A striking feature of the internal capsule during early development is that it is full of small neurones. Later, this group of neurones, called the perireticular thalamic nucleus, appears to have reduced in size, and only a few scattered cells are seen. In an effort to understand better the developmental history of the perireticular nucleus this study examines: i) the period of cell generation in the nucleus, ii) the magnitude of cell loss in the nucleus, and iii) the subsequent fate of cells in the nucleus during development. The perireticular cells are generated very early in development, being among the first generated in the thalamus (rats: E13-14; cats: E21-30). In rats, the first perireticular cells are generated at about the same developmental stage as the first subplate cells, which are among the first generated cells of the cortex: in cats, the first perireticular cells are generated well before those in the subplate (E24-30). In rats, the number of perireticular cells during developmental peaks at P5 (approximately 30,000) and then declines sharply (approximately 98%) by P15 (approximately 750), when adult-like patterns are seen. This dramatic loss of perireticular cells is due to both cell death and a migration of cells into the adjacent globus pallidus. The majority of the perireticular cells which migrate into the globus pallidus, however, are likely to die also. The presence of pyknotic profiles (indicators of dying cells) in the rat perireticular nucleus points to cell death as a contributor to the reduction in cell number during development. In this study, a period of relatively high pyknotic profile incidence (number of pyknotic cells per 1,000 "living" cells) is recorded in the perireticular nucleus over a 5 day period, from P2 to P7 (13.5-15.5). Similar values and patterns are recorded in the reticular nucleus and globus pallidus, except that in these structures, a period of relatively high pyknotic profile incidence (15-20) occurs over a shorter period (3 days; P2-5). Previous studies have suggested that some perireticular cells migrate into and settle within the adjacent globus pallidus. This study, with the use of long-term survivals after tracer injections in rats, shows that none (or very few) of these perireticular cells which migrate into the globus pallidus survive into more mature postnatal stages. Tracer (biotinylated dextran) was injected into the sensory nuclei of the dorsal thalamus at early stages (P7) and the rats were allowed to survive for either a day thereafter (to P8) or until well after the period of cell death was complete (to P16 or P21). In the short-term survivals (to P8), there are many dextran-labelled cells seen in the globus pallidus and in the perireticular nucleus. In the long-term survivals (to P16 or P21), by contrast, there are no dextran-labelled cells apparent in the globus pallidus or in the perireticular nucleus. It is likely that these cells in the globus pallidus, as with those in the perireticular nucleus, undergo cell death during development.

Herein, we describe the existence of distinct colonies of transient microglial cells that reside in well-defined zones of the forebrain white matter. Rats, aged at postnatal day (P) 0, P2, P5, P7, P10, P15 or adult, were anaesthetised with halothane gas, and various neural centres were injected unilaterally with the tracer biotinylated Dextran. The neural centres injected were cingulate or sensorimotor cortices, ventral nuclei of the dorsal thalamus, and the pontine reticular formation of the brainstem. Rats were allowed to survive to various stages, from 4 hours to 21 days, after the injection. They were then anaesthetised with sodium pentobarbitone, and their brains were aldehyde-fixed and processed by using standard methods. The following is a description of what is seen after injections at P0, P2, P5, P7, P10; we saw no labelled cells (described below) in the rats injected at P15 or adult. From 2 to 21 days after an injection of dextran into the above-mentioned centres, labelled microglial cell colonies, identified by using double-labelling with anti-OX-6 or Griffonia simplicifolia (Bandeiraea; isolectin B4), were seen in small isolated zones in the forebrain white matter. These colonies were in the corpus callosum, the dorsal and ventral regions of the external capsule, and the internal capsule. A striking feature of these labelled microglial cell colonies was that they were seen on both sides of the brain. Thus, regardless of the location of the injection site in either the cortex, thalamus, or brainstem, the same microglial cell colonies were labelled with dextran in the forebrain white matter. After injections of different coloured fluorescent dextrans into the cortex and into the brainstem of the same animal, many double-labelled cells in each of the colonies were seen. From our short-term survival cases (4 hours to 1 day), a rather strict sequence or progression of labelling of the colonies across the white matter from the injection site was seen; in general, the microglial cell colonies closest to the injection site became labelled well before (about a day) those further away. These results lead us to suggest that the microglial cells in each colony become labelled after a slow diffusion of the tracer through the extracellular space from the injection site.

