Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum

Prenatal cerebellar development was investigated with three approaches. In normal embryos sectioned in three planes morphological and cytological changes were determined at daily intervals beginning on embryonic day 13 (E13). A similar series of X-irradiated embryos was used to study changes in neuroepithelial organization and in the location of primitive (radiosensitive) or differentiated cells. Finally, to quantify the time of origin of different classes of cerebellar neurons with the progressively delayed labelling procedure, we used autoradiograms from adult rats whose mothers were injected with two successive daily doses of ³H-thymidine on overlapping days from day E13 on. The cerebellar anlage was delineated in the dorsal metencephalon by the collapse of its ventricular lining after X-irradiation. This “collapsing neuroepithelium” was located laterally on day E13, then it spread medially and reached the midline on day E16. Deep nuclear neurons began to differentiate on day E13, with two-thirds forming on day E14; Purkinje cell formation peaked on day E15, with a few cells still forming on day E16. It was postulated that the deep nuclear neurons settled first in the superficial “nuclear zone,” and that the Purkinje cells gathered temporarily in the underlying “transitory zone,” adjacent to the collapsing neuroepithelium. In the next period of cerebellar development four major events were recognized. (1) Beginning on day E17 the cells of the nuclear and transitory zones became intermingled. It was postulated that the Purkinje cells were migrating radially through the ranks of the stationary deep nuclear neurons and assembled under the spreading canopy of a fibrous plexus and the external germinal layer. (2) It was also on day E17 that the external germinal layer began to form as one of the prongs of the “germinal trigone” in the posteroventral aspect of the cerebellum. On the succeeding days the external germinal layer spread over the surface of the cerebellum; in the vermis in a rostral direction. (3) Two cell types destined to settle in the future granular layer, the pale cells and the Golgi cells, began to form at a relatively slow rate on day E19. Chronological considerations suggested that they were generated in the regressing, noncollapsing neuroepithelium of the cerebellar ventricle. (4) From the beginning (day E17) of its genesis posteroventrally, the primitive cerebellar cortex bridged the midline. As the fused cortex spread rostrally, the vertical ventricular cleft separating the underlying portions of the cerebellum became shallower and then disappeared; the process was completed in the anterior cerebellum by day E22. By the time of birth the maturation of the neurons of the deep nuclei appeared advanced but the maturation of the prenatally produced neurons of the cortex does not start until after birth when a new class of neurons is generated in the external germinal layer.     

Embryonic development of the rat cerebellum. II. Translocation and regional distribution of the deep neurons

In thymidine radiograms and plastic-embedded sections, the migration of cerebellar deep neurons was traced from their germinal source to their final settling sites. The route proved to be roundabout and three developmental events could be distinguished during the process. First, between days E14 and E16, transversely oriented cells of the nuclear transitory zone move in an arc from the ventrolateral neuroepithelium of the lateral cerebellar primordium in a medial direction. Second, between days E16 and E18, the cells of the rostral component of the nuclear transitory zone assume a longitudinal orientation. We postulated that this is the period of axonogenesis, the longitudinally oriented cells issuing efferents that join the superior cerebellar peduncle ipsilaterally and the transversely oriented cells (representing the neurons of the caudal fastigial nucleus) sending decussating fibers to the uncinate fasciculus (the hook bundle of Russell). Third, between days E18 and E21, the earlier-produced superficial cells of the nuclear transitory zone and the later-produced deep cells of the cortical transitory zone (the young Purkinje cells) exchange positions. The descent of the deep neurons is in the direction of the fibers of the inferior cerebellar peduncle, which becomes distributed throughout the cerebellum on day E17. The ascent of the Purkinje cells is in the direction of the external germinal layer, which begins to spread from caudal to rostral on day E17. The three deep nuclei, the lateral (dentate), interpositus, and medial (fastigial), can be distinguished before their descent into the depth of the cerebellum, and by day E22 a small-celled and a large-celled subdivision is identifiable in each nucleus.     

