according to Skinner's formula42. The animals were anaesthetized with Equithesin (3 ml/kg, i.p.) before being placed in a stereotaxic frame. A 1% aqueous solution of WGA-HRP complex (Sigma) was pressure-injected through a micropipette of 10 /~m calibre at the tip, fitted to a 1/~1 Hamilton syringe. The doses were increased according to the age of the animals (0.025, 0.035, 0.05, 0.1 and 0.15/d on days 1, 7, 14, 21 and adults, respectively). The survival times were 24 h for animals aged 1-14 days and 48 h for the others. After this period, the rats were anaesthetized and transcardially perfused for 2 min with saline solution, followed by 2.5% buffered glutaraldehyde for 1 h and 5% buffered sucrose for another 1/2 h. The brains were dissected and left overnight in 25% buffered sucrose at 4 °C. Frozen sections 40/~m thick were serially cut in a sliding microtome and the histochemical reaction for WGA-HRP carried out by the tetramethylbenzidine (TMB) procedure 31. One out of two sections was counterstained with
The results we describe are from rats whose injections were centered in the head of the striatum, although even in these cases some H R P diffusion was always present, especially in n e w b o r n and 7-day-old rats. The diffusion caused some staining in the cortex and corpus callosum along the intracerebral track of the micropipette, and sometimes it r e a c h e d the nucleus accumbens septi. All the descriptions are based on at least two animals p e r age group.
Retrograde transport of HRP A f t e r injecting W G A - H R P in the striatum of n e w b o r n rats, there was considerable cortical c o n t a m i n a t i o n which m a d e it difficult to decide if the cortical cell bodies were labelled by W G A - H R P t r a n s p o r t e d from the striatum or from the c o n t a m i n a t e d zones o f the cortex. W h e n a vertical a p p r o a c h was used, the tracer diffused ,to the prefrontal cortex whereas a lateral p e n e t r a t i o n contaminated an area of the parietal cortex next to the injection site. A f t e r the two kind of injections there was a lack of coincidence in the cortical labelling but labelled somata were always l o c a t e d n e a r the m i c r o p i p e t t e track and also symmetrically to this a r e a on the contralateral side (Fig. 1A,B). W h e n the tracer was injected into the cortex alone, the p a t t e r n of cortical labelling (Fig. 1C) was similar to that found after vertical injections into the striatum thus suggesting that the labelling came from the cortex rather than from the striatum. H o w e v e r , the labelling of these neuronal bodies was r e d u c e d to a few grains of p e r o x i d a s e - r e a c t i o n p r o d u c t scattered into the cytoplasm. A t this age, r e t r o g r a d e t r a n s p o r t was clearly o b s e r v e d in the thalamus (Fig. 1). T h e i n t r a l a m i n a r group of nuclei in particular parafascicular, central m e d i a l and paracentral nuclei - - were the most intensely labelled structures after the injection of peroxidase into the striatum. In addition, s o m e neurons located in the ventral (ventro lateral, ventro p o s t e r i o r complex and ventro medial nuclei) and midline nuclear groups also transp o r t e d W G A - H R P , but the n u m b e r of labelled cells and the intensity of the labelling were lower than those observed in the intralaminar group. There were some differences in the topography of the thalamic labelling according to which approach was used for the injections. When a vertical approach was used, some neurons of the mediodorsal nucleus appeared labelled, and the labelling of -
-
45
C
Fig. 1. Semi-schematic drawings of frontal sections from newborn rat brains showing anterograde (.) and retrograde (e) transport after injections of WGA-HRP in the striatum by vertical (A) or lateral (B) penetrations and also after injection restricted to the cortex (C). Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
both the ventral and midline groups was more intense than with lateral injections of label. Injections of W G A H R P in the cortex resulted in less labelling of the thalamus. Most stain was seen in the parafascicular nucleus and the ventral group whereas only a few neurons were labelled in the midline nuclear group. In all cases the neurons s e e m e d quite i m m a t u r e with r o u n d s o m a t a and they lacked dendritic trees (Fig. 2A). The a m o u n t of tracer inside the neuronal bodies was less than in the following weeks. The most intense r e t r o g r a d e transport to the brain stem was seen in the substantia nigra. In newborns the
distinction b e t w e e n pars c o m p a c t a and reticulata is difficult to m a k e partly because the myelination is not yet complete. Intense axonal t r a n s p o r t was c o n c e n t r a t e d in the ventrolateral part of the substantia nigra and small labelled cell bodies, with p o o r l y d e v e l o p e d dendritic trees could be clearly distinguished in the medial zones of this area (Fig. 2B). In contrast, n e u r o n s labelled in the raphe nuclei were few and the a m o u n t of tracer r e t r o g r a d e l y t r a n s p o r t e d was scarce (Fig. 2C). T h e labelling p a t t e r n of the brain stem was quite similar regardless of the injection approach. A n i m a l s that received cortical injections alone did not transport tracer to the brain stem. A t
46
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N
c
Fig. 3. Semi-schematic drawings from brain sections of a 7-day-old rat showing anterograde (.) and retrograde (e) transport after injecting WGA-HRP in the head of the caudate nucleus. Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
Fig. 2. Dark-field micrographsshowingperoxidase labellingof thalamus (A), substantia nigra (13)and raphe nuclei(C) in areas correspondingto the insetsof Fig. IA. Bar = 100/~m.
this age the neurons of the substantia nigra were the most intensely stained followed by those of the raphe nuclei and the thalamic ones. At the end of the first week, in addition to some cortical labelling around the micropipette path, there were labelled somata in regions of the ipsilateral ricocortex fairly distant from the injection tract in both the medial-lateral and rostro-caudal directions (Fig. 3). These observations indicate, perhaps, the beginning of retrograde transport from the striatum. Labelled neurons in the cortex were located predominantly in layers III and
47
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I( i I
A
I
B
Fig. 4. Semi-schematic drawings of brain sections from 14-day-old rats showing anterograde (.) and retrograde (e) transport after injecting W G A - H R P in the head of the caudate nucleus (A) and cortex (B) by a vertical approach. Bar = l mm. (See abbreviation list in legend Fig. 7.)
