Evidence for high molecular weight immunoreactive thyrotropin-releasing hormone (TRH) precursor forms in the developing mouse hypothalamus. Simultaneous immunolocalization with TRH in cultured neurons.

Two antisera (Anti-P7 and Anti-P10) were raised against (-Gln-His-Pro-Gly-) elongated peptides: P7 Gln-His-Pro-Gly-Lys-Arg-Phe) and P10 (Ser-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Phe). They recognized TRH extended peptides but not TRH. A RIA against P7 and a highly sensitive enzyme immunoassay against P10 were used to identify two major high mol wt forms of 25-35 K and 6-8 K in chromatography fractions of adult and fetal mouse as well as adult rat hypothalami. The existence of the largest form was confirmed by immunoblotting with Anti-P7. During mouse hypothalamus development in vivo and in vitro, the ratio of TRH content vs. P10-associated immunoreactivity increased several times. This suggests that these Pro-TRH peptides are precursors of TRH biosynthesis and indicate an acceleration of TRH processing during development. Double immunostaining with A-TRH and A-P7 of hypothalamic cells taken on the 16th fetal day and cultured for 6, 12, and 18 days in vitro (DIV) revealed three populations of neurons: 1) a very minor population (approximately 2%) of small round cells positive with A-TRH only; 2) a major population of neurons positive with both A-TRH and A-P7. 3) multipolar neurons positive with A-P7 only (up to approximately 45% after 18 DIV). The respective distribution of TRH and P7 along neurites also varied with time in culture. Whatever perikarya staining, TRH was restricted to short neurites and growth cones before synapse formation and, during synapse development, to varicosities and terminal boutons. However even at the latest stage examined some varicosities and terminal boutons were positive with A-P7 only. These results suggest a preferential processing of pro-TRH at a post-Golgi step during axonal transport to growth cones and synaptic boutons.

Alpha and gamma motoneurons in the peroneal nuclei of the cat spinal cord: an ultrastructural study.

The aim of the present study was to investigate whether ultrastructural features can be used as a guide to identify alpha- and gamma-motoneurons among the intermediate-size neurons of the peroneal motor nuclei. The peroneus brevis and peroneus tertius muscles of adult cats were injected with horseradish peroxidase, and motoneurons labeled by retrograde axonal transport were examined by electron microscopy. In both nuclei, the distributions of cell-body diameters, measured in the light microscope, were bimodal covering the range of 28-84 microns, with a trough around 50 microns. The sample of 25 motoneurons selected for the ultrastructural study included not only large (presumed alpha) and small (presumed gamma) neurons but also intermediate-size cell bodies with diameters in the 40-60 microns range. For each motoneuron, 2-5 profiles were reconstructed from ultrathin sections taken at 6-8 microns intervals. Synaptic boutons were counted and their lengths of apposition were measured. On the basis of three criteria, namely: (1) bouton types present on the membrane, (2) percentage of membrane length covered by synapses, and (3) the aspect of the nucleolus, all the examined motoneurons, including those with intermediate sizes, fell into one of two categories. Fourteen motoneurons, with cell-body diameters in a range of 55-84 microns, were contacted by all types of boutons (mainly S-type with spherical vesicles, F-type with flattened vesicles, and C-type with subsynaptic cistern); the synaptic covering of the somatic membrane was over 40% and the nucleus contained a vacuolated nucleolus. These were considered alpha-motoneurons. Eleven motoneurons, with only S and F boutons, a synaptic covering under 30%, a compact nucleolus and a cell-body diameter ranging between 28 and 50 microns, were considered gamma-motoneurons. No other combination of the three criteria was observed. These results show that unequivocal distinction of alpha- and gamma-motoneurons is possible in the peroneal nuclei, on the basis of morphological differences independent of cell-body size.

The dendrites of single brain-stem motoneurons intracellularly labelled with horseradish peroxidase in the cat. An ultrastructural analysis of the synaptic covering and the microenvironment.

Two laryngeal motoneurons intracellularly stained with horseradish peroxidase were studied ultrastructurally. The precise position of the ultrastructural observations made along the dendrites was obtained from the computer-reconstruction of the motoneurons in three dimensions. The shape and the size of the synaptic boutons, the percentage of membrane covered by bouton appositions and active zones, the number of boutons per 100 microns2 (packing density) were analysed on the soma and on the labelled dendrites at different distances from the soma up to 1000 microns. The results revealed no important regional differences in the mean length of synaptic apposition. The packing density was in the range of 9.3-14.9 boutons per 100 microns2 and was not correlated with the distance from the soma. The percentage apposition covering was higher on the soma and the proximal part of the dendrites than on the remaining part of the dendritic arborization. Close appositions between labelled dendrite and unlabelled somata and/or dendrites together with dendro-dendritic synapses suggested the possibility that the dendrites may be involved in local cell-to-cell communication. Microdendrites emerging from the soma or the proximal dendrites were contacted by synaptic boutons which may be more efficient as revealed by computation.

