Biomedical subjects

P Levitt

Publications and source records attributed to P Levitt.

At least 55 records · Page 3Linked to original sources

PubMed1997

Patterning and specification of the cerebral cortex.

Regionalization of the cerebral cortex occurs during development by the formation of anatomically and functionally discrete areas of the brain. Descriptive evidence based on expression of molecules and structural features suggests that an early parcelation of the cerebral wall may occur during fetal development. Experimental strategies using tissue transplants and cell culture models have explored the nature of the timing of areal specification. New signaling systems displaying the sensitivity of precursor cells to environmental cues that define the fate of neurons destined for specific areas of the cortex have been discovered. Studies in the field now suggest mechanisms of regulating cell phenotype in the cortex that are common to all parts of the neuraxis.

Animals↗

PubMed1997

Signaling pathways that regulate specification of neurons in developing cerebral cortex.

The expression of the limbic system-associated membrane protein (LAMP), a marker of specific functional regions of the cerebral cortex, has been used to determine the environmental signals that regulate cortical regionalization. Transplant and cell culture studies have shown previously that the fate of precursor cells, based on LAMP expression, is amenable to regulation by exposure to novel environmental stimuli. This has been demonstrated in vitro to be dependent upon exposure to transforming factor-alpha and collagen type IV. Results following exposure to the inductive signals for a specific duration indicate a cell cycle dependence on the decision to become a limbic or nonlimbic cortical neuron. It appears, therefore, that areal and laminar fates are both influenced by mechanisms that specify commitment early in cortical development.

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PubMed1997

Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory.

Progenitor cells in the early developing cerebral cortex produce neurons destined for discrete functional areas in response to specific inductive signals. Using lineage analysis, we show that cortical progenitor cells at different fetal ages retain the memory of an area-specific inductive signal received in vivo, even though they may pass through as many as two cell cycles in the absence of the signal in culture. When exposed to inductive signals in vitro, only those progenitors that progress through at least one complete cell cycle alter their areal phenotype. Our findings suggest that induction of an areal phenotype is linealy inherited, with the phenotype specified prior to the final cell cycle.

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PubMed1996

The alpha 1 subunit of soluble guanylyl cyclase is expressed prenatally in the rat brain.

The mRNA encoding the alpha 1 subunit of soluble guanylyl cyclase (alpha 1sGC) was identified in a different-display screening for genes spatially and temporally regulated during the development of fetal rat brain. The initially isolated fragment of the 3' untranslated region was used for in situ hybridization and to produce full-length cDNA clones by hybridization screening of cDNA libraries and by RACE (rapid amplification of cDNA ends), respectively. In situ hybridization analysis that alpha 1sGC was absent at embryonic day 12 (E12), but by E14-E15, the forebrain exhibited dense expression in the developing striatum, medial cerebral wall containing the presumptive hippocampus, cerebellar neuroepithelium, and roof plate. Weaker expression was observed in the septum, epithalamus, ventral thalamus, pineal gland and retina. This pattern is largely maintained and refined at E18, with additional expression domains in the olfactory tubercle, nucleus accumbens, zona incerta and neocortex. During early postnatal development, the adult pattern is expressed, as previously reported. The unexpected, early expression of alpha 1sGC, in conjunction with the known absence of its heterodimeric partner, the beta subunit of sGC, from the developing rodent brain during fetal ages raises potentially novel functional roles of the alpha 1 subunit during ontogeny, and might imply the existence of an alternative beta subunit specific for the prenatal brain.

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PubMed1996

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): I. Adult rat brain.

The search for molecular markers common to neural structures that are functionally related has become an attractive strategy for neurobiologists interested in identifying mechanisms involved in the formation of patterned connections. One such molecule is the limbic system-associated membrane protein (LAMP), a 64-68 kDa glycoprotein that is expressed in the soma and dendrites of subpopulations of adult neurons in the brain that are functionally associated with classic limbic structures. Such patterned molecular specificity is established prenatally; LAMP is detected during development on the surface of neurons, axonal membranes and pathfinding growth cones. This molecule has now been cloned (lamp) and has been shown to be highly conserved in rat and human. It is a new immunoglobulin superfamily member that has three Ig domains and a glycosyl-phosphatidylinositol (GPI) anchor to the cell membrane. In this study, the distribution of the lamp transcript in the adult rat brain was determined by using in situ hybridization. Generally, the distribution of lamp corresponds well with that of the LAMP protein. Within the cerebral cortex, the transcript is more abundant in areas that are associated with learning/memory and viscerosensory tasks. It is less abundant in somatic sensory and motor areas. The lamp transcript is also ubiquitous in the basal forebrain, amygdala, and preopticohypothalamic areas. In short, the lamp transcript is expressed heavily in areas of the forebrain and diencephalon that have been classically considered limbic and sparsely or moderately in nonlimbic midbrain and hindbrain regions. Correlative analysis of the connectivity patterns of the regions that express greater amounts of the transcript is consistent with a stronger limbic-associated function relative to the regions expressing less lamp. These quantitative differences may be significant in determining the function of LAMP in the adult brain.