Neurogenetic and morphogenetic heterogeneity in the bed nucleus of the stria terminalis

Neurogenesis and morphogenesis in the rat bed nucleus of the stria terminalis (strial bed nucleus) were examined with [3H]thymidine autoradiography. For neurogenesis, the experimental animals were the offspring of pregnant females given an injection of [3H]thymidine on 2 consecutive gestational days. Nine groups of embryos were exposed to [3H]thymidine on E13-E14, E14-E15,... E21-E22, respectively. On P60, the percentage of labeled cells and the proportion of cells originating during 24-hour periods were quantified at six anteroposterior levels in the strial bed nucleus. On the basis of neurogenetic gradients, the strial bed nucleus was divided into anterior and posterior parts. The anterior strial bed nucleus shows a caudal (older) to rostral (younger) neurogenetic gradient. Cells in the vicinity of the anterior commissural decussation are generated mainly between E13 and E16, cells just posterior to the nucleus accumbens mainly between E15 and E17. Within each rostrocaudal level, neurons originate in combined dorsal to ventral and medial to lateral neurogenetic gradients so that the oldest cells are located ventromedially and the youngest cells dorsolaterally. The most caudal level has some small neurons adjacent to the internal capsule that originate between E17 and E20. In the posterior strial bed nucleus, neurons extend ventromedially into the posterior preoptic area. Cells are generated simultaneously along the rostrocaudal plane in a modified lateral (older) to medial (younger) neurogenetic gradient. Ventrolateral neurons originate mainly between E13 and E16, dorsolateral neurons mainly between E15 and E16, and medial neurons mainly between E15 and E17. The youngest neurons are clumped into a medial "core" area just ventral to the fornix. For morphogenesis, pregnant females were given a single injection of [3H]thymidine during gestation, and their embryos were removed either 2 hours later (short survival) or in successive 24-hour periods (sequential survival). The embryonic brains were examined to locate areas of intensely labeled cells in the putative neuroepithelium of the strial bed nucleus, to trace migratory waves of young neurons, and to establish their final settling locations. Two different neuroepithelial sources produce neurons for the strial bed nucleus. The anterior strial bed nucleus is generated by a neuroepithelial zone at the base of the inferior horn of the lateral ventricle from the anterior commissural decussation area forward to the primordium of the nucleus accumbens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of the rat thalamus: II. Time and site of origin and settling pattern of neurons derived from the anterior lobule of the thalamic neuroepithelium

Short-survival, sequential, and long-survival thymidine radiograms of rat embryos, fetuses, and young pups were analyzed in order to examine the time of origin, settling pattern, and neuroepithelial site of origin of the anterior thalamic nuclei--the lateral dorsal (lateral anterior), anterodorsal, anteroventral and anteromedial nuclei--and of two rostral midline structures--the anterior paraventricular and paratenial nuclei. The neurons of the lateral dorsal nucleus are generated over a 3-day period between days E14-E16 and their settling pattern displays a combined lateral-to-medial and dorsal-to-ventral neurogenetic gradient. The bulk of the neurons of the anteroventral nucleus are generated over a 3-day period between days E15-E17 and settle with an oblique lateral-to-medial and ventral-to-dorsal neurogenetic gradient. The bulk of the neurons of the anteromedial nucleus are generated over a 2-day period between days E16-E17 and show the same settling pattern as the anteroventral nucleus. The neurons of the anterodorsal nucleus are generated over a 3-day period between days E15-E17 and show a lateral-to-medial neurogenetic gradient. The bulk of the neurons of the central part and lateral part of the paraventricular nucleus are generated over a 2-day period (E16-E17 and E17-E18, respectively) and each part displays a ventral-to-dorsal neurogenetic gradient. Finally, the bulk of the neurons of the paratenial nucleus are generated over a 4-day period between days E15-E18 and settle with a lateral-to-medial neurogenetic gradient. Observations are presented that the anterior thalamic nuclei, constituting the distinct "limbic thalamus," derive from a discrete neuroepithelial source. This is the crescent-shaped germinal matrix lining the diencephalic (medial) wall of the hitherto unrecognized anterior transitional promontory, which we call the anterior thalamic neuroepithelial lobule. On day E16 three migratory streams leave the anterior neuroepithelial lobule and, on the basis of their labeling pattern in relation to the neurogenetic gradients of the anterior thalamic nuclei, they are identified, from dorsal to ventral, as the putative migratory streams of the anterodorsal, anteroventral, and lateral dorsal nuclei. On day E17 the putative migratory stream of the anteromedial nucleus appears to leave the same neuroepithelial region that on the previous days was the source of the anteroventral nucleus. Dorsally, two neuroepithelial patches persist after day E17 and these are identified as the putative cell lines of the anterior paraventricular and paratenial nuclei.     

