Comparison of the neuritogenic activity of cyclic nucleotides and skeletal muscle-conditioned medium on ciliary ganglia in vitro

The parasympathetic ciliary ganglion (CG) of the embryonic chick in vitro, is unresponsive to Nerve Growth Factor and has been reported to form neurites only in response to neurotrophic factors derived from striated muscle or optic tissues. We investigated a possible role of cyclic adenosine-3',5'-monophosphate (cAMP) in the neurotrophic activity of skeletal muscle-conditioned medium (SCM) on CG explants. We showed that treatment with cAMP or dibutyryl cAMP stimulated neuritogenesis of CG explants in a dose-dependent fashion. SCM and cyclic nucleotide stimulation produced distinctly different types of neuritic growth. The growth cones of SCM-stimulated neurites were observed consistently to contact other cells or processes whereas nucleotide-stimulated neurites were not associated with other cells. These observations suggested that the two neuritogenic agents do not act through identical mechanisms, a conclusion supported by experiments demonstrating that stimulation of the CG with dibutyryl cAMP enhanced ornithine decarboxylase (ODC) activity relative to controls, whereas stimulation with SCM had no effect on ODC activity. We conclude that although cAMP does exhibit neuritogenic activity on the CG in vitro, it does not appear to be involved directly in the neurite formation elicited by SCM. It is feasible that the two types of neuritic growth which we described are representative of the two populations of CG neurons. Because only one of these neuronal populations innervates striated muscle in vivo, SCM and cAMP might act on different neurons.     

New approach to study of in vitro toxicity of multiple sclerosis and other sera

The in vitro effects of MS and control sera were quantified by the measurement of radiolabel released from myelinated cultures of rat cerebellum and compared with a visual assessment of myelin damage. Radiolabel release gave a sensitive index of serum effects in vitro which was free of the score assignment decisions that are associated with the visual assessment of myelin damage. Examination of the patterns of radiolabel release elicited by MS and control sera on cultures labelled with either L-[5-3H]tryptophan or galacto-D-[6-3H]cerebroside indicates that MS serum effects are not simply a stronger expression of the weak control serum effects.     

The development of immunoreactivity for neuron-specific enolase of preoptic and septal neurons in dissociated cultures

The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 daysin vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbancy measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 daysin vitro. This value remained constant up to 24 daysin vitro. The density of the immunoreaction product increased dramatically from 13 to 17 daysin vitro and was still higher at 24 daysin vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained.Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturationin vitro.     

Ultrastructural features of hippocampal CA1 synapses with respect to synaptic enhancement following repeated PKA activation

We reported previously that repeated activations, but not a single activation, of cyclic AMP-dependent protein kinase (PKA), led to a slowly developing (requiring approximately 1 week to develop) long-lasting (lasting > or = 3 weeks) enhancement of synaptic transmission efficiency in the organotypic slice culture of the rat hippocampus. It was accompanied by an increase in the number of synapses identified immunohistochemically. To answer the question of whether the "perforated synapse", which is known to occur transiently after the induction of long-term potentiation (LTP) in combination with the enlargement of postsynaptic density (PSD), is involved also in this slow/persistent synaptic enhancement, we examined the ultrastructural changes after the repeated activations of PKA. The answer was partially yes (occurrence of perforated synapses was increased) but partially no (the increase in the number of perforated synapses was not transient but persistent; mean apparent size of PSD did not increase). These results suggest that the mechanism of the slow/persistent synaptogenesis shares limited features with the mechanism of the quick/transient morphogenesis after LTP.     

Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia which leads to high economic losses in the swine industry worldwide. Vaccination against this pathogen is hampered by the occurrence of 15 serotypes, and commonly used whole cell bacterin vaccines are not sufficiently cross-serotype protective. In addition, for generating and maintaining specified pathogen-free herds it is desirable to use DIVA (differentiating infected from vaccinated animals) vaccines. Based on a detergent wash extraction of outer membrane associated proteins and secreted proteins we developed a DIVA vaccine using the immunogenic ApxII toxin which is present in 13 of the 15 A. pleuropneumoniae serotypes as the DIVA antigen. The apxIIA gene was deleted in one strain each of serotypes 1, 2, and 5 using a single-step transconjugation system, and equal parts of detergent washes from these strains served as the vaccine antigen. After intramuscular immunisation all pigs developed a strong humoral immune response to the vaccine antigen and showed no reactivity in an ApxIIA ELISA. Upon challenge all pigs were completely protected from clinical symptoms in trials with a homologous (serotype 2) as well as with a heterologous strain (serotype 9); in addition, colonisation of the challenge strain was clearly reduced but not abolished completely. As a result of the highly efficient protection, however, immunised pigs did not develop antibodies to the DIVA-antigen at levels detectable by ELISA but only by a more sensitive Western blotting approach, thereby demonstrating the challenge in developing appropriate marker vaccines for the livestock industry.     

Comparison of in vitro demyelination and cytotoxicity of humoral factors in multiple sclerosis and other neurological diseases

The distribution and nature of serum factors causing in vitro demyelination and glial lysis were investigated in multiple sclerosis (MS), other neurological diseases (OND), ill control and control groups. MS sera were unique in affecting only CNS myelin and glia whereas stroke and Guillain-Barré syndrome (GBS) sera brought changes to both CNS and PNS tissue. Through both visual scoring of myelin damage and the quantitative measurement of radiolabel release from cerebellar cultures, it was evident that the MS and OND groups have similar myelino- and cytotoxic effects. This may reflect MS and OND sera sharing similar humoral factors. 74% MS, 68% OND and 22% of control scores were above a score threshold designed to exclude culture handling trauma effects. When classified by their current disease state MS patients with severe and mild disease yielded higher in vitro scores than did those with moderate disease who comprised an older age group. No other clinical features of MS patients gave any association with in vitro serum effects. The rare demonstration of bound Fab IgG in cultures after MS serum tests indicates that immune mechanisms are unlikely to make a large contribution to serum-induced demyelination and cellular change in vitro.     

Reduction of neurotoxicity of mercuric chloride and glutamate by a membrane-permeating thiol reagent in vitro

Recent evidence suggests that inorganic mercuric chloride (MC) potentiates glutamate (GLU) excitotoxicity, probably by disturbing the astrocytic GLU transport. In primary astrocytic cultures GLU transport inhibition was prevented by a cell membrane-permeating thiol (SH) reagent, dithiothreitol (DTT). In this study, excitotoxic neuronal lesions produced in an organotypic culture of rat cerebellum by combined application of individually subtoxic doses of GLU (100μ ) and MC (1 μ ) were reduced when DTT (1 m ) was added, but not with a non-permeating SH reagent, reduced glutathione (GSH, 1 m ). The results support the view that MC neurotoxicity is mediated by its interaction with intramembraneous SH groups involved in astrocytic GLU transport.     

Cluster formation in monolayer cultures of normal and diseased human muscle

Earlier reports suggested that when muscle from patients with Duchenne muscular dystrophy was enzymatically dissociated and cultured, multilayered clumps of cells were found. These “clusters” were not present in control cultures, and were thought to be pathognomic for Duchenne dystrophy. In view of the importance of this finding to diagnostic and experimental procedures, we undertook to repeat the work. Cultures of dissociated muscle were established from nine samples of normal muscle and from 25 biopsies from patients with neuromuscular disorders including two with Duchenne muscular dystrophy. Multilayered clusters of cells were found in cultures set up from two of the normal muscle samples and in three of the diseased muscle samples which did not include those from Duchenne muscular dystrophy. Cluster formation does not, therefore, appear to be pathognomic for Duchenne dystrophy as the phenomenon, although it probably occurs in many Duchenne cultures, can also occur in cultures of dissociated muscle from other sources.