Amygdala↗

PubMed1996

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): II. Fetal rat brain.

The limbic system-associated membrane protein (LAMP) is a 64-68 kDa neuronal surface glycoprotein expressed in cortical and subcortical regions of the limbic system of the adult and developing rat central nervous system (CNS). LAMP is a member of the immunoglobulin superfamily of cell adhesion molecules with three Ig domains and is highly conserved between rat and human. In this study, the temporal and spatial pattern of lamp gene expression during fetal rat development was analyzed by using Northern blot analysis and in situ hybridization. In Northern blot analysis, two lamp mRNA transcripts, 1.6 kb and 8.0 kb, identical in size to those present in the adult rat nervous system, were detected in developing neural tissue. In situ hybridization analysis showed close correlation, though not identity, between the expression of lamp mRNAs and the distribution of LAMP in limbic regions of the developing rat CNS, indicative of a more complex regulation of gene expression than was previously thought to be the case. The expression of lamp mRNAs is first detected on about embryonic day (E) 13. The hybridization signal is not seen in the proliferative ventricular zone at any level of the neuraxis, indicating that lamp is expressed in postmitotic neurons. In the cerebral cortex, lamp mRNAs are expressed in limbic cortical regions, such as the perirhinal cortex, prefrontal cortex, and cingulate cortex. In the hippocampus, the hybridization signal is observed in Ammon's horn by E18. The neostriatum, amygdaloid complex, and most hypothalamic areas express lamp mRNAs from early stages (E13-E14) in a pattern consistent with the onset of neurogenesis. The emerging patterns of lamp expression at the outset are similar to those seen in adult hypothalamus and dorsal thalamus. Although the hybridization signal is observed in some nonlimbic areas, including midbrain and hindbrain structures, intense labeling is evident in more classic limbic regions. The high levels of expression of lamp in limbic regions, beginning in early developmental stages, combined with the results of previous functional in vitro and in vivo studies, support a role for LAMP as a recognition molecule involved in the formation of limbic connections.

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PubMed1996

Short exposure to methylazoxymethanol causes a long-term inhibition of axonal outgrowth from cultured embryonic rat hippocampal neurons.

Methylazoxymethanol (MAM) is an alkylating agent that is used to induce microencephaly by killing mitotically active neuroblasts. We found that at later developmental times, MAM exposure can result in abnormal fiber growth in vivo. However, there have not been any previous studies on the effects of MAM on differentiating neurons. We examined the outcome of short exposure to MAM on postmitotic embryonic hippocampal cultures during the establishment of axonal polarity. At 0, 1, or 2 days in vitro (DIV), neurons were treated with 0.1 nM-1 microM MAM for 3 hr and then transferred to glial conditioned media. At 3 DIV, the cells were fixed and analyzed by immunofluorescent staining for neuron viability and differentiation. Control cells initiate several minor processes; one process elongates rapidly at about 1 DIV eventually becoming an axon, while extensive dendritic growth occurs after 3-4 DIV. Neurons treated with 1 microM MAM at 0 or 1 DIV showed a marked inhibition of neurite growth and withdrawal of axons without affecting cell viability. These cells continued to show minimal neurite outgrowth at 7 DIV, even when transferred to a glial coculture. In contrast, cells treated initially with MAM, after neuronal polarity is established at 2 DIV, showed no effect on axonal growth. To determine the effects of MAM on the neuronal cytoskeleton, we examined the in vitro assembly of brain microtubules in a one cycle assay. Exposure to MAM depleted the soluble pool of proteins, including microtubule-associated protein 1B (MAP1B) and MAP2, which are required for neurite outgrowth, through a nonspecific process. Under non-saturating conditions, there were no changes in the total amount of microtubules assembled or the coassembly of MAP1B and MAP2 in the presence of MAM. These results demonstrate that MAM can directly affect differentiating neurons, indicating that an early disruption of axonal outgrowth may have long-term effects.

Alkylating Agents↗

PubMed1996

Restricted expression of the actin-regulatory protein, tropomyosin, defines distinct boundaries, evaginating neuroepithelium, and choroid plexus forerunners during early CNS development.

