Biomedical subjects

A E Schaffner

Publications and source records attributed to A E Schaffner.

18 recordsLinked to original sources

PubMed1995

Acetylcholine esterase and peripherin mRNA level decrease in wobbler mouse.

Homozygote wobbler mice develop motoneurone degeneration. Throughout development the expression of choline acetyltransferase, of trkC receptor and F3 adhesion molecule genes is similar in wobbler and wild-type spinal cord. Acetylcholinesterase mRNA level instead is decreased to about 50% with respect to wild-type values in one forth of P5 and P10 wobbler progeny, putative wr/wr individuals; at P21 its expression is equally highly reduced in known homozygotes and it is reduced to 35% of normal values in about one half of the progeny, putative heterozygotes. Thus, similarly to medium neurofilament gene over-expression, reduced acetylcholinesterase gene expression is an early molecular marker for the wobbler mutation before onset of the illness.

Acetylcholinesterase↗

PubMed1995

Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons.

We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36-48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

Animals↗

PubMed1994

Correlation of gp140trk expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord.

During rat embryogenesis, fibers containing nerve growth factor (NGF) are present near the target destinations of migratory spinal neuroblasts, suggesting that diffusible gradients of NGF provide signals to newly generated neurons in the developing cord. In vitro, pM concentrations of NGF induce neuroblast chemotaxis (directed migration along a chemical gradient), indicating evoked motility is mediated by high-affinity receptors. Binding of 125I-labelled NGF to fetal cord cells provides additional evidence that rat spinal neuroblasts express the high-affinity receptors; however, their presence has not been directly demonstrated. In the present study, we used immunocytochemistry to show that the high-affinity NGF receptor protein, gp140trk (trk) is detectable in embryonic spinal tissue sections and in cord dissociates. Correlation of trk expression with NGF-induced chemotaxis revealed that both the receptor protein expression and functional responses to NGF develop along a ventro-dorsal gradient that parallels the in vivo pattern of neurogenesis and migration. Analysis of the temporal changes in trk immunoreactivity demonstrated that expression of gp140trk is bimodal, possibly reflecting multiple effects of NGF during development. Chemotaxis to NGF was blocked by nM concentrations of the kinase inhibitor, K252a, suggesting that NGF stimulates motility via high-affinity receptors coupled to kinase activity. Elevated 3',5'-cyclic adenosine monophosphate (cAMP) also attenuated NGF-induced chemotaxis, presenting preliminary evidence that protein kinase A (PKA) may regulate motility responses to NGF.

Animals↗

PubMed1994

Fast presynaptic GABAA receptor-mediated Cl- conductance in cultured rat hippocampal neurones.

1. Hippocampal neurones cultured from the 18-day-old embryonic rat for 3 days to 3 weeks were recorded with Cl(-)-filled patch pipettes. Spontaneous synaptic currents, which reversed at the equilibrium potential for Cl- ions (ECl) and were blocked by the GABAA (gamma-aminobutyric acid) receptor antagonists bicuculline or picrotoxin, were recorded in every culture. At 25 degrees C and -80 mV they decayed with a time constant > or = 20 ms that invariably increased at positive potentials. After 2 weeks, 50-75% of all neurones were GABA immunoreactive. 2. In pairs-recordings, coincident synaptic currents in both cells were either spontaneous or evoked by stimulation of one cell. In the presence of tetrodotoxin and using pipettes containing lidocaine (lignocaine) N-ethyl bromide, coincident spontaneous Cl- transients still occurred in both neurones far more frequently than expected by chance. 3. Holding the potential of one neurone at a positive value reversed the synaptic transients in that cell and, in half of the cells, increased the frequency of coincident events in both cells. 4. In neurones where depolarization increased the frequency of coinciding events and all regenerative current apparent at the soma was abolished, short depolarizing pulses occasionally evoked all-or-none, pre- and postsynaptic currents with matching transmission failures and identical delays in transmission. 5. The results suggest that the same pulse of GABA simultaneously activates GABAA receptor-coupled Cl- channels on both sides of the same synaptic cleft, producing immediate auto-transmission in the absence of collaterals or interneurones.

