Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation.

Although neuron generation is precisely regulated during ontogeny, little is known about underlying mechanisms. In addition, relationships between precursor proliferation and the apparent sequence of developmental processes, including cell migration, neurite elaboration, transmitter expression and synaptogenesis remain unknown. To address these issues, we used a fully defined neuronal cell culture system derived from embryonic rat sympathetic ganglia (DiCicco-Bloom, E., and I. B. Black. 1988. Proc. Natl. Acad. Sci. USA. 85:4066-4070) in which precursors enter the mitotic cycle. We now find that, in addition to synthesizing DNA, neuroblasts also underwent division in culture, allowing analysis of developmental relationships and mitotic regulation. Our observations indicate that mitotic neuroblasts expressed a wide array of neuron-specific characteristics including extension of neuritic processes with growth cones, elaboration of neurotransmitter enzyme, synthesis and transport of transmitter vesicles and organization of transmitter release sites. These data suggest that neuroblasts in the cell cycle may simultaneously differentiate. Consequently, the apparent sequence of ontogenetic processes is not an immutable, intrinsic neuronal program. How, then, are diverse developmental events coordinated? Our observations indicate that neuroblast mitosis is regulated by a small number of epigenetic factors, including insulin and EGF. Since these signals also influence other processes in developing neurons, epigenetic regulation normally may synchronize diverse ontogenetic events.

Stages of neurotransmitter development in autonomic neurons.

The ontogeny of neurotransmitters in autonomic neurons proceeds through the successive stages of early expression, definitive expression, modulation, and regulation, extending from embryonic life to maturity. Although different extracellular signals influence development at different stages, a number of signals that influence development continue to govern transmitter function during maturity. The sequential ontogenetic stages parallel the progressive restriction of mutability of phenotypic expression; however, some degree of neuronal mutability appears to persist through maturity.

Plasticity of substance P in mature and aged sympathetic neurons in culture.

The effect of age on the plasticity of the putative peptide neurotransmitter substance P (SP) was examined in the rat superior cervical sympathetic ganglion. Explantation of ganglia from 6-month-old rats to serum-supplemented culture resulted in a tenfold increase in SP concentration, reproducing results previously obtained for ganglia from neonatal rats. Veratridine prevented the increase in SP concentration in adult ganglia, and tetrodotoxin blocked the veratridine effect, suggesting that membrane depolarization and sodium influx prevented the rise in the SP content of adult ganglia as well as of neonatal ganglia. However, the time courses of the increase in the amount of the peptide differed in neonatal and mature ganglia, suggesting that some aspects of regulation may differ in the two. The effects of aging on neural plasticity were further analyzed by explanting ganglia from 2-year-old rats. No significant increase in SP concentration was observed in these ganglia. Remarkable plasticity thus seems to persist in mature neurons but may be deficient in aged sympathetic neurons.

Catecholamine enzymes in the degenerative neurological disease idiopathic orthostatic hypotension.

Discrete brain areas and sympathetic ganglia obtained at autopsy from patients with idiopathic orthostatic hypotension were assayed for tyrosine hydroxylase and dopamine beta-hydroxylase. Dopamine beta-hydroxylase activity was decreased 7.5-fold in sympathetic ganglia, while tyrosine hydroxylase activity was reduced more than 50-fold in the pontine nucleus locus coeruleus. These observations indicate that noradrenergic neurons of both brain and ganglion are affected in idiopathic orthostatic hypotension, but suggest that the central and peripheral biochemical deficits differ.

Impulse activity differentially regulates [Leu]enkephalin and catecholamine characters in the adrenal medulla.

Regulation of the putative peptide neurohumour [Leu]enkephalin and the catecholaminergic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase was examined in the rat adrenal medulla in vivo and in vitro. Surgical denervation of the adrenal gland or pharmacologic blockade of synaptic transmission, treatments known to decrease catecholamine traits, increased [Leu]enkephalin content. Medullas explanted to culture exhibited a 50-fold rise in [Leu]enkephalin in 4 days, whereas tyrosine hydroxylase remained constant, and phenylethanolamine-N-methyltransferase decreased to a new baseline level. Veratridine-induced depolarization prevented the accumulation of [Leu]enkephalin, an effect that was blocked by tetrodotoxin, which antagonizes transmembrane Na+ influx. These studies suggest that enkephalinergic and catecholamine characters are differentially regulated by impulse activity and depolarization in the adrenal medulla.

Substance P and somatostatin regulate sympathetic noradrenergic function.