In the hindbrain, rhombomeres represent morphological units that develop characteristic, segment-specific structures. Similar segments, known as prosomeres, have been proposed to exist in the forebrain. The neuroepithelial cells of the sharp boundary regions that form the borders between many segments often exhibit distinct shapes, reflecting unique cytoskeletal organization. The present investigation examined the expression of one family of actin-binding, regulatory proteins, the tropomyosins (TM), in boundaries. We found that high molecular weight TMs selectively concentrate in boundary cells and other neuroepithelial zones that exhibit unique cell shapes and movements. Specific TM expression is found at hindbrain boundaries as early as embryonic day 10 in the rat, whereas rhombomeres themselves were TM-negative. Highly restricted TM localization also defined some prosomere boundaries in the early forebrain, particularly those exhibiting unique cell shapes. Furthermore, several regions of the neuroepithelium that evaginate are TM-immunoreactive, including tuberal and preoptic neuroepithelium. Most striking, a subpopulation of neuroepithelial cells in the medial telencephalic wall expresses TM, apparently marking the neuroepithelial region that gives rise to the choroid plexus at least 2 d before its formation. This suggests that the medial cerebral wall is not entirely dedicated to generating cells that comprise allocortex. TM expression in the choroid plexus is maintained through initial evagination and appearance in all ventricles. The spatially restricted expression of TMs implicates that this actin-binding protein is involved in the dynamic regulation of cell shape or motility associated with boundary formation and morphogenesis of the neuroepithelium during critical stages of brain development.

Actins↗

PubMed1996

cDNA cloning and structural analysis of the human limbic-system-associated membrane protein (LAMP).

The limbic-system-associated membrane protein (LAMP) is a 64-68-kDa neuronal surface glycoprotein distributed in cortical and subcortical regions of the limbic system. The human LAMP gene was cloned by RT-PCR using human cerebral cortex mRNA and oligodeoxyribonucleotide (oligo) primers derived from the rat lamp cDNA sequence. The human and rat LAMP cDNAs showed 94% identity at the nucleotide (nt) level, and the encoded 338-amino-acid (aa) polypeptides shared 99% sequence identity. All the important features of LAMP were conserved: (i) the deduced aa sequence reflecting a glycosyl-phosphatidylinositol (GPI)-anchor, (ii) eight putative N-linked glycosylation sites, and (iii) conserved pairs of Cys forming three internal repeats characteristic of the immunoglobulin superfamily (IgSF). Northern blot analysis indicated the presence of two mRNA transcripts in the human brain of a size identical to those identified in adult rat brain. These data indicate that LAMP is a highly conserved new member of the IgSF which, together with the opioid-binding cell adhesion molecule (OBCAM) and neurotrimin, comprises a new subfamily that has been designated as IgLONs. With a unique distribution in limbic structures, LAMP may play an important role in limbic system development and function, as suggested by previous in vitro and in vivo functional studies.

Amino Acid Sequence↗

PubMed1996

Requirement of a corepressor for Dr1-mediated repression of transcription.

A Dr1-associated polypeptide (DRAP1) was isolated from HeLa cells and found to function as a corepressor of transcription. Corepressor function requires an interaction between DRAP1 and Dr1. Heterodimer formation was dependent on a histone fold motif present at the amino terminus of both polypeptides. Association of DRAP1 with Dr1 results in higher stability of the Dr1-TBP-TATA motif complex and precluded the entry of TFIIA and/or TFIIB to preinitiation complexes. DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index. Analysis of DRAP1 in the developing brain of rat demonstrated undetectable levels of DRAP1 in actively dividing cells but high levels of DRAP1 expression in differentiated non dividing cells. Dr1 was immunodetected in all cells analyzed. A model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed.

Amino Acid Sequence↗

PubMed1996

Environmental signals influence expression of a cortical areal phenotype in vitro independent of effects on progenitor cell proliferation.

We have shown previously that, in vitro, cortical progenitor cells isolated from specific locations of the cerebral wall can adopt area-specific fates, assayed by expression of the limbic system-associated membrane protein (LAMP; R. T. Ferri and P. Levitt, Cereb. Cortex 3, 187-198, 1993). Progenitors destined to produce LAMP neurons, however, will differentiate to express the limbic molecular phenotype if grown with TGFalpha and collagen type IV (R. T. Ferri and P. Levitt, Development 121, 1151-1160, 1995), while other signals fail to induce LAMP. The present study used BrdU labeling of progenitor cells to examine whether modulation of LAMP expression was paralleled by predictable changes in cell proliferation. The general pattern of proliferation is similar under a variety of culture conditions: approximately half the cells are mitotic, and activity is always highest during the first 24 hr in vitro, with little cell division occurring by the third day. Moreover, the rate of proliferation, in the presence or absence of TGFalpha, is the same on all substrates tested, with the exception of laminin. The TGFalpha/collagen type IV signaling system that induces LAMP expression by the precursors has no modulating effect on their proliferative kinetics. Nonetheless, only progenitors that are mitotically active respond to LAMP-inducing signals; only 60% of the neurons, representing those that have divided at least once in culture, can be induced to express LAMP. The data suggest that while specific signals affect choice of area phenotype during the cell cycle, they do so in the absence of major changes in proliferative behavior.