Organization of projections from the inferior olive to the cerebellar nuclei in the rat

The detailed organization of projections from the inferior olive to the cerebellar nuclei of the rat was studied by using anterograde tracing. The presence of a collateral projection to the cerebellar nuclei could be confirmed, and a detailed organization was recognized at the nuclear and subnuclear level. Olivary projections to the different parts of the medial cerebellar nucleus arise from various parts of the caudal half of the medial accessory olivary nucleus. The interstitial cell groups receive olivary afferents from the intermediate part of the medial accessory olive and from the dorsomedial cell column. A mediolateral topography was noted in the projections from the rostral half of the medial accessory olive to the posterior interposed nucleus. Olivary projections to the lateral cerebellar nucleus are derived from the principal olive according to basically inversed rostrocaudal topography. Projections from the dorsomedial group of the principal olive to the dorsolateral hump were found to follow a basically rostrocaudal topography. The anterior interposed nucleus receives olivary afferents from the dorsal accessory olive. Its rostromedial parts are directed to the lateral part of the anterior interposed nucleus and its caudolateral part reach the medial anterior interposed nucleus. No terminal arborizations in the cerebellar nuclei were found to originate from (1) the dorsal fold of the dorsal accessory olive, which resulted in projections to the lateral vestibular nucleus and (2) the dorsal cap of Kooy. It was noted that the olivary projection to the cerebellar nuclei is strictly reciprocal to the nucleo-olivary projection as described by Ruigrok and Voogd (1990). Moreover, it is suggested that the olivonuclear projection adheres to the organization of the climbing fiber projection to the cerebellar cortex and to the corticonuclear projection, thus, establishing and extending the detailed micromodular organization of the connections between inferior olive and cerebellum.     

Projections of the vestibular and cerebellar nuclei in Rana pipiens

The efferent projections and cytoarchitecture of the vestibulocerebellar region were examined to determine the nuclear boundaries and potential homologies. The anterior portion of the vestibular complex projects to the ipsilateral oculomotor and trochlear nuclei and is the major source of commissural fibers. Neurons in the rostromedial portions of the complex project to the contralateral trochlear nucleus. Large neurons in the ventrolateral portion of the complex give rise to a bilateral vestibulospinal pathway. Medium-sized neurons in the neuropil and small neurons in the central gray giving rise to bilateral projections to the spinal cord and oculomotor nuclei as well as commissural and ipsilateral cerebellar efferents. Projections from the nucleus of the cerebellum reach the contralateral spinal cord and cerebellar nucleus and there is also a bilateral projection to the ventral rhombencephalic and mesencephalic basal plates. The medial portion of the nucleus gives rise to commissural, ipsilateral mesencephalic and contralateral spinal projections. The lateral portion of the nucleus projects to the contralateral ventral mesencephalon. On the whole, the results of this investigation substantiate the division of the anuran vestibular complex in anurans into nuclei which may be homologous to the superior nucleus and nucleus of Deiters in mammals. The case for distinct descending and medial nuclei is less compelling. Further, it appears possible to divide the nucleus of the cerebellum into medial and lateral components whose connectivity is similar to that of reptiles and to a lesser extent mammals.     

Pallidal and cerebellar afferents to pre-supplementary motor area thalamocortical neurons in the owl monkey: a multiple labeling study

In the present study, we determined where thalamic neurons projecting to the pre-supplementary motor area (pre-SMA) are located relative to pallidothalamic and cerebellothalamic inputs and nuclear boundaries. We employed a triple-labeling technique in the same owl monkey (Aotus trivirgatus). The cerebellothalamic projections were labeled with injections of wheat germ agglutinin conjugated to horseradish peroxidase, and the pallidothalamic projections were labeled with biotinylated dextran amine. The pre-SMA was identified by location and movement patterns evoked by intracortical microstimulation and injected with the retrograde tracer cholera toxin subunit B. Brain sections were processed sequentially using different chromogens to visualize all three tracers in the same section. Alternate sections were processed for Nissl cytoarchitecture or acetylcholinesterase chemoarchitecture for nuclear boundaries. The cerebellar nuclei primarily projected to posterior (VLp), medial (VLx), and dorsal (VLd) divisions of the ventral lateral nucleus; the pallidum largely projected to the anterior division (VLa) of the ventral lateral nucleus and the parvocellular part of the ventral anterior nucleus (VApc). However, we also found zones of overlapping projections, as well as interdigitating foci of pallidal and cerebellar label, particularly in border regions of the VLa and VApc. Thalamic neurons labeled by pre-SMA injections occupied a wide band and were especially concentrated in the VLx and VApc, cerebellar and pallidal territories, respectively. Labeled thalamocortical neurons overlapped cerebellar inputs in the VLd and VApc and overlapped pallidal inputs in the VLa and the ventral medial nucleus. The results demonstrate that inputs from both the cerebellum and globus pallidus are relayed to the pre-SMA.     

Degeneration of the cerebellar system in X-chromosome-linked copper malabsorption.

Examination of the cerebellar system of 5 autopsied patients with X-chromosome-linked copper malabsorption led to the discovery that among cerebellar afferent systems, only the dorsal spinocerebellar tract showed consistent degenerative changes. Cerebellar cortical lesions comprised granule cell loss and Purkinje cell abnormalities that included nuclear and cytoplasmic degenerative changes with cell loss and deficient dendritic arborization as well as the presence of somatic sprouts. Perisomatic baskets were often absent in the area of sprouting Purkinje cells. Cerebellar cortical topographical analysis revealed relative sparing of the caudal portion of the cerebellum. The nodulus was intact in all patients. The dentate nucleus and the superior cerebellar peduncle were preserved. The red nucleus was consistently degenerated.     

Development of the preoptic area: time and site of origin, migratory routes, and settling patterns of its neurons

Neurogenesis and morphogenesis in the rat preoptic area were examined with [3H]thymidine autoradiography. For neurogenesis, the experimental animals were the offspring of pregnant females given an injection of [3H]thymidine on two consecutive gestational days. Nine groups were exposed to [3H]thymidine on embryonic days E13-E14, E14-E15, E21-E22, respectively. On postnatal day P5, the percentage of labeled cells and the proportion of cells originating during 24-hr periods were quantified at four anteroposterior levels in the preoptic area. Throughout most of the preoptic area there is a lateral to medial neurogenetic gradient. Neurons originate between E12-E15 in the lateral preoptic area, between E13-E16 in the medial preoptic area, between E14-E17 in the medial preoptic nucleus, and between E15-E18 in the periventricular nucleus. These structures also have intrinsic dorsal to ventral neurogenetic gradients. There are two atypical structures: (1) the sexually dimorphic nucleus originates exceptionally late (E15-E19) and is located more lateral to the ventricle than older neurons; (2) in the median preoptic nucleus, where older neurons (E13-E14) are located closer to the third ventricle than younger neurons (E14-E17). For an autoradiographic study of morphogenesis, pregnant females were given a single injection of [3H]thymidine during gestation, and their embryos were removed either two hrs later (short survival) or in successive 24-hr periods (sequential survival). Short-survival autoradiography was used to locate the putative neuroepithelial sources of preoptic nuclei, and sequential survival autoradiography was used to trace the migratory waves of young neurons and their final settling locations. The preoptic neuroepithelium is located anterior to and in the front wall of the optic recess. The neuroepithelium lining the third ventricle is postulated to contain a mosaic of spatiotemporally defined neuroepithelial zones, each containing precursor cells for a specific structure. The neuroepithelial zones and the migratory waves originating from them are illustrated. Throughout most of the preoptic area, neurons migrate predominantly laterally. The older neurons in the lateral preoptic area migrate earlier and settle adjacent to the telencephalon. Younger neurons migrate in successively later waves and accumulate medially. The sexually dimorphic neurons are exceptional since they migrate past older cells to settle in the core of the medial preoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional and cellular distribution of protein kinase C in rat cerebellar Purkinje cells

