Development of the diencephalon in the rat. V. Thymidine-radiographic observations on internuclear and intranuclear gradients in the thalamus.

Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational days 13 and 14 (E13 + 14) until the day before birth (E21 + 22). Internuclear and intranuclear cytogenetic gradients were examined in radiograms of the thalamus sectioned in the coronal, sagittal and horizontal planes. There was a precise and segregated lateral-to-medial gradient between and within the habenular nuclei. In the ventral thalamus the reticular nucleus had a lateral-to-medial gradient, the subthalamic nucleus a laterodorsal-to-medioventral gradient. There was a caudal-to-rostral gradient between the medial geniculate and dorsal lateral geniculate nuclei, and between the pars posterior and pars anterior of the lateral nucleus. A clear intranuclear gradient could not be detected in the sensory relay nuclei with the exception of the medial geniculate nucleus. A lateral-to-medial internuclear gradient was seen between the relay nuclei and the intralaminar nuclei, and between the latter and some of the midline nuclei. On the basis of a consideration of the time of origin and time span of production of neurons of various thalamic nuclei, and taking into account some of the recognizable internuclear and intranuclear gradients, the thalamus was divided into five principal cytogenetic components; the epithelamus, the ventral thalamus, the dorsal thalamus, the medial thalamus, and the posterior thalamus. The epithalamic nuclei form over a protracted period resembling the nuclei of the hypothalamus. The nuclei of the ventral thalamus are generated early and over a relatively long period. The dorsal thalamus consists of the relay nuclei and the intralaminar nuclei; they form rapidly and ahead of the medial thalamus. The medial thalamus was subdivided into the earlier-forming anteromedial nuclei and the latest-forming midline nuclei. The posterior thalamus was not examined in detail.     

Development of the diencephalon in the rat. II. Correlation of the embryonic development of the hypothalamus with the time of origin of its neurons

The development of the nuclei of the hypothalamus was examined in normal and X-irradiated embryos from day 13 (E13) to the day before birth (E22). The diencephalic neuroepithelium was subdivided into three lobes (dorsal, medial, and ventral) and two lobules (superior and inferior). The hypothalamus is derived from the ventral lobe and the inferior lobule. The ventral neuropithelial lobe generates the neurons of most of the early arising hypothalamic structures, including those of the lateral tier nuclei associated with the medial forebrain bundle, and the heterogeneous intermediate tier nuclei. A specialized neuroepithelial region lining the diamond shaped ventricle produces the early neurohypophysial magnocellular neurons; the neurons of the paraventricular nucleus remain at the site, whereas the neurons of the supraoptic nucleus could be traced migrating laterally. The neurons of the late arising hypophysiotropic area of the posterior hypothalamus are derived from components of the inferior neuroepithelial lobule: the dorsomedial and ventromedial nuclei apparently from a shared matrix in the main portion of the inferior lobule; the tuberomammillary-arcuate complex from its posteroventral recess. The triple-decked and sequentially produced components of the mammillary system may arise from separate neuroepithelial sites. The autoradiographic results of the previous study (Altman and Bayer, '78a) showed that the structural and functional heterogeneity of the mature hypothalamus is paralleled by cytogenetic heterochronicity; the present embryonic observations indicate that many of the distinguishable components of the hypothalamus arise from a mosaic of heterogeneous neuroepithelial sites.     

Development of the diencephalon in the rat. IV. Quantitative study of the time of origin of neurons and the internuclear chronological gradients in the thalamus.

Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational days 13 and 14 (E13 + 14) until the day before birth (E21 + 22). With this progressively delayed comprehensive labelling procedure we determined the time of origin of neurons in the nuclei of the epithalamus, thalamus, and ventral thalamus. The zona incerta, subthalamic nucleus, reticular nucleus, posterior nucleus, and ventral lateral geniculate nucleus are composed of the earliest arising neurons (E13, or before, to E15). The neurons of the lateral habenular nucleus are produced between days E13--16. The neurons of the medial geniculate and lateral geniculate nuclei, the ventrobasal and ventrolateral complexes, and the nucleus lateralis, pars posterior, arise rapidly on days E14--15; the medial geniculate nucleus with a peak on day E14, the others with a peak on day E15. Neurons of a group of nuclei, with ill-defined boundaries medial to the sensory relax nuclei, arise apparently on days E15--16, with a peak on day E15; these may represent the intralaminar nuclei. The next group is generated on days E15--16 but with peak formation time on day E16; this includes the anteroventral, anterodorsal, anteromedial and mediodorsal nuclei. The rhomboid, reuniens and paratenial nuclei, and the paraventricular nucleus, pars anterior, arise next (E16--17). The medial habenular nucleus forms last and over a protracted period (E15--19). With their lengthy generation time the lateral and medial habenular nuclei resemble more the nuclei of the hypothalamus than the nuclei of the dorsal thalamus.     

The diencephalon of the opossum in stereotaxic coordinates. II. The ventral thalamus and hypothalamus

This is a second paper on the diencephalon of the opossum (see Oswaldo-Cruz and Rocha-Miranda, '67), providing information on cell type and nuclear configuration of the ventral thalamus and hypothalamus (including area preoptica). Stereotaxic coordinates, resulting from a statistical survey, are given.     

Development of commissural neurons in the embryonic rat spinal cord.

Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.     

Ontogeny of somatostatin gene expression in rat diencephalon.

Somatostatin gene expression is first detectable, using in situ hybridization, on the 14th fetal day in rat diencephalon. Other structures expressing somatostatin messenger RNA include the anterior basal periventricular nucleus, amygdalo-hippocampal complex, dorsolateral thalamus and distinct areas in the parieto-frontal cortex. Semiquantitative analysis reveals that somatostatin synthesis increases progressively throughout the last third of fetal life and onto postnatal life.     

Development of the rat thalamus: I. Mosaic organization of the thalamic neuroepithelium

Short-survival, sequential, and long-survival thymidine radiograms of rat embryos, fetuses and young pups were analyzed in order to delineate the boundaries of the proliferative thalamic neuroepithelium, describe its early transformations, identify its regional divisions, and, finally, attempt to relate its distinct neuroepithelial components to specific thalamic nuclei that they supply with neurons. On day E13 the thalamic neuroepithelium consists of two divisions, the rostral lobe and the caudal lobe, and interposed between the two is a small transient structure, the reticular protuberance. By day E14 the rostral lobe has become partitioned into the anterior lobule and the reticular lobule, and the caudal lobe into the intermediate lobule and the posterior lobule. By day E15 these four lobules have become further partitioned into sublobules, characterized as regional eversions and inversions (concavities and convexities) of the thalamic neuroepithelium. Several of these sublobules are still recognizable on day E16 but progressively disappear thereafter. In this introductory paper, some evidence is presented in support of the hypothesis that the identified thalamic sublobules represent putative cell lines committed to produce neurons for specific, early-generated thalamic nuclei. Detailed documentation of the evidence on which the identifications are based is provided in subsequent papers of this series which deal with the early development of specific thalamic regions and nuclei. In our attempt to identify these putative cell lines, we sought to meet the following criteria: (1) a good match between the time course of mitotic activity in a neuroepithelial sublobule and the birth days of neurons in the nucleus that it is postulated to supply with neurons, (2) relative proximity between the putative neuroepithelial source and the thalamic target structure, and, where possible, (3) the tracing of migrating cells from the germinal source to its destination. Using these criteria we have made the following tentative identifications. The early derivatives of the anterior thalamic lobules are the sublobules (committed cell lines) of the anterior thalamic nuclei, and of the central lateral and mediodorsal nuclei. The early derivatives of the reticular lobule and reticular protuberance are the sublobules of the reticular nuclear complex. The early derivatives of the intermediate lobule are the sublobules of the ventrolateral and ventrobasal nuclei. Finally, the early derivatives of the posterior lobule are the sublobules of the dorsal geniculate, ventral geniculate, and medial geniculate nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of the diencephalon in the rat. III. Ontogeny of the specialized ventricular linings of the hypothalamic third ventricle