Animals↗

PubMed1996

Normal development of tyrosine hydroxylase and serotonin immunoreactive fibers innervating anterior cingulate cortex and visual cortex in rabbits exposed prenatally to cocaine.

Anterior cingulate cortex develops abnormally in rabbits exposed to cocaine in utero but visual cortex is normal. The interactions of cocaine with the dopamine, norepinephrine and serotonin systems makes each a potential candidate for influencing these developmental effects. Here, we report no differences in the distribution and density of serotonin and tyrosine hydroxylase-immunoreactive fibers in the anterior cingulate cortex in postnatal rabbits exposed prenatally to cocaine or saline. Because the pattern and extent of cortical innervation by these systems appears normal, the data suggest that the effects of prenatal exposure to cocaine may involve changes in monoamine signaling distinct from morphological reorganization of the cortical afferents.

Animals↗

PubMed1996

Dopamine receptors mediate differential morphological effects on cerebral cortical neurons in vitro.

A morphogenic role of neurotransmitters during cellular differentiation in vitro has been demonstrated in recent years. Using in situ hybridization, we confirm the presence of the D1 receptor at E16 and show additionally that the transcript is relatively widespread and present in both proliferative and differentiating areas of the cerebral wall. Because DA receptor expression precedes the arrival of presynaptic terminals during forebrain development, we examined the role of DA in cerebral cortical neuron differentiation in vitro, using immunohistochemical markers of dendrites, microtubule-associated-membrane protein 2 (MAP2) and axons, neurofilament protein (NF-H). Neurite length, cell size, and cell viability in response to D1 and D2 receptor agonists SKF38393 and quinpirole, respectively, and to DA were analyzed in neurons obtained from embryonic (E) day 16 rats. We have shown that 1) paradoxically, DA at different concentrations can either stimulate or inhibit neurite outgrowth; 2) there is a bimodal pattern of DA-induced axonal outgrowth, i.e., at low and high doses; 3) D2 receptor activation induces neurite outgrowth while D1 receptor activation is inhibitory; 4) D2-mediated neurite elongation is preferentially axonal while D1 receptor activation reduces both axonal and dendritic outgrowth; 5) low doses of DA promote the expression of cytoskeletal components of axonal maturation; and 6) D1 receptor activation decreases neuronal size. We suggest that DA may influence cellular differentiation and circuitry formation early in development of the cerebral cortex through receptor-mediated effects on process outgrowth, which could lead to effects on circuit formation.

Animals↗

PubMed1996

Limbic system-associated membrane protein (LAMP) in primate amygdala and hippocampus.

The distribution of the limbic system-associated membrane protein in the amygdaloid complex and hippocampal formation of cynomolgus monkeys (Macaca fascicularis) was studied with immunohistochemical procedures. A highly complex and heterogeneous staining pattern is encountered in the macaque amygdala. The basal, lateral, and accessory basal nuclei display the most intense immunostaining with local heterogeneities. The lateral division of the central nucleus also stains intensely, whereas the medial division of the central nucleus and the medial nucleus are more weakly stained. The dorsal division of the bed nucleus-amygdala continuum (extended amygdala) is strongly immunoreactive. The hippocampus displays the strongest immunoreactivity encountered so far in the primate brain. The intensity of the immunostaining is highest in the cornu Ammonis (Ammon's horn; CA1-CA3 fields) and gradually decreases toward the dentate gyrus or the subicular area. In the hippocampus proper, the stratum radiatum, the pyramidal layer, the stratum oriens, and the alveus all display intense immunoreactivity. The immunostaining is much less prominent in the dentate gyrus, whose granule cell layer is completely devoid of labeling. In the subicular area, there is a lateromedial decreasing gradient in immunostaining intensity, the subiculum being moderately stained and the parasubiculum weakly stained. These results reveal that the limbic system-associated membrane protein labels structures that form the core of the limbic system in primates. Within each of these structures, however, the labeling is highly heterogeneous and appears to be confined to specific functional domains.