48 V but some stained neurons were in layer VI. Most were pyramidal neurons of medium or large size. in the contralateral cortex, the labelling extended through symmetrical areas but smaller than in the ipsilateral side (Fig. 3). Labelled cells on the contralateral side were less numerous than on the ipsilateral side and they were restricted to layers III and V. The nuclear groups labelled in the thalamus were the same as in newborn rats, but the intensity of the labelling was higher. Furthermore, the number and maturity of labelled cells were greater and the nuclei more clearly delimited. The intralaminar nuclei, in particular the central medial and the parafascicular nuclei, exhibited the strongest reaction. Some nuclei of the ventral group, the ventrolateral and the ventroposterior, showed moderate transport of peroxidase, whereas only a few labelled neurons were found in the lateral group. At the mesencephalic level, the substantia nigra exhibited the strongest labelling (Fig. 3). The neuronal bodies were mainly concentrated in the pars compacta of the substantia nigra, which by now is clearly differentiated from the pars reticulata. Some neurons were located in the ventral tegmentai area and the retrorubral field. Transport to the raphe nuclei was still quite weak (Fig. 3). Two weeks after birth, the peroxidase injections affected the rostral 2/3 of the striatum and the cortex surrounding the injection path. The most striking observation at this age was the retrograde labelling of the neocortex, which was greatly increased in both hemispheres. The ipsilateral labelling extended from the midline to the basal aspects of the neocortex whereas on the contralateral side it was less intense and restricted to more rostral regions (Fig. 4A). The labelled neurons in the medial and rostral zones of the ipsilateral cortex were situated in layers III, V and VI (Fig. 5A) whereas they were located mainly in layer V in the posterior and lateral regions. Some peroxidase positive neurons were observed for the first time in the lateral amygdaloid nucleus of the ipsilateral side (Fig. 4A). At this age, neuronal cell bodies were clearly labelled for the first time in the globus pallidus and nucleus entopeduncularis (Fig. 5B). The labelling of the thalamus was more intense in the parafascicular and the central medial nuclei than in the ventral group, where the transport was unevenly distributed and affected mainly the ventromedial, ventrolateral and ventroposterior nuclei. In some animals, retrograde transport to the posterior nuclear group was also observed. The neurons of the substantia nigra were intensely filled with W G A - H R P , as were some neuronal bodies located in the tegmental ventral area and the retrorubral field. These neurons showed morphological features of adult animals. The cells seen in the caudal linear and dorsal raphe nuclei were located in the
Fig. 5. Dark-field micrographs showing peroxidase labelling of the ipsilateral cortex (A), entopeduncular nucleus (B) and raphe nuclei (C) in areas corresponding to the insets of Fig. 4A. Neuronal bodies in the entopeduncular nucleus are pointed by arrows. Bar = 100pm.
49
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Dr
Fig. 6. Semi-schematic drawings from brain sections of a 21-day-old rat showing anterograde (.) and retrograde (e) transport after injecting W G A - H R P in the head of the caudate nucleus. Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
f
Fig. 7. Dark-field micrographs showing peroxidase labelling of the ipsilateral cortex (A), lateral amygdaloid nucleus (B), and raphe nuclei (C) in areas corresponding to the insets of Fig. 6. Bar = 100 #m. Ac, accumbens nucleus; Dr, dorsal raphe nuclei; IC, internal capsule; It, intralaminar thalamic nuclei; La, lateral amygdaloid nucleus; Pf, parafascicular nucleus; Pt, pyramidal tract; SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticulata; Vt, ventral thalamic nuclei.
51) dorso-lineal and medio-caudal nuclei (Figs. 4A and 5C). The animals which received injections in the cortex exhibited transport of peroxidase into areas surrounding the injection site and in symmetrically situated areas on the contralateral side. Some of the nuclei which were labelled in the thalamus, such as the mediodorsal, parafascicular and ventroposterior, were the same as those that were stained following striatal injections. In these animals, there was no significant retrograde transport either to the substantia nigra or the raphe (Fig. 4B). The injections performed from day 21 onward (Fig. 6), were centered in the head of the striatum and only the overlying cortex showed contamination. The cortical hemispheres were clearly labelled, but with more staining on the ipsi- than on the contralateral side (Figs. 6 and 7A). The lateral amygdaloid nucleus was the most densely labelled structure of the amygdaloid complex (Figs. 6 and 7B) and some labelled neurons were observed for the first time in the contralateral nucleus. A great number of neurons were now densely filled by the W G A - H R P in the substantia nigra, retrorubral field and ventral tegmental area. At this age a few labelled neurons were seen in the contralateral side. Well-defined retrograde labelling was also observed in dorsal and caudal linear raphe nuclei (Figs. 6 and 7C).