The development of the specialized linings of the hypothalamic third ventricle was examined autoradiographically in mature rats that were labelled with ³H-thymidine during the developmental period, and in a closely spaced series of embryonic and infant rats. We distinguished in mature rats, apart from the typical ependymal wall, three specialized linings: the convoluted ependyma, the laminated epithelium, and the tanycytic epithelium. The ventricular wall of most of the anterior hypothalamus, and of the dorsal portion of the posterior hypothalamus, is composed of ciliated ependymal cells and most of them are generated several days before birth, soon after the cessation of neurogenesis in the adjacent hypothalamic nuclei. The cells of the rostral convoluted ependyma adjacent to the paraventricular nucleus are produced at about the same time as the neighboring cells of the smooth ependyma. Its cells come from the same germinal region that we have assumed to generate the neurons of the magnocellular neurohypophysial secretory system. The structural differentiation of the convoluted ependyma starts after birth and is completed by the beginning of the second week. Many of the ependymal cells of the laminated epithelium are produced postnatally, and the production of the specialized cells that form a parallel subependymal row extends into the third week. These cells appear to arise from the same matrix that generates earlier the neurons of the dorsomedial and ventromedial hypothalamic nuclei; their structural differentiation begins during the second week. Also the cells of the tanycytic epithelium are produced mostly postnatally, predominantly during the first week. They appear to arise from the same matrix that generated earlier the neurons of the hypophysiotropic tuberomammillary and arcuate nuclei. It is postulated that these three specialized ventricular linings are specifically related to the three components of the endocrine hypothalamus with which they have shared neuroepithelial sites of origin.     

Postnatal development of calbindin and parvalbumin immunoreactivity in the thalamus of the rat

The maturation of the calcium binding proteins calbindin-D28k (CB) and parvalbumin (PV) during the first 3 postnatal weeks was studied in the rat thalamus using immunohistochemistry. These two proteins display a non-homogeneous distribution in the adult thalamus. In the rat, CB is mainly localized in the neurons and neuropil of the thalamic midline, intralaminar, and ventromedial nuclei, as well as in the posterior complex. At birth, CB-immunoreactive cell bodies were evident in thalamic midline structures, and especially in the nucleus reuniens. The number of thalamic CB-positive cell bodies, as well as the intensity of the neuropil immunostaining, increased progressively in the first postnatal weeks. This quantitative increase was first apparent in the midline structures and then in the other thalamic territories which are CB-positive in adulthood, and followed a mediolateral gradient. The mature pattern was achieved by the end of the third postnatal week. In the adult rat thalamus the neurons of the reticular nucleus display PV-immunostaining and PV-positive fibers densely innervate most of the dorsal thalamic domains. PV-immunoreactivity was clearly evident at birth in the cell bodies of the reticular nucleus. The density of PV-containing fibers increased progressively after birth in the dorsal thalamus, with a lateromedial gradient. At the end of the third postnatal week the ventroposterior (VP) complex appeared heavily innervated by PV-positive fibers, whose density in more medial structures was still lower than in the adult thalamus. A transient hyperinnervation of PV-immunoreactive fibers, displaying a dishomogenous organization in distinct segments, was observed in VP, and especially in the ventroposteromedial nucleus, during the second postnatal week. Altogether these findings indicate that the maturation of CB and PV requires postnatally a relatively prolonged period of time. The possible involvement of these proteins in different functional aspects of thalamic neuronal maturation is discussed.     

