Ras reduces L-type calcium channel current in cardiac myocytes. Corrective effects of L-channels and SERCA2 on [Ca(2+)](i) regulation and cell morphology.

Heart failure is associated with dysregulation of intracellular calcium ([Ca(2+)](i)), reduction in myofibrils, and increased activation of Ras, a regulator of signal-transduction pathways. To evaluate the potential effects of Ras on [Ca(2+)](i), we expressed constitutively active Ras (Ha-Ras(V12)) in cardiac myocytes and monitored [Ca(2+)](i) via fluorescence and electrophysiological techniques. Ha-Ras(V12) reduced the magnitude of the contractile calcium transients. Unexpectedly, however, calcium loading of the sarcoplasmic reticulum was increased, suggesting that Ha-Ras(V12) introduces a defect in excitation-calcium release coupling. Consistent with this idea, L-channel calcium currents were reduced by Ha-Ras(V12), which also downregulated the activity of the L-channel gene promoter. Coexpression of L-channels and SERCA2 largely corrected Ha-Ras(V12)-induced dysregulation of [Ca(2+)](i). Furthermore, whereas Ha-Ras(V12) downregulated myofibrils, this effect was blocked by coexpression of L-channels. These results suggest that Ras downregulates L-channel expression, which may play a pathophysiological role in cardiac disease.

Competitive interactions during dendritic growth: a simple stochastic growth algorithm.

A simple growth algorithm is presented that deals with one feature of dendritic growth, the distance between branches. The fundamental assumption of our growth algorithm is that the lengths of dendritic segments are determined by the branching characteristics of the growing neurite. Realistic-appearing dendritic trees are produced by computer simulations in which it is assumed that: (1) growth of individual neurons occurs only at the tips of each growing neurite; (2) the growing neurite can either branch (as a bifurcation) or continue to elongate; (3) events at any one growing tip do not affect the events at any other growing tip; and (4) the probability of branching is a function only of the distance grown either from the cell body (if branching has not occurred) or from the previous branch point. An analytic solution of a differential equation based on these same assumptions produces a distribution of dendritic segment lengths that accurately fits an experimentally determined distribution of dendritic segment lengths of reconstructed neurons, accounting for about 89% of the sample variance. Our analysis indicates that, immediately following branching, the temporary suppression of further branching during dendritic growth may be an important mechanism for regulating the distance between branches.

Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of mice.

The output potential of proliferating populations in either the developing or the adult nervous system is critically dependent on the length of the cell cycle (T(c)) and the size of the proliferating population. We developed a new approach for analyzing the cell cycle, the 'Saturate and Survive Method' (SSM), that also reveals the dynamic behaviors in the proliferative population and estimates of the size of the proliferating population. We used this method to analyze the proliferating population of the adult dentate gyrus in 60 day old mice of two inbred strains, C57BL/6J and BALB/cByJ. The results show that the number of cells labeled by exposure to BUdR changes dramatically with time as a function of the number of proliferating cells in the population, the length of the S-phase, cell division, the length of the cell cycle, dilution of the S-phase label, and cell death. The major difference between C57BL/6J and BALB/cByJ mice is the size of the proliferating population, which differs by a factor of two; the lengths of the cell cycle and the S-phase and the probability that a newly produced cell will die within the first 10 days do not differ in these two strains. This indicates that genetic regulation of the size of the proliferating population is independent of the genetic regulation of cell death among those newly produced cells. The dynamic changes in the number of labeled cells as revealed by the SSM protocol also indicate that neither single nor repeated daily injections of BUdR accurately measure 'proliferation.'

Barrels in somatosensory cortex of normal and reeler mutant mice.

The pattern of projection of the ventrobasal thalamic complex (VB) upon the neocortical barrel field of normal and reeler mutant mice was determined by orthograde degeneration experiments and by Timm's histochemical method. In both reeler and normal mice the thalamic terminals are concentrated at the midcortical zone dominated by granule cells and the adjacent zone dominated by medium-sized pyramidal cells. The medium-sized pyramids are principally supragranular in normal cortex but infragranular in the reeler. Throughout the barrel fields of reeler and normal mice thalamic terminals are organized as a mosaic of radially oriented columns coextensive in the tangential plane with barrels whose cross-sectional areas and shapes are similar in both genotypes. The radial extent of the columns in reeler is 2-3 times that in normal cortex. These observations suggest that the tangential organization of the thalamocortical projection is normal in reeler and that thalamic terminals are distributed among the same neuronal classes in normal and reeler mice despite cell malposition in the mutant.

