Multiple Parallel Pathways Research Articles

Bone stem cells.

Osteoblasts are the skeletal cells responsible for synthesis, deposition, and mineralization of the extracellular matrix of bone. By mechanisms that are only beginning to be understood, stem and primitive osteoprogenitors and related mesenchymal precursors arise in the embryo and at least some appear to persist in the adult organism, where they contribute to replacement of osteoblasts in bone turnover and in fracture healing. In this paper, the nature of these cells, whether they constitute a stem cell pool or a committed progenitor pool, and aspects of their apparent plasticity are discussed. Current understanding of differential expression of osteoblast-associated genes during osteoprogenitor proliferation and differentiation to mature matrix synthesizing osteoblasts is summarized. Finally, evidence is discussed that supports the hypothesis that the mature osteoblast phenotype is heterogeneous with subpopulations of osteoblasts expressing only subsets of the known osteoblast markers, raising also the possibility of multiple parallel differentiation pathways and perhaps even different progenitor pools. J. Cell. Biochem. Suppls. 30/31:73-82, 1998. © 1998 Wiley-Liss, Inc.

A hierarchical neuronal network for planning behavior.

Planning a goal-directed sequence of behavior is a higher function of the human brain that relies on the integrity of prefrontal cortical areas. In the Tower of London test, a puzzle in which beads sliding on pegs must be moved to match a designated goal configuration, patients with lesioned prefrontal cortex show deficits in planning a goal-directed sequence of moves. We propose a neuronal network model of sequence planning that passes this test and, when lesioned, fails in a way that mimics prefrontal patients' behavior. Our model comprises a descending planning system with hierarchically organized plan, operation, and gesture levels, and an ascending evaluative system that analyzes the problem and computes internal reward signals that index the correct/erroneous status of the plan. Multiple parallel pathways connecting the evaluative and planning systems amend the plan and adapt it to the current problem. The model illustrates how specialized hierarchically organized neuronal assemblies may collectively emulate central executive or supervisory functions of the human brain.

Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion.

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these "stress-activated" MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/ SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.

Modeling blood flow heterogeneity.

It has been known for some time that regional blood flows within an organ are not uniform. Useful measures of heterogeneity of regional blood flows are the standard deviation and coefficient of variation or relative dispersion of the probability density function (PDF) of regional flows obtained from the regional concentrations of tracers that are deposited in proportion to blood flow. When a mathematical model is used to analyze dilution curves after tracer solute administration, for many solutes it is important to account for flow heterogeneity and the wide range of transit times through multiple pathways in parallel. Failure to do so leads to bias in the estimates of volumes of distribution and membrane conductances. Since in practice the number of paths used should be relatively small, the analysis is sensitive to the choice of the individual elements used to approximate the distribution of flows or transit times. Presented here is a method for modeling heterogeneous flow through an organ using a scheme that covers both the high flow and long transit time extremes of the flow distribution. With this method, numerical experiments are performed to determine the errors made in estimating parameters when flow heterogeneity is ignored, in both the absence and presence of noise. The magnitude of the errors in the estimates depends upon the system parameters, the amount of flow heterogeneity present, and whether the shape of the input function is known. In some cases, some parameters may be estimated to within 10% when heterogeneity is ignored (homogeneous model), but errors of 15-20% may result, even when the level of heterogeneity is modest. In repeated trials in the presence of 5% noise, the mean of the estimates was always closer to the true value with the heterogeneous model than when heterogeneity was ignored, but the distributions of the estimates from the homogeneous and heterogeneous models overlapped for some parameters when outflow dilution curves were analyzed. The separation between the distributions was further reduced when tissue content curves were analyzed. It is concluded that multipath models accounting for flow heterogeneity are a vehicle for assessing the effects of flow heterogeneity under the conditions applicable to specific laboratory protocols, that efforts should be made to assess the actual level of flow heterogeneity in the organ being studied, and that the errors in parameter estimates are generally smaller when the input function is known rather than estimated by deconvolution.

Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.

We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.