We have explored two aspects of internal capsule development that have not been described previously, namely, the development of glia and of blood vessels. To these ends, we used antibodies to glial fibrillary acidic protein (GFAP) and to vimentin (to identify astrocytes and to radial glia) and Griffonia simplicifolia (lectin; to identify microglia and blood vessels). Further, we made intracardiac injections of Evans Blue to examine the permeability of this dye in the vessels of the internal capsule during neonatal development. Our results show that large numbers of radial glia, astrocytes and microglia are not labelled with these markers in the white matter of the internal capsule until about birth; very few are labelled earlier, during the critical stages of corticofugal and corticopetal axonal ingrowth (E15-E20). The large glial labelling in the internal capsule at birth is accompanied by a dense vascular innervation of the capsule; as with the glia, very few labelled patent vessels are seen earlier. After intracardiac injections of Evans Blue, we find that the blood vessels of the internal capsule are not particularly permeable to Evans Blue. At each age examined (P0, P5, P15), blood vessels are outlined very clearly and there is no diffuse haze of fluorescence within the extracellular space, which is indicative of a leaky vessel. There are three striking differences between the glial environment of the internal capsule and that of the adjacent thalamus. First, the internal capsule is never rich with radial glial fibres (vimentin- and GFAP-immunoreactive) during development (except at P0), whereas the thalamus has many radial fibres from very early development (E15-E17). Second, astrocytes (vimentin- and GFAP-immunoreactive) first become apparent in the internal capsule (E20-P0) well before they do in the thalamus (P15). Third, the internal capsule houses a large transient population of amoeboid microglia (P0-P22), whereas the thalamus does not; only ramified microglia are seen in the thalamus. In summary, our results indicate that all three types of glia in the internal capsule are associated closely with the vasculature, suggesting they may play a role in the development of the blood-brain barrier among the vessels in this white matter region of the forebrain.

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30-P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20-E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.

A striking feature of the internal capsule during early development is that it is full of small neurones. Later, this group of neurones, called the perireticular thalamic nucleus, appears to have reduced in size, and only a few scattered cells are seen. In an effort to understand better the developmental history of the perireticular nucleus this study examines: i) the period of cell generation in the nucleus, ii) the magnitude of cell loss in the nucleus, and iii) the subsequent fate of cells in the nucleus during development. The perireticular cells are generated very early in development, being among the first generated in the thalamus (rats: E13-14; cats: E21-30). In rats, the first perireticular cells are generated at about the same developmental stage as the first subplate cells, which are among the first generated cells of the cortex: in cats, the first perireticular cells are generated well before those in the subplate (E24-30). In rats, the number of perireticular cells during developmental peaks at P5 (approximately 30,000) and then declines sharply (approximately 98%) by P15 (approximately 750), when adult-like patterns are seen. This dramatic loss of perireticular cells is due to both cell death and a migration of cells into the adjacent globus pallidus. The majority of the perireticular cells which migrate into the globus pallidus, however, are likely to die also. The presence of pyknotic profiles (indicators of dying cells) in the rat perireticular nucleus points to cell death as a contributor to the reduction in cell number during development. In this study, a period of relatively high pyknotic profile incidence (number of pyknotic cells per 1,000 "living" cells) is recorded in the perireticular nucleus over a 5 day period, from P2 to P7 (13.5-15.5). Similar values and patterns are recorded in the reticular nucleus and globus pallidus, except that in these structures, a period of relatively high pyknotic profile incidence (15-20) occurs over a shorter period (3 days; P2-5). Previous studies have suggested that some perireticular cells migrate into and settle within the adjacent globus pallidus. This study, with the use of long-term survivals after tracer injections in rats, shows that none (or very few) of these perireticular cells which migrate into the globus pallidus survive into more mature postnatal stages. Tracer (biotinylated dextran) was injected into the sensory nuclei of the dorsal thalamus at early stages (P7) and the rats were allowed to survive for either a day thereafter (to P8) or until well after the period of cell death was complete (to P16 or P21). In the short-term survivals (to P8), there are many dextran-labelled cells seen in the globus pallidus and in the perireticular nucleus. In the long-term survivals (to P16 or P21), by contrast, there are no dextran-labelled cells apparent in the globus pallidus or in the perireticular nucleus. It is likely that these cells in the globus pallidus, as with those in the perireticular nucleus, undergo cell death during development.