Peptidergic-noradrenergic interactions were examined in explants of rat sympathetic superior cervical ganglia and in cultures of dissociated cells. The putative peptide transmitters substance P and somatostatin each increased the activity of the catecholamine-synthesizing enzyme tyrosine hydroxylase after 1 week of exposure in culture. Maximal increases occurred at 10(-7) molar for each peptide, and either increasing or decreasing the concentration reduced the effects. Similar increases in tyrosine hydroxylase were produced by a metabolically stable agonist of substance P, while a substance P antagonist prevented the effects of the agonist. The data suggest that the increased tyrosine hydroxylase activity was mediated by peptide interaction with specific substance P receptors and that peptides may modulate sympathetic catecholaminergic function.

Maternal glucocorticoid hormones influence neurotransmitter phenotypic expression in embryos.

Treatment of pregnant rats with reserpine prevented the normal disappearance of catecholamine fluorescence in presumptive neuroblasts of the embryonic gut. These cells normally express the noradrenergic phenotype transiently during embryonic development. The effect of reserpine was reproduced by treating mothers with hydrocortisone acetate. Moreover, the reserpine effect was blocked by treatment with dexamethasone, which inhibits the stress-induced increase in plasma glucocorticoids, and by mitotone, which causes adrenocortical cytolysis. It is concluded that reserpine, through the mediation of maternal glucocorticoid hormones, alters the phenotypic expression of these embryonic neuroblasts.

Substance P in principal sympathetic neurons: regulation by impulse activity.

Regulation of the putative peptide neurotransmitter substance P was examined in the superior cervical sympathetic ganglion of the neonatal rat. Surgical decentralization (denervation) of the superior cervical ganglion increased ganglion substance P content. In cultured ganglia, the amount of substance P increased more than 50-fold after 48 hours, and this rise was dependent on protein and RNA synthesis. Veratridine prevented the increase in substance P in vitro, and tetrodotoxin blocked the veratridine effect; this suggests that sodium influx and membrane depolarization prevent substance P elevation. Immunohistochemical analysis of cultured ganglia indicated that substance P was present in the perikarya of principal sympathetic neurons and in ganglionic nerve processes. Transsynaptic impulses, through the mediation of postsynaptic sodium influx, may decrease substance P in sympathetic neurons.

Biochemistry of information storage in the nervous system.

The use of molecular biological approaches has defined new mechanisms that store information in the mammalian nervous system. Environmental stimuli alter steady-state levels of messenger RNA species encoding neurotransmitters, thereby altering synaptic, neuronal, and network function over time. External or internal stimuli alter impulse activity, which alters membrane depolarization and selectively changes the expression of specific transmitter genes. These processes occur in diverse peripheral and central neurons, suggesting that information storage is widespread in the neuraxis. The temporal profile of any particular molecular mnemonic process is determined by specific kinetics of turnover and by the geometry of the neuron resulting in axonal transport of molecules to different synaptic arrays at different times. Generally, transmitters, the agents of millisecond-to-millisecond communication, are subject to relatively long-lasting changes in expression, ensuring that ongoing physiological function is translated into information storage.

Neurotransmitter plasticity at the molecular level.

Contrary to long-held assumptions, recent work indicates that neurons may profoundly change transmitter status during development and maturity. For example, sympathetic neurons, classically regarded as exclusively noradrenergic or cholinergic, can also express putative peptide transmitters such as substance P. This neuronal plasticity is directly related to membrane depolarization and sodium ion influx. The same molecular mechanisms and plastic responses occur in mature as well as developing neurons. Further, contrary to traditional teaching, adult primary sensory neurons may express the catecholaminergic phenotype in vivo. Transmitter plasticity is not restricted to the peripheral nervous system: ongoing studies of the brain nucleus locus ceruleus in culture indicate that specific extracellular factors elicit marked transmitter changes. Consequently, neurotransmitter expression and metabolism are dynamic, changing processes, regulated by a variety of defined factors. Transmitter plasticity adds a newly recognized dimension of flexibility to nervous system function.

NT-3 stimulates sympathetic neuroblast proliferation by promoting precursor survival.

Although proliferation is fundamental to the generation of neuronal populations, little is known about the function of trophic mechanisms during neurogenesis. We now describe a novel role for neurotrophin-3 (NT-3): the neurotrophin stimulates proliferation of sympathetic neuroblasts through trophic mechanisms. NT-3 promotes survival of the dividing precursors, but does not directly stimulate mitosis. NT-3 trophic effects differ markedly from those of the sympathetic mitogen, insulin. Furthermore, whereas NT-3 exhibits trophic activity for dividing neuroblasts, nerve growth factor characteristically promotes survival of postnatal sympathetic neurons. The stage-specific activity of NT-3 and nerve growth factor in culture parallels the sequence of trkC and trkA receptor gene expression detected in vivo. Thus, neurotrophins apparently serve as trophic factors during ontogeny, acting sequentially during establishment of individual populations.

Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor.

Mounting evidence indicates that extracellular factors exert proliferative effects on neurogenetic precursors in vivo. Recently we found that systemic levels of basic fibroblast growth factor (bFGF) regulate neurogenesis in the brain of newborn rats, with factors apparently crossing the blood-brain barrier (BBB) to stimulate mitosis. To determine whether peripheral bFGF affects proliferation during adulthood, we focused on regions in which neurogenesis persists into maturity, the hippocampus and the forebrain subventricular zone (SVZ). In postnatal day 1 (P1) rats, 8 hr after subcutaneous injection (5 ng/gm body weight), bFGF increased [(3)H]thymidine incorporation 70% in hippocampal and SVZ homogenates and elicited twofold increases in mitotic nuclei in the dentate gyrus and the dorsolateral SVZ, detected by bromodeoxyuridine immunohistochemistry. Because approximately 25% of proliferating hippocampal cells stimulated in vivo expressed neuronal traits in culture, bFGF-induced mitosis may reflect increased neurogenesis. bFGF effects were not restricted to the perinatal period; hippocampal DNA synthesis was stimulated by peripheral factor in older animals (P7-P21), indicating the persistence of bFGF-responsive cells and activity of peripheral bFGF into late development. To begin defining underlying mechanisms, pharmacokinetic studies were performed in P28 rats; bFGF transferred from plasma to CSF rapidly, levels rising in both compartments in parallel, indicating that peripheral factor crosses the BBB during maturity. Consequently, we tested bFGF in adults; peripheral bFGF increased the number of mitotic nuclei threefold in the SVZ and olfactory tract, regions exhibiting persistent neurogenesis. Our observations suggest that bFGF regulates ongoing neurogenesis via a unique, endocrine-like pathway, potentially coordinating neuron number and body growth, and potentially providing new approaches for treating damaged brain during development and adulthood.

Rab3A is required for brain-derived neurotrophic factor-induced synaptic plasticity: transcriptional analysis at the population and single-cell levels.

Brain-derived neurotrophic factor (BDNF) modulates synaptic strength in hippocampal neurons, in addition to promoting survival and differentiation. To identify genes involved in trophic regulation of synaptic plasticity, we have used a multidisciplinary approach of differential display and family-specific slot blots in combination with whole-cell patch-clamp recordings of dissociated hippocampal neurons. Three hour exposure to BDNF elicited a 2.6-fold increase in synaptic charge and a concomitant induction of 11 genes as revealed by differential display, including the small GTP-binding vesicular trafficking protein Rab3A and the enzyme guanylate cyclase (GC). Slot blot analysis on a population of neurons confirmed an average of 3.1-fold induction of these clones. In contrast, individual pyramidal-like neurons that were first characterized electrophysiologically in the presence of BDNF and subjected to transcriptional analysis displayed more robust increases (4.8-fold), emphasizing the neuronal heterogeneity. Transcriptional changes of Rab3A and GC were accompanied by translational regulation, shown by Western blot analysis. Furthermore, a number of GC-associated and Rab3A effector molecules were induced by BDNF at either the gene or protein levels. The functional role of Rab3A in BDNF-induced synaptic plasticity was assessed using cells derived from Rab3A knock-out mice. These neurons failed to show an increase in synaptic charge in response to BDNF at 10 min; however a late response to BDNF was detected at 20 min. This late response was similar in time course to that induced by postsynaptic activation of glutamate receptors. Our results demonstrate a requirement for Rab3A and may reveal a temporal distinction between presynaptic and postsynaptic mechanisms of BDNF-induced synaptic plasticity associated with learning and memory.

Neurogenesis in neonatal rat brain is regulated by peripheral injection of basic fibroblast growth factor (bFGF).

