Survival and neurotransmitter plasticity in cultured rat colonic myenteric neurons

The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso-N-acetyl-d,l-penicillamine (SNAP, a NO donor) or N-nitro-l-arginine methyl ester (l-NAME, a NOS inhibitor) affect neuronal survival.

After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or l-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or l-NAME did not change the percentage of neurons expressing NOS.

In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.     

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.     

Membrane properties of cultured rat sympathetic neurons: Morphological studies of adrenergic and cholinergic differentiation

Dissociated neurons from the newborn rat superior cervical ganglion were grown under conditions which lead to either adrenergic or cholinergic differentiation. Lectins and toxins were used to detect differences in the cell membrane associated with transmitter status, age of the neurons, or location on the neurons. These ligands were made visible in the light or electron microscope by coupling to rhodamine or colloidal gold. The density of binding sites for concanavalin A (Con A), ricin (RCA₆₀), and wheat germ agglutinin (WGA) increased with age in culture on both adrenergic and cholinergic cells. Soybean agglutinin (SBA) binding increased about threefold on adrenergic axons, but failed to increase on neurons induced to become cholinergic by medium conditioned by rat heart cells (CM). The effect of CM on SBA binding paralleled previously described effects of CM on transmitter production; the CM binding pattern developed slowly and was not readily reversible. Mature adrenergic neurons also appeared to bind more WGA than neurons in CM cultures. Tetanus toxin gold binding was uniform, but low, on axons of adrenergic and cholinergic neurons at all ages. In contrast, cholera toxin binding decreased with age on adrenergic axons. Binding sites for SBA and tetanus toxin were found to be less numerous on the cell body surface than on the axonal surface. Thus growth in CM induces fundamental changes in the phenotype of developing sympathetic neurons involving the cell membrane as well as transmitter choice. Differences also appear with maturation and between axonal and somatic cell surface membranes.     

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.     

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.     

Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons

Although the cellular and molecular mechanisms underlying the delayed-type hypersensitivity (DTH) reaction have been investigated, the functions of infiltrating leukocytes and skin resident cells in the elicitation phase of the DTH reaction are not completely understood. To gain more insight into the role of these cells in the DTH reaction, we identified about 250 cDNA fragments showing elevated expression during the DNCB-induced guinea pig skin DTH reaction by differential display analysis. Characterization of 50 of them led to the identification of 28 genes whose expression was elevated in the DNCB-induced DTH reactive tissue. Sequencing of the 28 cDNA fragments and homology search analysis demonstrated that 10 of them represented known genes, some of which, in particular elafin (an elastase inhibitor) and ferritin, are considered to play roles in the DTH reaction. The other 18 fragments are probably derived from unknown genes. Cloning of the cDNAs of one of these genes indicated that it is that for guinea pig tryptophanyl-tRNA synthetase (WRS), a protein found to be induced by interferon-gamma and upregulated during the late stages of mononuclear phagocyte maturation in vitro. Strong induction of the WRS gene during the DTH reaction suggests its involvement in the in vivo immune response.     

Early presynaptic changes during plasticity in cultured hippocampal neurons

Long-lasting increase in synaptic strength is thought to underlie learning. An explosion of data has characterized changes in postsynaptic (pstS) AMPA receptor cycling during potentiation. However, changes occurring within the presynaptic (prS) terminal remain largely unknown. We show that appearance of new release sites during potentiation between cultured hippocampal neurons is due to (a) conversion of nonrecycling sites to recycling sites, (b) formation of new releasing sites from areas containing diffuse staining for the prS marker Vesicle-Associated Membrane Protein-2 and (c) budding of new recycling sites from previously existing recycling sites. In addition, potentiation is accompanied by a release probability increase in pre-existing boutons depending upon their individual probability. These prS changes precede and regulate fluorescence increase for pstS GFP-tagged-AMPA-receptor subunit GluR1. These results suggest that potentiation involves early changes in the prS terminal including remodeling and release probability increase of pre-existing synapses.     

Anti-homeostatic synaptic plasticity of glycine receptor function after chronic strychnine in developing cultured mouse spinal neurons

In this study, we describe a novel form of anti-homeostatic plasticity produced after culturing spinal neurons with strychnine, but not bicuculline or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Strychnine caused a large increase in network excitability, detected as spontaneous synaptic currents and calcium transients. The calcium transients were associated with action potential firing and activation of gamma-aminobutyric acid (GABA(A)) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as they were blocked by tetrodotoxin (TTX), bicuculline, and CNQX. After chronic blockade of glycine receptors (GlyRs), the frequency of synaptic transmission showed a significant enhancement demonstrating the phenomenon of anti-homeostatic plasticity. Spontaneous inhibitory glycinergic currents in treated cells showed a fourfold increase in frequency (from 0.55 to 2.4 Hz) and a 184% increase in average peak amplitude compared with control. Furthermore, the augmentation in excitability accelerated the decay time constant of miniature inhibitory post-synaptic currents. Strychnine caused an increase in GlyR current density, without changes in the apparent affinity. These findings support the idea of a post-synaptic action that partly explains the increase in synaptic transmission. This phenomenon of synaptic plasticity was blocked by TTX, an antibody against brain-derived neurotrophic factor (BDNF) and K252a suggesting the involvement of the neuronal activity-dependent BDNF-TrkB signaling pathway. These results show that the properties of GlyRs are regulated by the degree of neuronal activity in the developing network.     

