Dissociated Cell Culture System Research Articles

Developmental changes in the responsiveness of rat spiral ganglion neurons to neurotrophic factors in dissociated culture: differential responses for survival, neuritogenesis and neuronal morphology

The way that the development of the inner ear innervation is regulated by various neurotrophic factors and/or their combinations at different postnatal developmental stages remains largely unclear. Moreover, survival and neuritogenesis in deafferented adult neurons is important for cochlear implant function. To address these issues, developmental changes in the responsiveness of postnatal rat spiral ganglion neurons (SGNs) to neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were examined by using a dissociated cell culture system. SGNs at postnatal day (P) 0, P5 and P20 (young adult) were cultured with the addition of NT-3, BDNF, or LIF or of a combination of NT-3 and BDNF (N + B) or of NT-3, BDNF and LIF (ALL factors). SGNs were analyzed for three parameters: survival, longest neurite length (LNL) and neuronal morphology. At P0, SGNs required exposure to N + B or ALL factors for enhanced survival and the ALL factors combination showed a synergistic effect much greater than the sum of the individual factors. At P5, SGNs responded to a wider range of treatment conditions for enhanced survival and combinations showed only an additive improvement over individual factors. The survival percentage of untreated SGNs was highest at P20 but combinations of neurotrophic factors were no more effective than individual factors. LNL of each SGN was enhanced by LIF alone or ALL factors at P0 and P5 but was suppressed by NT-3, BDNF and N + B at P5 in a dose-dependent manner. The LNL at P20 was enhanced by ALL factors and suppressed by N + B. Treatment with ALL factors increased the proportion of SGNs that had two or more primary neurites in all age groups. These findings suggest that NT-3, BDNF, LIF and their combinations predominantly support different ontogenetic events at different developmental stages in the innervation of the inner ear.

Establishment of dissociated cell culture system of inner ear sensory epithelial cells

Establishment of dissociated cell culture system of inner ear sensory epithelial cells

The Corticostriatal System in Dissociated Cell Culture

The sparse connectivity within the striatum in vivo makes the investigation of individual corticostriatal synapses very difficult. Most studies of the corticostriatal input have been done using electrical stimulation under conditions where it is hard to identify the precise origin of the cortical input. We have employed an in vitro dissociated cell culture system that allows the identification of individual corticostriatal pairs and have been developing methods to study individual neuron inputs to striatal neurons. In mixed corticostriatal cultures, neurons had resting activity similar to the system in vivo. Up/down states were obvious and seemed to encompass the entire culture. Mixed cultures of cortical neurons from transgenic mice expressing green fluorescent protein with striatal neurons from wild-type mice of the same developmental stage allowed visual identification of individual candidate corticostriatal pairs. Recordings were performed between 12 and 37 days in vitro (DIV). To investigate synaptic connections we recorded from 69 corticostriatal pairs of which 44 were connected in one direction and 25 reciprocally. Of these connections 41 were corticostriatal (nine inhibitory) and 53 striatocortical (all inhibitory). The observed excitatory responses were of variable amplitude (−10 to −370 pA, n = 32). We found the connections very secure – with negligible failures on repeated stimulation (approximately 1 Hz) of the cortical neuron. Inhibitory corticostriatal responses were also observed (−13 to −314 pA, n = 9). Possibly due to the mixed type of culture we found an inhibitory striatocortical response (−14 to −598 pA, n = 53). We are now recording from neurons in separate compartments to more closely emulate neuroanatomical conditions but still with the possibility of the easier identification of the connectivity.

A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division

Drosophila melanogaster is a key model system that has greatly contributed to the advance of developmental biology through its extensive and sophisticated genetics. Nevertheless, only a few in vitro approaches are available in Drosophila to complement genetic studies in order to better elucidate developmental mechanisms at the cellular and molecular level. Here we present a dissociated cell culture system generated from the optic lobes of Drosophila larval brain. This culture system makes it feasible to study the proliferative properties of Drosophila postembryonic Nbs by allowing BrdU pulse and chase assays, as well as detailed immunocytochemical analysis with molecular markers. These immunofluorescence experiments allowed us to conclude that localization of asymmetric cell division markers such as Inscuteable, Miranda, Prospero and Numb is cell autonomous. By time-lapse video recording we have observed interesting cellular features of postembryonic neurogenesis such us the polarized genesis of the neuroblast progeny, the extremely short ganglion mother cell (GMC) cell cycle, and the last division of a neuroblast lineage. The combination of this cell culture system and genetic tools of Drosophila will provide a powerful experimental model for the analysis of cell cycle and asymmetric cell division of neural progenitor cells.