Ultrastructural and electrophysiological properties of accessory abducens nucleus motoneurones: an intracellular horseradish peroxidase study in the cat.

The physiological and morphological (light and electron microscopy) properties of six retractor bulbi motoneurones were analysed using the technique of intracellular recording and intracellular labelling with horseradish peroxidase. The retractor bulbi motoneurones were identified by antidromic invasion and orthodromic responses following stimulation of trigeminal afferents were studied. Two of these motoneurones were examined ultrastructurally. Terminal boutons forming synapses with labelled soma, labelled proximal and distal dendrites were characterized. Serial sections allowed the axon hillock to be analyzed and the initial segment of a presumed motoneurone to be observed in the section where the injected motoneurone was described. The ultrastructure of unidentified elements observed in the accessory abducens nucleus is stressed.

Gaba-like immunoreactive terminals on lumbar motoneurons of the adult cat. A quantitative ultrastructural study.

The aim of this ultrastructural study was to analyse quantitatively the distribution of gamma-aminobutyric acid (GABA)-like immunoreactivity in axon terminals apposed to somatic and proximal dendritic membranes of cat motoneurons in lumbar column 2. Preembedding immunocytochemistry was used to count the GABAergic terminals contacting profiles of eighteen alpha-and six gamma-motoneurons. Of the 1293 terminals counted on the somatic and proximal dendritic compartments of alpha-motoneurons, 197 were GABAergic. In contrast, a total number of only 62 terminals were counted on gamma-motoneurons, of which 8 were GABAergic. These populations of GABAergic terminals were less numerous than the population of glycinergic terminals observed in a previous study. The morphometric characteristics of GABAergic synapses were analyzed using postembedding immunocytochemistry. Most of the GABAergic terminals contained pleomorphic vesicles (F-type boutons, flattened or pleomorphic vesicles). All terminals presynaptic (P boutons) to large terminals containing sphericle vesicles (M-type boutons, characteristic of alpha-motoneurons), were GABA-immunopositive. These results suggest that there are different distributions of the GABAergic control of excitability on gamma- and alpha-motoneurons. GABA appears to be strongly involved in post-synaptic inhibition of alpha-motoneurons, whereas gamma-motoneurons receive very few GABAergic inhibitory inputs. Morphological correlates of GABAergic presynaptic inhibition were seen on alpha- but not on gamma-motoneurons.

Postnatal development of alpha- and gamma-peroneal motoneurons in kittens: an ultrastructural study.

Motoneurons innervating the peroneus brevis muscle of 1 week- and 3 week-old kittens were retrogradely labelled by HRP and examined by electron microscopy. At 1 week the distribution of mean cell body diameters was unimodal. Consequently alpha- and gamma-motoneurons could not be identified by their size. The aim of this study was to see whether the alpha- and gamma-motoneurons of kittens could be identified using the combination of ultrastructural criteria previously defined in the adult cat. Using these three criteria it was not possible to distinguish all the motoneurons as either alpha- or gamma in the kitten and a fourth criterion (frequency of F bouton profiles) was added to aid identification. However, with these four criteria, at 1 week six of 21 motoneurons and at 3 weeks two of 18 could still not be clearly identified as alpha or gamma (four were tentatively considered to be gamma, and four could not be identified). The maturation of alpha-motoneurons between 1 week and the adult was accompanied by an increase in somatic membrane area and a significant decrease in the somatic packing density of F boutons. On gamma-motoneurons there was a decrease in the somatic packing density of F boutons between 1 and 3 weeks. However, the numbers of F and S boutons remained stable for both motoneuron types. Age-related changes in apposition and active zone lengths of F and S boutons characterize the synaptic rearrangements which are occurring during the postnatal development of motoneurons.

Distribution of glycinergic terminals on lumbar motoneurons of the adult cat: an ultrastructural study.

The distribution of glycine-like immunoreactivity on cat lumbar motoneurons was examined in electron microscopy, using pre-embedding immunocytochemistry. In the dorsolateral portion of the ventral horn, numerous labeled axon terminals were presynaptic to somatic and dendritic profiles of alpha-motoneurons. Most of the glycinergic boutons contained pleomorphic vesicles and showed symmetrical contacts. On the somatic and proximal dendritic compartments, glycinergic terminals accounted for, respectively, 24.6 and 26.6% of the total number of terminals. There were very few glycinergic terminals on gamma-motoneurons. Immunoreactive axons, dendrites and cell bodies were also observed near the motoneurons. These results support the view that glycine plays a major role in the inhibition of alpha-motoneurons and suggest that inhibitory mechanisms occur on the soma as well as on dendrites.