Ultrastructural studies on the intracellular fate of 125I-nerve growth factor in cultured rat sympathetic neurons

Primary cell cultures of sympathetic neurons from rat were exposed to 125I-nerve growth factor (NGF) and the fate of the NGF in the cell was followed using electron microscopic autoradiography. The intracellular localization of NGF was determined in the cell bodies and in the proximal neurites of neurons that had been grown in three-chamber dishes, following 5 or 24 hr of retrograde transport of NGF from the distal portions of the neurites. Label in the proximal neurites was predominantly associated with lysosomes and multivesicular bodies (MVBs), and at 5 hr elongated tubular elements were especially heavily labeled. Most of the label in the cell bodies was concentrated in lysosomes and MVBs. Lysosomes accounted for the largest fraction (45-60%) of the grains in the cell body, with a labeling density (LD = % grains/% area) of 3-5, while MVBs accounted for 5-10% of the grains with an LD of 5-20. We observed no evidence of nuclear labeling after 5 or 24 hr of retrograde transport. Mass cultures of neurons were incubated for 22 hr with NGF in the presence of the lysosomal inhibitors chloroquine (CQ, 0.05 mM) or methylamine (MA, 10 mM). In both agents the lysosomes were swollen with membranous material but still sequestered NGF, especially in CQ where the lysosomes were associated with almost 65% of the grains and had an LD of 6. CQ and MA had different effects on the morphology of the MVBs: in CQ they were few in number and compact while in MA they were numerous and appeared swollen and vacuolated. We observed no evidence for the nuclear accumulation of NGF even in the presence of the lysosomotropic agents.     

Distinct spatial localization of specific mRNAs in cultured sympathetic neurons

We examined the subcellular distribution of specific mRNAs in cultured sympathetic neurons. Under appropriate conditions, sympathetic neurons extend both axons and dendrites that are distinguishable by light microscopic and immunocytochemical criteria. In situ hybridization revealed a differential localization of mRNA within dendrites. mRNA encoding MAP2 was abundant in cell bodies and distributed nonhomogeneously throughout the dendritic compartment, but was not detected in axons. In contrast, mRNAs encoding GAP-43 and alpha-tubulin were restricted to the cell body and largely excluded from dendrites as well as axons. Detergent extraction revealed that most dendrite-associated mRNA encoding MAP2 was associated with the Triton X-100 insoluble fraction of the cell. The subset of mRNAs present in the dendritic compartment may encode proteins involved in the morphogenesis and remodeling of dendrites.     

Characterization of the vesicular monoamine transporter in cultured rat sympathetic neurons: persistence upon induction of cholinergic phenotypic traits

The binding of [3H]dihydrotetrabenazine ([3H]TBZOH), a specific ligand of the reserpine-sensitive monoamine transporter in brain and adrenal medulla storage vesicles, has been measured in cultured sympathetic neurons from newborn rat in relation to their neurotransmitter phenotype. As shown previously, neurons cultured in the absence of muscle-conditioned medium displayed high activities in catecholamine synthesizing enzymes and low levels of choline acetyltransferase, and neurons cultured in conditioned medium displayed the reverse pattern (J. P. Swerts, A. Le Van Thai, A. Vigny, and M. J. Weber, Dev. Biol. 100, 1-11, 1983). The density of [3H]TBZOH binding sites as well as their subcellular distribution were identical in both types of cultures. Two other structures rich in choline acetyltransferase, the electric organ of Torpedo and the ciliary ganglion of the chick embryo did not contain measurable amounts of [3H]TBZOH binding sites, suggesting that the monoamine transporter is not an ubiquitous component of cholinergic synaptic vesicles. These data suggest that the synthesis of the monoamine transporter in sympathetic neurons is not coregulated with the syntheses of the three norpinephrine synthesizing enzymes. It is proposed that the same population of synaptic vesicles can accumulate acetylcholine or catecholamine, depending only upon which neurotransmitter synthesizing enzymes are expressed by sympathetic neurons.     