Animals↗

PubMed1993

Frequency modulation of transmitter release.

In 1952 Fatt and Katz recorded at a frog neuromuscular junction while stimulating the nerve and found "... that successive endplate potential responses varied in a step-like manner, corresponding to units of miniature endplate potentials" (J Physiol 117, 109-128). This led them to propose that fast neuromuscular transmission is 'quantal'. Quantal release is now commonly ascribed to a vesicular form of neurosecretion since vesicles have routinely been visualized in presynaptic terminals. The vesicular hypothesis (Del Castillo and Katz, 1955) assumes that quanta, or 'transmitter packets of standard size', are assembled and stored in the numerous vesicles routinely identified in micrographs of virtually all central and peripheral presynaptic nerve terminals. Simply stated, this model predicts that each one of the miniature synaptic signals (MSSs) follows from the exocytosis of one vesicle's contents. However, the time required for membrane fusion preceding exocytosis (Almers and Tse, 1990) and the variability in MSS amplitude and time course (Vautrin et al, 1992a,b) cannot readily be reconciled by a simple, exocytotic model of quantal release from preloaded vesicles. These difficulties with the original model have led us to re-evaluate MSSs generated at the classical peripheral synapse, the cholinergic neuromuscular junction of the mouse diaphragm, as well as at central synapses between embryonic hippocampal neurons mediated by gamma-aminobutyric acid (GABA). At these synapses, the release of GABA is also assumed to have classical quantal properties like peripheral acetylcholine release (Edwards et al, 1990). Our results show that at both synapses, progressive alterations in elementary signal properties can be induced in a remarkably rapid manner. The original report of preferred amplitudes and intervals in the spontaneous miniature signals (Fatt and Katz, 1952) has repeatedly been confirmed and is here incorporated into a dynamic model of fast synaptic transmission. Although MSSs exhibit variable rise-times and peak amplitudes, they can both be described in terms of synchronization of transmitter release. We have reviewed many experimental findings, which together strongly suggest that the original interpretation of Fatt and Katz (1952) regarding MSSs as reflecting the non-propagated 'neurogenic' activity of 'terminal spots' may be a useful concept to pursue since it may help to explain part of the underlying molecular basis of quantal release.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Electrophysiological properties of early postnatal rat septal neurons in short-term culture.

Electrophysiological properties of septal neurons dissociated from PN1-PN7 rats were examined between 1 and 5 days in vitro (DIV) with whole-cell patch-clamp recording. The neurons had RMPs in the range -40 to -80 mV, resistances 0.5-1.5 G omega, and 50-90 mV action potentials. By 2-3 DIV, most neurons were spontaneously active, with some cells exhibiting rhythmic firing patterns. Depolarizations from -80 mV holding potential elicited TTX-sensitive inward Na+ currents, and transient and sustained outward K+ currents. Pharmacological dissection of the outward currents in PN6/7 neurons suggest the presence of multiple types of K+ currents (IA, IC, IK). L-type Ca2+ currents were observed in all PN6/7 neurons examined but were not always detectable in PN1/2 neurons. All PN1/2 and PN6/7 neurons were sensitive to glutamate and GABA but did not respond to ACh or NE applications. Responses to GABA were excitatory i.e., characteristic of immature neurons. Comparison of the results obtained in this study with the properties of adult neurons characterized in vivo or in brain slice preparations in vitro, suggest that septal neurons from PN1-PN7 rats are still in the process of differentiation of their mature electrical and chemosensitive membrane properties.

Adenosine Triphosphate↗

PubMed1992

Two classes of spontaneous GABA-mediated miniature synaptic currents in cultured rat hippocampal neurons.

Amplitude and time course of spontaneous gamma-aminobutyric acid (GABA)-mediated miniature postsynaptic currents (MPSCs), recorded in cultured embryonic hippocampal neurons in presence of either tetrodotoxin (TTX) or increased external [Mg2+/Ca2+] ratio, revealed that they form two classes. The distribution of the most commonly recorded MPSCs was skewed both in terms of peak amplitude and rise-time (skew-MPSCs, mode: 70-120 pS). Another, less frequent class (mode: 1-3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonically attenuated. Bell-MPSCs did not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.

Animals↗

PubMed1990

Electrical and chemical excitability appear one week before birth in the embryonic rat spinal cord.

Embryonic rat spinal cord cells were acutely dissociated with the enzyme papain, stained with a voltage-sensitive oxonol dye and incubated with various pharmacological agents. Changes in the fluorescence intensity and, by inference, membrane potential of the cells were analyzed in a flow cytometer. Veratridine caused depolarization of the cells in a TTX-sensitive manner from as early as embryonic day 13. Depolarizing responses to muscimol and kainate appeared slightly later, at embryonic days 14 and 15, and were blocked by the antagonists bicuculline and CNQX, respectively. Responses to veratridine and kainate did not occur in sodium-free medium. The emergence of these excitable membrane properties coincides with postmitotic differentiation and synaptic development in the embryonic spinal cord.

Animals↗

PubMed1988

Flow cytometric analysis of membrane potential in embryonic rat spinal cord cells.

Flow cytometric analysis of membrane potential in suspensions of embryonic rat spinal cord cells was carried out in a fluorescence-activated cell sorter (FACS) using anionic voltage-sensitive, fluorescent dyes (oxonols). The FACS or flow cytometer is an analytical instrument that measures optical properties of large cell populations at a single cell level of resolution. The incorporation of oxonol allows relative measurements of membrane potential, since the partition of oxonol within the plasmalemma is directly related to the degree of cell depolarization. Incubation of cells in elevated K+ concentrations or with the Na+ channel agonist batrachotoxin (BTX) changed the fluorescence intensity distribution pattern of the live-cell population; these changes were consistent with the depolarizing effects of these manipulations. Fluorescence shifts were either undetectable or minimal in the dead-cell population. The BTX-induced shift was blocked by tetrodotoxin (TTX) and was reversed in Na+-free medium, indicating embryonic expression of functional Na+ channels. Fluorescence microscopy of sorted cells showed that live cells typically exhibited circumferential ring-like patterns, whose intensities were enhanced under depolarizing conditions. The results show that flow cytometry combined with oxonol dyes can be used to measure the relative membrane potential of large numbers of individual central nervous system cells. The analysis of the changes in the distributions of these membrane potentials can be used to reveal the development of functional ion conductance mechanisms.

Animals↗

PubMed1987

Developmental regulation of somatostatin gene expression in the brain is region specific.

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.

Animals↗

PubMed1987

Fluorescence-activated cell sorting of embryonic mouse and rat motoneurons and their long-term survival in vitro.

Motoneurons from embryonic mice and rats were labeled with retrogradely transported succinyl wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC). After dissociation of the spinal cord, fluorescent motoneurons were isolated by flow cytometry. Sorted motoneurons were maintained for as long as 6 weeks in vitro on monolayers of astrocytes in muscle-conditioned medium. Immunocytochemical staining of the cultures for various neuronal antigens suggested that sorted motoneurons are receptive to GABAergic and glycinergic, as well as cholinergic, innervation. Many of the sorted cells were also labeled intracellularly with antibodies to choline acetyltransferase (ChAT) and GABA.

4-Aminopyridine↗

PubMed1986

The developmental appearance of alpha-bungarotoxin binding sites on rodent spinal cord neurons in cell culture.

[125I]alpha-Bungarotoxin specifically binds to a subpopulation of rodent spinal cord neurons in vitro. Binding first becomes apparent between 1 and 2 weeks in culture and then increases dramatically after 3 weeks. Similarly, cell suspensions from freshly dissociated embryonic spinal cords do not bind toxin whereas cell suspensions from 1 week old neonates demonstrate specific binding of [125I]alpha-bungarotoxin. In vitro, binding is inhibited more effectively in the presence of nicotinic rather than muscarinic agents. Autoradiography of [125I]alpha-bungarotoxin binding to 4-week-old cultures revealed a uniform labeling pattern over cell somas and processes. Although the relation of toxin binding to functional acetylcholine receptors is not known, the appearance of toxin binding sites may have some developmental significance for the maturation of cholinergic transmission or the maintenance of synaptic connections.

Animals↗

PubMed1986

Monoclonal antibodies to the dentate gyrus: immunocytochemical characterization and flow cytometric analysis of hippocampal neurons bearing a unique cell-surface antigen.

Monoclonal antibodies were generated using 5 d neonatal rat dentate gyrus as immunogen. One antibody of this panel, G6E3, recognized a cell-surface protein with an Mr = 43,000 that was found only in the nervous system. The antigen was expressed as early as embryonic day 13 in the rat in both the brain and spinal cord. In the adult rat the antigen was demonstrated immunohistochemically to be restricted to dentate gyrus granule, hippocampal pyramidal, and cerebellar Purkinje neurons. These results suggested that the antigen recognized by G6E3 may be developmentally regulated. Moreover, G6E3 did not appear to bind to mitotic cells, implying that the antigen was expressed after the terminal mitosis. The antibody also bound to hippocampal and cerebellar cells from mouse brain, including the reeler mutant, and rat hippocampal neurons in vitro. Double-labeling experiments performed on embryonic rat hippocampal cultures with G6E3 and antibodies to neuron-specific enolase (NSE) or anti-glutamic acid decarboxylase (GAD) revealed that only NSE-positive cells were immunoreactive for G6E3 and, while G6E3-positive cells were decorated with GAD-positive boutons, their cell bodies did not contain GAD. With the use of a fluorescence-activated cell sorter it was possible to analyze the immune reaction on embryonic and postnatal hippocampal cells and to sort G6E3-positive neurons for maintenance in vitro.

Animals↗

PubMed1984

Three cholinergic neuroblastoma hybrid cell lines that form few synapses on myotubes are deficient in acetylcholine receptor aggregation molecules and large dense core vesicles.

Three neuroblastoma X glioma hybrid cell lines that synthesize and release acetylcholine but that form few or no synapses with cultured skeletal muscle cells lack two characteristics of neuroblastoma or hybrid cell lines that do form many synapses with myotubes: large dense core vesicles and the ability to increase the number of nicotinic acetylcholine receptor clusters on co-cultured myotube membranes. Functional synapse formation on myotubes was increased by co-culturing myotubes and cells from one of the defective lines with neuroblastoma cells that induce myotube acetylcholine receptor aggregation but which synthesize little or no acetylcholine.

Animals↗

PubMed1984

Synapse repression in cell culture.

The phenomenon of synapse repression was investigated in cell culture. It was found that the number of synapses between ciliary ganglion neurons and myotubes was reduced by the concomitant presence of ventral spinal cord neurons. Neurons from dorsal root ganglia, dorsal spinal cord or cortex did not cause a reduction. Conditioned medium from ventral spinal cord-myotube co-cultures was without effect suggesting the absence of soluble 'repression factors'. The addition of D-tubocurarine partially reversed the repression, indicating that the phenomenon has both activity-dependent and activity-independent aspects.

Animals↗

PubMed1984

Carotid body cell culture and selective growth of glomus cells.

Cells of the fetal carotid body have been obtained by enzymatic digestion and maintained in culture both as single cells and as clusters for up to 2 mo. The glomus cells in culture synthesize catecholamines and adenine nucleotides, as determined by histochemical methods, and contain characteristic dense-core vesicles. Their growth requirements are different from other cells of neural crest origin in that they do not depend on nerve growth factor (as do sympathetic neurons) or corticosteroids (as do SIF cells) for survival. Neither hypoxia nor hypercarbia affects survival or relative preponderance of glomus cells in culture. Tyrosine-free medium, which selects for cells containing tyrosine hydroxylase, eliminates most of the nonadrenergic cells, thereby providing a culture dramatically enhanced in glomus cells.

Animals↗

PubMed1982

Conditioned medium from cultures of embryonic neurons contains a high molecular weight factor which induces acetylcholine receptor aggregation on cultured myotubes.

The developmental mechanisms involved in the formation of stable arrays of postsynaptic neurotransmitter receptors near sites of neurotransmitter release are essentially unknown. However, several recent studies have shown that cells or tissues of neural origin produce macromolecular factors which induce an increase in the number of acetylcholine (ACh) receptors or the number of receptor aggregates on cultured embryonic myotubes. We have tested primary cultures of embryonic neurons and other cell types for the release of an ACh receptor aggregation factor. Conditioned medium from the cultures was applied to cultures of embryonic rat myotubes for 1 day; ACh receptors on the myotubes were stained with tetramethylrhodamine-labeled alpha-bungarotoxin and ACh receptor aggregation activity, defined as the change in the number of receptor aggregates per myotube, was assayed. Aggregation activity with a molecular weight greater than or equal to 50,000 was released by cultures of neurons from sympathetic ganglia, dorsal root ganglia, spinal cord, and cerebellum. Little or no activity was released by glial or other non-neuronal cultures. Release of aggregation activity by different neuronal cell types varied by up to an order of magnitude; however, this variation was not well correlated with the differences in ACh synthesis. The factor(s) in neuronal cell conditioned medium induced a rearrangement of pre-existing receptors at the cell surface, and its action was not dependent on new protein synthesis. The results of this study are consistent with the idea that one or more receptor aggregation factors secreted by neurons are involved in the organization of neurotransmitter receptors during synapse formation in vivo.

Animals↗

PubMed1981

Separation of cell types from embryonic chicken and rat spinal cord: characterization of motoneuron-enriched fractions.

Single cell suspensions prepared from embryonic chick or rat spinal cords were separated into morphologically and functionally distinct subpopulation based on their buoyant densities The lightest fraction (F-1) was highly enriched for cells containing the enzyme choline acetyltransferase (CAT), a marker for developing motoneurons. The morphology biochemistry, and in vitro development of this and other spinal cord cell fractions isolated by the outlined procedure were investigated. Spinal cords, dissected from 6-day chick or 12-day rat embryos, were dissociated with trypsin and applied to iso-osmotic metrizamide density gradients. After brief centrifugation, biochemical analysis revealed that cholinergic cells migrated to lower densities than other spinal cord cells. The use of discontinuous density gradients allowed rapid and simple isolation of three fractions of viable cells (designated F-1 to F-3, lowest to highest density). Characterization of chicken and rat embryo cell fractions gave similar results. The cells in Fraction 1 were large with prominent nuclei and nucleoli, while those in F-2 and F-3 were distinctly smaller. Fraction 1 was highly enriched for cholinergic cells. The CAT specific activity (CAT/cell) was increased 400% in Fraction 1 compared to unfractionated cells, while CAT specific activity in F-2 and F-3 was reduced to 25% and less than 4% that of unfractionated cells, respectively. The recovery of cholinergic cells using this procedure was much better than with other published procedures; greater than half the spinal cord CAT activity was routinely recovered in the enriched fraction. The cholinergic-enriched cells (F-1) were unique in their in vitro growth characteristics. All fractions had neuronal cells, while non-neuronal cells were distributed primarily in F-3, fewer in F-2, and were essentially absent from F-1. Neurons in F-2 and F-3 remained viable under a variety of conditions, most of which were not supportive of F-1 cell survival. The cholinergic-enriched F-1 cells survived and developed only in the presence of muscle cells or in muscle-conditioned medium on highly adhesive substrata. Large, multipolar neurons predominated under these conditions. The method described provides a means of characterizing the factors involved in the development of distinct populations of cells from the embryonic spinal cord.

Animals↗