Michel Dolivo

Triiodothyronine Has Diverse and Multiple Stimulating Effects on Expression of the Major Myelin Protein Genes

Abstract: If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin‐associated glycoprotein (Mag), proteolipid protein (Plp), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half‐lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein‐specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15‐day‐old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A ‘floating fraction’ was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.

Neuronal organization of the stapedius reflex pathways in the rat: a retrograde HRP and viral transneuronal tracing study

The location of stapedius motoneurons in the rat was determined with horseradish peroxidase (HRP) retrograde tracing techniques. After injection of free HRP or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the stapedius muscle on one side, labeled neurons were seen ipsilaterally in a region ventromedial to the rostral half of the facial motor nucleus (VII), extending rostrally to the caudal part of the superior olivary complex (SOC). These labeled neurons, located outside the SOC and facial motor nuclei themselves, constitute the pool of stapedius motoneurons, in agreement with previous descriptions for other species. In order to identify the origin of some inputs to the stapedius motoneurons, injections of herpes virus suis were performed in the stapedius muscle. After replication in the motoneurons, the viruses are transported transneuronally to some premotor neurons, as previously reported in other systems. The presence of the virus was detected by immunofluorescence in neurons corresponding to the stapedius motoneurons labeled with HRP or WGA-HRP. In addition, infected neurons were seen bilaterally at the level of the SOC, in the mediotrapezoid region, where no labeled cells were observed following HRP or WGA-HRP injections in the stapedius muscle. These neurons were considered as infected transneuronally and therefore providing inputs to the pool of stapedius motoneurons. No virus could be detected in cochlear nucleus neurons. These data are consistent with previous observations in the rabbit based on lesion experiments, suggesting that neurons at the level of the SOC are involved in the reflex arc of middle ear muscles.

Ultrastructure and function in sympathetic ganglia isolated from rats infected with pseudorabies virus

(1) After inoculation of the pseudorabies virus in the anterior chamber of the eye of the rat, virions can be found only in the neurons of the superior cervical sympathetic ganglion and in the sensory ganglion of the fifth nerve on the inoculated side. Other nervous structures--central or peripheral--are not infected. (2) It is shown that the retrograde axonal flow carries the virus from the eye to the sympathetic neurons. (3) The ultrastructure of the infected neuron has been studied at various intervals after inoculation and at different stages of the viral replication. (4) Excised infected ganglia in vitro show a spontaneous electrophysiological activity that can be recorded on both the post- and preganglionic nerve. Such an activity has never been seen in normal excised ganglion of rat. (5) The shape and frequency of the electrophysiological discharges recorded on the postganglionic nerve have been analyzed at various intervals after inoculation. (6) Correlations established between the ultrastructure, the effect of various drugs and the electrophysiological activity permit the proposal of various hypothesis about the abnormal activity of the infected neurons.

Tensor tympani reflex pathways studied with retrograde horseradish peroxidase and transneuronal viral tracing techniques

The neural pathway involved in activation of the tensor tympani (TT) muscle was studied in the rat using retrograde HRP and transneuronal viral tracing techniques. The pool of TT motoneurons labeled with HRP was located ipsilaterally under the anterior third of the trigeminal motor nucleus and extended rostrally towards the lateral lemniscus. The origin of the inputs to these motoneurons was then determined using transneuronal viral transport: presumably transneuronally infected neurons appeared bilaterally in the vicinity of the superior olivary complex, mainly in between the two nuclei of the trapezoid body. The present data are consistent with previous conclusions based on lesion experiments that the TT reflex loop is made up of a chain of 4 neurons.