Anterograde transport of HRP At birth, anterograde transport from the striatum (Fig. 1) was present in the three main projection areas - globus pallidus, entopeduncular nucleus and substantia nigra. The most relevant changes observed during the first postnatal month (Figs. 3, 4 and 6) were quantitative, with an increase in both the area covered by W G A - H R P filled axons and by the intensity of the transport inside them. Anterograde labelling probably transported from cortical areas was seen in the thalamus and along the corticospinal tract. DISCUSSION
Cortico-striatal projections The labelling of neuronal somata observed in the ipsiand contralateral cortices of newborn animals is suggestive of transport from cortical regions rather than from the striatum for the following reasons: First, the location of the labelled areas differed depending on whether a penetration was made vertical or lateral although the tracer was presumably injected into the same area of the caudate nucleus. Second, there was lack of anterograde transport to the striatum after peroxidase injection into the cortex. Third, contralateral labelling was symmetrical and proportional to the amounts of W G A - H R P deposited in the cortex when the cortex was deliberately injected.
As transport from the striatum to the cortex was not clearly observed in our experiments until the end of the first week we conclude that the cortico-striatal pathway is established around this time. In support of this conclusion is the report that glutamatergic receptors in rat striatum are not mature until the 7th postnatal day; and prior to this age the amounts of glutamate, [3H]kainate binding and [3H]glutamate uptake are very small in the striatum and the striatal neurons are almost insensitive to kainate, a toxic agent that binds to glutamate receptors 5. The labelled areas described in our results correspond to those previously found to project to the head of the caudate nucleus 27,52'53 whereas areas that preferentially project to the ventral striatum, such as the subicutum TM, were not labelled. The extension of the cortical areas labelled on the contralateral hemisphere was always less than on the ipsilateral side. This is in agreement with the reported existence of bilateral projections from prelimbic, motor and cingulate areas but only ipsilateral from somatosensory and visual cortices 11. The labelling of other areas such as the claustrum and the amygdala was also observed on the 14th postnatal day, giving further support to the idea that the connections between the telencephalon and the striatum are established in the rat during the second postnatal week. The amygdaloid projection to the striatum is not as dense as the projection to the accumbens, but it is clear in the rat and cat that it is restricted to the ventral half and medial edge of the caudate nucleus 23'26'3s'40. Thus the labelling observed in our experiments may be reinforced by some W G A - H R P diffusion to the accumbens. With regard to the position of labelled neurons in the cortex, at the end of the second week these cells were grouped in a wide band that extended between layers II and VI with a maximal density in layers III to V. On the contralateral side, there was a similar distribution but the number and intensity of labelled cells were smaller. This arrangement remained almost unchanged in older animals and agrees with previous reports from adult rats 6, and cats 13. Nevertheless, the possibility of some extrastriatal contamination being responsible for the labelling of cortical cells in layers other than layer V cannot be completely ruled out.
Reciprocal connections between pallidum and striatum As noted in the Introduction, the equivalent of the primate pallidum in rodents is divided into the globus pallidus and the nucleus entopeduncularis. This division is based not only on topography but also on functional differences. Thus, the nucleus entopeduncularis, together with the pars reticulata of the substantia nigra, is considered to be an important efferent pathway from the striatum to other structures of diencephalon and brain
51 stem, whereas the globus pallidus represents the first station of an indirect circuit that reaches the nucleus entopeduncularis and the nigra through the subthalamic nucleus 2. It would be interesting to know if the striatal connections with these two zones have a different timing of maturation. Our observations showed that the efferents from the striatum to the globus pallidus and entopeduncular nucleus were present already at birth whereas the pallidal afferents to the striatum were only distinguished from the 14th postnatal day onwards. At this time, labelled cell bodies could be seen in the periphery of the entopeduncular nucleus (which were not labelled when only cortical injections of W G A - H R P were made), therefore confirming the existence of an entopedunculostriatal pathway described by Takada and Hattori some years ago 4s. These neurons seem to innervate both the striatum and the lateral habenula, and it has been suggested that they may play a role in integrating motor and limbic functions at the basal ganglia level. The interpretation of labelled neuronal cell bodies in the globus pallidus of the youngest animals was made difficult by the fact that there is intense anterograde transport as well as transport caused by contamination from the striatum, making it impossible to decide whether there is any retrograde transport before the second postnatal week. Thalamo-striatal projection Our results showed clearly that the thalamic afferents to the striatum are well established from birth, in particular those to the intralaminar group which is considered to be the main source of afferents. Labelling was also present in neuronal somata located in lateral, ventral and midline groups. Nevertheless, as the extension and the precision of the injections were influenced by both the growth of the caudate head and the colateralization of some neurons that project simultaneously to cortex and striaturn 22, our data do not reflect the maturation of these projections with an absolute topographical accuracy and must therefore be considered as a preliminary description. A complete description requires a more specific study.
REFERENCES 1 Adinolfi, A.M., Fisher, R.S., Levine, M.S., Hull, C.D. and Buchwald, N.A., Postnatal ontogeny of connectivityin the basal ganglia of the cat: afferents of the substantia nigra and ventral tegmental area, Soc. Neurosci. Abstr., 8 (1982) 964. 2 Albin, R.L., Young, A.B. and Penney, J.B., The functional anatomy of basal ganglia disorders, Trends Neurosci., 12 (1989) 366-375. 3 Anden, N.E., Carlsson, A., Dahlstr6m, A., Fuxe, K., Hillarp, N.A. and Larsson, K., Demonstration and mapping out of nigroneostriatal dopamine neurons, Life Sci., 3 (1964) 523-530. 4 Beckstead, R.M., The thalamostriatal projection in the cat, J. Comp. Neurol., 223 (1984) 313-346.