Tracing neural pathways in snail olfaction: From the tip of the tentacles to the brain and beyond

The anatomical organization of the olfactory system of terrestrial snails and slugs is described in this paper, primarily on the basis of experiments using the African snail Achatina fulica. Behavioral studies demonstrate the functional competence of olfaction in mediating food finding, conspecific attraction, and homing. The neural substrate for olfaction is characterized by an extraordinarily large number of neurons relative to the rest of the nervous system, and by the fact that many of them are unusually small. There exist multiple serial and parallel pathways connecting the olfactory organ, located at the tip of the tentacle, with integrative centers in the central nervous system. Our methods of studying these pathways have relied on the selective neural labels horseradish peroxidase and hexamminecobaltous chloride. One afferent pathway contains synaptic glomeruli whose ultrastructure is similar to that of the glomeruli seen in the mammalian olfactory bulb and the insect olfactory lobe. All of the olfactory neuropils, but especially the tentacle ganglion, contain large numbers of morphologically symmetrical chemical synapses. The procerebrum is a unique region of the snail brain that possesses further features analogous with olfactory areas in other animal groups. Olfactory axons from the tentacle terminate in the procerebrum, but the intrinsic neurons do not project outside of it. An output pathway from the procerebrum to the pedal ganglion has been identified and found to consist of inter-ganglionic dendrites. The major challenge for future studies is to elucidate the pattern of connectivity within, rather than between, the various olfactory neuropils.

Distribution and morphology of area 17 neurons that project to the cat's extrastriate cortex

We used intracellular dye injections in lightly fixed cortical slices to examine the distribution and morphology of area 17 neurons that project to extrastriate cortex. Both the projection to the medial bank of the posterior lateral suprasylvian sulcus (PMLS) and the projection to area 18 arise from a number of different morphological types distributed throughout layers 2-6. The majority are found in the superficial layers and include large, medium, and small pyramidal neurons. Some are also found in the deep layers and they include very large, pyramidal neurons as well as some heretofore undescribed, fusiform neurons. The projection to area 18 contains two types not found in the projection to PMLS: spinous stellate neurons in layer 4 and inverted pyramidal neurons in layer 3. Finding that a variety of morphological types contribute projections to a single cortical area raises the possibility that corticocortical projections, like retinogeniculate and geniculocortical projections, comprise multiple parallel pathways with different physiological properties and patterns of termination. Finding that the projection to area 18 contains morphological types that do not project to PMLS indicates that the projections from area 17 are likely to contribute to the functional specialization of extrastriate visual areas.

Distributed processing of sensory information in the leech. II. Identification of interneurons contributing to the local bending reflex

Isolated midbody ganglia of the leech Hirudo medicinalis were surveyed for interneurons contributing to the dorsal component of the local bending reflex, i.e., to the excitation of dorsal excitatory motor neurons that follows stimulation of dorsal mechanoreceptors responsive to pressure (P cells). Nine types of local bending interneuron could be distinguished on physiological and morphological grounds--8 paired and 1 unpaired cell per ganglion. Synaptic latencies from sensory neurons to interneurons were consistent with a direct or possibly disynaptic pathway. Connections between interneurons appeared to be rare and hyperpolarization of individual interneurons during local bending produced small but reliable decrements in motor neuron response, suggesting that multiple parallel pathways contribute to the behavior. Paradoxically, most interneurons received substantial inputs from ventral as well as dorsal mechanoreceptors, indicating that interneurons that were distinguished by their contribution to dorsal local bending were, in fact, active in ventral and lateral bends as well. The capacity to detect a particular stimulus and produce the appropriate response cannot be localized to particular types of interneuron; rather, it appears to be a distributed property of the entire local bending network.

Clonal analysis of mechanisms of murine T helper cell collaboration with effector cells of macrophage lineage.

