Interaction with membrane remnants of target myotubes maintains transmitter sensitivity of cultured neurons.

Parasympathetic neurons, when cultured alone, lose sensitivity to acetylcholine, but if striated muscle is included in the culture, neuronal chemosensitivity is maintained. The membrane remnants of myotubes ruptured by osmotic shock also supported the responsiveness of the cultured neurons to transmitter, whereas muscle-conditioned medium or membrane remnants of nonmuscle embryonic skin cells did not support this responsiveness. The regulation of chemosensitivity by contact of neurons with the target cell membrane may be important in the formation and maintenance of neuronal circuitry.

Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function.

Urethral obstruction produces increased voiding frequency (0.7 +/- 0.06 to 1.1 +/- 0.08 h-1) and hypertrophy of the urinary bladder (89 +/- 1.7 to 708 +/- 40 mg) with profound increments in the dimensions of afferent (4, 6) and efferent neurons (299 +/- 4.7 to 573 +/- 8.6 microns2) supplying this organ in the rat. We discovered that hypertrophied bladders of rat and human contain significantly more nerve growth factor (NGF) per milligram wet weight, protein, and DNA than normal bladders. The temporal correlation between NGF content, neuronal hypertrophy, and bladder weight was consistent with a role for this growth factor in the neurotrophic effects associated with obstruction. Autoimmunity to NGF abolished the hypertrophy of NGF-sensitive bladder neurons in the pelvic ganglion after obstruction. Relief of urethral obstruction reduced bladder size (349 +/- 78 mg), but neuronal hypertrophy (460.2 +/- 10.2 microns2) and elevated NGF levels were only partially reversed. Bladder hypertrophy (133 +/- 4.3 mg) induced by osmotic diuresis slightly increased ganglion cell area (365.2 +/- 6.1 microns2) and only doubled NGF content of the bladder. These findings provide important new evidence that parenchymal cells in the hypertrophied bladder can synthesize NGF and possibly other molecular messengers that act to alter the size and function of neurons in adult animals and man.

Mitochondrial DNA-depleted neuroblastoma (Rho degrees) cells exhibit altered calcium signaling.

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Mitochondrial impact on nerve growth factor production in vascular smooth muscle-derived cells.

Ht30/=Ht5). Cells with reduced mitochondrial activity also showed abnormal responses to the stimulation of NGF output. Thrombin and phorbol ester elevated NGF production from Ht100, Ht30 and Ht10 cells, but not from Ht5 cells. Ht30 cells, despite secreting less NGF basally than Ht100 cells, reached a similar or greater NGF output upon stimulation. Mitogens increased NGF output and NGF mRNA levels with the largest effect on NGF protein in Ht30 cells. Free radical production and the ability of cells to respond to NGF-inducing agents were related. These data suggest that chronic impairment of mitochondrial function associates with disturbances in cellular production of a signaling protein.

Nerve growth factor synthesis in vascular smooth muscle.

Details of the interdependent, trophic relation between smooth muscle and its neural innervation are not well known despite suggestions that neural influences may contribute significantly to hypertensive and other cardiovascular disease. Vascular smooth muscle is a major target of innervation by neurons of the sympathetic nervous system. Sympathetic neurons depend on a constant supply of the potent neurotrophic peptide nerve growth factor. Nerve growth factor regulates an impressive list of neuronal and perhaps muscle properties, yet its source in vessels and the determinants of its synthesis are not known. We have taken advantage of the cytoarchitecture of the aorta to demonstrate that vascular smooth muscle cells synthesize nerve growth factor. The survival of cultured sympathetic neurons is supported in a nerve growth factor-dependent manner by co-culture with pure rat aortic vascular smooth muscle cells. Furthermore, pure smooth muscle cell cultures contain nerve growth factor-specific messenger RNA. Levels of messenger nucleic acid coding for nerve growth factor in smooth muscle are regulated by contractile agonists (angiotensin II, arginine vasopressin) and the adrenergic agonist phenylephrine. This suggests a link between muscle activity and growth factor production. Secretion of nerve growth factor protein by vascular smooth muscle was measured using a sensitive two-site immunoassay. Secretion is highest during muscle growth. Secretion is elevated by angiotensin II and arginine vasopressin but slightly inhibited by phenylephrine. These results suggest that cultured vascular smooth muscle can serve as a useful model in which to study the cellular regulation of trophic factor synthesis in health and disease.

Increased nerve growth factor mRNA stability may underlie elevated nerve growth factor secretion from hypertensive vascular smooth muscle cells.

Altered nerve growth factor (NGF) regulation has been linked to the pathophysiology of hypertension. Vascular smooth muscle cells from an inbred hypertensive, but normoactive rat strain (WKHT) secreted NGF at a greater rate than from a hyperactive, normotensive strain (WKHA). Exposure to phorbol ester increased NGF secretion rates from WKHT by 400-800% but not from WKHA vascular muscle. NGF secretion rates from both WKHT and WKHA vascular cells were elevated by co-application of platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) by 300-1000%. This response was partially attenuated by actinomycin D, an inhibitor of RNA transcription. These results suggest that regulation of NGF production does not occur solely at the level of transcription and post-transcriptional mechanisms operate. Analysis of NGF mRNA stability in the two strains following PDGF and TGF-beta1 treatment showed that NGF mRNA in WKHT had a half-life of 126.2+/-11.68 min while in WKHA vascular smooth muscle cells, the half-life was 47. 33+/-11.98 min. In addition to increased NGF mRNA stability in WKHT vascular muscle, these cells have an increased translational efficiency of NGF protein; elevated synthesis of NGF protein per unit NGF mRNA. Differences in signaling pathways may result in increased NGF mRNA stability and translational efficiency that may account for the elevated NGF protein in WKHT vascular smooth muscle cells.

Ionic excitation of a clone of mouse neuroblastoma.

The electrical properties and the possible regulation of these properties were studied by means of intracellular microelectrode recordings in cells of mouse neuroblastoma clone N1E-115. This clone has high levels of tyrosine hydroxylase and regulates this enzyme. Cells treated for 24 h with 4 muM aminopterin followed by at least 5 days in culture developed rhythmic discharge of action potentials when superfused with phosphate-buffered saline containing less than 0.2 mM calcium or less than 0.2 mM calcium and zero potassium. This ionic excitation occurred in no cells at less than 5 days after treatment with aminopterin but at 5 days or more after treatment, 20% of cells responded to low calcium while 52% responded to low calcium and zero potassium. Concomitant with the development of a susceptibility to ionic excitation was an increase in the average resting membrane potential and morphologic maturation. This ionic excitation of cultured mouse neuroblastoma cells may be useful for studying biochemical events associated with repetitive discharge of action potentials.

Muscle membrane preparation restores sensitivity to acetylcholine in cultured chick ciliary ganglion neurons.

Ciliary ganglion (CG) neurons grown in culture in the absence of muscle cells rapidly lose sensitivity to acetylcholine (ACh), while neurons grown in the presence of muscle or muscle cell membranes maintain sensitivity to ACh for extended periods of time. The present study examined whether exposure to muscle membrane preparation or stimulation of cAMP-dependent processes could restore sensitivity to ACh in cultured neurons which had lost responsiveness to ACh. CG neurons from 11- to 14-day-old chick embryos were grown on collagen substrate in the absence of muscle cells. Sensitivity to ACh was assessed by measuring peak current responses following application of ACh (IACh) to neurons under whole-cell voltage clamp. In control cultures IACh decreased from an average of 837 pA the day of plating to 145 pA following 4 days in culture. Stimulation of cAMP-dependent processes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) or 8'Br-cAMP and IBMX had variable effects on IACh. These treatments increased peak IACh in some neurons maintained in culture for less than 48 h. Treatment with these agents decreased peak IACh in cultures which were more than 48 h old. Exposure of neurons, which had lost sensitivity to ACh in culture, to muscle membranes increased IACh 2- to 3-fold over 24 to 48 h. This membrane-induced restoration of sensitivity to ACh was blocked by exposure to the protein synthesis inhibitor cycloheximide. Stimulation of cAMP-dependent processes in neurons exposed to muscle membrane decreased IACh. In conclusion, these results indicate that some element associated with the membranes of muscle cells has the ability to restore ACh responsiveness to CG neurons which have become insensitive to ACh in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor responsiveness of cultured major pelvic ganglion neurons from the adult rat.