Connections between mesencephalic structures and striatum The feed-back circuit established between striatum and substantia nigra (pars compacta and adjacent region of ventral tegmental area), is already present at birth since both anterograde and retrograde transport was evident in our experiments at this age. The anterograde labelling of the pars reticulata and neighboring ventral tegmental area was also quite clear and intense at birth, indicating that the connectivity between the striatum and the mesencephalon probably starts in the prenatal period as suggested earlier by several authors using fluorescence and immunohistochemical methods 1'29. Retrograde transport to the raphe nuclei was seen in a small amount at birth and increased during the following weeks. It will be interesting to know if the cells transporting peroxidase to the raphe nuclei at birth are those of the non-serotoninergic component, which was proposed to be dopaminergic ~6. This would confirm a sequential development in the innervation of the striatum from the brain stem, as suggested by previous histofluorescence studies 29.
In summary, our results provide a picture of the development of striatal connectivity in rats that is in contrast with that depicted in cats and monkeys, in which all the connections are present at birth 13'21. In the rat, only subcortical connections were detected in the newborn animal, and clearly labelled neuronal bodies were found in thalamus, nigra and raphe nuclei. By the end of the first week cortical projections began transporting peroxidase, while the maturation of areas previously innervated continued. The second week was featured by spectacular increment in retrograde transport to the cortex and the maturation of some important loops as demonstrated by the labelling of cell bodies in the pallido-entopeduncular complex and also by an increase in perikaryal size and dendritic tree expansion in the neurons of other nuclei. By the third week, the connectivity patterns were similar to that of the adult.
Acknowledgements. This research was supported by CAICYT Grants PB 86/0338 and PB 86/0276.
5 Campochiaro, P. and Coyle, J.T., Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2025-2029. 6 Cospito, J.A., Kultas-Ilinsky, K., Synaptic organization of motor corticostriatal projections in the rat, Exp. Neurol., 72 (1981) 257-266. 7 Cospito, J.A., Levine, M.S. and Adinolfi, A.M., The organization of developing precruciate corticostriate projections in kittens, Exp. Neurol., 67 (1980) 447-452. 8 CueUo, A.C. and Paxinos, G., Evidence for a long leuenkephalin striopallidal pathways in rat brain, Nature, 271 (1978) 178-180. 9 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I.
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53 49 Van der Kooy, D., The organization of the thalamic, nigral and raphe cells projecting to the medial vs lateral caudate-putamen in rat. A fluorescent retrograde double labeling study, Brain Res., 169 (1979) 381-387. 50 Van der Kooy, D., Coscina, D.V. and Hattori, T., Is there a non-dopaminergic nigrostriatal pathway?, Neuroscience, 6 (1981) 345-357. 51 Vandermaelen, C.P., Kocsis, J.D. and Kitai, S.T., Caudate afferents from the retrorubral nucleus and other midbrain areas in the cat, Brain Res. Bull., 3 (1978) 639-644. 52 Veening, J.G., Cornelissen, F.M. and Lieven, P.A.J.M., The
topical organization of the afferents to the caudatoputamen of the rat. A horseradish peroxidase study, Neuroscience, 5 (1980) 1233-1268. 53 Webster, K.E., Cortico-striate interrelations in the albino rat, J. Anat., 95 (1961) 532-544. 54 Wilson, C.J., Morphology and synaptic connections of crossed corticostriatal neurons in the rat, J. Comp. Neurol., 263 (1987) 567-580. 55 Wilson, C.J., Postsynaptic potentials evoked in spiny neostriatal projection neurons by stimulation of ipsilateral and contralateral neocortex, Brain Res., 367 (1986) 201-213.
43
BRESD 51171
Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase Carlos Ifiiguez 1, Joaquin De Juan 1, Ali A1-Majdalawi 2 and Manuel J. Gayoso 2 IDepartarnento de Histologia, Universidad de Alicante, Alicante (Spain) and 2Departamento de Biologia Celular, Universidad de Valladolid, Valladolid (Spain) (Accepted 17 July 1990) Key words: Basal ganglia; Caudate nucleus; Striatum; Wheat germ agglutinin-horseradish peroxidase transport; Postnatal development
This paper deals with the postnatal development of afferent and efferent connections of the rat striatum as revealed by the transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). Tracer was injected weekly from birth to the end of the first postnatal month in the head of the caudate nucleus. To control for transport from cortical areas contaminated by the micropipette, injections in newborn rats were made by either vertical or lateral penetrations. In addition some newborn and 14-day-old animals were injected only in the cortex. The results showed that at birth there was retrograde transport to the thalamus, substantia nigra and raphe nuclei. Labelling in the cortex was seen at birth but was probably due to cortical contamination. Transport from the striatum was clearly established on day 7, when a few labelled neurons were observed on both the ipsi and contralateral sides. These neurons increased in number and were distributed through layers III to VI by day 14. At this time labelled cell bodies were observed in the claustrum and lateral amygdaloid nucleus as well as in the globus pallidus and entopeduncular nucleus. On day 21 the contralateral labelling of the lateral amygdaloid nucleus was apparent. The anterograde transport from the striatum to globus paUidus, entopeduncular nucleus and substantia nigra was already visible at birth although its intensity increased during the first postnatal month. INTRODUCTION The rat striatum is an essential c o m p o n e n t of the basal ganglia. This area is sometimes referred to as the dorsal striatum since the nucleus accumbens septi and some areas of the olfactory tubercle are also considered to be striatal structures and constitute the ventral striatum 19. In this p a p e r we will refer to the so-called dorsal striatum as the striatum. O u r morphological knowledge of the connectivity of the basal ganglia has increased with i m p r o v e m e n t of tract-tracing techniques, together with identification of n e u r o t r a n s m i t t e r s by immunocytochemistry. This has also led to a b e t t e r understanding of the role p l a y e d by the striatum in the functioning of the brain 2'x6"19. T h r e e inputs (cortical, thalamic and nigrostriatal) have been classically considered as the main source of afferents to the striatum. The whole neocortex projects to the striatum in a topographically o r d e r e d m a n n e r 27"5z'53 and glutamate is the c o m m o n l y accepted n e u r o t r a n s m i t t e r for these afferents 14'43. Neurons from the amygdaloid complex that project to the striatum release somatostatin, cholecystokinin (CCK) and p e r h a p s vasoactive intestinal p e p t i d e (VIP) 32"39, whereas neurons from the claustrum and the a d j a c e n t piriform cortex release C C K 32. The