Short-latency synaptic patterns among cat peroneal motoneurons.

Motoneurons innervating peroneal muscles in the cat leg (PB, PT and PL, respectively, for peroneus brevis, tertius and longus) were examined for their connections with afferents from these and other leg muscles and with cutaneous afferents. The aim was to investigate (1) whether inputs from nearby muscles and cutaneous areas are likely to assist or oppose the excitation elicited in peroneal motoneurons by PB contractions, and (2) whether reflex connectivity might allow distinction of alpha (i.e. motoneurons innervating skeletal muscle fibres) and beta (i.e. motoneurons innervating both skeletal and intrafusal muscle fibres) subgroups among PB and PT motoneurons. In the three peroneal pools, every motoneuron had excitatory monosynaptic connections with Ia afferents from each of the three peroneal muscles, and nearly every motoneuron received di- or trisynaptic excitation from low-threshold cutaneous afferents in sural or superficial peroneal nerves. Inputs from these sources might facilitate the contraction-induced positive feedback. In contrast, the patterns of short-latency synaptic connections with group I afferents from pretibial flexor and post-tibial extensor muscles were heterogeneous among peroneal motoneurons but did not point to any specific beta pattern.

Inhibition of poliovirus RNA synthesis as a molecular mechanism contributing to viral persistence in the mouse central nervous system.

Many survivors of poliomyelitis, several decades after the acute phase of the disease, develop a set of new muscle symptoms called post-polio syndrome. The persistence of poliovirus (PV) in the central nervous system (CNS) may be involved in the aetiology of this syndrome. By using a mouse model, we have shown that PV persists in the CNS of paralysed mice for over a year after the acute disease. Detection of PV plus- and minus-strand RNAs in the spinal cord of paralysed mice suggested continuous PV RNA replication in the CNS. However, infectious PV particles could not be recovered from homogenates of CNS from paralysed mice beyond 20 days post-paralysis, indicating that PV replication was restricted. In an attempt to identify the molecular mechanism by which PV replication was limited, PV plus- and minus-strand RNA levels were estimated in the CNS of persistently infected mice by a semi-quantitative RT-nested PCR method. Results revealed that RNA replication was inhibited at the level of plus-strand RNA synthesis during persistent infection. Similar results were obtained in neuroblastoma IMR-32 cell cultures persistently infected with PV Restriction of PV RNA synthesis could be involved in persistence by limiting PV replication.

[Recent findings on the external oculomotor nucleus: the center for horizontal ocular movement]. / Données récentes concernant le noyau oculomoteur externe: centre du mouvement oculaire horizontal.

Various neurobiological methods allowing the analysis of electrophysiological and morphological properties of a cell population within the central nervous system will be illustrated in the study of the abducens nucleus. Intracellular recordings provided the functional identification of the different types of cells within the abducens nucleus. Antidromic excitation, refractoriness, conduction velocity and frequency of discharges indicated the properties of the abducens motoneurones. Various types of afferent stimulations were applied to study the synaptic responses of the neurones. A dye was then electrophoretically injected and the labelled neurone observed with a light microscope. Full reconstruction of dendritic arborization and of axon trajectory was performed. The morphological study has shown the heterogeneity of the population of the motor nucleus, and the ultrastructural features indicated a complex synaptic organisation. The origin of some afferent terminals was determined.

Ultrastructural study of vestibular and reticular projections to the abducens nucleus.

The origin of the synaptic boutons in the abducens nucleus was studied following lesions of the contralateral medial vestibular nucleus, the ipsilateral paramedian pontine reticular formation and the contralateral dorsomedial part of the reticular formation caudal to the abducens nucleus. Lesions in the rostral part of the contralateral medial vestibular nucleus resulted in degeneration of boutons located mainly on dendritic processes. On the other hand, lesions in both ipsilateral and contralateral reticular formations provoked degenerating terminals on the somata of the abducens neurones and on proximal dendrites in the abducens nucleus beneath of genu of the facial nerve.

Ultrastructural observations of the abducens nucleus of the cat after injection of horseradish peroxydase into the lateral rectus muscle.

The ultrastructure of cat abducens motoneurones is described, after labelling by retrograde axonal transport of horseradish peroxydase. The motoneurones were densely covered with synaptic boutons of both the "terminal" and "en passant" types and axo-axonal synapses were observed on the initial segment of motoneurone axons. Within the population of axons forming the VIth nerve, multiple branching was encountered and axons closely apposed at the nodes of Ranvier were seen.

The fine structure of neurones and cellular relationships in the abducens nucleus in the cat.

Light and electron microscope studies of the abducens nucleus in the cat have disclosed two populations of neurones: large neurones 25 to 75 micrometer in diameter and small neurones 10 to 25 micrometer in diameter. Characteristic of the large neurones are a highly developed granular endoplasmic reticulum and a large number of axo-somatic synapses. The small neurones have a poorly developed granular endoplasmic reticulum and comparative by few axo-somatic synapses. Injection of horseradish peroxidase (HRP) into the ipsilateral lateral rectus muscle has been used to identify abducens motoneurones which represent 65% of the total number of cells in the abducens nucleus and form part of the large cell population. The remaining unlabelled large neurones (30% of the large cell population) probably represent motoneurones of the retractor bulbi muscles, abducens motoneurones which have failed to transport the HRP, and possibly internuclear neurones. The small neurones (5% of the total cell population) were never labelled by HRP. Axo-somatic and axo-dendritic synapses are numerous. Many of the synaptic terminals form contacts with two or more different postsynaptic elements. Axo-axonic synapses and synapses on the initial segments of large neurones are also present. Nodal synapses and branching of myelinated axons are common findings. Finally, extended areas of close apposition between dendritic membranes are frequently observed.

Age-related appearance of tubular aggregates in the skeletal muscle of almost all male inbred mice.

Tubular aggregates (TAs) which have been recently observed in a few mouse myopathies are identical to those described in human diseases. In this study we show that TAs are also found in the skeletal muscle of almost all normal inbred mice strains. In these inbred strains of mice the presence of TAs is shown to be related to both age and sex. Nine different muscles were stained with the modified Gomori trichrome method to reveal the general morphology of the muscles. Anti-SERCA1 ATPase was used to confirm that the TAs were in fact accumulations of sarcoplasmic reticulum and anti-MyHC IIB to demonstrate that these accumulations were found exclusively in the type IIB muscle fibers. An ultrastructural study confirmed the observations revealed by light microscopy that the TAs were derived from the sarcoplasmic reticulum. TAs were never observed in female inbred mice and were only found in type IIB glycolytic muscle fibers of male inbred mice. Therefore when analyzing the effect of genetic knock out and knock in experiments on the muscle phenotype of transgenic mice one should be aware that the presence of these aggregates is a non-specific phenomenon induced by inbreeding.

Sensitive trace gas detection with near-infrared laser diodes and an integrating sphere.

We describe a simple spectrometer for sensitive trace gas detection in the atmosphere. A communication laser diode is used as a light source, and a commercial integrating sphere is used as a multipass absorption cell. We developed a theoretical formulation of the relative absorption of the optical power by trace gases in the sphere and applied it to two kinds of experimental result: one that is concerned with a structureless broad absorption band of butane with the use of a 1.2-µm multimode laser diode, and one that is related to the study of an isolated and sharp rovibrational line of water vapor in air at atmospheric pressure with the use of an 830-nm single-mode laser diode. With equivalent path lengths of several meters obtained with a 10-cm-i.d. integrating sphere we can demonstrate the usefulness of such a device as a broadband multipass cell for the measurement of small absorptions.

Anti-5-hydroxytryptamine antibodies: studies on their cross-reactivity in vitro and their immunohistochemical specificity.

Antibodies to 5-hydroxytryptamine (5-HT) were obtained from 4 rabbits after injections of 5-HT coupled to bovine serum albumin by means of paraformaldehyde (PF). Two methods were used to monitor the development of antibodies (AB): the one based on the "in vitro" competitive binding properties of the antibodies with 3(H)5-HT, the other, on their "in situ" binding properties to endogenous 5-HT, using the peroxidase-anti-peroxidase immunohistochemical technique, applied to paraffin embedded sections of cat brainstem. No pharmacological processing, detergents or proteolytic enzymes were used. The specificity of the antiserum was tested by competitive procedures with 20 analogs using the "in vitro" and "in situ" techniques. "In vitro" studies were performed with 5-HT free analogs and with analogs previously coupled with PF to lysine. Radioimmunological tests showed that the antibodies recognize mainly the ethylamine (CH2-CH2-NH2)-chain of the free analogs and that the best specificity was obtained with the 5-HT conjugate (5-HT-lysine-PF). The results suggest that the hapten is coupled through the phenolic positions C4 or C5. The "in situ" immunohistochemical extinction assays also revealed a distinct specificity for 5-HT. Possible optical and ultrastructural applications are illustrated in the raphé nuclei of the cat. These results confirm the reliability of radioimmunological tests for studying the specificity of AB directed against haptens, provided that haptens and analogs tested were first chemically transformed to resemble the immunogen (herewith lysine-PF coupling) with regard to its antigenic structure.

The climbing fibers of the cerebellar cortex, their origin and pathways in cat.

The sources and pathways of the climbing fibers to the cerebellar posterior vermis were studied with comibined electrophysiological and anatomical methods in cats. Recording from identified cerebellar Purkinje cells, monosynaptic climbing fiber (CF) responses have been obtained both for stimulation of the inferior olive (IO) and various parts of the brain stem (BS). CF responses were found to of three types, IO only, BS only or both IO and BS. However the responses to BS stimulation were very few in number in comparison with IO or IO and BS types of responses. The latencies of the responses were shorter for the BS cases consistent with their distance from the cerebellum. A comparison of latencies and the relative responsiveness of the different area of the brain stem which were studied, indicate that part of the CF ascend through the pontine region and enter the cerebellum by way of the medium and superior penduncles. This finding is confirmed by the results of anatomical studies in which degenerating fibers were found in the molecular layer (using the Nauta technique) after lesion of the brachium pontis but not after lesions of the medial portion of the pons. Similarly, injection of radioactive leucine into the pontine nuclei failed to show any labeled fibers in the molecular layer. Horseradish peroxidase (HRP) was injected into localized regions of the posterior vermis after total bilateral destruction of the inferior peduncles. Large numbers of positive, marked cells were still found in the inferior olive. It is concluded that nearly all, if not all, the climbing fibers originate in the inferior olive and that they ascend to the cerebellum by way of all the peduncles.

Persistent poliovirus infection in mouse motoneurons.

Poliovirus (PV) is the causal agent of paralytic poliomyelitis. Many survivors of the acute disease, after decades of clinical stability, develop new muscular symptoms called postpolio syndrome. It has been hypothesized that the persistence of PV in the spinal cord is involved in the etiology of this syndrome. To investigate the ability of PV to persist in the spinal cord after the onset of paralysis, we exploited a mouse model in which most animals inoculated with a mouse-adapted mutant survived after the onset of paralysis. Light microscopy and ultrastructural immunohistochemical studies and reverse transcription followed by nested PCR performed on spinal cord from paralyzed mice demonstrated that PV persisted in the mouse spinal cord for at least 12 months after the onset of paralysis. This mouse model provides a new tool for studying poliomyelitis evolution after the onset of paralysis.

Poliovirus induces apoptosis in the mouse central nervous system.

Poliovirus (PV) is the etiological agent of human paralytic poliomyelitis. Paralysis results from the destruction of motoneurons, a consequence of PV replication. However, the PV-induced process leading to the death of motoneurons is not well known. We investigated whether PV-induced central nervous system (CNS) injury is associated with apoptosis by using mice as animal models. Transgenic mice expressing the human PV receptor were infected intracerebrally with either the neurovirulent PV-1 Mahoney strain or a paralytogenic dose of the attenuated PV-1 Sabin strain. Nontransgenic mice were infected with a mouse-adapted PV-1 Mahoney mutant. DNA fragmentation was demonstrated in CNS tissue from paralyzed mice by visualization of DNA oligonucleosomal laddering and by enzyme-linked immunosorbent assay. Viral antigens and DNA fragmentation detected by the in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling technique were colocalized in neurons of spinal cords from paralyzed mice. In addition, morphological changes characteristic of cells undergoing apoptosis were observed in motoneurons by electron microscopy. Thus, we show that PV multiplication and CNS injury during paralytic poliomyelitis are associated with apoptosis.

A Combined Analysis of Laser Optogalvanic and Fourier Transform Emission Spectra of NH2 near its Barrier to Linearity.

The Ã2A1-&Xtilde;2B1 electronic transition of NH2 is reinvestigated in the region of its barrier to linearity. A detailed rotational analysis is performed on its laser optogalvanic spectrum recorded in the region 11 300-14 300 cm-1 and on its near infrared Fourier transform emission spectrum. Many new excited levels are identified resulting from more than 2900 assigned lines. A number of them are high levels of &Xtilde;2B1. A set of molecular parameters has been obtained. Numerous perturbations are detected and discussed. This analysis confirms the predictions obtained from previous calculations and allows the estimation of their accuracy. Copyright 1999 Academic Press.