The bladder and other pelvic viscera are innervated in the rat by the major pelvic ganglion (MPG), a mixed sympathetic/parasympathetic population of neurons that participates in lower urinary pathophysiology. Neurons from the MPG of adult females were removed, dissociated and cultured in order to test retention of the neuronal phenotype and whether they responded to Nerve Growth Factor (NGF). The bladder-specific subset of MPG neurons were distinguished by retrograde labeling prior to culture. The adult ganglionic neurons adapted to culture with greater than 80% survival in the best cases. The cultured neurons retained excitability, as determined by measuring voltage-activated ionic currents. They were positive for neuron-specific beta-tubulin and many retained immunoreactivity for characteristic peptides and transmitter synthetic enzyme. The proportion of neurons in the different categories tested varied somewhat from that in vivo, but there was no evidence of selective death of a particular population. The cultured MPG neurons were responsive to NGF and anti-NGF antibody. NGF supported neuronal survival and expression of tyrosine hydroxylase. Added NGF also affected the expression of neuropeptide Y. Hypertrophied neurons from animals with experimental bladder outlet obstruction demonstrated increased responsiveness to NGF. The data suggest that NGF participates in adult neural plasticity due to continued responsiveness to the factor. Furthermore, questions concerning regulation of MPG neurons may be addressed in vitro.

Long-term survival and development of dissociated parasympathetic neurons in culture.

Ciliary ganglion neurons from chick embryo were grown in cell culture following enzymatic and mechanical dissociation. The culture conditions necessary for long-term (greater than 21 days) survival of the isolated neurons were investigated. Neurons could be cultured with and without non-neural cells by adjusting the culture substrate. Chick embryo extract was found to be essential in the growth medium, and the inclusion of horse serum had an additional beneficial effect. Also, non-neural cells increased the survival of these neurons in culture. Thus, there appear to be several factors involved in the survival of these neurons in culture. Assay of choline acetyltransferase activity revealed a 100-fold increase in activity over the first two weeks in vitro, and the developmental pattern of the enzyme activity was initially similar to that seen in vivo. The cultured neurons also retained many of the electrophysiological properties of ganglionic neurons in vivo. These included normal resting transmembrane potential, action potential amplitude and an afterpotential. The passive membrane properties of the cultured neurons (membrane resistance, capacitance and time constant) differed somewhat from those of neurons in ganglia. These results suggest that if proper culture conditions are provided, these parasympathetic neurons adapt well to cell culture and develop many of the properties of normal ciliary ganglion neurons in vivo.

Neurally mediated hyperactive voiding in spontaneously hypertensive rats.

The development of hypertension in spontaneously hypertensive rats (SHR) and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor (NGF) and altered patterns of innervation. The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy. Voiding behavior was monitored using specially designed metabolic cages. NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA. NGF mRNA in cultured bladder smooth muscle cells (BSMCs) was quantified using reverse transcriptase PCR. Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine (NE) content in bladders with high performance liquid chromatography. SHRs voided more frequently than WKY rats. NGF content was higher in bladders from adult SHRs when compared to Wistar-Kyoto normotensive rats (WKYs). No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs. However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs. SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats. The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs. Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation.

Chronic reduction in complex I function alters calcium signaling in SH-SY5Y neuroblastoma cells.

Sporadic, non-familial Parkinson's disease is characterized by a 15-30% reduction in complex I activity of the electron transport chain. A pharmacological model of reduced complex I activity was created by prolonged treatment of SH-SY5Y cells with low doses (5-20 nM) of rotenone, a selective inhibitor of complex I. Short-term (less than 2 week) exposure to rotenone did not influence calcium signaling, production of reactive oxygen species, or mitochondrial morphology. However, following 2 weeks of rotenone exposure, SH-SY5Y cells showed unusual calcium dynamics, specifically multiple calcium responses to carbachol, a muscarinic agonist. These secondary calcium responses were not seen in control SH-SY5Y cells and were dependent upon calcium influx. Mitochondrial membrane potential was also reduced in low dose rotenone-treated cells. These results demonstrate that a chronic, partial reduction in complex I activity, such as that seen in Parkinson's disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival.

Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures (0-96 h) of dorsal root ganglion (DRG) neurons from aged (>22 months) Fisher 344 x Brown Norway F1 hybrid rats, compared to young (4-6 month) and middle-aged (14 month) animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful DRG neurons.

Soluble and membrane-bound factors together account for target dependence of cultured parasympathetic neurons.

The survival of avian ciliary ganglion (CG) neurons in culture depends upon an exogenous supply of trophic factor(s). Skeletal muscle, a normal ganglionic target tissue, is a well documented provider of survival-promoting activity, although the molecular basis for this ability to foster neuronal survival has not been thoroughly investigated. To identify the source of skeletal muscle support, dissociated neurons were plated into microwells containing either: a basal, trophically deficient medium; live pectoral muscle myotubes; medium conditioned by myotubes; membrane remnants of osmotically lysed myotubes; or, membrane remnants and conditioned medium. Neurons remaining in culture were counted after 1, 2, 5, and 7 days. The results reveal that neuronal survival is supported by both muscle conditioned medium and the membrane remnants of cultured myotubes. Each of these alone provides for only partial survival, while both combine to equal the activity of live myotubes. Treatment of the lysed membranes with either 1.5 M NaCl and/or 15 U heparin removed only 50-60% of the activity, suggesting that multiple factors are involved in the neuronal support obtained from lysed myotubes. This is in contrast to fibroblast remnants, which support some neuronal survival, but whose activity is wholly removed by NaCl. Conditioned medium also contains a heparin binding component which accounts for approximately 60% of its activity. These results indicate that full trophic support from the cultured target tissue requires at least two distinct active agents. The experiments further suggest that the target-derived factors responsible for neuronal survival in culture, and perhaps in vivo, are both soluble and membrane-associated molecules.

NGF, bFGF and CNTF increase survival of major pelvic ganglion neurons cultured from the adult rat.

The responsiveness of cultured major pelvic ganglion (MPG) neurons, isolated from adult rats, to nerve growth factor (NGF), basic fibroblastic growth factor (bFGF) and ciliary neuronotrophic factor (CNTF) was tested using in vitro survival assay. MPG neurons respond to NGF with increased survival (+35 +/- 13.3%, mean +/- S.E.), a response completely blocked by antibodies specific to NGF. bFGF (+85 +/- 9.6%) and CNTF (+10.5 +/- 0.5%) also augment survival of MPG neurons in vitro. The effect of bFGF was partially blocked by bFGF antibody. Anti-NGF antibody reduced neuronal survival by 25 +/- 4.1% in conditioned medium from cultures of bladder smooth muscle, suggesting bladder produces NGF. Combining antibodies against NGF and bFGF reduced survival by 19 +/- 0.5% in medium supplemented with bladder extracts, suggesting the extracts contain neurotrophic activity in addition to NGF. These results support the hypothesis that neurons regulating bladder function respond to NGF and other growth factors. Therefore, previously documented changes in bladder neurotrophic factors following hypertrophy, inflammation and injury may elicit growth or change in the autonomic nervous system.

Characterization of laterality of innervation of the rat bladder.

Retrograde axonal tracers were used to assess the laterality of bladder innervation which is unknown. Fluoro-Gold and Fast Blue were injected into the left and right bladder walls, respectively of male Wistar rats. The major pelvic (MPG), pelvic accessory (PAG), and dorsal root (DRG) ganglia were then removed and analyzed. 59% and 33% of the total labelled MPG cells were found in the ipsilateral and contralateral MPG (efferents), respectively. 65% and 31% of the total labelled PAG cells were found in the ipsilateral and contralateral PAG (efferents) respectively. The L1/L2 and L6/S1 DRG each received similar amount of ipsilateral innervation (45-52%) based on total labelled cells at each DRG level. 8%, 4%, and 4% of labelled cells in the MPG, PAG and DRG, respectively, contained both Fluoro-Gold and Fast Blue consistent with bilateral input originating from a single neuron. Immunohistochemistry showed that 40% of retrogradely labelled PAG cells were positive for tyrosine hydroxylase, far exceeding what has been shown for the MPG. Thus, assuming limited diffusion of tracers, the bladder receives a substantial crossed innervation especially in bladder afferents. This redundancy and bilateral neural input may contribute to preservation of bladder function and sensation even after extensive pelvic surgery.

Regulation of cholinergic development by target contact.

Avian ciliary ganglion neurons in cell culture have been used as a model neuronal system to examine the developmental role of direct contact with an appropriate target tissue. Live myotubes, which are immediately innervated, and their membrane remnants, on which neurons form terminal structures, were both found to stimulate or accelerate the acquisition or retention of important parameters of neuronal function. Contact with the target membrane supports survival, aids retention of active nicotinic receptors to acetylcholine, and accelerates the acquisition of adult transmitter synthetic capacity. These results emphasize the multifaceted nature of neurodevelopment, with soluble protein factors, membrane-bound elements, and other functional events all acting in concert in the embryonic nervous system.