thalamic p r o j e c t i o n is also t o p o g r a p h i c a l l y organized and originates mainly in the i n t r a l a m i n a r nuclei, although some of its fibres arise from the ventral, posterior and midline neuronal groups 4'49'52. A n o t h e r group of diencephalic afferents comes from the globus pallidus and e n t o p e d u n c u l a r nucleus 24"36'44'48 but the neuroactive substances used by these two pathways have not been elucidated. The main afferents from the brain-stem originate in neuronal groups A9, A10 and A 8 of D a h l s t r 6 m and Fuxe 9 located in the pars c o m p a c t a of the substantia nigra, t e g m e n t a l ventral area and r e t r o r u b r a l field respectively. All are d o p a m i n e r g i c 3"47'51 although a small n o n - d o p a m i n e r g i c c o m p o n e n t has been found 46"5°. In addition, a serotoninergic p r o j e c t i o n reaches the striatum from the raphe nuclei, which also has a non-serotoninergic c o m p o n e n t 16'33'45. The a f o r e m e n t i o n e d afferents end mainly in the ipsilateral striatum, but bilateral projections from the cortex and the m e s e n c e p h a l o n have been described 6"1s'3°'54"55. The efferents from the striatum are directed towards the substantia nigra, globus pallidus and nucleus e n t o p e d u n c u l a r i s (globus pallidus lateralis and medialis in primates) 12'a7"41. All these pathways use y - a m i n o b u t y r i c acid ( G A B A ) as the neur o t r a n s m i t t e r but the coexistence of different peptides
Correspondence: C. Ifiiguez, Departamento de Histologia, Apdo. Correos 374, E-03080 Alicante, Spain. 0165-3806/90/$03.51) © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
44 has been d e t e c t e d in them. Thus, enkephalin is present in the pallidal projection 8"~° whereas substance P and dynorphin have been associated with the fibres that end in the nucleus entopeduncularis and the pars reticulata of the substantia nigra 37.
Giemsa to facilitate the identification of brain structures 2~'. Pairs of adjacent sections, one showing the HRP reaction and the other counterstained, were drawn using a camera lucida attached to a microscope under dark- and bright-field illumination.
In contrast with data from adults, knowledge about the d e v e l o p m e n t of these pathways is scarce. Most of these studies have been carried out in the cat, an animal which is born with fairly m a t u r e striatal connections and, according to both electrophysiological and morphological results, the afferents from the cortex, thalamus and midbrain as well as the efferents from the caudate are present and functional at birth 1"7"28"34'35. The results
RESULTS
o b t a i n e d in kittens with wheat germ a g g l u t i n i n - h o r s e radish peroxidase ( W G A - H R P ) transport showed that there is no postnatal addition of afferents to the striatum and the extent of the axonal arborizations is relatively similar in neonates and adults 13. With respect to this, the lack of d a t a in the rat is a gap to be filled in our k n o w l e d g e since this animal is very commonly used in neuroscience and its striatal d e v e l o p m e n t could be s o m e w h a t different to the cat as h a p p e n s in other areas of the brain. The aim of the present work was therefore to establish w h e t h e r all the striatal connections in the rat transport H R P at birth and to describe their postnatal d e v e l o p m e n t and maturation. The results are c o m p a r e d with d a t a from o t h e r animals.
MATERIALS AND METHODS Albino Wistar rats, ages 1, 7, 14, and 21 days, in groups of four plus two adults, were used for the experiments. The animals received WGA-HRP injections in the head of the caudate, chosen to diminish the risk of contamination on non-striatal structures. All the injections were made via vertical penetrations except for newborn animals in which both vertical and lateral penetrations were made because brain immaturity at this age allows WGA-HRP diffusion to adjacent cortical areas. Nevertheless, as some contamination along the trajectory of the micropipette could not be avoided, two additional rats received cortical injections on days 1 and 14 to compare the transport of the tracer with those injected in the striatum. The stereotaxic coordinates for adult animals were calculated from the atlas of KOnig and Klippe125, and corrected for the different ages according to Skinner's formula42. The animals were anaesthetized with Equithesin (3 ml/kg, i.p.) before being placed in a stereotaxic frame. A 1% aqueous solution of WGA-HRP complex (Sigma) was pressure-injected through a micropipette of 10 /~m calibre at the tip, fitted to a 1/~1 Hamilton syringe. The doses were increased according to the age of the animals (0.025, 0.035, 0.05, 0.1 and 0.15/d on days 1, 7, 14, 21 and adults, respectively). The survival times were 24 h for animals aged 1-14 days and 48 h for the others. After this period, the rats were anaesthetized and transcardially perfused for 2 min with saline solution, followed by 2.5% buffered glutaraldehyde for 1 h and 5% buffered sucrose for another 1/2 h. The brains were dissected and left overnight in 25% buffered sucrose at 4 °C. Frozen sections 40/~m thick were serially cut in a sliding microtome and the histochemical reaction for WGA-HRP carried out by the tetramethylbenzidine (TMB) procedure 31. One out of two sections was counterstained with
The results we describe are from rats whose injections were centered in the head of the striatum, although even in these cases some H R P diffusion was always present, especially in n e w b o r n and 7-day-old rats. The diffusion caused some staining in the cortex and corpus callosum along the intracerebral track of the micropipette, and sometimes it r e a c h e d the nucleus accumbens septi. All the descriptions are based on at least two animals p e r age group.