Many major diseases of human brain involve deficiencies of select neuronal populations. As one approach to repair, we examined regulation of neurogenesis directly in vivo, employing postnatal day 1 (P1) cerebellar cortex, which is composed primarily of granule neurons and dividing precursors. We focused on basic fibroblast growth factor (bFGF), which stimulates precursor mitosis in culture and which is highly expressed in cerebellum during neurogenesis. Subcutaneous injection of bFGF increased [3H]thymidine ([3H]dT) incorporation, a marker for DNA synthesis, by 50% in whole cerebellar homogenates, suggesting that peripherally administered factor altered ongoing neural proliferation. Further, assay of isolated granule precursors revealed a 4-fold increase in [3H]dT incorporation following in vivo bFGF treatment, indicating that granule neuroblasts were the major bFGF-responsive population. Morphologic analysis indicated that twice as many granule precursors were in S-phase of the mitotic cycle after peripheral bFGF. To determine whether other neurogenetic populations respond to peripheral bFGF, we examined additional brain regions in vivo. bFGF stimulated DNA synthesis by 68% in hippocampus, and by > 250% in pontine subventricular zone (SVZ). In contrast, incorporation was not altered in basal pons or cerebral cortex, regions in which neurogenesis has already ceased. To define potential direct actions of peripherally administered factor, 125I-bFGF was used to study distribution. Intact 18 kDa 125I-bFGF was recovered from brain following peripheral injection, suggesting that the factor acted directly to stimulate mitosis in dividing neuroblasts. The stimulation of neuronal proliferation by exogenous bFGF suggests that the factor normally regulates neurogenesis, and provides new therapeutic approaches to promote functional recovery from nervous system diseases.

Mitotic neuroblasts determine neuritic patterning of progeny.

Neuronal precursor proliferation and axodendritic outgrowth have been regarded as strictly sequential, with process formation presumably beginning after mitotic activity ceases. We now report that sympathetic precursors in vitro often elaborate long neurites before dividing. Of 437 neuroblasts observed in 48 time-lapse recordings, 42 neuroblasts divided. Thirty (71%) of these mitotic neuroblasts had neurites prior to cytokinesis. "Paramitotic" neurites were found to contain microtubules (MTs), indicating that precursors elaborate neuritic cytoskeleton during proliferation. Remarkably, the precise neuritic pattern exhibited by parental neuroblasts was consistently reproduced by daughter cell pairs. Preservation of neuritic morphology occurred through asymmetric division, with individual neurites allocated to specific daughter cells. Paramitotic neurites either remained intact throughout mitosis (12 of 65), or "retracted" into the soma during prophase and then "regrew" within minutes after cytokinesis (53 of 65). "Retraction" and "regrowth" involved resorption of cytoplasm into the soma, then refilling of residual cell membrane, resulting in recapitulation of the parental neurite pattern. Paramitotic neuritogenesis appears to be intrinsically driven, but is responsive to environmental signals. The culture substrate influenced neurite length, but not the response of paramitotic neurites during mitosis or the preservation of neuritic morphology. However, the incidence of neurite-bearing neuroblasts increased from 38 +/- 1.3% to 94 +/- 1.1% with growth factor treatment. The surprisingly high incidence of paramitotic neurites and the fidelity with which patterning was conserved across cell generations raise the possibility that mitotic precursors engage in pathfinding. Our studies suggest a novel link between neurogenesis and cytoarchitectonic patterning.

Embryonic sensory development: local expression of neurotrophin-3 and target expression of nerve growth factor.

Development and maintenance of peripheral sensory and sympathetic neurons are regulated by target-derived neurotrophins, including nerve growth factor (NGF). To determine whether trophins are potentially critical prior to and during target innervation, for neuronal survival or axon guidance, in situ hybridization was performed in the rat embryo. We examined the expression of genes encoding NGF, neurotrophin-3 (NT-3), and their putative high-affinity receptors, trk A and trk C, respectively. Trks A and C were detected in dorsal root sensory ganglia (DRG) on embryonic day 12.5 (E12.5), implying early responsiveness to NGF and NT-3. NGF mRNA was expressed in the central spinal cord target and by the peripheral somite, at this early time, which thereby may function as a transient "guidepost" target for sensory fibers. Somitic expression was transient and was undetectable by E17.5. NT-3 was expressed in the DRG itself from E13.5 to 17.5, suggesting local transient actions on sensory neurons. NT-3 was also expressed in the ventral spinal cord at low levels on E13.5. We examined the trigeminal ganglion to determine whether cranial sensory neurons are similarly regulated. Trk A was detected in the trigeminal ganglion, while NGF was expressed in the central myelencephalon target, paralleling observations in the DRG and spinal cord. However, NT-3 and trk C were undetectable, in contrast to DRG, suggesting that the environment or different neural crest lineages govern expression of different trophins and trks. Apparently, multiple trophins regulate sensory neuron development through local as well as transient target mechanisms prior to innervation of definitive targets.

Hippocampal regulation of the survival and morphological development of locus coeruleus neurons in dissociated cell culture.

The influence of target structures on neural development, originally described for the peripheral nervous system, has more recently been investigated in the central nervous system. We sought to determine whether targets regulate the development of the locus coeruleus, with its diffuse and complex projections in marked contrast to the simpler neural circuits of the peripheral nervous systems. Dissociated locus coeruleus cells were grown alone or with the hippocampus in serum-free, chemically defined medium that minimized non-neuronal growth. Coculture with the hippocampus resulted in a significant increase in locus coeruleus tyrosine hydroxylase activity. Elevated tyrosine hydroxylase activity was accompanied by a commensurate increase in the number of tyrosine hydroxylase-immunoreactive cells, suggesting hippocampal enhancement of locus coeruleus survival. Furthermore, when hippocampal cells were added to locus coeruleus dissociates at zero time, or after two days, tyrosine hydroxylase-positive cell number was increased only by hippocampal cells added initially, suggesting that the target does indeed foster survival. The apparent target enhancement of locus coeruleus survival seems to be selective since total protein and total neuron number, estimated with neuron-specific enolase immunocytochemistry, were not affected by the hippocampus. Coculture with the hippocampus also increased the length and complexity of locus coeruleus cell processes. Neither the increase in tyrosine hydroxylase cell number nor the changes in morphology could be attributed to nonspecific effects of the increased cell density in cocultures. Our observations thus suggest that the target hippocampus influences the survival and neurite elaboration of afferent locus coeruleus neurons.

Multiple ephrins regulate hippocampal neurite outgrowth.

Increasing evidence indicates that the eph family of ligands and receptors guides the formation of topographic maps in the brain through repulsive interactions. For example, we have recently found that in the hippocamposeptal system, the ligand ephrin-A2, which is expressed in an increasing gradient from dorsal to ventral septum, selectively induces pruning of topographically inappropriate medial hippocampal axons. The recent detection of ephrins A3 and A5, as well as A2, in the septum raised critical functional questions. Do the ligands act combinatorially, ensuring appropriate three-dimensional spatiotemporal projection, or do they exert entirely distinct actions in addition to guidance mechanisms? To approach these alternatives, we cloned mouse ephrin-A2 and compared the activities of the three ligands. Here, we show that these ligands reduce the number of hippocampal neurites in a similar fashion. The effect was regionally specific; medial hippocampal neurites were reduced 1.5- to 1.8-fold, whereas lateral hippocampal neurites were not significantly affected, conforming to topographic projection in vivo. Furthermore, we found that ephrins regulated neurite number in a stage-specific fashion, affecting E19 hippocampal neurites more than E16 neurites. Our observations suggest that all three septal ephrins, A2, A3, and A5, play spatiotemporally specific roles in guiding topographic projections from the hippocampus.

Postmortem changes in brain catecholamine enzymes.

Postmortem changes in the activities of tyrosine hydroxylase, dopa decarboxylase, and dopamine-beta-hydroxylase were examined in various areas of rat brain. Tyrosine hydroxylase activity decreased in an exponential fashion with a half-time of two to four hours in caudate-putamen, substantia nigra, and locus ceruleus. Dopa decarboxylase activity remained within 20% of control values at five hours in these areas, but then decreased precipitously. Dopamine-beta-hydroxylase activity remained within 20% of control for at least 20 hours after death.

Localization of high-affinity and low-affinity nerve growth factor receptors in cultured rat basal forebrain.

Previous work has indicated that nerve growth factor specifically and selectively increases choline acetyltransferase and acetylcholinesterase in organotypic cultures of rat basal forebrain-medial septal area. To determine whether these actions are potentially receptor-mediated, organotypic and dissociated basal forebrain-medial septal area cultures were examined. Two independent methods, [125I]nerve growth factor binding and immunocytochemistry with a monoclonal nerve growth factor receptor antibody (192-IgG), detected specific receptors. The nerve growth factor receptors were localized to two different cellular populations: flat, large, non-neuron-like cells, and small, round, process-bearing, neuron-like cells. Dissociation studies with [125I]nerve growth factor suggested that high-affinity receptors were localized to the neuron-like population, while only low-affinity receptors were localized to the non-neuron-like cells. We tentatively conclude that nerve growth factor may elicit cholinergic effects by directly binding to high-affinity receptors on neurons. To begin examining receptor regulation, cultures were exposed to exogenous, unlabeled nerve growth factor continuously for 10 days before binding studies were performed. Prior exposure to nerve growth factor did not alter binding characteristics of the receptor, using the present methods.