Amygdala↗

PubMed1995

Intrauterine cocaine exposure of rabbits: persistent elevation of GABA-immunoreactive neurons in anterior cingulate cortex but not visual cortex.

The effects of prenatal cocaine exposure on the development of the rabbit cerebral cortex were studied. Two cortical areas were compared: primary visual cortex (VC) and anterior cingulate cortex (ACC). ACC was selected because behavioral deficits observed in cocaine-exposed infants suggest the involvement of ACC. In addition, ACC receives dense dopaminergic innervation and cocaine's action in inhibiting the re-uptake of dopamine is believed to underly the rewarding properties of cocaine. VC was selected as a control area because there is no evidence of behavioral deficits associated with visual perception in cocaine-exposed infants, and because VC receives minimal dopaminergic innervation. Two aspects of cortical development were studied: (i) cortical morphology, growth and cytoarchitectonic organization; and (ii) the development of the GABAergic neurotransmitter system. Measures of postnatal cortical growth, including cortical lamination, cell number and soma size, were compared in cocaine-exposed or control (saline) rabbits aged P5-P60. There was no difference between cocaine and saline animals in any of these parameters, and cortical cytoarchitecture appeared normal. However, despite the absence of major abnormalities in cortical development, we found that the number of GABA-immunoreactive neurons in cocaine-exposed animals was significantly higher than normal in ACC. This effect was highly consistent, was present in all laminae and at all ages studied, and persisted into maturity (P60). In contrast, in VC, the number of GABA-immunoreactive neurons in cocaine-exposed animals did not differ from normal. We suggest that increased GABA immunoreactivity may reflect a compensatory response to excessive excitatory input to ACC. A change in the balance of excitation and inhibition in ACC, reflecting 'noisy' or dysfunctional intracortical circuitry, may underly the emotional lability and attentional deficits characteristically described in infants exposed in utero to cocaine.

Animals↗

PubMed1995

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Animals↗

PubMed1995

Molecular contributions to cerebral cortical specification.

Evidence is accumulating that decisions of cell fate and commitment to specific regional phenotypes in the cerebral cortex occur through cell interactions that likely begin early in development, perhaps in the proliferative zone. We have focused on the development of the limbic cortex in rats, which includes areas involved in both cognitive and autonomic functions and is marked by expression of the limbic system-associated membrane protein. Transplantation studies show that precursor cells are sensitive to environmental cues which can control expression of area-specific phenotypes, including limbic system-associated protein synthesis and connectivity patterns, but early postmitotic neurons faithfully express traits based on their origin in the donor. We have studied this sensitive period of decision making in vitro. Molecules from the epidermal growth factor family influence dramatically the fate of precursor cells, but only in the presence of matrix molecules. In vivo, both the epidermal growth factor receptor and collagen type IV are expressed in the progenitor cell pool indicating that they can directly affect the initial decisions in differentiation. We suggest that early patterns of gene expression, influenced by environmental cues, are likely to provide a specific framework for subsequent decisions that lead to establishing cortical areas.

Animals↗

PubMed1995

Distribution of limbic system-associated membrane protein immunoreactivity in primate basal ganglia.

The limbic system-associated membrane protein is a 64,000-68,000 mol.wt molecule known to be preferentially expressed by neurons in limbic structures of rats and cats. The present immunohistochemical study describes the distribution of this protein in the basal ganglia of Macaca fascicularis. The ventral striatum of the cynomolgus monkey displays a very intense immunostaining, whereas the dorsal striatum is much more weakly stained, except for some small zones scattered in the caudate nucleus and, to a lesser extent, in the putamen. These protein-rich zones are in register with striosomes, as visualized on adjacent sections immunostained for calbindin. At pallidal levels, immunostaining for the protein is observed only in the subcommissural regions, at the ventromedial tip of the internal pallidum, and in the caudoventral portion of the external pallidum. At nigral levels, the immunostaining is highly heterogeneous with a marked decreasing rostrocaudal gradient. The staining is most intense in nigral regions that receive striatal inputs and are enriched with calbindin. Nigral sectors populated by dopaminergic neurons, as visualized on adjacent sections immunostained for tyrosine hydroxylase, are largely devoid of immunoreactivity. In contrast, the immunostaining is uniformly intense in the ventral tegmental area. This study provides the first neuroanatomical evidence for teh existence of the limbic system-associated membrane protein in primate brain. It reveals that this glycoprotein is distributed in a highly heterogeneous manner in primate basal ganglia, where it preferentially labels regions that are anatomically and functionally linked to the limbic system.

Animals↗