Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos

We compared neuronal growth patterns in primary cultures prepared by dissociating 3-day-old chick embryos, either whole embryo (E3WE) or head only (E3H) and plating the dispersed cells onto Petri dishes coated with either poly-L-lysine, collagen or laminin. The culture medium was Dulbecco's Modified Eagle's Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS). As we have previously described, in E3WE cultures on poly-L-lysine the neuronal primary growth patterns were aggregation with neuritic fasciculation, presence of growth cones with microspikes and very few flat cells. In contrast with cultures grown on poly-L-lysine, in cultures grown on collagen or laminin the distinct growth pattern was extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells. When 5% FCS was used, as compared to 10% FCS, neuronal aggregates were fewer and smaller on poly-L-lysine; on collagen or laminin a tendency to aggregate was observed. Several differences were observed in the E3H cultures when compared to E3WE: (a) aggregates were less numerous with the prevailing pattern being a web-like, self-contained aggregate; (b) aggregates connected with other aggregates or flat cells were rare and the aggregate adhesivity was minimized; (c) neurons on collagen or laminin formed networks with the exception of a few, small aggregates displaying no fasciculation; (d) flat cells did not form a monolayer but islets which hosted the neuronal meshy networks. We attribute these differences in the growth patterns between the various types of cultures to be the combined result of a variety of environmental signals, derived from the provided substrata, the serum and the nonneuronal cell factors and cell surface, all primarily regulating neuronal adhesivity.     

Transplantation of embryonic chick spinal cord into transection adult chicken spinal cords: a useful model for transplantation research

Adult chicken spinal cords were completely transected under direct visualization. One week later, embryonic chick spinal cords 6 days, 9 days, or 11 days old were implanted into the site of transection. The animals were allowed to survive for 2 months and then killed and the spinal cords were prepared for histologic analysis. The survival and outgrowth of the embryonic transplants were compared with regard to the age of the implant, and its effects on the host tissue. Six-day embryonic tissue survived and integrated far better than 9-day or 11-day, and adult spinal cord subjacent to the early age embryo showed fewer degenerative changes. The chick embryo may provide a model for use in CNS transplantation.     

Growth patterns of primary cultures dissociated from 3-day-old chick embryos: morphological and biochemical comparisons

Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.     

The onset and development of descending pathways to the spinal cord in the chick embryo

The ontogenetic development of afferent (supraspinal and propriospinal) as well as efferent (ascending) fiber connections of the spinal cord was examined following the injection of horseradish peroxidase (HRP) or wheat germ agglutinin HRP (WGA-HRP) into the cervical and lumbar spinal cords (or brains) of embryos ranging in age from 4 to 14 days of incubation. A few cells were first reliably retrogradely labelled in the pontine reticular formation on embryonic day (E) 4 and E5 following the injection of WGA-HRP into the cervical and lumbar spinal cord, respectively. Propriospinal projections to the lumbar spinal cord, originating from brachial spinal cord, were found by E5, and from the cervical spinal cord by E5.5. Ascending fibers arising from neurons in the lumbar spinal cord could be followed to rostral mesencephalic levels in E5 embryos. Thus, the earliest supraspinal, propriospinal, and ascending fiber connections appear to be formed almost simultaneously. Retrogradely labelled cells were found in the raphe, reticular, vestibular, interstitial, and hypothalamic nuclei in E5.5 embryos following lumbar injections of WGA-HRP. Except for neurons in cerebellar nuclei, all the cell groups of origin that project to the cervical spinal cord of posthatching chicks were also retrogradely labelled by E8. There was a delay in the time of appearance of the projections from various regions of the brain stem to the lumbar versus the cervical spinal cord, ranging from 0.5 to 7 days, but typically of about 3 days duration. A large number of cells located in the ventral hypothalamic region, just dorsal to the optic chiasma, were found to be labelled following cervical HRP injection between E6 and E10. These cells may represent transient projections that are present only during embryonic stages since no labelled cells were found in this region in the newly-hatched chick.     

Development and distribution of substance P in the spinal cord and ganglia of embryonic and newly hatched chick: an immunofluorescence study

The development and distribution of substance P (SP) immunoreactivity were studied in the spinal cord and ganglia of embryonic and newly hatched chick by using the indirect immunofluorescence method. Substance P immunoreactivity was first detected in the spinal cord at embryonic stages 18-20 (incubation day 3). Before stage 32 (day 7), it was mainly found in regions corresponding to the dorsolateral funiculus and Lissauer's tract. Subsequently, SP fibers appeared in the dorsal horn. By stage 38 (day 11), they were demonstrated almost throughout the gray matter, but mostly in laminae I and II. During this period, however, many SP-positive cells were found just ventral to the central canal at the thoracic level, although a few were also detected in other areas throughout the cord. In the white matter, very dense longitudinal SP fibers were observed in Lissauer's tract and the dorsolateral funiculus, where extremely dense plexuses of SP immunoreactivity were also detected around a group of nonimmunoreactive cell bodies. At later stages, no remarkable differences were noticed in the distribution of SP fibers, but the SP-positive cells decreased gradually in number and disappeared after hatching. However, they reappeared following colchicine treatment. In the spinal ganglia, SP immunoreactivity appeared initially at stage 25 (day 4). It was mostly located in small neurons of the mediodorsal region. These cells also decreased in number from later stages but increased by colchicine treatment after hatching. The development and distribution of SP immunoreactivity in the spinal cord and ganglia were generally comparable at all levels examined, except where indicated.     

Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures

The phospholipid and phospholipid fatty acid compositions of mixed murine spinal cord neuronal cultures are reported. The phospholipid composition was primarily comprised of ethanolamine glycerophospholipids (44.8%) and choline glycerophospholipids (43.5%). Plasmalogens made up 29.1% of the ethanolamine glycerophospholipids (13.0% of the total phospholipids) and 4.5% of the choline glycerophospholipids (1.9% of the total phospholipids). Other phospholipids ranged from 2.9% for sphingomyelin to 1.0% for phosphatidylinositol 4-phosphate. The fatty acid compositions of the ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylserine, and phosphatidylinositol were also determined. The choline glycerophospholipids were the most saturated and contained the smallest amount of polyunsaturated fatty acids. The ethanolamine glycerophospholipids were the most unsaturated and contained the highest amount of polyunsaturated fatty acids. The phospholipids contained minimal amounts of 20:3 n-9 (Mead acid) and are not considered polyunsaturated fatty acid deficient. Thus, for the mixed neuronal spinal cord cultures, the phospholipid fatty acid compositions were not polyunsaturated fatty acid deficient and contained a large amount of polyenoic fatty acids of both the n-3 and n-6 series. © 1993 Wiley-Liss, Inc.     

The critical period for ethanol effects on cholinergic neuronal expression in neuroblast-enriched cultures derived from 3-day-old chick embryo: NGF ameliorates the cholinotoxic effects of ethanol

Studies from our laboratory have established that ethanol exerts morphological and biochemical neurotoxic effects during early neuroembryogenesis in the chick brain both in ovo and in culture. In the present study, we further localized the critical period for ethanol effects on cholinergic neuronal expression using neuroblast-enriched cultures derived from 3-day-old chick embryos. Moreover, we report that NGF attenuated the cholinotoxic effects of ethanol. We used the following experimental paradigms: cultures treated with ethanol alone either C0-C3 or C4-C10; NGF alone CO-C4 or C4-C10; ethanol and NGF given concomitantly; ethanol given first then replaced with NGF in the medium; or NGF given first then replaced with ethanol in the medium. The results revealed:

1. (1) the cholinotoxic effect of ethanol occurs between culture days CO and C4 with day 3 appearing to be most critical.

2. (2) similarly, the critical period for the cholinotoxic effects of NGF is during early neuroblast differentiation, culture days CO-C4.

3. (3) NGF can prevent the cholinotoxic effects of ethanol only if both ethanol and NGF are given concomitantly or if ethanol is given first, then culture is replaced with NGF-containing medium.

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor.

A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non-motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive.     

Correlation between morphological and biochemical effects of ethanol on neuroblast-enriched cultures derived from three-day-old chick embryos.

We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth. In this study we examined the direct effects of ethanol exposure on developing neuroblasts in culture. Neuroblast-enriched cultures derived from 3-day-old whole chick embryos were grown in the presence of ethanol at doses ranging from 12.5 to 50 mM from culture day 3-14. Cholinergic and GABAergic phenotypic expression were both significantly reduced following ethanol exposure as assessed by the activities of choline acetyltransferase and glutamate decarboxylase, respectively. Morphometric analysis of the growth patterns showed significant differences between control and ethanol-treated cultures. Control cultures exhibited the characteristic pattern of growth consisting of neuronal aggregation with neuritic arborization, i.e., neuritic bundles and fasciculation. Cultures grown in ethanol from culture day 3 consisted of aggregates that measured significantly greater in size than those observed in control cultures. In addition, in ethanol-treated cultures, the primary pattern of neuritic bundles was replaced by a complex network of individual neurites radiating from the central aggregate, forming a defined "neuritic field." Morphometric analysis revealed that both neurite number and neurite length were significantly reduced in ethanol-treated cultures. The biochemical data confirm earlier reports from this laboratory suggesting that ethanol exposure during early embryogenesis alters the normal neuronal pattern of phenotypic expression. In addition, we have presented evidence in this study that ethanol alters the morphological growth patterns of developing neurons. Although ethanol does not alter the ability of these cells to aggregate, there is a significant alteration in neuritic outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Illusive transience of parvalbumin expression duringembryonic development of the primate spinal cord

Parvalbumin has been located by pre-embedding light- and electron microscopic immunohistochemical techniques in the spinal cords of monkey fetuses (Macaca fasciculata), ranging from E70 to E 123, and in young (P20) and young adult (3 years) Macaque monkeys. During the time window investigated, the main developmental events of parvalbumin-containing neural elements are that parvalbumin-positive dorsal root collaterals establish intercellular networks first around nerve cells of Clarke's nucleus, then in the motoneuron pool and finally in the upper dorsal horn. In each of these areas, location of the parvalbumin-positive network is gradually shifted from medial to lateral. Whenever an intercellular network is established, nerve cells innervated by parvalbumin-positive terminals of dorsal root collaterals start to express parvalbumin. Immunoreactivity of dorsal root axons is transient; it disappears first from the intercellular networks and, afterwards, also from the dorsal columns. However, the pericellular synaptic terminals and their post-synaptic nerve cells express parvalbumin into adulthood. It is concluded that some of the large (Type A) dorsal root ganglion cells are the first ones in the spinal reflex pathway to express parvalbumin, which is elicited and gradually increased in nerve cells synaptically innervated by parvalbumin-positive axon terminals. This seems to represent a specific case of activation (or desinhibiton) of the genome. Apparent "transience" of parvalbumin is due to the specific geometry of primary sensory neurons equipped with extremely long axonal processes, and the consequent specialities of axonal transport characteristics.     

Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs.

Motoneurons of adult frog spinal cord have been retrogradely labeled with the carbocyanine derivative diI. Spinal cords were then dissociated and the labeled motoneurons partially purified by centrifugation over a bovine serum albumin (BSA) density gradient. The resulting cell suspension was plated on a substrate of innervated muscle extracellular matrix (ECM) to which the motoneurons attached and extended processes. Labeled adult motoneurons survived for more than 4 weeks in a defined medium in the absence of added serum or growth factors. These cultures of adult motoneurons provide a favorable preparation for studying molecular factors that influence process outgrowth and synapse formation.     

First step of selective motoneuron axonal growth: selective outgrowth at discrete sites in the spinal cord

Selective axonal growth at a series of choice points along pathways is essential for the establishment of precise motoneuron projections. To reveal some of the molecules responsible for this selective growth of motoneuron axons, this study investigates the phenomenon of why motoneurons extend axons outside the spinal cord, whereas interneurons do not. Axonal growth in the chick embryonic spinal cord at early stages of development was examined immunohistochemically. MAb SC1 staining of serial sections selectively revealed the entire distribution of motoneuron axons in the embryo. In the cervical segments, some motoneurons, called dorsal motoneurons, extended axons outside the cord via the dorsal root entry zone. The axons of both dorsal and ventral motoneurons were distributed only in the anterior half of the sclerotome; inside the cord motoneuron cell bodies and axons were distributed evenly along the anterior-posterior axis. Motoneurons and interneurons extended axons during the same period, and although the growth cones of both were intermixed in the same locations, only motoneuron axons grew out from the cord. The outgrowth of all st 19 motoneurons from the cord through either the dorsal or ventral exit zone strongly suggests that extension of their axons outside the cord is a selective rather than random process.     

The actions of neuropeptides on dorsal horn neurons in the rat spinal cord slice preparation: an intracellular study

Responses of dorsal horn neurons to bath application of substance P, somatostatin and enkephalin were studied by intracellular recording in the neonatal spinal cord slice preparation. Substance P depolarized dorsal horn neurons and increased their excitability. The depolarization was most commonly associated with an increase in neuronal input resistance. Somatostatin and enkephalin hyperpolarized dorsal horn neurons and caused reduction or abolition of spontaneous firing. While the hyperpolarization produced by enkephalin was always associated with a fall in neuronal input resistance, in the case of somatostatin the similar effect was less consistently observed.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

The effect of cortisol on gamma-glutamyl transpeptidase activity in the glycogen body and lumbosacral segments of developing chick spinal cord

Gamma-glutamyl transpeptidase (GGT) activity is heterogeneously distributed between the lumbosacral chick spinal cord and the adjacent glycogen body during late embryonic and early posthatch development and displays hormonal sensitivity. As GGT activity may reflect the cellular transport of some amino acids, the transiently high activity of this enzyme in the glycogen body suggests that these cells play an important role in amino acid transport and metabolism at a time coincident with the initial phase of myelination in the embryonic chick spinal cord.     

Distribution of substance P and vasoactive intestinal polypeptide neurons in the chicken spinal cord, with notes on their postnatal development

The distribution of substance P (SP) and vasoactive intestinal polypeptide (VIP) was investigated by immunohistochemistry in the adult chicken spinal cord. By using colchicine treatment, populations of neurons containing either SP or VIP was observed in several regions of the spinal cord. SP neurons were found dorsal to the central canal (CC) and in lamina IV throughout the cord. However, at the thoracic level, numerous relatively larger SP perikarya were located ventral to the CC and aligned on either side of the midline. The distribution of SP fibers is very similar to that reported previously in mammals: they were mostly observed in laminae I and II, in Lissauer's tract, in the dorsolateral funiculus, and dorsal to the CC. In addition, two dense plexuses of SP fibers were noticed in lamina IV. VIP neurons were located mainly in lamina I, in the nucleus of the dorsolateral funiculus, and in the lateral portion of the neck of the dorsal horn throughout the spinal cord. At the thoracic level, many also were located lateral to the CC. Occasionally, single VIP neurons also were encountered dorsal to the CC, in laminae II-IV, and in the intermediate zone. VIP fibers were observed in similar numbers at all spinal levels, occurring mainly in laminae II (probably I) and III, dorsal to the CC, and in the intermediate zone. In addition, examination of the developing chick spinal cords showed similar results as in adult chickens.     

Ethanol increases cholinergic and decreases GABAergic neuronal expression in cultures derived from 8-day-old chick embryo cerebral hemispheres: interaction of ethanol and growth factors.

We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth both in ovo and in vitro. Moreover, we have found that growth factors significantly attenuate the in ovo neurotoxicity produced by ethanol. In this study, we further examined the direct effects of ethanol exposure on neuron-enriched cultures derived from 8-day-old chick embryo cerebral hemispheres consisting primarily of differentiated neurons. In addition, we examined the interaction of ethanol and nerve growth factor (NGF) or epidermal growth factor (EGF) when the growth factors were given concomitantly with ethanol. Choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) were used as markers for cholinergic and GABAergic neuronal phenotypic expression, respectively. We found that ethanol alone enhanced ChAT and reduced GAD activities in a dose-dependent manner. NGF and EGF given alone enhanced the expression of both neuronal phenotypes. When NGF was given concomitantly with ethanol at C4-8 the decline in GAD produced by ethanol was reversed. The effects of concomitant administration of ethanol and growth factors on ChAT activity revealed that ethanol interfered with the increases produced by the growth factors and especially with NGF when given alone. We conclude from these findings that ethanol may interfere with neuronal phenotypic expression by altering neuronal responsiveness to neurotrophic signals important for neuronal differentiation.     

Depression of benzodiazepine binding and diazepam potentiation of GABA-mediated inhibition after chronic exposure of spinal cord cultures to diazepam

Cultures of fetal mouse spinal cord were exposed to 12.6 μM (3.6 μg/ml) diazepam for 7 days. After drug removal, benzodiazepine receptor binding was assayed on intact cells and intracellular recordings of diazepam effects on GABA-mediated inhibitory responses were obtained. The biochemical and electrophysiological data revealed significant and parallel reductions in both receptor binding and pharmacological action on GABA responses which did not return to control levels until 3–4 days after removal of diazepam. The results indicate that chronic exposure of spinal cord cultures to diazepam results in a reversible down-regulation of diazepam binding and function.     

Blocking weight-induced spinal cord injury in rats: effects of TRH or naloxone on motor function recovery and spinal cord blood flow

The ability of thyrotropin releasing hormone (TRH) or naloxone to reduce the motor function deficit and to improve the spinal cord blood flow (SCBF) was investigated in a rat spinal cord compression injury model. Spinal cord injury was induced by compression for 5 min with a load of 35 g on a 2.2 x 5.0 mm sized compression plate causing a transient paraparesis. One group of animals was given TRH, one group naloxone and one group saline alone. Each drug was administered intravenously as a bolus dose of 2 mg/kg 60 min after injury followed by a continuous infusion of 2 mg/kg/h for 4 h. The motor performance was assessed daily on the inclined plane until Day 4, when SCBF was measured with the 14C-iodoantipyrine autoradiographic method. It was found that neither TRH nor naloxone had promoted motor function recovery or affected SCBF 4 days after spinal cord injury.