In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.

Hypoxic excitation in neurons cultured from the rostral ventrolateral medulla of the neonatal rat.

Neurons within cardiorespiratory regions of the rostral ventrolateral medulla (RVLM) have been shown to be excited by local hypoxia. To determine the electrophysiological properties of these excitatory responses to hypoxia, we developed a primary dissociated cell culture system to examine the intrinsic response of RVLM neurons to hypoxia. Neonatal rat neurons plated on medullary astrocyte monolayers were studied using the whole cell perforated patch-clamp technique. Sodium cyanide (NaCN, 0.5-10 mM) was used, and membrane potential (V(m)), firing frequency, and input resistance were examined. In 11 of 19 neurons, NaCN produced a V(m) depolarization, an increase in firing frequency, and a decrease in input resistance, suggesting the opening of a cation channel. The hypoxic depolarization had a linear dose response and was dependent on baseline V(m), with a greater response at more hyperpolarized V(m). In 8 of 19 neurons, NaCN produced a V(m) hyperpolarization, decrease in firing frequency, and variable changes in input resistance. The V(m) hyperpolarization exhibited an all-or-none dose response and was independent of baseline V(m). These differential responses to NaCN were retained after synaptic blockade with low Ca(2+)-high Mg(2+) or TTX. Thus hypoxic excitation 1) is maintained in cell culture, 2) is an intrinsic response, and 3) is likely due to the increase in a cation current. These hypoxia-excited neurons are likely candidates to function as central oxygen sensors.

Ciliary activity in differentiating and reactivated human respiratory epithelial cells.

Experimental studies of mucociliary clearance, especially those involving drug effects, suffer from the difficulty of determining whether drugs act directly on ciliary motility or whether their effects are indirect, acting via changes in cell metabolism, viscous load, or alternative mechanisms. The present study provides a solution to this problem by comparing the motile characteristics of ciliated cells that have differentiated in primary cell culture with those of demembranated cilia reactivated with MgATP. Human respiratory epithelial cells (REC) were dissociated from trachea, bronchus, nasal polyps, or turbinates and then placed in a dissociated cell culture system. Thirty-three percent of the dissociated cells contain beating cilia. Following 1 week in culture, the REC dedifferentiated, but then redifferentiated within 96 hours after they were brought to an air interface. The cilia on such cells beat with planar waves consisting of power and recovery strokes. Beat frequencies at 20 degrees C were 15+/-2.3 Hz. Ciliary beating often was coordinated both within and between cells with a defined antilaeoplectic pattern of coordination. Either fresh or cultured cells could be demembranated with Triton X-100 and reactivated with MgATP. The activity of these reactivated models was equivalent to those observed in living cells. The authors have demonstrated that ciliary beat frequency of demembranated human respiratory epithelial cells can be modulated by MgATP and can be adjusted to the same level of activity as measured in living cells. This allows them to selectively test whether a drug's effects on mucociliary transport are the result of direct interactions with the ciliary apparatus or are produced through other indirect mechanisms.

Properties of depolarization-induced suppression of inhibitory transmission in cultured rat hippocampal neurons.

Properties of depolarization-induced suppression of inhibitory transmission ("DSI") in cultured rat hippocampal neurons were examined, by recording inhibitory postsynaptic currents (IPSCs) evoked by single presynaptic neurons. In about 40% of the inhibitory synapses, transient suppression of IPSCs was induced by applying a depolarizing pulse (to 0 mV, > 2 s) to the postsynaptic neuron, which was identified to be gamma-aminobutyric acid dependent (GABAergic) in some pairs, and to be glutamatergic in some other pairs. This depolarization-induced suppression of IPSCs, "DSI", was Ca2+ dependent, and was associated with an increase in the paired-pulse ratio. Phorbol esters had an occluding effect on DSI when applied to the bath solution, but not to the pipette solution used for the postsynaptic neuron. Application of the opioid receptor antagonist naloxone had no effect on DSI. The present study demonstrates that a pair of cultured hippocampal neurons can exhibit DSI similar to that previously reported to occur in hippocampal slices; this suggests that a dissociated cell culture system can provide a useful model system for the study of DSI. Furthermore, it was found that inhibitory neurons, in addition to excitatory ones, can induce DSI, and that a process sensitive to phorbol esters, but not activation of opioid receptors, is involved in DSI.

Cell Cultures of Rat Suprachiasmatic Nucleus: Circadian Oscillation of Vasopressin Release

A dissociated cell culture system has been developed for the suprachiasmatic nucleus (SCN), in which release of vasopressin showed a clear circadian oscillation. The oscillation peaked at early subjective day and appeared within one day in culture. This system may provide a valuable model for the study of cellular and molecular mechanisms of the mammalian circadian pacemaker.

Neurotrophins and Basic Fibroblast Growth Factor Induce the Differentiation of Calbindin-Containing Neurons in the Cerebral Cortex

Lineage studies have recently shown that the expression of calcium-binding proteins in neurons of the cerebral cortex is not genetically programmed and is likely to be induced by external factors. Current hypotheses suggest that basic fibroblast growth factor (bFGF) and a number of neurotrophins play important roles in the proliferation and differentiation of cortical progenitor cells to a particular lineage. Using a dissociated cell culture system, we found that bFGF and the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor differentially affect the expression of the calcium-binding protein calbindin in selective neuronal subpopulations in the developing cerebral cortex. Specifically, BDNF and NT-3 greatly promoted the morphological differentiation of a relatively small, early-generated population of GABAergic neurons and induced the expression of calbindin in these cells. Furthermore, treatment with BDNF, NT-3, and bFGF produced an two- to threefold increase in the number of newly generated calbindin-positive neurons. The effect of bFGF was more striking in earlier (E14) than later (E16) ages, whereas the action of neurotrophins was independent of the age from which the cultures were prepared. Switching experiments combined with BrdU incorporation have suggested that NT-3 acts on postmitotic neurons rather than on proliferating progenitors to induce calbindin expression and that its action is mediated via trk receptors. Application of retroviral vectors in culture resulted in the presence of neuronal clones that were predominantly heterogeneous with regard to calbindin expression, suggesting, in agreement with our earlierin vivostudies, that the expression of this calcium-binding protein is not lineage dependent. Our results characterize the roles of BDNF, NT-3, and bFGF in the expression of calbindin in developing neocortical neurons.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

Electrophysiological properties of identified postnatal rat hippocampal pyramidal neurons in primary culture

Electrophysiological experiments were performed on primary cell cultures of retrogradely labelled postnatal rat hippocampal neurons. Rhodamine microspheres injected into the dorsal fornix clearly labelled the pyramidal cell layer of the hippocampus while excluding the other hippocampal layers and the dentate gyrus. In dissociated cell culture, the labelled cells were easily identified by fluorescence microscopy. Anti-neuron-specific enolase and anti-glial fibrillary acidic protein antibody staining confirmed that the labelled cells were neurons. The input resistance decreased from 2 GΩ to 450 MΩ, the input capacitance increased from 25 pF to 75 pF, the percentage of cells showing repetitive action potentials increased from 6% to 30%, and both peak GABA and glutamate responses increased over 100% during the 0 to 10 days time period investigated. This increase in chemosensitivity can be accounted for by an increase in cell size without an increase in the specific amino acid gated channel density. The subset of hippocampal neurons identified by the retrograde tracer technique are similar to non-labelled neurons with respect to the electrophysiological and pharmacological variables investigated. Nevertheless, it is likely that identified neurons may possess unique properties not evident in this study and refinement of the dissociated cell culture system using identified neuronal subpopulations may facilitate investigations looking at neuronal interactions such as synapse formation.

Light and electron microscopic analysis of insulin binding sites on neurons in dissociated brain cell cultures

The distribution of insulin binding sites on primary cultured neurons and glia from the fetal rat was examined by the immunoperoxidase method using a specific insulin receptor antiserum. Light and electron microscopic analysis revealed a homogenous distribution of insulin binding sites on selective neuron-like cells of the dissociated cell culture system. To determine the influence of medium insulin on the distribution of insulin binding sites, dissociated cell cultures were maintained in the presence or absence of porcine insulin for varying time periods. We observed a significant increase in the number of insulin stained neuron-like cells maintained in insulin free defined medium compared to neuron-like cells maintained in insulin supplemented defined medium. Further, we examined the distribution of insulin binding sites after incubation with the antibody, which has agonistic properties in peripheral tissues, for varying time periods prior to fixation. Under these conditions, the light microscopic analysis revealed a heterogeneous (patchy) distribution of immunoreactive insulin binding sites, suggesting that the ligand receptor complex migrates. These results demonstrate the presence and distribution of insulin binding sites on neurons maintained in vitro, and provide morphological evidence to support a functional role for insulin in CNS tissues.