Protein kinase C (PCK) is a family of isoforms that are implicated in subcellular signal transduction. The authors investigated the distribution of several PKC isoforms (PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon) within major cerebellar cell types as well as cerebellar projection target neurons, including Purkinje neurons, cerebellar nuclear neurons, and secondary vestibular neurons. PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon are found within the cerebellum. Of these isoforms, PKC-gamma and PKC-delta are highly expressed in Purkinje cells. PKC-gamma is expressed in all Purkinje cells, whereas the expression of PKC-delta is restricted to sagittal bands of Purkinje cells in the posterior cerebellar cortex. In the lower folia of the uvula and nodulus, Purkinje cell expression of PKC-delta is uniformly high, and the sagittal banding for PKC-delta expression is absent. Within the cerebellar nuclei, PKC-delta-immunolabeled axons terminate within the medial aspect of the caudal half of the ipsilateral interpositus nucleus. PKC delta-immunolabeled axons also terminated within the caudal medial and descending vestibular nuclei (MVN and DVN, respectively), the parasolitary nucleus (Psol), and the nucleus prepositus hypoglossi (NPH). PKC-gamma-immunolabeled axons terminated in all of the cerebellar nuclei as well as in the lateral and superior vestibular nuclei and the MVN, DVN, Psol, and NPH. The projection patterns of PKC-immunolabeled Purkinje cells were confirmed by lesion-depletion studies in which unilateral uvula-nodular lesions caused depletion of PKC-immunolabeled terminals ipsilateral to the lesion in the vestibular complex. These data identify circuitry that is unique to cerebellar-vestibular interactions.     

Cerebellar efferents in the lizard Varanus exanthematicus. II. Projections of the cerebellar nuclei

The projections of the cerebellar nuclei have been studied in the lizard Varanus exanthematicus with various experimental anatomical techniques. In anterograde degeneration experiments (lesions of the cerebellar peduncle) both ascending and decending contralateral projections were found. Ascending fibers which could be traced from the cerebellar commissure ventralward decussated at the level of the trochlear and oculomotor nuclei. These fibers coursed rostralward to the mesodiencephalic junction. With anterograde tracing techniques (3H-leucine and HRP) this tract was found to terminate in the nucleus ruber and the interstitial nucleus of the fasciculus longitudinalis medialis. Moreover, retrograde tracer studies (HRP, "Fast Blue") showed that this tract appeared to arise mainly in the lateral cerebellar nucleus. With both anterograde degeneration and tracing techniques (3H-leucine and HRP) a bundle of fibers could be followed, which decussates in the basal part of the cerebellum and passes dorsally around the contralateral medial cerebellar nucleus to the lateral side of the brainstem. This contralaterally descending projection system was found, lateral to the vestibular nuclear complex, and as far caudally as the descending vestibular nucleus, to terminate on various vestibular nuclei. Horseradish peroxidase studies showed that this contralaterally descending projection system originates mainly in the medial cerebellar nucleus, but ipsilaterally descending projections were also found. With the fluorescent double labeling technique ("Fast Blue" and "Nuclear Yellow") the projections of the cerebellar nuclei described above were confirmed. Furthermore, double labeling revealed neurons in both cerebellar nuclei (especially the medial nucleus) that project to both the mesencephalon and the cervical spinal cord. The present results indicate that the efferent connections of the cerebellar nuclei in the lizard Varanus exanthematicus are organized as two main projections, an ascending projection comparable to the mammalian brachium conjunctivum arising in the lateral cerebellar nucleus, and a descending projection comparable to the mammalian hook bundle (fasciculus uncinatus), originating mainly in the medial cerebellar nucleus. Such projections are common for terrestrial vertebrates.     

Origins of cerebellar mossy and climbing fibers immunoreactive for corticotropin-releasing factor in the rabbit

Corticotropin-releasing factor (CRF) has been implicated by both anatomical and physiological techniques as a potential cerebellar transmitter or modulator. In the present experiment, with the aid of immunohistochemistry, we have described specific cerebellar afferent pathways in the rabbit in which CRF is located. CRF-immunoreactive climbing fibers were present in the molecular layer throughout the cerebellum, but especially in lobules 8–9a. All inferior olivary neurons were CRF-immunoreactive. In lobules 8–9a, CRF-immunoreactive mossy fibers were organized in sagittal bands. The highest density of CRF-immunoreactive mossy fiber terminals was observed in the granule cell layer of lobules 8–9a and the flocculus. No CRF-immunoreactive perikarya were located in rabbit cerebellum. The brainstem origin of CRF-immunoreactive mossy fiber terminals was suggested by numerous CRF-immunoreactive perikarya located in the medial, lateral and descending vestibular nuclei, nucleus prepositus hypoglossi, nucleus x, paramedian reticular nucleus, gigantocellular reticular nucleus, lateral reticular nucleus, and raphe nuclei. Using double label experiments, we investigated the specific CRF afferent projection to the flocculus and posterior vermis. Horseradish peroxidase (HRP) injections into the posterior vermis double labeled CRF-immunoreactive neurons in the caudal medial and descending vestibular nuclei and nucleus prepositus hypoglossi. HRP injections into the flocculus double labeled more CRF-immunoreactive neurons in the nucleus prepositus hypoglossi than in the vestibular nuclei. HRP injections into either the posterior vermis or flocculus double labeled CRF-immunoreactive neurons in the paramedian reticular nucleus, nucleus reticularis gigantocellularis, and raphe nuclei. These data suggest that CRF may play an important role in vestibularly related functions of the cerebellum. © 1993 Wiley-Liss, Inc.     

The origin of serotoninergic afferents to the cat's cerebellar nuclei

In the cat, serotoninergic (5HT) axons and terminals form a dense plexus that is present throughout the granule cell and Purkinje cell layers of the cerebellar cortex and all of the cerebellar nuclei. The intent of the present study was to identify the source of 5HT fibers in the cerebellar nuclei. The medial, interposed, and lateral cerebellar nuclei were selectively injected with either rhodamine or fluorescein-labeled latex microspheres that were retrogradely transported to brainstem neurons. Transverse sections of the brainstem were processed with a primary antibody to 5HT and secondary antibody tagged with either rhodamine or fluorescein. The location of neurons containing both serotonin-like immunoreactivity and retrogradely transported microspheres was plotted. All three of the cerebellar nuclei receive 5HT afferents from the nucleus locus coeruleus, the dorsal raphe nucleus, and the dorsal tegmental nucleus. In addition, the medial nucleus receives projections from the superior central nucleus, the nucleus raphe obscurus, the nucleus raphe magnus, and the periolivary reticular formation. The interposed nuclei receive additional projections from the nucleus raphe magnus, whereas the lateral nucleus receives additional projections from the superior central nucleus. In conclusion, the 5HT projections to the cerebellar nuclei do not appear to be collaterals of those projecting to the cortex (Kerr and Bishop, J Comp Neurol 304:502–515, 1991). These findings suggest that, although the cortex and nuclei are anatomically and physiologically related, they do not process all information in parallel. © Wiley-Liss, Inc.     

Differentiation of Purkinje cells in cerebellar slice cultures: an immunocytochemical and Golgi EM study

In the rat central nervous system, the cerebellar cortex has a stereotypical cytoarchitecture and a characteristic connectivity pattern, both mainly formed post-natally. Organotypic cultures of immature cerebellar tissue were used to study the formation of the cerebellar lamination and the differentiation of Purkinje cells in the absence of their extracerebellar afferents. The lamination was retained in the majority of the cerebellar cultures and most Purkinje cells were aligned. Axonal profiles of Purkinje cells, immunolabelled for UCHT1 or anti-calbindin D-28 k, followed pathways similar to those in vivo cerebellum. The dendrites were orientated towards the superficial layer except of those neurons which were ectopically positioned. Unlike in vivo, the dendritic arborization of Golgi-impregnated/gold-toned or immuno-stained Purkinje cells was reduced and the dendritic spines were often elongated. Somatic spines, a morphological feature of immature Purkinje cells persisted even after 4 weeks in culture. We conclude that the Purkinje cells in organotypic cultures send their axon to the correct target region independent of their local position. In contrast the dendritic orientation and differentiation is influenced by the cellular environment and by specific synaptic interaction.     

Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat

Bromodeoxyuridine (BrdU) is broadly used in neuroscience to study embryonic development and adult neurogenesis. The potential toxicity of this halogenated pyrimidine analogue is frequently neglected. In this study, we administered BrdU in small doses by the progressively delayed cumulative labeling method to immunocytochemically tag different cerebellar cell types with antibodies to specific markers and BrdU in the same section. The well-known structure of the cerebellum made it possible to ascertain several toxic effects of the treatment. Time-pregnant rats were given five or six injections of 5 or 6 mg of BrdU ( approximately 12-20 mg/kg) at 8-hour intervals over 2 successive days between day 11 and 21 of pregnancy (E11-E12 to E20-E21), and the adult progeny was processed by immunocytochemistry. We demonstrate that this treatment effectively labeled distinct cerebellar cell populations but produced striking defects in the proliferation, migration, and settling of the Purkinje cells; reduced the size of the cerebellar cortex and nuclei; produced defects in the patterning of foliation; and also affected litter size, body weight, and mortality of the offspring. The observed toxic effects were consistent within individual treatment groups but varied between different treatment groups. Treatment with BrdU at the peak of neurogenesis of cerebellar projection neurons (E14) produced the most severe malformations. We observed no overt effects on the timing of neurogenesis for cerebellar neurons and glia across experimental groups. In conclusion, BrdU is a useful tool to study neural development, but its cytotoxicity represents a serious pitfall particularly when multiple doses are used to label cells.     

Organization of projections from the gracile, medial cuneate and lateral nuclei in the North American opossum. Horseradish peroxidase study of the cells projecting to the cerebellum, thalamus and spinal cord

Using the horseradish peroxidase technique on the North American opossum, we were able to locate the neurons within the dorsal column and lateral cuneate nuclei which innervate the cerebellum and thalamus as well as those within the dorsal column nuclei which project spinalward. The medial and lateral cuneate nuclei supply axons to the anterior lobe, the paramedian lobule and the pyramis of the cerebellum and the lateral nucleus provides an additional projection to the uvula. The cerebellar projections from these nuclei arise from neurons located rostral to the obex. The thalamic projections from the gracile and medial cuneate nuclei originate from neurons throughout their rostral to caudal extent, although most of them are located just rostral to the obex. Neurons within the lateral cuneate nucleus which innervate the thalamus are found at intermediate rostrocaudal levels where most of them approximate the medial cuneate nucleus. The medial cuneate also projects to at least lumbar levels of the spinal cord in the opossum and neurons giving rise to such connections are found at the level of the obex and caudal to it. Neurons within the dorsal part of the dorsal column nuclei were labelled only after thalamic injections. Our results in the opossum are compared with those obtained in several placental mammals.     