Growth cones of cultured sympathetic neurons contain adrenergic vesicles

The growth cones of dissociated rat sympathetic neurons developing in culture were fixed with potassium permanganate to visualize vesicular stores of norepinephrine through the formation of granular precipitates. It was found that growth cones contain numerous small granular vesicles (SGV) 40-60 nm in diameter. The majority of the SGV was present in the varicosity of the growth cone but SGV also occurred in filopodia. The SGV appeared in clusters or scattered throughout the varicosity. Treatment of the cultured neurons, before fixation, with reserpine, which depletes catecholamine stores by blocking uptake into vesicles, resulted in the presence of small clear vesicles. In contrast, growth cones of nonadrenergic sensory neurons dissociated from dorsal root ganglia and fixed with permanganate lacked SGV and possessed small clear vesicles. These observations indicate that the growth cones of cultured sympathetic neurons contain norepinephrine, suggest that the norepinephrine is stored in synaptic vesicles, and raise the question whether this transmitter plays a role in early axon-target cell interactions during synapse formation.     

The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and alpha-internexin along>85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and alpha-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins.     

Differences in monoamine oxidase activity between cultured noradrenergic and cholinergic sympathetic neurons

Two types of monoamine oxidase activity (MAO-A and MAO-B) help regulate the levels of biogenic amines such as catecholamines and serotonin. Although MAO-A has greater activity toward most catecholamines than MAO-B, no direct experiments have determined the types and levels of MAO activity that are normally expressed in noradrenergic neurons. Noradrenergic neurons from neonatal rat superior cervical ganglia were isolated and cultured under conditions that permit either continued expression of the noradrenergic phenotype or promote a transition to a predominantly cholinergic phenotype. After 14-21 days in vitro, neurons from both types of cultures were assayed for the type and amount of monoamine oxidase activity using tryptamine, a common substrate for both MAO-A and MAO-B. Neurons cultured under noradrenergic conditions expressed sevenfold greater MAO activity than neurons cultured under cholinergic conditions. Essentially all MAO activity in the noradrenergic cultures was inhibited by preincubation with 10(-8)-10(-9) M clorgyline, which indicated that this activity was primarily MAO-A. Cultures grown under cholinergic conditions exhibited 6- to 10-fold lower MAO-A activity and an 8- to 10-fold lower level of catecholamine synthesis from labeled precursors compared to neurons grown under noradrenergic conditions. These results directly demonstrate that high MAO-A activity is expressed in noradrenergic neurons in vitro. The corresponding decreases in both MAO-A specific activity and catecholamine synthesis as neurons become cholinergic in vitro suggest that the expression of the noradrenergic phenotype involves the coordinate regulation of degradative as well as synthetic enzymes involved in catecholamine metabolism.     

Persistence of an amine uptake system in cultured rat sympathetic neurons which use acetylcholine as their transmitter

Cultures of dissociated rat superior cervical ganglion neurons (SCGN) were treated with the sympatholytic agent, guanethidine. When treated within the first couple of weeks in vitro, the neurons were rapidly destroyed. The cells grew less susceptible to the toxic effects of guanethidine with age in vitro. Moreover, the apparent affinity, Km, of the transport molecule for norepinephrine (NE) and guanethidine remained essentially unchanged between 2 and 7 wk in culture, as did the maximum velocity of transport (Vmax). This is at a time when previous studies have shown these neurons to be using acetylcholine (ACh) as their neurotransmitter. Cultures which were grown without supporting cells and from which cholinergic synaptic interactions were recorded physiologically were processed for autoradiography after incubation with [3H]NE. All cell bodies and processes seen had silver grains accumulated over them. These experiments show that sympathetic neurons in vitro maintain their amine uptake system relatively unchanged, even though they use ACh as their transmitter. The implications of these findings are discussed.     

Axonal agranular reticulum and synaptic vesicles in cultured embryonic chick sympathetic neurons

Cultured chick embryonic sympathetic neurons contain an extensive axonal network of sacs and tubules of agranular reticulum. The reticulum is also seen branching into networks in axon terminals and varicosities. The axonal reticulum and perikaryal endoplasmic reticulum resemble one another in their content of cytochemically demonstrable enzyme activities (G6Pase and IDPase) and in their characteristic membrane thicknesses (narrower than plasma membrane or some Golgi membranes). From the reticulum, both along the axon and at terminals, there appear to form dense-cored vesicles ranging in size from 400 to 1,000 Å in diameter. These vesicles behave pharmacologically and cytochemically like the classes of large and small catecholamine storage vesicles found in several adrenergic systems; for example, they can accumulate exogenous 5-hydroxydopamine. In addition, dense-cored vesicles at the larger (1,000 Å) end of the size spectrum appear to arise within perikaryal membrane systems associated with the Golgi apparatus; this is true also of very large (800–3,500 Å) dense-cored vesicles found in some perikarya.     

Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons.

Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.