The molecular basis and genetic restrictions of collaboration between Th cells and macrophages (Mo) and the numbers of types of collaboration in the Ag-specific cellular immune response were analyzed. Using the response of cloned Ag-specific T cells we examined the mechanisms of induction of the macrophage procoagulant response. Two generic types of collaboration were identified. One was mediated by the lymphokine monocyte procoagulant inducing factor (MPIF) and the second mechanism was by apparent contact collaboration. The lymphokine MPIF was produced by T cells and cloned CD4+ T cells after specific Ag stimulation. Cloned CD8+ cells, most of which also exhibited cytolytic activity, produced little MPIF. There was no evident restriction of the response of Mo of different MHC or background genes. In the second collaborative pathway a subset of CD4+ cloned Th cells were able to directly collaborate by an apparent contact mechanism with Mo for the procoagulant response. There was no correlation of this latter capacity with MPIF production. In addition abrogation of protein synthesis and lymphokine production by Ag-driven clones did not abrogate the direct cell association type of collaboration. Both forms of collaboration were equally efficient across MHC incompatibility barriers and different genetic background. We conclude that there are two independent and parallel Th:Mo collaborative pathways for Ag-driven responses in this limb of the cellular immune response, i.e., a MPIF lymphokine pathway and a contact pathway, and that there are quantitative and qualitative clonal differences in the use of these two pathways. We suggest that the existence of multiple parallel pathways for cellular collaboration may occur more widely in the Th:Mo limb of the immune response in respect to other Mo effector molecules and should be explored to understand the orchestration of this limb of the immune response.

Expression of granulocytic functions by leukemic promyelocytic HL-60 cells: differential induction by dimethylsulfoxide and retinoic acid

Recently, a novel approach has been used in the treatment of leukemia: induction of the leukemic cells to undergo terminal differentiation. Based on its in vitro ability to induce differentiation in several myeloid leukemic cell lines, retinoic acid (RA) has been applied clinically in cases of myelodysplastic syndromes and acute myeloid and promyelocytic leukemia. In the present study we have determined in detail the ability of RA to induce expression of granulocytic functions in a human promyelocytic leukemia cell line (HL-60) and compared it with that of dimethylsulfoxide (DMSO). Several granulocytic characteristics (phagocytosis, surface adherence and generation of free radicals in response to phorbol-ester) were induced to the same degree by both agents. Other normal neutrophil functions, including lysozyme accumulation, spontaneous migration, chemotactic activity toward zymosan-activated serum (containing C 5a), the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and spontaneous motility in semi-solid medium were induced by DMSO, but they were absent or incompletely expressed in RA-induced cells. In contrast, only RA induced migration toward leukotriene B 4 (LTB 4). Simultaneous treatment with RA and DMSO proved synergistic with respect to morphological maturation and several functions (e.g. NBT reduction), but complementary stimulation of other activities (e.g. chemotaxis, lysozyme content) could not be demonstrated. Furthermore, characteristics induced by DMSO (i.e., expression of C 5a and FMLP receptors and accumulation of lysozyme) were inhibited by the addition of RA. The results suggest that the inducer determines not only the lineage specificity of the differentiation process, but also affects the expression of cellular functions and characteristics specific to a particular lineage. It may be that induction of differentiation in HL-60 cells does not involve a ‘master switch’ mechanism operating through sequential activation of specific genes, but rather multiple parallel pathways, each independently acted upon by the inducer. The possibility of using a combination of inducers which will complement each other's actions should be considered when differentiation-inducing therapy is indicated.

Double-labeled metabolic maps of memory.

The physical changes representing a memory are believed to be localized to specific neurons, widely distributed in multiple parallel pathways in the brain. 2-Fluorodeoxyglucose, labeled with two discriminable radioactive tracers, was used to construct quantitative metabolic maps in split-brain cats during a visual task. One side of the brain served to estimate the metabolic variability of nonspecific influences. The other side was used to map metabolic changes related to the presence of previously learned visual cues, as well as changes related to nonspecific influences, in the same periods of time. When the two sides were compared, between 5 million and 100 million neurons (depending upon the significance level selected) were identified in which activity increased during presentation of the familiar cues. The wide distribution of these neurons throughout the brain is compatible with prior evidence of a distributed memory system. However, the large number of neurons involved is difficult to reconcile with theories in which individual neurons are dedicated to specific memories.