Development of the precerebellar nuclei in the rat: I. The precerebellar neuroepithelium of the rhombencephalon

Short-survival thymidine radiograms from rat embryos aged 13-19 days were analyzed to delineate the precerebellar neuroepithelium of the rhombencephalon. The original definition of the term "rhombencephalon" was modified to refer only to the unique dorsal portion (surface plate) of the medulla and pons where the neural groove fails to fuse and, instead, the medullary velum covers the rhomboid lumen of the fourth ventricle. Initially, the neuroepithelial tissue of the rhombencephalon consists of a pair of rostral and caudal bridgeheads: the former the primary neuroepithelium of the cerebellum and the latter the primary neuroepithelium of the octavo-precerebellar system. The spatial relationship between the cerebellar and precerebellar neuroepithelia soon changes as a result of ongoing morphogenetic events, such that the cerebellar primordium assumes a dorsal position and the precerebellar primordium a ventral position, and the distance between the two decreases. Concurrently the tela choroidea invaginates into the fourth ventricle and a secondary precerebellar neuroepithelium develops. The rostral portion of the secondary precerebellar neuroepithelium grows forward along the choroid plexus and forms the medial recess of the anterior fourth ventricle, while its caudal portion grows in the opposite direction beneath the medullary velum and forms the rostral wall of the posterior fourth ventricle. Evidence will be presented in the succeeding papers that the primary precerebellar neuroepithelium first generates the neurons of the inferior olive that migrate by a circumferential intramural (parenchymal) route to their destination. Next, the neurons of the lateral reticular and external cuneate nuclei are generated. These migrate by a posterior extramural (superficial) route and settle contralaterally. Subsequently, the primary precerebellar neuroepithelium produces the neurons of the nucleus reticularis tegmenti pontis and these form the anterior extramural migratory stream and settle ipsilaterally. Finally, the secondary precerebellar neuroepithelium produces the latest generated neurons of the basal pontine gray that follow the anterior extramural stream and settle ipsilaterally.     

Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex

Motor commands computed by the cerebellum are hypothesized to use corollary discharge, or copies of outgoing commands, to accelerate motor corrections. Identifying sources of corollary discharge, therefore, is critical for testing this hypothesis. Here we verified that the pathway from the cerebellar nuclei to the cerebellar cortex in mice includes collaterals of cerebellar premotor output neurons, mapped this collateral pathway, and identified its postsynaptic targets. Following bidirectional tracer injections into a distal target of the cerebellar nuclei, the ventrolateral thalamus, we observed retrogradely labeled somata in the cerebellar nuclei and mossy fiber terminals in the cerebellar granule layer, consistent with collateral branching. Corroborating these observations, bidirectional tracer injections into the cerebellar cortex retrogradely labeled somata in the cerebellar nuclei and boutons in the ventrolateral thalamus. To test whether nuclear output neurons projecting to the red nucleus also collateralize to the cerebellar cortex, we used a Cre‐dependent viral approach, avoiding potential confounds of direct red nucleus‐to‐cerebellum projections. Injections of a Cre‐dependent GFP‐expressing virus into Ntsr1‐Cre mice, which express Cre selectively in the cerebellar nuclei, retrogradely labeled somata in the interposed nucleus, and putative collateral branches terminating as mossy fibers in the cerebellar cortex. Postsynaptic targets of all labeled mossy fiber terminals were identified using immunohistochemical Golgi cell markers and electron microscopic profiles of granule cells, indicating that the collaterals of nuclear output neurons contact both Golgi and granule cells. These results clarify the organization of a subset of nucleocortical projections that constitute an experimentally accessible corollary discharge pathway within the cerebellum. J. Comp. Neurol. 523:2254–2271, 2015. © 2015 Wiley Periodicals, Inc.     

小鼠小脑皮质的生长发育

目的：探讨小鼠小脑皮质的组织发生过程。方法：应用光镜和电镜技术对胚胎和生后小脑皮质进行形态学观察,对各层厚度和细胞密度进行测量。结果：胚胎12d（E12）小脑原基有室管膜层、套层和边缘层构成,约出生当日（P0）出现外颗粒层、分子层、Purkinje细胞层和内颗粒层。外颗粒层P6／7达最厚,至P20消失。P0至P30内颗粒层细胞逐步分化发育成熟,Purkinje细胞树突树逐渐形成,约P7时Purkinje细胞排列成单层。结论：E12至P0片层化小脑主要经历了细胞增殖、分化与迁移;P0至P30片层化结构逐渐发育成熟,外颗粒层消亡以细胞迁移和凋亡为主,其他各层细胞主要经历了分化发育与凋亡。