The second, intralaminar thalamo-cortical projection system.

In the marmoset (Callithrix jacchus), HRP and 3H-apo-HRP were injected into various cortical regions and the positions of labelled neurons in the non-specific, intralaminar thalamic nuclei (N. centralis and centre m edian ) were investigated. Although neuron populations projecting to the different cortical regions overlap widely, a coarse topology exists inasmuch as intralaminar neurons projecting to the posterior cortex were located more rostrally and those projecting to the anterior cortex were located more caudally in the intralaminar complex. With injections into nearby cortical regions of the parieto-temporal association cortex with HRP and 3H-apo-HRP, respectively, no double labelled cells were found in the intralaminar nuclei, although the fields of labelled cells completely overlapped. Also in the specific projection nuclei no double labelled cells were encountered. About 10-20% of the thalamo-cortical projection cells are located in the intralaminar nuclei. Some functional aspects of this second thalamo-cortical projection system are discussed.

Overexpression of p27 Kip 1, probability of cell cycle exit, and laminar destination of neocortical neurons.

Neocortical projection neurons arise from a pseudostratified ventricular epithelium (PVE) from embryonic day 11 (E11) to E17 in mice. The sequence of neuron origin is systematically related to mechanisms that specify neuronal class properties including laminar fate destination. Thus, the neurons to be assembled into the deeper layers are the earliest generated, while those to be assembled into superficial layers are the later generated neurons. The sequence of neuron origin also correlates with the probability of cell cycle exit (Q) and the duration of G1-phase of the cell cycle (T(G1)) in the PVE. Both Q and T(G1) increase as neuronogenesis proceeds. We test the hypothesis that mechanisms regulating specification of neuronal laminar destination, Q and T(G1) are coordinately regulated. We find that overexpression of p27(Kip1) in the PVE from E12 to E14 increases Q but not T(G1) and that the increased Q is associated with a commensurate increase in the proportion of exiting cells that is directed to superficial layers. We conclude that mechanisms that govern specification of neocortical neuronal laminar destination are coordinately regulated with mechanisms that regulate Q and are independent of mechanisms regulatory to cell cycle duration. Moreover, they operate prior to postproliferative mechanisms necessary to neocortical laminar assembly.

Corticospinal tract collaterals to the dorsal column nuclei of cats. An anatomical single and double retrograde tracer study.

A double-labelling anatomical strategy employing horseradish peroxidase and tritiated, enzymatically inactive horseradish peroxidase allowed simultaneous visualization of corticospinal neurones and cortical neurones projecting to the dorsal column nuclei in cats. By this approach it is shown that although most cortical fibres to these nuclei are not branches of corticospinal axons, neurones projecting to both targets are present in all areas of the sensorimotor cortex and especially in area 3a. Thus, cortical control upon the dorsal column nuclei is mediated via descending fibres that differ as to their origin and to their branching pattern.

Descending projections from brainstem and sensorimotor cortex to spinal enlargements in the cat. Single and double retrograde tracer studies.

Single and double retrograde tracer techniques were employed in cats to investigate: (1) the topographical relationships between supraspinal neurons projecting to either the brachial or lumbosacral enlargement, (2) the distribution and relative frequency of single supraspinal neurons which project to both enlargements by means of axonal branching. In one group of cats large injections of horseradish peroxidase (HRP) were made throughout either the brachial or lumbosacral enlargement. The results from these experiments support recent observations on the multiplicity of brainstem centers giving origin to descending spinal pathways and provide evidence for a population of corticospinal neurons in area 6. In a second set of experiments, HRP was injected in one enlargement, and 3H-apo-HRP (enzymatically inactive) was injected in the other enlargement. Relatively large numbers of neurons with collateral projections to both enlargements (double-labeled) were observed in the medullary and pontine reticular formation, the medial and inferior vestibular nuclei bilaterally, the ipsilateral lateral vestibular nucleus, Edinger-Westphal nucleus, caudal midline raphe nuclei and nuclear regions surrounding the brachium conjunctivum. By contrast, double-labeled neurons were infrequently observed in the red nucleus and sensorimotor cortex, contralateral to the injections. In the red nucleus, lateral vestibular nucleus and sensorimotor cortex, neurons projecting to the brachial enlargement were largely segregated topographically from neurons projecting to the lumbosacral enlargement. However, there was some overlap, and double-labeled neurons were consistently observed within the region of overlap. In the sensorimotor cortex, the overlap between brachial- and lumbar-projecting neurons was most prominent in areas 4 and 3a, along the cruciate sulcus, but also involved other cytoarchitectonic regions in the medial aspect of the hemisphere.

Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase.

Two S-phase markers for in vivo studies of cell proliferation in the developing central nervous system, tritiated thymidine ((3)H-TdR) and bromodeoxyuridine (BUdR), were compared using double-labeling techniques in the developing mouse cortex at embryonic day 14 (E14). The labeling efficiencies and detectability of the two tracers were approximately equivalent, and there was no evidence of significant tracer interactions that depend on order of administration. For both tracers, the loading time needed to label an S-phase cell to detectability is estimated at <0.2 h shortly after the injection of the label, but, as the concentration of the label falls, it increases to approximately 0.65 h after about 30 min. Thereafter, cells that enter the S-phase continue to become detectably labeled for approximately 5-6 h. The approximate equivalence of these two tracers was exploited to observe directly the numbers and positions of nuclei entering (labeled with the second tracer only) and leaving (labeled with the first tracer only) the S-phase. As expected, the numbers of nuclei entering and leaving the S-phase both increased as the interval between the two injections lengthened. Also, nuclei leaving the S-phase rapidly move towards the ventricular surface during G2, but, unexpectedly, the distribution of the entering nuclei does not differ significantly from the distribution of the nuclei in the S-phase. This indicates that: (1) the extent and rate of abventricular nuclear movement during G1 is variable, such that not all nuclei traverse the entire width of the ventricular zone, and (2) interkinetic nuclear movements are minimal during S-phase.

CNS development: an overview.

The basic principles of the development of the central nervous system (CNS) are reviewed, and their implications for both normal and abnormal development of the brain are discussed. The goals of this review are (a) to provide a set of concepts to aid in understanding the variety of complex processes that occur during CNS development, (b) to illustrate how these concepts contribute to our knowledge of the normal anatomy of the adult brain, and (c) to provide a basis for understanding how modifications of normal developmental processes by traumatic injury, by environmental or experiential influences, or by genetic variations may lead to modifications in the resultant structure and function of the adult CNS.

Dorsal column nuclei and ascending spinal afferents in macaques.

Cell populations and thalamic projections of the dorsal column nuclei in macaques have been investigated in the medullae of normal animals and of animals with injections of horseradish peroxidase in the nucleus ventralis posterolateralis. In the same species, the course, distribution and origin of ascending non-primary pathways to the dorsal column nuclei have been demonstrated with the aid of degeneration methods, 3H-amino acid autoradiography and retrograde axonal transport of horseradish peroxidase. Non-primary afferents to the gracile and cuneate nuclei ascend mainly in the dorsal columns and, to a lesser extent, in the dorsal part of the lateral funiculus. Afferents originating from lumbar segments and ascending in the lateral funiculus terminate mainly in the rostral part of the gracile nucleus while those ascending in the dorsal columns distribute throughout most of the rostrocaudal extent of the same nucleus. Afferents from brachial levels terminate mainly in the cuneate nucleus and in the external cuneate nucleus. Degeneration and autoradiographic material concurrently demonstrate that non-primary afferents to the cuneate nucleus terminate preferentially within certain cytoarchitectonic subdivisions of this nucleus. Ascending spinal afferents to the dorsal column nuclei originate mainly from the ipsilateral dorsal horn, particularly from its medial part at upper cervical levels and from a band of gray, throughout the cord, largely corresponding to lamina IV and adjacent laminae. Large neurons along the lateral border of the ventral horn at lumbar levels may also contribute non-primary afferents to the ipsilateral dorsal column nuclei. These anatomical results provide some cues to a revised view of the organization of the dorsal column nuclei in monkeys and, taken together with recent electrophysiological and clinical data, contribute to a re-evaluation of some functional aspects of the dorsal column-medial lemniscal system of primates.

Local homogeneity of cell cycle length in developing mouse cortex.

We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the proliferating cells in the ventricular zone during early development of the cerebral cortex.

Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex.

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

Postembryonic neurogenesis in the procerebrum of the terrestrial snail, Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70-80%) of the snail procerebrum are produced during the first 1-2 months of posthatching development.

Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization.

Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalomocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex.