Biomedical subjects

P Levitt

Publications and source records attributed to P Levitt.

At least 19 recordsLinked to original sources

PubMed1997

Limbic system-associated membrane protein (LAMP) induces neurite outgrowth and intracellular Ca2+ increase in primary fetal neurons.

The ability of cell adhesion molecules (CAMs) to transduce cell surface signals into intracellular responses is critical for developing neurons, particularly during axonal pathfinding and targeting. It has been suggested that different CAMs can promote neuronal outgrowth via activation of common neuronal CAM-specific second-messenger pathways, although the elements involved in this cascade could differ. Limbic system-associated membrane protein (LAMP), a member of the Ig superfamily, is a molecule that promotes cell adhesion and neurite outgrowth from specific populations of fetal neurons. In the present study, we show that LAMP can induce several types of calcium (Ca2+) signals. Neurite outgrowth is promoted if fetal hippocampal neurons are grown on lamp-transfected CHO cells. This LAMP-induced outgrowth of neurons is mediated in part through activation of L-type Ca channels. Application of soluble LAMP to cultures of fetal hippocampal neurons caused a sustained (up to 60 min) elevation of intracellular Ca2+ as measured by fluo-3 fluorescence on a confocal microscope. The number of responding hippocampal neurons was initially low, but increased with age in culture and the [Ca2+]i elevation was only partially decreased by an L-type Ca(2+)-channel blocker. In contrast, at all times in culture, only a small fraction of neurons from visual cortex responded to LAMP application and only with transient elevation of cytosolic Ca2+ (< 15 min). Based on these observations, LAMP appears to function primarily through homophilic interactions and acts in part by modulating intracellular Ca2+ levels during neurite outgrowth by increasing the Ca2+ influx through L-type calcium channels, but has additional effects on intracellular Ca2+ signaling at later developmental stages.

Animals↗

PubMed1997

Cocaine administration in pregnant rabbits alters cortical structure and function in their progeny in the absence of maternal seizures.

Previous studies have reported that cocaine exposure in utero results in structural and functional alterations in the development of the anterior cingulate cortex (ACC). In the present study, the effects of maternal cocaine dosage and of cocaine-elicited maternal seizures on the progeny were studied. The incidence of maternal generalized tonic clonic seizures (GTCSs) elicited by cocaine was recorded. No GTCSs were elicited in pregnant rabbits by doses of 2 or 3 mg/kg of cocaine, but GTCSs were sometimes elicited by the highest dose (4 mg/kg per injection). We analyzed the offspring of cocaine-exposed and control animals using three assays of ACC development: (i) the structure of apical dendrites of pyramidal neurons, (ii) the distribution of a calcium binding protein (parvalbumin) in the dendrites of GABAergic neurons, and (iii) coupling of D1-like receptors and their G proteins. In all progeny of rabbits exposed to 3 or 4 mg/kg of cocaine during pregnancy, there was a significant change in the structure of apical dendrites, a significant increase in the number of dendrites of GABAergic neurons which were parvalbumin immunoreactive, and a significant reduction in D1/G protein coupling. In assays of apical dendrites, the effects on offspring of rabbits given 2 mg/kg cocaine were as pronounced as in offspring of rabbits given 3 or 4 mg/kg, but the effects on parvalbumin immunoreactivity and D1/G protein coupling were reduced at this low dose. Thus, previous findings of ACC developmental abnormalities in offspring of rabbits given a dose of 4 mg/kg were replicated, the effects were shown to be dose-related and to be independent of maternal seizures. A mechanism by which dysfunction of the D1 receptor system could mediate cocaine-associated changes in all three parameters of ACC structure and function is discussed.

Animals↗

PubMed1997

New evidence for neurotransmitter influences on brain development.

The early appearance of monoamine systems in the developing mammalian CNS suggests that they play a role in neural development. We review data from two model systems that provide compelling new evidence of this role. In one model system-in utero exposure to cocaine-specific and robust alterations are seen in dopamine-rich areas of the cerebral cortex, such as the anterior cingulate cortex: D1 receptor-G protein coupling is greatly reduced, the GABAergic system is altered and pyramidal dendrites undergo excessive growth. In a second model system-a transgenic mouse line in which the gene that encodes monoamine oxidase A (MAOA) is disrupted, resulting in excessively high 5-HT levels-barrels fail to form in the developing somatosensory cortex. Both models reveal the effects of very early manipulation of monoamines on forebrain development, and the long-term anomalies that persist into adulthood.

Animals↗

PubMed1997

Dynamic expression suggests multiple roles of the eph family receptor brain-specific kinase (Bsk) during mouse neurogenesis.

The eph family ligands and receptors have been implicated in mediating topographic neuron-target interactions. We recently isolated Bsk, a new member of the eph family receptors, and showed that it is expressed primarily in the brain. To investigate the role of Bsk in the development of the nervous system, we examined the temporal and spatial patterns of Bsk expression using in situ hybridization. We report here that Bsk expression exhibits dynamic changes during embryogenesis. In early embryos, Bsk is widely transcribed in the nervous system, including the forebrain, midbrain, hindbrain and spinal cord. Bsk expression in the midbrain, hindbrain and spinal cord, however, gradually decreases while in the forebrain increases over time. By embryonic day 18, the most intense Bsk expression was found in the limbic system. High levels of the expression in the limbic system persisted throughout post-natal development and remained stable in the adult up to 24 month. The topography of Bsk expression is in the form of gradients in several regions of the brain, including the lateral septum, spinal cord, as well as the hippocampus. Selective expression was also observed in Purkinje cells. Our findings on the topography of Bsk expression provide support to potential roles of Bsk in topographic projection. Our analyses further suggest that there may be other novel functions of Bsk in early neurogenesis in addition to potential roles in topographic mapping.

Animals↗

PubMed1997

Response diversity and the timing of progenitor cell maturation are regulated by developmental changes in EGFR expression in the cortex.

Early cortical progenitor cells of the ventricular zone (VZ) differ from later progenitor cells of the subventricular zone (SVZ) in cell-type generation and their level of epidermal growth factor receptors (EGFRs). To determine whether differences in their behavior are causally related to EGFR number/density, we introduced extra EGFRs into VZ cells with a retrovirus in vivo and in vitro. This results in premature expression of traits characteristic of late SVZ progenitor cells, including migration patterns, differentiation into astrocytes, and proliferation of multipotential cells to form spheres. The choice between proliferation and differentiation depends on ligand concentration and progenitor cell age and may reflect different thresholds of stimulation. The level of EGFRs expressed by progenitor cells in the cortex may therefore contribute to the timing of their maturation and choice of response to pleiotropic environmental signals.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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↗