Expression of calbindin immunoreactivity by subpopulations of primary sensory neurons in chick embryo dorsal root ganglion cells grown in coculture or conditioned medium

Primary sensory neurons which innervate neuromuscular spindles in the chicken are calbindin-immunoreactive. The influence exerted by developing skeletal muscle on the expression of calbindin immunoreactivity by subpopulations of dorsal root ganglion (DRG) cells in the chick embryo was tested in vitro in coculture with myoblasts, in conditioned medium (CM) prepared from myoblasts and in control cultures of DRG cells alone. Control cultures of DRG cells grown at the 6th embryonic day (E6) did not show any calbindin-immunostained ganglion cell. In coculture of myoblasts previously grown for 14 days, about 3% of calbindin-immunoreactive ganglion cells were detected while about 1% were observed in some cultures grown in CM. Fibroblasts from various sources were devoid of effect. Skin or kidney cells were more active than myoblasts to initiate calbindin expression by subpopulations of DRG cells in coculture or, to a lesser degree, in CM. The results suggest that cellular factors would rather induce calbindin expression in certain sensory neurons than ensure a selective neuronal survival.     

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.     

Influence of environmental factors on the expression of phenotypic characters by chicken dorsal root ganglion cells in culture

Primary sensory neurons were grown under four conditions of culture. The influence of nonneuronal cells, horse serum or both was studied on the phenotypic expression of certain neuronal subpopulations. The number of neurons expressing acetylcholinesterase, alpha-bungarotoxin-binding sites or a high uptake capacity for glutamine was enhanced by nonneuronal cells. The horse serum increases the neuronal subpopulation exhibiting a carbonic anhydrase activity. Certain phenotypic changes fit conditions consistent with an epigenetic induction rather than a cell selection.     

Calbindin-immunoreactive sensory neurons of dorsal root ganglion project to skeletal muscle in the chick

In the chicken dorsal root ganglia, two neuronal subpopulations referred to as A1 and B1 share in common an immunoreactivity to antisera raised to calbindin D-28k but are distinguished by their cytological and ultrastructural characteristics. To determine the peripheral targets innervated by calbindin-immunoreactive neurons in lumbosacral dorsal root ganglia, cryostat sections of various hindlimb tissues were treated with anticalbindin antisera. Calbindin-immunostained axons were clearly detected in skeletal muscle. Large myelinated nerve fibres and afferent axon terminals in neuromuscular spindles were calbindin-immunoreactive; thin unmyelinated nerve fibres were also immunostained in nerve bundles of the perimysium. Since motoneurons and neurons of the autonomic nervous system were devoid of calbindin immunostaining, it was suggested that the immunoreactive axons found in skeletal muscle originate from sensory neurons expressing a calbindin immunoreaction in the dorsal root ganglia. This hypothesis was corroborated after introduction of wheat germ agglutinin coupled with horseradish peroxidase or colloidal gold particles into the sartorius muscle. The retrogradely transported tracer was collected only in ganglion cell bodies which displayed the ultrastructural characteristics of A1 and B1 sensory neurons. On the basis of calbindin immunoreaction and of tracer retrograde transport, it is concluded that ganglion cells of subclasses A1 and B1 contribute to the sensory innervation of skeletal muscle in the chicken.     

Neuronal phenotypes in mouse dorsal root ganglion cell cultures: Enrichment of substance P and calbindin D-28k expressing neurons in a defined medium

The primary sensory neurons in mouse dorsal root ganglia consist of diversified subpopulations which express distinct phenotypic characteristics such as substance P or calbindin D-28k. To determine whether neuronal phenotypes are altered or not in in vitro cultures carried out in a defined synthetic medium, dissociated dorsal root ganglion cells from newborn mice were grown in the alpha-modified minimum essential medium either supplemented with 10% fetal calf serum or serum-free. About 80% of the neurons survived after 5 days of culture in both media, but only 35% or 65% were rescued after 12 days in serum-free or fetal calf serum supplemented medium, respectively. The neuronal subpopulations expressing substance P or calbindin D-28k displayed similar morphological properties in both media and a higher resistance to culture conditions than the whole neuronal cell population, especially in serum-free medium. It is therefore concluded that a defined synthetic medium offers reproducible conditions to culture dorsal root ganglion cells for at least 5 days, stimulates the expression of substance P and enriches preferentially neuronal phenotypes expressing substance P or calbindin D-28k, for a longer period of culture.     

Death of sensory ganglion neurons after acute withdrawal of nerve growth factor in dissociated cell cultures

The time course of dependence on nerve growth factor (NGF) for survival in sensory neurons in vitro was examined with microscopic and biochemical methods. Primary dorsal root ganglion (DRG) cultures from embryonic-day-15 (E-15) and day-19 (E-19) rats were maintained with standard dissociated cell culture techniques in the absence of most non-neuronal cells. After various times in culture, neurons were acutely deprived of neurotrophic support by changing to NGF-free medium and adding NGF antiserum to eliminate any residual NGF. Neuronal cultures were examined with phase microscopy; and, their metabolic activity was measured with a protein assay at various time points after NGF deprivation. E-15 neurons grown in culture for 5 days were exquisitely sensitive to acute NGF deprivation. By 12 h after NGF deprivation, neuronal morphology was severely disrupted and the majority of neurons appeared dead. E-15 neurons grown in culture for 8 or 11 days showed progressively less dependence on NGF for survival. These older neurons did not die until 24 and 48 h, respectively, following NGF withdrawal. Neurons grown in culture for 20 days did not show any morphologic changes by phase microscopy up to 4 days after NGF deprivation. Protein incorporation progressively decreased between 12 and 48 h after NGF withdrawal in E-15 neurons grown in culture for 5, 8, or 11 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

A procedure for selecting and culturing subpopulations of neurons from rat dorsal root ganglia using magnetic beads

Current protocols for preparing primary sensory neuron cultures are inadequate when studying individual subpopulations of dorsal root ganglion (DRG) neurons. The DRG is made up of a heterogeneous population of cells, making it difficult to study treatment effects on any given population in mass cultures. Thus, we describe a procedure using magnetic beads from Dynal to select and plate viable populations of neurons based on expression of specific cell surface markers. We show that, by the use of the lectin IB4, we can select a highly enriched viable subpopulation of GDNF-responsive DRG neurons, leaving a viable population of non-selected IB4-ve, Trk+ve neurons. Key factors for successful cultures are (i) quick and careful dissection of DRGs from 4- to 5-week-old Sprague-Dawley rats, (ii) adequate removal of debris and non-neuronal contamination and (iii) gentle handling of bead-bound cells during selection.     

Triiodothyronine Exerts a Trophic Action on Rat Sensory Neuron Survival and Neurite Outgrowth through Different Pathways

Apart from several growth factors which play a crucial role in the survival and development of the central and peripheral nervous systems, thyroid hormones can affect different processes involved in the differentiation and maturation of neurons. The present study was initiated to determine whether triiodothyronine (T₃) affects the survival and neurite outgrowth of primary sensory neurons in vitro. Dorsal root ganglia (DRG) from 19-day-old embryos or newborn rats were plated in explant or dissociated cell cultures. The effect of T₃ on neuron survival was tested, either in mixed DRG cell cultures, where neurons grow with non-neuronal cells, or in neuron-enriched cultures where non-neuronal cells were eliminated at the outset. T₃, in physiological concentrations, promoted the growth of neurons in mixed DRG cell cultures as well as in neuron-enriched cultures without added nerve growth factor (NGF). Since neuron survival in neuron-enriched cultures cannot be promoted by endogenous neurotrophic factors synthesized by non-neuronal cells, the increased number of surviving neurons was due to a direct trophic action of T₃. Another trophic effect was revealed in this study: T₃ sustained the neurite outgrowth of sensory neurons in DRG explants. The stimulatory effect of T₃ on nerve fibre outgrowth was considerably reduced when non-neuronal cell proliferation was inhibited by the antimitotic agent cytosine arabinoside, and was completely suppressed when the great majority of non-neuronal cells were eliminated in neuron-enriched cultures. These results indicate that the stimulatory effect of T₃ on neurite outgrowth is mediated through non-neuronal cells. It is conceivable that T₃ up-regulates Schwann cell expression of a neurotrophic factor, which in turn stimulates axon growth of sensory neurons. Together, these results demonstrate that T₃ promotes both survival and neurite outgrowth of primary sensory neurons in DRG cell cultures. The trophic actions of T₃ on neuron survival and neurite outgrowth operate under two different pathways.     

Developmental capacities of avian embryonic dorsal root ganglion cells: neuropeptides and tyrosine hydroxylase in dissociated cell cultures

Dorsal root ganglia (DRG) from quail embryos of 10-15 days of incubation (E10-15) contain a subpopulation of cells, distinct from postmitotic neurons, that can, under suitable conditions of culture in vitro, differentiate into neuron-like cells that display a variety of adrenergic properties, including tyrosine hydroxylase (TH) immunoreactivity (Xue et al., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8800-8804). The present study was undertaken to determine whether other markers typical of autonomic sympathetic nerve cells are also expressed in the same system. Cells immunoreactive for vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were found to differentiate continually from non-dividing precursors in all cultures of dissociated E10 quail DRG grown in the presence of chick embryo extract. Whereas VIP was already present (in a minute number of cells) in DRG in situ, NPY could not be detected before 3 days of culture, when it appeared simultaneously with TH. Double immunostaining experiments showed that most VIP-positive cells and about half the NPY-positive cells also displayed TH-immunoreactivity. On the other hand, there was no overlap between the substance P-containing neuronal population and any of the cells containing TH, NPY or VIP. These observations are pertinent to the problem of the segregation of autonomic and sensory cell lines during peripheral nervous system ontogeny.     

Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in areas of the brain, including the septal nucleus. Certain growth factors may promote the long-term survival of this subpopulation of neurons at risk. This study was undertaken to characterize growth factors' long-term effects on survival and development of neurons expressing the calcium-binding protein calbindin. In order to accomplish this, embryonic day 16 rat septal neurons were grown in bilaminar culture with astrocytes and in the absence of serum. These cultures were chronically treated with estrogen (Es), insulin-like growth factors I/II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Insulin-like growth factor II significantly increased the number of neurons immunoreactive for calbindin by 155%, suggesting either an increase in the survival of this subpopulation or an increase in the percentage of cells expressing calbindin. Chronic treatment with NGF, IGF-II, and Es significantly increased the number of primary neuritic processes on calbindin-positive neurons, whereas NGF and Es caused significant increases in the number of secondary processes and in the total lengths of the neuritic processes. Thus, effects of IGF-II, estrogen, and NGF on survival and maintenance of this neuronal subpopulation may be dependent on alterations in neurons which are immunopositive for calbindin.     

Age-dependent changes in the capacity of rat sympathetic neurons to form dendrites in tissue culture

We compared the ability of prenatal and postnatal rat sympathetic neurons to form dendrites in tissue culture. Dendrites were distinguished from axons by light microscopic criteria after intracellular dye injection and by differential immunostaining with antibodies to microtubule-associated protein-2 and to both non-phosphorylated and phosphorylated forms of the M and H neurofilament subunits. When maintained in the absence of serum and non-neuronal cells, most (72%) prenatal neurons were unipolar and had only an axon. In contrast, most (89%) neurons derived from postnatal ganglia were multipolar and extended both axons and dendrites. The dendritic morphology of postnatal neurons was usually simple with cells commonly having 2-5 short (50-200 microns), relatively unbranched dendrites. Thus, as the development of the dendritic arbor progresses in situ, sympathetic neurons acquire an enhanced ability to extend dendrites in tissue culture. To determine whether changes in the capacity to develop dendrites might occur with aging in vitro, ganglia were removed from prenatal rats and grown as explants for 3 weeks in the presence of non-neuronal cells; under these conditions, prenatal neurons within the explant became multipolar. When neurons derived from aged explants were subsequently maintained in dissociated cell culture, most formed dendrites. In cultures treated with an antimitotic agent, neurons typically had 1-4 unbranched dendrites; greater amounts of dendritic growth occurred in cultures in which ganglionic non-neuronal cells were allowed to proliferate. We conclude that: (1) the acquisition of the capacity to form dendrites in dissociated cell culture does not require either normal afferent input or physical contact with the target tissue; and (2) even after aging in vitro, sympathetic neurons remain responsive to the dendrite-promoting activity of ganglionic non-neuronal cells.     