Retrograde transport of HRP A f t e r injecting W G A - H R P in the striatum of n e w b o r n rats, there was considerable cortical c o n t a m i n a t i o n which m a d e it difficult to decide if the cortical cell bodies were labelled by W G A - H R P t r a n s p o r t e d from the striatum or from the c o n t a m i n a t e d zones o f the cortex. W h e n a vertical a p p r o a c h was used, the tracer diffused ,to the prefrontal cortex whereas a lateral p e n e t r a t i o n contaminated an area of the parietal cortex next to the injection site. A f t e r the two kind of injections there was a lack of coincidence in the cortical labelling but labelled somata were always l o c a t e d n e a r the m i c r o p i p e t t e track and also symmetrically to this a r e a on the contralateral side (Fig. 1A,B). W h e n the tracer was injected into the cortex alone, the p a t t e r n of cortical labelling (Fig. 1C) was similar to that found after vertical injections into the striatum thus suggesting that the labelling came from the cortex rather than from the striatum. H o w e v e r , the labelling of these neuronal bodies was r e d u c e d to a few grains of p e r o x i d a s e - r e a c t i o n p r o d u c t scattered into the cytoplasm. A t this age, r e t r o g r a d e t r a n s p o r t was clearly o b s e r v e d in the thalamus (Fig. 1). T h e i n t r a l a m i n a r group of nuclei in particular parafascicular, central m e d i a l and paracentral nuclei - - were the most intensely labelled structures after the injection of peroxidase into the striatum. In addition, s o m e neurons located in the ventral (ventro lateral, ventro p o s t e r i o r complex and ventro medial nuclei) and midline nuclear groups also transp o r t e d W G A - H R P , but the n u m b e r of labelled cells and the intensity of the labelling were lower than those observed in the intralaminar group. There were some differences in the topography of the thalamic labelling according to which approach was used for the injections. When a vertical approach was used, some neurons of the mediodorsal nucleus appeared labelled, and the labelling of -
-
45
C
Fig. 1. Semi-schematic drawings of frontal sections from newborn rat brains showing anterograde (.) and retrograde (e) transport after injections of WGA-HRP in the striatum by vertical (A) or lateral (B) penetrations and also after injection restricted to the cortex (C). Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
both the ventral and midline groups was more intense than with lateral injections of label. Injections of W G A H R P in the cortex resulted in less labelling of the thalamus. Most stain was seen in the parafascicular nucleus and the ventral group whereas only a few neurons were labelled in the midline nuclear group. In all cases the neurons s e e m e d quite i m m a t u r e with r o u n d s o m a t a and they lacked dendritic trees (Fig. 2A). The a m o u n t of tracer inside the neuronal bodies was less than in the following weeks. The most intense r e t r o g r a d e transport to the brain stem was seen in the substantia nigra. In newborns the
distinction b e t w e e n pars c o m p a c t a and reticulata is difficult to m a k e partly because the myelination is not yet complete. Intense axonal t r a n s p o r t was c o n c e n t r a t e d in the ventrolateral part of the substantia nigra and small labelled cell bodies, with p o o r l y d e v e l o p e d dendritic trees could be clearly distinguished in the medial zones of this area (Fig. 2B). In contrast, n e u r o n s labelled in the raphe nuclei were few and the a m o u n t of tracer r e t r o g r a d e l y t r a n s p o r t e d was scarce (Fig. 2C). T h e labelling p a t t e r n of the brain stem was quite similar regardless of the injection approach. A n i m a l s that received cortical injections alone did not transport tracer to the brain stem. A t
46
J
l ~
N
c
Fig. 3. Semi-schematic drawings from brain sections of a 7-day-old rat showing anterograde (.) and retrograde (e) transport after injecting WGA-HRP in the head of the caudate nucleus. Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
Fig. 2. Dark-field micrographsshowingperoxidase labellingof thalamus (A), substantia nigra (13)and raphe nuclei(C) in areas correspondingto the insetsof Fig. IA. Bar = 100/~m.
this age the neurons of the substantia nigra were the most intensely stained followed by those of the raphe nuclei and the thalamic ones. At the end of the first week, in addition to some cortical labelling around the micropipette path, there were labelled somata in regions of the ipsilateral ricocortex fairly distant from the injection tract in both the medial-lateral and rostro-caudal directions (Fig. 3). These observations indicate, perhaps, the beginning of retrograde transport from the striatum. Labelled neurons in the cortex were located predominantly in layers III and
47
l /
I( i I
A
I
B
Fig. 4. Semi-schematic drawings of brain sections from 14-day-old rats showing anterograde (.) and retrograde (e) transport after injecting W G A - H R P in the head of the caudate nucleus (A) and cortex (B) by a vertical approach. Bar = l mm. (See abbreviation list in legend Fig. 7.)