Paralytic tremor (pt) : a new allele of the proteolipid protein gene in rabbits

Abstract: Paralytic tremor (pt) is a sex‐linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents ∼30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP‐ and DM‐20‐specific messenger RNAs. Our results showed that both PLP and DM‐20 are affected and that the ratio DM‐20/PLP was higher in pt rabbits than in age‐matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T → A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Paralytic tremor (pt) rabbit: a sex-linked mutation affecting proteolipid protein-gene expression

Paralytic tremor (pt) is a neurological sex-linked recessive mutation in rabbits which is characterized by a coarse body tremor and limb paresis. Morphological studies showed that this mutation affects CNS myelination. Although the number of oligodendrocytes is not reduced, myelination is slower, irregular and defective. We have made a biochemical and molecular analysis of 4-wk-old mutant and normal rabbits. The amount of myelin in the mutant represents only approximately 25% of the normal level. Radioimmunoassay for myelin basic protein showed a reduction to approximately 40% in pt whole-brain homogenate but the difference was not significant in purified myelin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of brain homogenates followed by immunoblotting showed that all major myelin proteins are affected by the pt mutation, although to different degrees. While most of the myelin proteins are reduced to approximately 60-80% of the normal level, an important reduction to approximately 30%, was measured for the proteolipid protein (PLP). In purified myelin, the difference in PLP concentration was significant while the other specific proteins were less affected. A similar reduction in myelin-protein gene expression was detected at the mRNA level. Sex-linked transmission, low concentrations of PLP and its specific mRNA in the CNS indicate that the pt mutation primarily affects the expression of the Plp gene.

Neurohypophysial peptides depress cholinergic transmission in a mammalian sympathetic ganglion.

The actions of arginine-vasopressin (AVP) and oxytocin (OXT) were investigated in the rat superior cervical ganglion (SCG). At micromolar concentrations AVP decreased the amplitude of fast excitatory postsynaptic potentials (f-EPSPs) evoked by preganglionic stimulation and in many cells depolarized the postsynaptic membrane. Both effects were reversibly abolished by a potent vasopressor antagonist. The peptide decreased the frequency of spontaneous miniature EPSPs and the quantal content of the f-EPSPs without affecting the sensitivity of the ganglion cells to acetylcholine. OXT exerted the same effects as AVP but was less powerful. It was concluded that neurohypophysial peptides exert a dual pre- and post-synaptic action mediated by specific receptors.

Alteration of the electrophysiological activity in symphathetic ganglia infected with a neurotropic virus. I. Presynaptic origin of the spontaneous bioelectric activity

The bioelectric activity of the rat superior cervical ganglion (SCG) infected with pseudorabies virus (PRV) was examined in vitro 30-38 h after inoculation. Simultaneous intra- and extracellular recordings on the internal (ICN) and external carotid nerves (ECN) revealed a synchronized spontaneous activity. This synchronization can be ascribed either to the functional organization of the ganglion or to the mechanism of initiation itself. In the infected ganglia two categories of cells were observed: cells displaying abnormal spontaneous discharges, and silent cells whose electrophysiological behavior was similar to control cells. Spontaneously active cells showed intermittent spiking and bursting activity. The discharge pattern was associated with the firing rate of the emitting cell: sporadically active cells emitted single spikes whereas highly active cells fired bursts of action potentials (APs). Long lasting intracellular recordings demonstrated that the cells undergo gradual changes evolving from sporadic on to high activity. Spontaneous APs usually rode on prepotentials similar to the excitatory postsynaptic potentials (EPSPs). A comparative study of spontaneous prepotentials and orthodromically evoked EPSPs in the same cell demonstrated that the spontaneous prepotentials are real synaptic potentials. No pace-maker potentials were observed. The passive and active electrical membrane properties of spontaneously active neurons were not different from those of silent cells or control cells impaled in uninfected ganglia. D-Tubocurarine abolished the spontaneous activity in the whole ganglion. Ortho- and antidromic electrical stimulations of suprathreshold intensity elicited an evoked response in neurons displaying spontaneous activity, followed by a delayed burst whose shape was similar to the spontaneous burst of the cell. Stimuli of subthreshold intensities induced this delayed burst independently from the evoked response. We conclude that the spontaneous bioelectrical activity is of presynaptic, but not necessarily of preganglionic origin. The possible existence of a cholinergic intraganglionic pathway revealed by the viral infection is discussed.

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination, in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport.