Transient patterns of calbindin-D28k expression in the ...

Neuroscience Letters 220 (1996) 211–214

Transient patterns of calbindin-D28k expression in the developing striatum of man Kresˇimir Letinic´*, Ivica Kostovic´ Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Sˇalata 11, 10 000 Zagreb, Republic of Croatia Received 14 October 1996; revised version received 15 November 1996; accepted 15 November 1996

Abstract In fetal and adult human brains, calbindin immunoreactivity (CB-ir) of neostriatal neuropil showed inhomogeneous pattern. A mosaic of CB-ir neuropil patches matching the acetylcholinesterase (AChE)-reactive patches and most of the encapsulated cell-dense islands was distributed in a lighter stained background matrix. During infancy, the pattern of CB expression changed from one of CB-rich patches to one of CB-poor striosomes and rich matrix. Furthermore, we observed a steady development of population of medium-sized neurons in the striatal matrix; in addition, a transient CB-expression was found in cells of the ganglionic eminence and presumably in a subset of striatal interneurons.  1996 Elsevier Science Ireland Ltd. All rights reserved Keywords: Calcium binding protein; Acetylcholinesterase; Basal ganglia; Human

The main feature of the mammalian neostriatum is the compartmentalization of neuroactive substances and afferent and efferent connections into patch and matrix regions [11]. Calcium-binding protein calbindin (CB) is selectively expressed by medium-sized projection neurons and neuropil of the striatal matrix [7,8,11]. Calcium-binding proteins are important for the control of intracellular calcium [2]; bearing in mind the important roles calcium ions play in various aspects of neuronal development [2,19] we examined patterns of calbindin immunoreactivity (CB-ir) in the human neostriatum during prenatal and postnatal life. The postmortem brain tissue, without signs of neurological disorders, was obtained from six fetuses (postovulatory ages 12–28 weeks), two infants (newborn and 18 month) and two young adults (10 and 15 years) at the autopsy (postmortem time 6–12 h) and fixed by immersion for 12–24 h in 4% paraformaldehyde in phosphate buffer. Coronal 90 mm thick frozen sections were processed by the peroxidase immunochemistry and avidin-biotin technique (ABC; Vectastain Co.). We used the monoclonal anti-calbindin-D28K antibody (Sigma); for control we omitted primary antibody. acetylcholinesterase (AChE) * Corresponding author. Tel.: +385 1 466815; fax: +385 1 271195.

histochemistry was described previously [16]. The comparisons of CB and AChE-reactive compartments [13–15] and cellular islands [14,15] was made on pairs or triplets of adjacent sections. At 12 weeks of gestation (wg), the striatal anlage appeared devoid of CB-ir. In older fetuses (16–40 wg), transient CB-expression occurred in the ganglionic eminence (Fig. 1). Furthermore, CB-antibody apparently identified two groups of neurons in the fetal striatum; numerous small to medium-sized (somal diameter 4-12 mm), relatively lightly stained neurons in various stages of maturation (Fig. 2E–F), and much less numerous, larger (10–16 mm in diameter) and more darkly-stained neurons with long ramified processes (Fig. 2G–H). In all fetuses, neurons of the later group were phenotypically distinct and usually larger than the more common CB-ir cells. We also noted the absence of such cells in the postnatal life. First CB-ir cells were distributed in the dorsolateral putamen, however, by 25 wg they were spread throughout the caudoputamen. Furthermore, at 16–40 wg, a mosaic of highly reactive patches of neuropil (dimensions 200–500 mm) distributed in a lighter stained matrix appeared throughout the caudoputamen (Fig. 3B). CB-ir cell bodies were aligned with the zones of low CB (Fig. 2A–C). A striking match was found between the CB-ir and AChE-reactive

0304-3940/96/$12.00  1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (96 )1 3270-5

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K. Letinic´, I. Kostovic´ / Neuroscience Letters 220 (1996) 211–214

Fig. 1. Calbindin-positive cells in the ventricular (A) and subventricular (B) zone of the ganglionic eminence in the 28 week fetus. Scale bar, 25 mm.

