Reactive oxygen species modulate the differentiation of neurons in clonal cortical cultures

Reactive oxygen species (ROS) are important regulators of intracellular signaling. We examined the expression of ROS during rat brain development and explored their role in differentiation using cortical cultures. High levels of ROS were found in newborn neurons. Neurons produced ROS, not connected with cell death, throughout embryogenesis and postnatal stages. By P20, ROS-producing cells were found only in neurogenic regions. Cells with low levels of ROS, isolated from E15 brains by FACS, differentiated into neurons, oligodendrocytes, and astrocytes in clonal cultures. Neurons produced high ROS early in culture and later differentiated into two types: large pyramidal-like neurons that fired no or only a single action potential and smaller neurons that expressed nuclear calretinin and fired repeated action potentials. Antioxidant treatment did not alter neuron number but increased the ratio of small to large neurons. These findings suggest that modulation of ROS levels influences multiple aspects of neuronal differentiation.     

Xenopus embryonic spinal neurons recorded in situ with patch‐clamp electrodes – conditional oscillators after all?

The central pattern generator for swimming Xenopus embryo is organized as two half-centres linked by reciprocal inhibition. Microelectrode recordings suggest that Xenopus neurons are poorly excitable, necessitating a key role for postinhibitory rebound in the operation of the central pattern generator. However the Xenopus central pattern generator seems unusual in that the component neurons apparently have no intrinsic or conditional rhythmogenic properties. We have re-examined the firing properties of Xenopus embryo spinal neurons by making patch-clamp recordings in situ from intact spinal cord. Recordings made from 99 neurons were divided into three groups. Central pattern generator neurons overwhelmingly (44/51) fired trains of action potentials in response to current injection. Just over half of the sensory interneurons (13/22) also fired trains of action potentials. Neurons that received no synaptic inputs during swimming mostly fired just one or two action potentials (22/26). Thirty-four neurons were identified morphologically. Commissural (8/12) and descending (6/6) interneurons, key components of the spinal central pattern generator, fired repetitive trains of action potentials during current injection. Neurons that were not part of the central pattern generator did not demonstrate this preponderance for repetitive firing. Analysis of the interspike intervals during current injection revealed that the majority of central pattern generators, descending and commissural interneurons, could readily fire at frequencies up to twice that of swimming. We suggest that Xenopus neurons can be considered as conditional oscillators: in the presence of unpatterned excitation they exhibit an ability to fire rhythmically. This property makes the Xenopus embryonic central pattern generator more similar to other model central pattern generators than has hitherto been appreciated.     

Participation of neurons of the midbrain reticular formation of the cat in conditioned reflex activity

Spike discharges of mesencephalic reticular formation neurons were investigated during classical and instrumental conditional reflexes in chronic experiments on cats. Neurons were divided into several functional groups according to patterns of their responses. Two groups of neurons produced continuous tonic spike discharges. One of them was activated by positive conditional stimuli and did not respond to the same stimulus after extinction. Another group was involved in reaction only after extinction. Thus the neurons of these two groups developed additional tonic ascending action not only during fulfillment of conditional reflex but under its internal inhibition too. Neurons of the third group revealed double phasic responses: the first--to sound and the second--in connection with initiation of the conditioned movement. They decreased the level of the background and evoked activity during differentiation and extinction. Initial changes of reticular neuron responses were similar under conditioning and under pseudoconditioning. It is concluded that mesencephalic reticular formation takes an active and differential participation in elaboration, fulfillment and internal inhibition of classical and instrumental conditional reflexes.     

Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytes

Generation of reactive oxygen species (ROS) in brain tissue leads to neurodegeneration. The major source of ROS is the mitochondrial respiratory chain. We studied regulation of Ca2+ level, mitochondrial potential, and ROS generation in defined mixed hippocampal cell cultures exposed to glutamate (100 microM). Recordings were made from individually identified astrocytes and neurons to compare the physiologic responses in both cell types. Neurons identified by synaptotagmin immunoreactivity were characterized functionally by the fast Ca2+ increase with K+ (50 mM) stimulation, and the astrocytes identified by glial fibrillary acidic protein (GFAP) staining had the functional characteristic of a transient Ca2+ peak in response to ATP (10 microM) stimulation. We found that the glutamate-mediated Ca2+ response in neurons is due largely to influx of extracellular Ca2+. This is consistent with our finding that in cultured hippocampal neurons, stores depending on the activity of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump had a low Ca2+ content, regardless of whether the neurons were challenged or not with K+ before applying the SERCA inhibitor cyclopiazonic acid (CPA). Astrocytes displayed a large CPA-mediated Ca2 response, indicating a high level of Ca2+ load in the stores in astrocytes. Importantly, the rise in ROS generation due to glutamate application was cell-type specific. In neurons, glutamate induced a marked rise in generation of ROS, but not in astrocytes. In both astrocytes and neurons, the mitochondrial potential was increased in response to glutamate challenge. We conclude that in neurons, Ca2+ influx accounts for the increased ROS generation in response to glutamate. This might explain the high vulnerability of neurons to glutamate challenge compared to the vulnerability of astrocytes. The high resistance of astrocytes is accompanied by an efficient downregulation of cytosolic Ca2+, which is not found in neurons.     

Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation

Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.     

Spontaneous electrical activity induced by herpes virus infection in rat sensory neuron cultures

Dissociated cultures of rat dorsal root ganglion neurons were infected with a syncitial strain of herpes simplex virus type 1. Over 90% of neurons in infected cultures were spontaneously active and fired action potentials which, on membrane potential hyperpolarization, were replaced by depolarizing events similar to excitatory postsynaptic potentials. Amplitude analysis of these events produced populations described by the sum of several unitary events with Gaussian rather than binomial or Poisson distributions. Such spontaneous activity was blocked by tetrodotoxin but not by low calcium high magnesium solutions containing cadmium. Simultaneous recording from pairs of spontaneous active neurons revealed excitatory connections between cells. It is suggested that virus-induced fusion of nerve cell processes induces electrical coupling between sensory neurons, and that the resulting electrical network supports spontaneous activity.     

Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids.

Sexual differentiation of dopaminergic neurons was studied in gender-specific cultures. Dissociated cell cultures were prepared from di- or mesencephalon of gestational day 14 rat embryos and raised in the absence or presence of 17 beta-estradiol or testosterone for up to 13 days in vitro (DIV). Developmental profiles of levels of dopamine (DA) and metabolites as well as capacity for vesicular storage of the transmitter were determined by HPLC. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted. Higher levels of DA were measured in female than in male cultures of both brain regions. In mesencephalic cultures, the differences in DA levels were fully accounted for by sex differences in numbers of TH-IR cells, whereas no sex differences in cell numbers were found in diencephalic cultures. Dihydroxyphenylacetic acid (DOPAC) levels and vesicular storage capacity matured faster in mesencephalic than in diencephalic cultures, but no sex differences were observed. Homovanillic acid (HVA) could not be detected except in 13-DIV mesencephalic cultures. Hormonal treatment did not erase sexual differentiation of dopaminergic neurons. Irrespective of the gender, however, both steroids decreased DA and DOPAC contents in diencephalic cultures but not in mesencephalic cultures. It is proposed that sexual differentiation of dopaminergic systems proceeds in a region-specific fashion and that neurogenesis and development of various parameters of dopaminergic activity may be differentially affected. Sexual differentiation of dopaminergic neurons may be initiated independently of the action of gonadal steroid hormones and may subsequently be modified by differences in hormonal environment.     

Developmental effect of dimethyl sulfoxide on hypothalamo-neurohypophysial neurons in vitro

Primary dissociated cultures were established from diencephalic tissue of 14-day-old fetal rats. Neurons exhibiting immunocytochemical staining for neurophysin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio-immunometric quantitation of neuron-specific enolase. In contrast, in DMSO-treated cultures the number of neurophysin-immunoreactive neurons was more than doubled as compared to control cultures. [3H]Thymidine labeling and autoradiography in conjunction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time-course analysis of the action of DMSO revealed a 6-day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin-immunoreactive cells. These findings suggest that DMSO, which has previously been reported to have a differentiation-inducing effect on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.     

Newborn neurons acquire high levels of reactive oxygen species and increased mitochondrial proteins upon differentiation from progenitors

A population of embryonic rat cortical cells cultured in the presence of FGF2 and having neuronal morphology expressed higher levels of reactive oxygen species (ROS) than did progenitor cells, astrocytes, and several cell lines of neuronal and non-neuronal origin. ROS were assessed using 5-(and-6)-chlormethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCF-DA), and high levels persisted in the presence of antioxidants or lowered levels of ambient oxygen. Greater than 95% of high ROS-producing cells, isolated by fluorescence-activated cell sorting, expressed the neuronal marker beta III tubulin. These cells did not incorporate BrdU or express nestin, unlike low ROS-producing cells, 99% of which exhibited both of these characteristics. Upon growth factor removal, low ROS-expressing cells differentiated into neurons and astrocytes and these neurons expressed high levels of ROS, indicating that ROS accumulation accompanies the differentiation of progenitors into neurons. ROS levels were decreased by added superoxide dismutase and catalase, suggesting that both superoxide and hydrogen peroxide contribute to the ROS signal. High ROS-expressing cells also contained higher levels of several mitochondrial respiratory chain components. Although ROS have been associated with conditions that lead to cell death, our results and recent studies on the role of ROS as regulators of signal pathways are consistent with the possibility that ROS play a role in the development of the neuronal phenotype. Moreover, the differential production of ROS provides a useful method to isolate from mixed populations cells that are highly enriched for either progenitor cells or neurons.     

A subpopulation of embryonic telencephalic neurons survive and develop in vitro in response to factors derived from the periphery

Denervated chick muscle contains factors that enhance neurite outgrowth in cultures of embryonic chicken spinal neurons. Chromatography of muscle extract on a column of DEAE-Sepharose yielded a fraction which retained most of the starting neurite-promoting activity. This DEAE fraction was tested for its activity on neurons from other regions of the central nervous system of 5-day-old chicken embryos. Both neurite outgrowth and survival of telencephalic neurons in vitro were greatly enhanced when the DEAE fraction was added at protein concentrations around 1 microgram/ml. When cultures were prepared from embryos later than 6 days in ovo, the effects of the DEAE fraction progressively diminished with age. Neurons from the embryonic diencephalon, mesencephalon and rhombencephalon were not responsive to the DEAE fraction, although they all developed neurites on a laminin substratum. Similar neurite-promoting activities for telencephalic neurons were found in extracts of neonatal brain, liver and heart, but not lung.     

Suprachiasmatic neurons in tissue slices from ovariectomized rats: Electrophysiological and neuropharmacological characterization and the effects of estrogen treatment

Single-unit activity and unit responses to putative neurotransmitters were recorded from suprachiasmatic nucleus (SCN) neurons in brain tissue slices from ovariectomized rats either treated or untreated with estrogen. Altogether, 204 units were studied from estrogen-treated and untreated preparations, and at the resting state, 37% of these units fired regularly, 57% fired irregularly, and 6% were silent but evokable by electrical stimulation. Most of the irregular units fired continuously (n = 100), while the rest fired intermittently (n = 12) or phasically (n = 4). Neurons with different types of firing patterns also varied significantly in resting firing rate and in responsiveness to transmitters and to estrogen treatment. The average resting firing rate decreased significantly from regular, irregular and continuous, intermittent, to silent units. Acetylcholine (ACh) and/or serotonin (5-HT) injected directly into the perfusion chamber evoked responses from more irregular (69% of 61 units) than regular units (20% of 46 units). None of the 5 silent units tested was activated by ACh or 5-HT. Responses to ACh (predominantly inhibitory) and 5-HT (predominantly excitatory) seen here in vitro were opposite to those observed in vivo with iontophoretic application, and were not reversed or abolished by the blockade of synaptic transmission. Comparisons of data between the two types of preparations showed that only the responsiveness of the irregular units to ACh and to 5-HT were significantly different: both types of responsiveness were higher in estrogen-treated than in untreated preparations. No significant difference was found in the responsiveness of regular units, or in firing patterns or firing rate. Thus, the present in vitro studies have demonstrated that SCN contains a heterogeneous population of neurons distinguishable by their electrophysiological and neuropharmacological characteristics, and that estrogen has a specific action on specific types of SCN neurons.     

Comparative effects of herpes simplex virus types 1 and 2 in organotypic cultures of mouse dorsal root ganglion.

Mature organized cultures of mouse dorsal root ganglion (MDRG) were infected with herpes simplex virus, type 1 (HSV 1) and type 2 (HSV 2). Onset of infectious virus production occurred faster and reached higher levels in HSV 2-infected cultures. Neurons, supporting cells and myelin were affected in both types of infection, but morphological changes occurred significantly earlier and more dramatically with the type 2 infection. The pattern of myelin changes was distinctly different in the two types of infection. Within 20 hours post infection nerve cells infected with HSV 2 developed several types of intranuclear inclusions consisting of membranes and filaments; no such neuronal inclusions were seen with HSV 1 infection. HSV 2 infection showed frequent, large, membranous inclusions in supporting cell nuclei whereas, only rare, small inclusions of this type were seen in supporting cells infected with HSV 1. The observations demonstrate that the two virus types produced different virus replication patterns and different morphologic changes in long term cultures of MDRG. There appears to be a differential response of neurons and non-neuronal elements to the virus in this tissue substrate. Viral latency was not induced in this system by direct inoculation of the virus under the conditions described.     

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage

We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.     

Effects of 5-bromodeoxyuridine on development of Mauthner's neuron and neural retina of Xenopus laevis embryos

Bromodeoxyuridine (BrUdR) injected into Xenopus embryos at stages before, during or after the last period of DNA synthesis in Mauthner's neurons had no serious effect on differentiation of Mauthner's neurons. However, the BrUdR caused widespread cell degeneration in the central nervous system. The action of BrUdR on retinal neuronal differentiation depended on dose and stage of infection. At all doses (0.005-0.9 microgram per injection), and all stages (22-29), the main effect was retinal cell degeneration. The drug produced complete absence of differentiated retinal neurons only when given at early stages (22-24) and at the highest doses (0.5-0.9 microgram) that enabled the embryo to survive to stage 44. At stages 22-24 at lower doses (0.005 or 0.05 microgram per injection) and at stages 26-29 at all doses, some retinal neurons differentiated, although retinal cell degeneration was the main effect. In these two systems BrUdR did not selectively inhibit neuronal differentiation.     

Neurotoxic effect induced by quinolinic acid in dissociated cell culture of mouse hippocampus

Quinolinic acid (QUIN), an endogenous convulsant that is a product of tryptophan metabolism, is suggested to belong to the pathogenic factors in some neurodegenerative disorders of the central nervous system (CNS). The aim of the present study was to evaluate the effect of QUIN on hippocampal nerve cells in dissociated cell culture at different periods of its development in vitro. The neurodegenerative effect of QUIN in vitro was found 1.5 to 2 hr after exposure to QUIN (500 microM) in differentiated hippocampal culture 20-21 days in vitro (DIV). The cytotoxic action depended on the nature of the interneuronal relations established in culture and on the period of neuronal development in vitro. Neurons in 11-DIV cultures were undamaged even 10 days after exposure to QUIN; nor were the individual, isolated neurons probably not connected synaptically with the other ones in 20-21 DIV cultures. Ultrastructural analysis of 20-21 DIV cultures exposed to QUIN showed acute swelling and destruction of postsynaptic elements and severe degeneration of individual nerve cells located in cellular aggregates, whereas presynaptic axon terminals remained intact. The results emphasize the utility of QUIN as a tool to model degenerative changes of CNS and confirm the participation of mature synaptic junctions in QUIN neurotoxicity.     

Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.     

Neuron-glial interactions involved in the regulation of glutamine synthetase

Cocultures of rat cortical astrocytes with cerebellar granule cell neurons, but not a variety of other cell types tested, resulted in an induction of glutamine synthetase (GS) mRNA over the basal levels expressed in pure astrocyte cultures. This induction involved both contact- and noncontact-mediated events and may be a result of astroglial differentiation promoted by interactions with the primary neurons. Astrocytes grown in the presence of the granule neurons (but not the other cell types tested) exhibited a more complex, process-bearing morphology typical of more differentiated cells. In addition, glial cell proliferation was inhibited not only by the presence of live granule cells, but also by fixed neurons and neuronal membranes. Under the same experimental conditions, GS mRNA was increased (two- to threefold) compared with the expression observed in pure astrocyte cultures. Because of the role of GS in glutamate metabolism and the influence of the glutamatergic granule neurons on glial GS mRNA levels, the effect of exogenous glutamate was examined. The addition of 100 microM glutamate to the culture medium resulted in an increase in GS mRNA in the astrocyte cultures similar to that observed in the cocultures, where the addition of glutamate did not further increase GS mRNA levels. These results provide further evidence for the importance of neuron-glial interactions in the regulation of glial gene expression.     

Protein kinase C activation causes neurite retraction via cyclinD1 and p60‐katanin increase in rat hippocampal neurons

Neurons are differentiated postmitotic cells residing in G₀ phase of the cell cycle and are unable to proceed through G₁ phase, in which cyclinD1 needs to be up‐regulated for initiation. Yet, a growing body of evidence has shown that cell cycle re‐activation via cyclinD1 up‐regulation drives neurons into apoptosis. By contrast, there is also evidence demonstrating cell cycle proteins playing roles in neuronal differentiation. cyclinD1 has been shown to be differently regulated by protein kinase C alpha (PKC‐α) in various mitotic cells. Based on these different effects, we investigated the role of PKC‐α on cyclinD1 regulation in hippocampal neurons. Neurons were treated with PKC activator, PMA, and analysed for subcellular distributions of PKC‐α and cyclinD1. Remarkably, PMA treatment increased nuclear PKC‐α and cyclinD1, but not PKC‐ε in hippocampal neurons. Increases in nuclear PKC‐α and cyclinD1 were accompanied by microtubule re‐organisation via increases in tau and retinoblastoma protein phosphorylation levels. Increased p60‐katanin and p53 changed the neuronal morphology into neurons with shorter, but increased number of side branches. Since up‐regulation of cell cycle is associated with apoptosis in neurons, we also analysed changes in Bax, Bcl‐2 early and PARP (poly(ADP‐ribose)polymerase), caspase3 late apoptotic markers. However, we did not observe any indication of apoptosis. These data suggest that in addition to their previously known roles in mitotic cells on cell cycle regulation, PKC‐α and cyclinD1 seem to be important for differentiation, and nuclear PKC‐α and cyclinD1 interfere with differentiation by promoting microtubule re‐organisation through PKC signaling without triggering apoptosis.     

Neuronal differentiation of the early embryonic auditory hindbrain of the chicken in primary culture

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.