Development of 'non-specific' cholinesterase-containing neurons in the dorsal thalamus of the rat

In adult rats, neurons displaying histochemical staining for 'non-specific' cholinesterase (ChE) are found 3 distinct regions of the dorsal thalamus: the thalamic reuniens nucleus (Re), the anterior dorsal nucleus (AD), and a region that includes the lateral part of the central lateral nucleus (CL) and the ventral portion of the lateral dorsal nucleus (LD). Normal development of ChE-positive neurons was studied with cholinesterase histochemical techniques in postnatal infant rats. Although ChE staining of capillary endothelium is detectable shortly after birth, ChE staining of neurons first occurs at about postnatal day 5 (PND 5) with light staining of AD and CL-LD. At PND 7, staining in AD and CL-LD has increased in intensity and staining also is present in neurons of the anterior ventral (AV) and ventral anterior (VA) nuclei. ChE staining of neurons in Re first appears at PND 10. The number of neurons staining for ChE in each of these nuclei, and also the intensity of staining in individual neurons, appear to increase during the next several days until about PND 14. After PND 14, ChE staining intensity in neurons of AD, Re, and CL-LD appears to plateau and the pattern of staining continues into adulthood. In contrast, ChE staining of neurons in VA declines markedly and only a very few neurons in the dorsal part of VA remain ChE-positive after PND 21. ChE staining of neuropil in AV increases markedly, obscuring somatal staining in this nucleus. These results are discussed in regard to transient and continued expression of ChE activity in the dorsal thalamus and possible functional roles of ChE.     

Connections between cells of the internal capsule,thalamus, and cerebral cortex in embryonic rat

The aim of our study is to understand the development of the earliest connections in the mammalian pallium by documenting the distribution of cells and fibres labelled from the dorsal and ventral thalamus, internal capsule, perirhinal, and dorsal cortex during the period between embryonic day (E) 14 and 17 by using carbocyanine dye tracing in fixed embryonic rat brains. Dye placed in the thalamus of E14 brains backlabels cells in the thalamic reticular nucleus and within the primitive internal capsule. Both anterograde and retrograde tracing confirmed that the first corticofugal projections reach the internal capsule by E14. At E15–E16, after the first cortical plate cells have migrated into the lateral cortex, some cells of the cortical plate and subplate and marginal zone, are backlabelled from the internal capsule, but still not from the dorsal thalamus, even with very long incubation periods. Crystal placement into the perirhinal cortex at E14–E15 labels numerous cells within the internal capsule, whereas no such cells are revealed from dorsal cerebral cortex until E17, suggesting that internal capsule cells establish early connections with the perirhinal and ventral but not dorsal cortex. We propose that the growth of axons from cortex to dorsal thalamus is delayed in two regions: first from E14–E15 at the lateral entrance of the internal capsule and then, from E16, closer to the thalamus, probably within the thalamic reticular nucleus. Subplate projections reach the proximity of the diencephalon at an early stage, but they might never enter the dorsal thalamus. J. Comp. Neurol. 413:1–25, 1999. © 1999 Wiley-Liss, Inc.     

Development of embryonic spinal cord transplants in the rat

Although fetal brain tissue, grafted into the CNS of neonatal and adult animals, has been shown to survive and differentiate, relatively little information has been obtained regarding the development of embryonic spinal cord transplants, especially in the injured host CNS. The survival and differentiation of fetal spinal cord transplants in either intracerebral cavities or the lateral ventricles of the adult rat brain were thus examined with light and electron microscopy. Approximately 90% of the spinal cord implants taken from 12-15-day fetuses persisted in either transplantation site with some surviving for as long as 8 months (latest interval studied). The survival rate was considerably lower (22%), however, with tissues obtained from older fetuses. Within 3 weeks, the transplants obtained from 12-15-day donors had become extensively myelinated and contained many neurons of different sizes, including some clusters of large neurons resembling ventral horn cells of the intact spinal cord. In addition, all of the mature grafts were characterized by multiple myelin-free regions of neuropil, containing many small neurons (20 micron in diameter). [3H]Thymidine labelling of the transplants and intact cords of the surviving littermates of the donor fetuses suggested that these myelin-free areas corresponded to the substantia gelatinosa of the adult spinal cord. In many cases, the transplants were confluent with the host CNS parenchyma without an intervening glial scar. Furthermore, multiple spinal cord transplants, placed into the same lesion site, were often fused, and injection of one of the transplants with horseradish peroxidase demonstrated many retrogradely labelled neurons in the adjacent implant. The results of this study suggest that some topographical features of the normal spinal cord may be represented in mature spinal cord transplants. In addition, these findings establish a basis for future investigations aimed at repair of the injured host spinal cord with homologous fetal tissue.     

Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex

Retinae from embryonic mice were transplanted to the occipital cortex of neonatal rats together with their normal target regions, tectum or diencephalon, from embryonic mice or rats. In control experiments, retinae were cotransplanted with embryonic rat occipital cortex. In over 80% of the experimental animals, both transplants differentiated and grew. Ganglion cells in the retinae cotransplanted close to tectum or diencephalon survived for at least 15 weeks. Their survival was associated with the development of a distinct optic fiber layer and outgrowth of axons from the transplanted mouse retina. Specific innervation of distinct patches within the cotransplanted rat tectum or diencephalon was demonstrated by the use of an anti-mouse antibody. The innervated regions, which could be as far away as 1.3 mm from the retinae, were correlated with cytological features of the cotransplanted tectum or diencephalon. By contrast, the host cortex was never innervated by the transplanted retinae. In the control animals in which the retinae were cotransplanted with occipital cortex and in four animals in which the cotransplants lay more than 2.7 mm apart, no ganglion cells were identified and there was no evidence of an optic fiber layer, outgrowth of axons, or innervation. These results support the idea that in order to survive, retinal ganglion cells need to innervate an appropriate target region. Further, the specific innervation of regions within the cotransplanted tectum or diencephalon suggests that these target regions are able to exert a tropic influence on the axons of retinal ganglion cells, even in the absence of many of the normal structure cues.     

Observations on the development of certain ascending inputs to the thalamus in rats. I. Postnatal development

We have studied the postnatal development of the major ascending afferents to the thalamus in postnatal rats using tetramethylbenzidine histochemistry following wheat germ agglutinin-conjugated horseradish peroxidase injections into either the dorsal column nuclei, the deep cerebellar nuclei, or the inferior colliculus. By the day of birth, the efferents from each of these regions have already entered, and arborized extensively within, their appropriate thalamic relay nuclei. However, the overall distribution of each of these ascending afferent systems differs dramatically from that seen in mature rats. In neonatal rats, a substantial proportion of the ascending axons extend beyond the thalamus and often enter the internal capsule, some bypassing the thalamus altogether. In addition, some of the axons which enter and arborize within the thalamus extend beyond their appropriate terminal field into adjoining thalamic nuclei. Retrograde tracing experiments utilizing Fast blue indicate that the cells of origin of these overshooting axons are distributed similarly to the cells of origin of the definitive thalamic afferents. These early erroneous projections are all subsequently eliminated and the characteristically restricted adult distribution of each afferent system is evident by P30. These results indicate that developmental overgrowths and targeting errors of thalamic afferent fibers are not unique to the visual system (where they have been documented previously), but may be a general feature in the development of these pathways.     

Ultrastructure of the axon reaction in the immature rat thalamus.

The axon reaction was studied using immature rats aged 4 or 14 days at the time of unilateral removal of the cingulate cortex. After survival times of one to 5 days they were sacrificed and the anterior thalamic nucleus was examined using ultrastructural methods. The degree of degenerative change was much more intense in the 4-day group where the center of the reactive area was characterized by pronounced lysis of the neuropil and the loss of most neurons. Dark neurons were observed in both groups but mitochondrial proliferation was restricted to the 14-day group. Evidence for microglial formation from pericytes was observed in the 4-day group while reactive astrocytes were observed in both groups. It was concluded that a rapid maturation of the thalamic injury response occurs between the 4th and the 14th day in the rat so that by the 14th day the response is similar in many respects to that of mature animals.     