Direct visualization of organelle movement along actin filaments dissociated from characean algae.

A system has been developed in which organelle transport can be studied without the influence of an organized cellular cytoplasm. Binding and continuous unidirectional movement of organelles along isolated cellular transport cables were directly visualized by video light microscopy after the dissociation of the cytoplasm of characean algae cells in a Ca2+-free buffer containing adenosine triphosphate. Individual organelles had more than one attachment site and moved at mean rates of 11.2 or 62.1 micrometers per second along multiple parallel pathways on each cable. Electron microscopy of these cables after direct freezing demonstrated that they consist of compact bundles of actin filaments. Under these conditions, characteristics of organelle movement should reflect directly the underlying molecular processes of binding and force generation.

Ascending projections to the medial geniculate body of the cat: evidence for multiple, parallel auditory pathways through thalamus

The sources of ascending input to the medial geniculate body (MGB) of the cat were studied using the retrograde transport of horseradish peroxidase (HRP). HRP injections were made iontophoretically through micropipettes which were also used to record physiological properties at the injection sites. This technique produced small injections which appeared to be restricted to single subnuclei. The tectothalamic projection of the auditory system was found to consist of at least four distinct and separate pathways. The ventral division of the MGB receives a topographical projection from the central nucleus of the inferior colliculus (ICC) which preserves tonotopicity and provides short latency, sharply frequency-tuned responses. The medial part of the ICC projects to the deep dorsal nucleus, which contains only units tuned to high frequencies. The major inputs to the caudodorsal nucleus (DC) stem from nucleus sagulum and the pericentral nucleus of the inferior colliculus (ICP). Units in DC and the ventrolateral nucleus, which also receive input from ICP, have very broad tuning properties and late, habituating responses. Injections of HRP into the medial division (MGM) produced labeled cells scattered throughout the external nucleus of the inferior colliculus and the ventral part of ICC. This widespread input is reflected in the wide range of auditory responses found in MGM. Auditory responses in the suprageniculate nucleus were poorly defined and many units did not respond to tonal stimuli; following HRP injections no filled cells were found in the inferior colliculus, but labeled cells were found in the deeper layers of the superior colliculus and in the interstitial nucleus of the brachium of the inferior colliculus. Together with recent findings on the auditory thalamocortical projection, these results provide evidence for multiple parallel auditory pathways through the thalamus.

Statistical analysis of multipath neural systems.

Peripheral sensory and motor systems may be characterized by models consisting of multiple parallel convergent pathways, each described by the same set of equations, but having different parameter values in each path. Such models, although deterministic, are best analyzed using a statistical approach, which is illustrated here by analysis of several simple multipath models composed of linear dynamic elements and static non-linear elements. Relationships between instantaneous means of signals at different points in such systems are used to show that a multipath system can exhibit behaviour which would not be expected from observations of individual pathways. Mechanisms for linearization of static nonlinearities are briefly described. Important implications for neurophysiologists are discussed.

Chapter 13 The Use of Mutants in Metabolic Studies

Publisher Summary This chapter discusses the use of mutants in metabolic studies. The use of mutants in metabolic studies is fundamental: (1) Mutants can be used to confirm, to disprove, or even to discover the existence of a metabolic pathway.(2) They can be used to establish the occurrence within the cell of multiple parallel or cross-linked pathways. Each metabolic system generally presents some peculiarities allowing the selection of mutants, and each mutant selected in turn can be a source for the selection of new mutants. Physiological studies profit at least as metabolic studies from the use of appropriate mutants. The physiological role of some key metabolites such as sterols, fatty acids, vitamins, and hemin can be studied by looking for the primary defect after starvation or for the effect of replacement of the metabolite by another one. The permeation mechanisms for a metabolite can be examined by the use of mutants unable to transform it, allowing the study of its entry and the size of its pool independently of its utilization. The physiological links between different cell functions such as RNA and protein biosynthesis can be investigated in thermosensitive mutants impaired in one of these processes.