High-affinity uptake of noradrenaline in quail dorsal root ganglion cells that express tyrosine hydroxylase immunoreactivity in vitro

During embryonic life, avian sensory ganglia contain cells with the potential to express, under appropriate experimental conditions, a number of properties characteristic of autonomic sympathetic neurons. Thus, cells capable of synthesizing noradrenaline (NA) from tyrosine differentiate when dorsal root ganglia (DRG) from 10-15 d embryonic quail are grown in culture (Xue et al., 1985a, b). In the present study, we show that cultures of DRG from 10 d embryos can take up 3H-NA by a high-affinity (Km = 1.0 microM), temperature-dependent process that can be inhibited by desmethylimipramine. By means of combined immunocytochemistry and autoradiography, it was demonstrated that the majority (70-80%) of the tyrosine hydroxylase (TH)-immunoreactive cells that developed in the cultures possessed a transport system for NA. Catecholamine (CA) uptake also occurred in a small, but relatively constant, number of TH-negative cells, but was absent from substance P-containing neurons. In contrast to TH, which appears only after 3-4 d in vitro, cells capable of taking up NA with high affinity were found in DRG cultures after only a few hours, and a small number (less than 0.5% of the total cell population) was detected in freshly removed, uncultured ganglia. Such cells did not react with antibodies directed against substance P or neurofilament proteins. We conclude that autonomic precursors are identifiable in a subset of non-neuronal DRG cells, prior to full expression of a noradrenergic phenotype, by their possession of a high-affinity uptake system for CA.     

NF-L and peripherin immunoreactivities define distinct classes of rat sensory ganglion cells.

Double immunofluorescence studies using antibodies against NF-L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin-positive (NF-L-negative), and 48% were large and NF-L-positive (peripherin-negative). About 6% were both peripherin- and NF-L-positive. All of the DRG neurons reacted with antibodies to NF-M and nonphosphorylation-dependent or phosphorylation-independent antibodies to NF-H. The neuropeptides were predominantly found in the peripherin-positive small cell population. Eighty-seven percent of the peripherin-positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18-24% of the NF-L-positive large-cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum-free medium, stably maintained exclusively peripherin-positive neurons, with about 5% of these containing coexistent NF-L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture.     

Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture

Capsaicin is a pungent pain-producing compound found in plants of the capsicum family; it exerts excitatory, desensitizing, and toxic effects on a subset of sensory neurons, including the polymodal nociceptor population. We have carried out a quantitative study of capsaicin-induced fluxes of sodium, guanidine, calcium, rubidium, and chloride ions in cultures of neonatal and adult rat DRG neurons, in conjunction with the use of a histochemical stain that identifies capsaicin-sensitive neurons by means of cobalt uptake. Those cells that take up cobalt in a capsaicin-dependent manner (EC50 = 0.2 microM) represent about 50% of the total neuronal population derived from neonatal DRGs on short-term culture. Overnight treatment of cultures with 2 microM capsaicin leads to the loss of the cobalt-staining subpopulation. The capsaicin-insensitive neurons contain immunoreactive neurofilament epitopes that are present in fewer than 10% of capsaicin-sensitive neurons. This observation provides indirect evidence that the sensitive cells correspond to the small, dark B-type neurons, which are negative for neurofilament immunoreactivity in vivo. A capsaicin-dependent calcium uptake (EC50 = 0.2 microM), as measured by 45Ca incorporation, is shown by a DRG neuronal subpopulation that, like the cobalt-staining population of DRG neurons, is lost after overnight capsaicin treatment (2 microM). Capsaicin application leads to the accumulation of millimolar levels of calcium within a few minutes. Cadmium and other divalent cations block capsaicin-induced calcium uptake, but little or no inhibition is seen with organic calcium channel antagonists. Mitochondria, rather than the endoplasmic reticulum, are the probable destination of the internalized calcium, because ruthenium red inhibits calcium uptake (IC50 = 0.05 microM), whereas methylxanthines are inactive. The subset of sensory neurons that takes up calcium also releases 86Rb when exposed to capsaicin (EC50 = 0.06 microM). No efflux of 36Cl ions could be induced by capsaicin. These cells also show a capsaicin-induced uptake of 22Na or 14C guanidine (EC50 = 0.06 microM). In contrast, chick DRG cells in culture showed no capsaicin-induced calcium or cobalt uptake. Primary cultures of rat superior cervical ganglion neurons and Schwann cells, and a number of neuronal cell lines, also failed to respond to capsaicin, as judged by the calcium, cobalt, or guanidine uptake assays.     

Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates

A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85–90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiences greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.     

Plasticity of the auditory brainstem: cochleotomy-induced changes of calbindin-D28k expression in the rat

Calbindin is a calcium binding protein that is characteristically expressed in several auditory brainstem nuclei during ontogeny and is thought to serve as a buffer, protecting cells against toxic levels of calcium. Upon maturation, calbindin is drastically reduced or entirely lost in many auditory nuclei. We made cochleotomies in mature rats to study effects of deafening and deafferentation on the expression of calbindin in the auditory brainstem. Following unilateral cochleotomy, we observed a substantial increase in the number of calbindin-immunoreactive fibers and boutons in the ventral subdivisions of the ipsilateral cochlear nucleus. At the same time, calbindin-positive astrocytes emerged in the dorsal and ventral cochlear nucleus. Beyond the immediately affected ipsilateral cochlear nucleus, we found calbindin-positive neurons in the lateral superior olive and in the central inferior colliculus, both contralateral to the operation. The loss of one cochlea reduces auditory input and puts the flow of neuronal activity originating in the two ears out of balance. Our findings indicate that the need for the neuronal networks in the auditory brainstem to adjust to this drastically changed pattern of sensory signals invokes the expression of calbindin in glial cells as well as in directly and indirectly affected neuronal cell populations.     

Complementary Distribution of Calbindin D-28k and Parvalbumin in the Basal Forebrain and Midbrain of the Squirrel Monkey

The distribution of cell bodies expressing either calbindin D-28k or parvalbumin immunoreactivity in the basal forebrain and midbrain of squirrel monkeys (Saimiri sciureus) was studied on contiguous sections incubated with monoclonal antibodies raised against calbindin or parvalbumin. In the nucleus accumbens, medium-sized calbindin-positive neurons formed two cell bridges joining the ventral part of the striatum to the olfactory tubercle, whereas medium-sized parvalbumin-positive cells in the same area were much less numerous and more uniformly distributed. The medial and dorsal septal nuclei contained a small number of elongated calbindin-positive neurons and only a few parvalbumin-immunoreactive cells. In the nucleus of the diagonal band of Broca, calbindin and parvalbumin were found to label two distinct but closely intermingled neuronal populations. In the striatum, medium-sized calbindin-immunoreactive cells occurred in very large numbers and appeared to be confined to the extrastriosomal matrix. Medium-sized, parvalbumin-immunoreactive neurons were also present in the striatum but they were less numerous than the calbindin-positive cells. The calbindin-positive neurons in the dorsal portion of the striatum were less intensely stained than those in the ventral portion, whereas this pattern did not occur for neurons expressing parvalbumin immunoreactivity. At the pallidal level, neurons in both segments were devoid of calbindin but displayed a very strong parvalbumin immunoreactivity. Most of the large neurons of the nucleus basalis of Meynert were strongly calbindin-immunoreactive and many of them invaded dorsally the medullary laminae of the pallidal complex. The neurons of the subthalamic nucleus were markedly enriched with parvalbumin but displayed only light calbindin staining. In the substantia nigra/ventral tegmental area complex, calbindin-immunoreactive cells abounded in the ventral tegmental area and in the dorsal tier of the pars compacta of the substantia nigra, but were absent in the ventral tier of the pars compacta and in the entire pars reticulata of the substantia nigra. In contrast, numerous parvalbumin-immunoreactive neurons occurred in the pars reticulata and pars lateralis, but none were found in the pars compacta and ventral tegmental area. These findings reveal that the patterns of calbindin and parvalbumin distribution in primate basal forebrain and midbrain are strikingly complementary, suggesting a synergistic role for these calcium-binding proteins in basal forebrain and midbrain function.     

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.     

Survival of chick embryo sympathetic neurons in cell culture

Changes in neuronal numbers during the development of the chick embryo paravertebral sympathetic nervous system have been examined using cell culture techniques. Early sympathetic ganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequently there is a large decrease in the number of neurons and an increase in the population of non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-day or older embryos than those from earlier stages of development.