Gliogenesis and ependymogenesis during embryonic development of the rat. An autoradiographic study

With the aid of [3H]thymidine autoradiography gliogenesis and ependymogenesis were studied in the brain of the rat during embryonic development. Gliogenesis was found to begin on day 17 of gestation in the caudal regions of the brain stem, and to spread rostrally. On days 20 and 21 of gestation gliogenesis reached a peak, and then declined. Ependymogenesis began earlier and showed the following pattern: day 14 of gestation in the 4th ventricle and cerebral aqueduct, day 15 in the 3rd ventricle, and day 17 in the lateral ventricles reaching a peak on different days in different sites. Both gliogenesis and ependymogensis continued up to the last day of gestation, day 22. Issues pertaining to gliogenesis and the formation of glioblasts, and the relationship between gliogenesis and ependymogenesis are discussed.     

Proliferation and differentiation of transplanted embryonic diencephalon in the brain of adult rats

The survival, proliferation potential, differentiation, and host tissue reaction of allografts of undifferentiated embryonal diencephalic tissue (E12.5, E17.5) transplanted into or around the third ventricle of adult rats were investigated. Rats harboring grafts were sacrificed at three, six, and nine weeks after transplantation. The proliferative activity of the grafts was assessed by injection of 5'-bromo-2'-deoxyuridine (BrdU) into pregnant rats before the removal of fetuses for transplantation, and staining the grafts using an anti-BrdU antibody. The proliferative activity of the transplanted grafts was evaluated by immunostaining using an anti-proliferating cell nuclear antigen (PCNA) antibody. The differentiation of the grafts into neurons was estimated by double immunostaining using anti-BrdU and anti-neuron-specific enolase (NSE) antibodies. The survival rate of the grafts was strongly related to the proliferative activity of the graft. Surviving E17.5 grafts contained immunoreactive BrdU cells. E12.5 grafts could survive without immunoreactive BrdU cells. Undifferentiated El2.5 grafts proliferated up to six weeks after transplantation. Thereafter, most graft cells differentiated into mature neurons. E12.5 diencephalic allografts survived well with minimal rejection reactions and resulted in substantial neurite ingrowth into the host brain, while El7.5 allografts caused substantial reactive gliosis and little ingrowth.     

Neural damage in the rat thalamus after cortical infarcts.

Histopathologic changes in the thalamus of 23 rats after somatosensory cortical infarction produced by middle cerebral artery occlusion were examined using the Fink-Heimer silver staining method, immunohistochemistry with antibodies against glial fibrillary acidic protein and laminin, and conventional stains. Middle cerebral artery occlusion produced cortical infarcts in the lateral parietal region, with variable involvement of the frontoparietal parasagittal sensorimotor cortex. Within 3 days after occlusion, massive terminal degeneration but no neuronal changes were apparent in the ipsilateral thalamus. By 1 week after occlusion, abnormal neurons with darkly stained, shrunken nuclei and atrophic perikarya were present in the ipsilateral thalamic nuclei. These neurons were densely argyrophilic in Fink-Heimer sections. Rats with small lateral parietal cortical lesions had degenerating neurons limited to the medial ventroposteromedial nucleus. Large lesions involving the parasagittal sensorimotor cortex resulted in widespread neuronal damage in the ventroposteromedial, ventroposterolateral, intralaminar, and posterior nuclear regions but nowhere else. Immunoreactivity to laminin antibody decreased, and astrocytic proliferation was abundant in affected thalamic areas. These findings are consistent with retrograde neuronal degeneration due to thalamocortical fiber damage in ischemic cortical regions. Such lesions remote from the infarct may influence functional recovery in patients with stroke.