48 V but some stained neurons were in layer VI. Most were pyramidal neurons of medium or large size. in the contralateral cortex, the labelling extended through symmetrical areas but smaller than in the ipsilateral side (Fig. 3). Labelled cells on the contralateral side were less numerous than on the ipsilateral side and they were restricted to layers III and V. The nuclear groups labelled in the thalamus were the same as in newborn rats, but the intensity of the labelling was higher. Furthermore, the number and maturity of labelled cells were greater and the nuclei more clearly delimited. The intralaminar nuclei, in particular the central medial and the parafascicular nuclei, exhibited the strongest reaction. Some nuclei of the ventral group, the ventrolateral and the ventroposterior, showed moderate transport of peroxidase, whereas only a few labelled neurons were found in the lateral group. At the mesencephalic level, the substantia nigra exhibited the strongest labelling (Fig. 3). The neuronal bodies were mainly concentrated in the pars compacta of the substantia nigra, which by now is clearly differentiated from the pars reticulata. Some neurons were located in the ventral tegmentai area and the retrorubral field. Transport to the raphe nuclei was still quite weak (Fig. 3). Two weeks after birth, the peroxidase injections affected the rostral 2/3 of the striatum and the cortex surrounding the injection path. The most striking observation at this age was the retrograde labelling of the neocortex, which was greatly increased in both hemispheres. The ipsilateral labelling extended from the midline to the basal aspects of the neocortex whereas on the contralateral side it was less intense and restricted to more rostral regions (Fig. 4A). The labelled neurons in the medial and rostral zones of the ipsilateral cortex were situated in layers III, V and VI (Fig. 5A) whereas they were located mainly in layer V in the posterior and lateral regions. Some peroxidase positive neurons were observed for the first time in the lateral amygdaloid nucleus of the ipsilateral side (Fig. 4A). At this age, neuronal cell bodies were clearly labelled for the first time in the globus pallidus and nucleus entopeduncularis (Fig. 5B). The labelling of the thalamus was more intense in the parafascicular and the central medial nuclei than in the ventral group, where the transport was unevenly distributed and affected mainly the ventromedial, ventrolateral and ventroposterior nuclei. In some animals, retrograde transport to the posterior nuclear group was also observed. The neurons of the substantia nigra were intensely filled with W G A - H R P , as were some neuronal bodies located in the tegmental ventral area and the retrorubral field. These neurons showed morphological features of adult animals. The cells seen in the caudal linear and dorsal raphe nuclei were located in the
Fig. 5. Dark-field micrographs showing peroxidase labelling of the ipsilateral cortex (A), entopeduncular nucleus (B) and raphe nuclei (C) in areas corresponding to the insets of Fig. 4A. Neuronal bodies in the entopeduncular nucleus are pointed by arrows. Bar = 100pm.
49
(
::
I
!
Dr
Fig. 6. Semi-schematic drawings from brain sections of a 21-day-old rat showing anterograde (.) and retrograde (e) transport after injecting W G A - H R P in the head of the caudate nucleus. Bar = 1 mm. (See abbreviation list in legend Fig. 7.)
f
Fig. 7. Dark-field micrographs showing peroxidase labelling of the ipsilateral cortex (A), lateral amygdaloid nucleus (B), and raphe nuclei (C) in areas corresponding to the insets of Fig. 6. Bar = 100 #m. Ac, accumbens nucleus; Dr, dorsal raphe nuclei; IC, internal capsule; It, intralaminar thalamic nuclei; La, lateral amygdaloid nucleus; Pf, parafascicular nucleus; Pt, pyramidal tract; SNc, substantia nigra, pars compacta; SNr, substantia nigra, pars reticulata; Vt, ventral thalamic nuclei.
51) dorso-lineal and medio-caudal nuclei (Figs. 4A and 5C). The animals which received injections in the cortex exhibited transport of peroxidase into areas surrounding the injection site and in symmetrically situated areas on the contralateral side. Some of the nuclei which were labelled in the thalamus, such as the mediodorsal, parafascicular and ventroposterior, were the same as those that were stained following striatal injections. In these animals, there was no significant retrograde transport either to the substantia nigra or the raphe (Fig. 4B). The injections performed from day 21 onward (Fig. 6), were centered in the head of the striatum and only the overlying cortex showed contamination. The cortical hemispheres were clearly labelled, but with more staining on the ipsi- than on the contralateral side (Figs. 6 and 7A). The lateral amygdaloid nucleus was the most densely labelled structure of the amygdaloid complex (Figs. 6 and 7B) and some labelled neurons were observed for the first time in the contralateral nucleus. A great number of neurons were now densely filled by the W G A - H R P in the substantia nigra, retrorubral field and ventral tegmental area. At this age a few labelled neurons were seen in the contralateral side. Well-defined retrograde labelling was also observed in dorsal and caudal linear raphe nuclei (Figs. 6 and 7C).
Anterograde transport of HRP At birth, anterograde transport from the striatum (Fig. 1) was present in the three main projection areas - globus pallidus, entopeduncular nucleus and substantia nigra. The most relevant changes observed during the first postnatal month (Figs. 3, 4 and 6) were quantitative, with an increase in both the area covered by W G A - H R P filled axons and by the intensity of the transport inside them. Anterograde labelling probably transported from cortical areas was seen in the thalamus and along the corticospinal tract. DISCUSSION
Cortico-striatal projections The labelling of neuronal somata observed in the ipsiand contralateral cortices of newborn animals is suggestive of transport from cortical regions rather than from the striatum for the following reasons: First, the location of the labelled areas differed depending on whether a penetration was made vertical or lateral although the tracer was presumably injected into the same area of the caudate nucleus. Second, there was lack of anterograde transport to the striatum after peroxidase injection into the cortex. Third, contralateral labelling was symmetrical and proportional to the amounts of W G A - H R P deposited in the cortex when the cortex was deliberately injected.