patches, and the majority of encapsulated cell-dense islands (Fig. 3). Finally, in the oldest infant and adults, zones of low CB (dimensions 500–1000 mm) aligned with the AChE-poor striosomes appeared against a darkly stained background (Fig. 2D and Fig. 4). Transient CB-ir in the ganglionic eminence may represent an early expression of CB in future medium-sized neurons of the matrix compartment; the prolonged presence of CB concurs with the protracted genesis of these neurons [4]. Alternatively, as calcium ions may modulate the mitotic process and cell movement [2,19], CB, with its calcium-binding ability, may participate in the regulation of cell proliferation and migration in the striatal proliferative zone. A striking feature of the fetal striatum is the marked variation in the developmental characteristics of

population of CB-ir neurons. Previous studies showed that the medium-sized spiny neurons exhibit the prolonged maturation resulting in several differentiation stages coexisting at the same fetal age [5]. Thus, most CB-ir striatal cells are probably medium-sized spiny neurons. However, some CB-ir cells were larger with morphological features suggestive of a striatal interneurons. The fact that we could not observe similar cells in the postnatal life suggests that CB-expression might occur transiently in striatal interneurons. The phenomenon of transient CB-expression has been reported in various brain areas [2] and may indicate a role of CB in the control of neuronal maturation. Furthermore, a detailed correspondence was found between the patchworks of AChE and CB neuropil staining, and the encapsulated cell-dense islands. Since the AChErich patches of the fetal striatum later become the AChEpoor striosomes of the mature striatum [10], the CB-ir patches and cellular islands are at the sites of future striosomes. Moreover, the islands match striosomes in the adult primate striatum [20]. CB-ir of the striatal neuropil could possibly reside in corticostriatal afferents. Despite of the early arrival of cortical input [9], CB-ir cells appear in the cortical plate relatively late [21], and CB-ir subplate neurons [21] apparently are not projection neurons [3]. Moreover, the diffuse-to-patchy transformation of the corticostriatal terminals [9] is in contrast to an opposite pattern of changes in AChE/CB staining. Heterogeneities in the CB-ir of the striatal neuropil could then be related to the dopaminergic fibre system, appearing in the striatal anlage as early as 9.5 wg [1], and showing patchy distribution in fetuses [17,18]. It was previously suggested that

Fig. 2. (A–D) Low power of a population of CB-ir striatal neurons in the 16 (A), 25 (B) and 40 (C) week fetus, and young adult (D). In (A–C) CB-ir cells are located outside of the patches (asterisks) of CB-ir neuropil. In (D) CB-ir cells reside in the CB-rich matrix, while CB-poor zone (only a part of which is visible and indicated by the large asterisks in (D)) is devoid of labeled cells; note also the clear border (row of small asterisks) that delimits the boundary between the two regions. (E–H) high power of several CB-ir cells taken from the 25 week fetus. Scale bar, (A–D) 100 mm;(E–H) 25 mm.


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Fig. 3. Relation of CB-ir patches to AChE-rich patches and encapsulated cell-dense islands. (A,B) Two serially adjoining sections from the 16 week fetus, chosen to demonstrate precise match between (A) AChE- and (B) CB-ir patches in the putamen (asterisks mark two such matches). (C,D) Correspondence between (C) CB-ir patches and (D) cell-dense islands of the putamen in serial section pair from the 28 week fetus (arrowheads mark matching cell-islands and CB-ir patches). Scale bar, (A–B) 1 mm; (C–D) 0.5 mm.

AChE-reactivity in the patches is contained within nigrostriatal fibers making up the dopamine islands in the developing mammalian striatum [10]. This possibility is supported by an apparent similarity between the trajectories of catecholamine fibres [17] and AChE-positive bands [6], reaching the developing striatum from the subthalamic region. Most notably, in fetuses CB is present in the substantia nigra and axonal fascicles streaming from this region toward the globus pallidus and striatum (unpublished). Supposing a linkage between AChE, CB and dopaminergic innervation, how to explain complex postnatal changes in the distribution of these transmitter markers? Namely, early in development dopaminergic fibers form islands in the caudoputamen, and later they spread through its full expanse [10,11]; in addition, there is a transition from a patchy distribution of AChE/CB to an adult pattern of densely stained matrix. Such outcome would be predicted if AChE and CB were contained within the nigrostriatal fibers projecting only to the matrix; when moving from patch to non-patch regions these fibers would leave behind AChE/CB-poor striosomes and also a more uniform dopaminergic innervation. Consistently, CB is selectively expressed in nigral projections to the matrix [8]. Alternatively, both AChE and CB could occur transiently in the patch fibers (actually, AChE-reactivity is a transient property of many growing fibre systems in the human

telencephalon [16]) and a progressive postnatal increase in AChE/CB staining outside of the patches might predominantly result from the maturation of cholinergic interneurons (the major source of striatal AChE [11,12] and CB-ir neurons [7,8]. In conclusion, several mechanisms could eventually lead to the mature striatal compartmentalization of the respective transmitter markers.

Fig. 4. Relation of CB-poor zones and AChE-poor striosomes during postnatal life. Matching fields from adjoining (A) AChE and (B) CB sections from the 18 month infant are shown. Note that two light CBir patches (surrounded by matrix of high reactivity) are aligned with AChE-poor striosomes. Asterisks mark corresponding points in the two photographs. Scale bar, 0.4 mm.

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