As transport from the striatum to the cortex was not clearly observed in our experiments until the end of the first week we conclude that the cortico-striatal pathway is established around this time. In support of this conclusion is the report that glutamatergic receptors in rat striatum are not mature until the 7th postnatal day; and prior to this age the amounts of glutamate, [3H]kainate binding and [3H]glutamate uptake are very small in the striatum and the striatal neurons are almost insensitive to kainate, a toxic agent that binds to glutamate receptors 5. The labelled areas described in our results correspond to those previously found to project to the head of the caudate nucleus 27,52'53 whereas areas that preferentially project to the ventral striatum, such as the subicutum TM, were not labelled. The extension of the cortical areas labelled on the contralateral hemisphere was always less than on the ipsilateral side. This is in agreement with the reported existence of bilateral projections from prelimbic, motor and cingulate areas but only ipsilateral from somatosensory and visual cortices 11. The labelling of other areas such as the claustrum and the amygdala was also observed on the 14th postnatal day, giving further support to the idea that the connections between the telencephalon and the striatum are established in the rat during the second postnatal week. The amygdaloid projection to the striatum is not as dense as the projection to the accumbens, but it is clear in the rat and cat that it is restricted to the ventral half and medial edge of the caudate nucleus 23'26'3s'40. Thus the labelling observed in our experiments may be reinforced by some W G A - H R P diffusion to the accumbens. With regard to the position of labelled neurons in the cortex, at the end of the second week these cells were grouped in a wide band that extended between layers II and VI with a maximal density in layers III to V. On the contralateral side, there was a similar distribution but the number and intensity of labelled cells were smaller. This arrangement remained almost unchanged in older animals and agrees with previous reports from adult rats 6, and cats 13. Nevertheless, the possibility of some extrastriatal contamination being responsible for the labelling of cortical cells in layers other than layer V cannot be completely ruled out.
Reciprocal connections between pallidum and striatum As noted in the Introduction, the equivalent of the primate pallidum in rodents is divided into the globus pallidus and the nucleus entopeduncularis. This division is based not only on topography but also on functional differences. Thus, the nucleus entopeduncularis, together with the pars reticulata of the substantia nigra, is considered to be an important efferent pathway from the striatum to other structures of diencephalon and brain
51 stem, whereas the globus pallidus represents the first station of an indirect circuit that reaches the nucleus entopeduncularis and the nigra through the subthalamic nucleus 2. It would be interesting to know if the striatal connections with these two zones have a different timing of maturation. Our observations showed that the efferents from the striatum to the globus pallidus and entopeduncular nucleus were present already at birth whereas the pallidal afferents to the striatum were only distinguished from the 14th postnatal day onwards. At this time, labelled cell bodies could be seen in the periphery of the entopeduncular nucleus (which were not labelled when only cortical injections of W G A - H R P were made), therefore confirming the existence of an entopedunculostriatal pathway described by Takada and Hattori some years ago 4s. These neurons seem to innervate both the striatum and the lateral habenula, and it has been suggested that they may play a role in integrating motor and limbic functions at the basal ganglia level. The interpretation of labelled neuronal cell bodies in the globus pallidus of the youngest animals was made difficult by the fact that there is intense anterograde transport as well as transport caused by contamination from the striatum, making it impossible to decide whether there is any retrograde transport before the second postnatal week. Thalamo-striatal projection Our results showed clearly that the thalamic afferents to the striatum are well established from birth, in particular those to the intralaminar group which is considered to be the main source of afferents. Labelling was also present in neuronal somata located in lateral, ventral and midline groups. Nevertheless, as the extension and the precision of the injections were influenced by both the growth of the caudate head and the colateralization of some neurons that project simultaneously to cortex and striaturn 22, our data do not reflect the maturation of these projections with an absolute topographical accuracy and must therefore be considered as a preliminary description. A complete description requires a more specific study.
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Connections between mesencephalic structures and striatum The feed-back circuit established between striatum and substantia nigra (pars compacta and adjacent region of ventral tegmental area), is already present at birth since both anterograde and retrograde transport was evident in our experiments at this age. The anterograde labelling of the pars reticulata and neighboring ventral tegmental area was also quite clear and intense at birth, indicating that the connectivity between the striatum and the mesencephalon probably starts in the prenatal period as suggested earlier by several authors using fluorescence and immunohistochemical methods 1'29. Retrograde transport to the raphe nuclei was seen in a small amount at birth and increased during the following weeks. It will be interesting to know if the cells transporting peroxidase to the raphe nuclei at birth are those of the non-serotoninergic component, which was proposed to be dopaminergic ~6. This would confirm a sequential development in the innervation of the striatum from the brain stem, as suggested by previous histofluorescence studies 29.
In summary, our results provide a picture of the development of striatal connectivity in rats that is in contrast with that depicted in cats and monkeys, in which all the connections are present at birth 13'21. In the rat, only subcortical connections were detected in the newborn animal, and clearly labelled neuronal bodies were found in thalamus, nigra and raphe nuclei. By the end of the first week cortical projections began transporting peroxidase, while the maturation of areas previously innervated continued. The second week was featured by spectacular increment in retrograde transport to the cortex and the maturation of some important loops as demonstrated by the labelling of cell bodies in the pallido-entopeduncular complex and also by an increase in perikaryal size and dendritic tree expansion in the neurons of other nuclei. By the third week, the connectivity patterns were similar to that of the adult.
Acknowledgements. This research was supported by CAICYT Grants PB 86/0338 and PB 86/0276.
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