Are locus coeruleus neurons involved in blinking?

To investigate the involvement of the noradrenergic locus coeruleus (LC) in the reflex blink circuit, c-Fos and neuronal tracer experiments were performed in the rat. LC neurons involved in reflex blink were localized by analyzing c-Fos protein expression after electrical stimulation of the supraorbital nerve. Subsequently, neuronal tracers were injected in two different nuclei which are part of the reflex blink circuit. Anterograde tracer experiments in the sensory trigeminal complex (STC) explored the trigemino-coerulear connection; retrograde tracer experiments in the latero-caudal portion of the superior colliculus (SC) established coerulear-collicular connections. The combination of retrograde tracer injections into the latero-caudal SC portion combined with electrical stimulation of the supraorbital nerve identified c-Fos positive LC neurons that project to the latero-caudal SC. Our results revealed the existence of a STC-LC-SC loop.

Reticulo-collicular and spino-collicular projections involved in eye and eyelid movements during the blink reflex.

Reflex blinking provides a useful experimental tool for various functional studies on the peripheral and central nervous system, yet the neuronal circuitry underlying this reflex is not precisely known. In the present study, we investigated as to whether neurons in the reticular formation and rostral cervical spinal cord (C1) may be involved in the blink reflex in rats. To this end we investigated c-Fos expression in these areas following supraorbital nerve stimulation combined with retrograde tracing of gold conjugated horse radish peroxidase (Gold-HRP) from the superior colliculus. We observed many double labeled neurons in the parvocellular reticular nucleus, medullary reticular formation, and laminae IV and V of C1. Thus, these brain regions contain neurons that may be involved in blink reflexes as well as eye movements, because they both can be activated following peri-orbital stimulation and project to the superior colliculus. Consequently, we suggest that the medullary reticular formation and C1 region play a central role in the coordination of eye and eyelid movements during reflex blinking.

Trigeminovestibular and trigeminospinal pathways in rats: retrograde tracing compared with glutamic acid decarboxylase and glutamate immunohistochemistry.

This study identified neurons in the sensory trigeminal complex with connections to the medial (MVN), inferior (IVN), lateral (LVN), and superior (SVN) vestibular nuclei or the spinal cord. Trigeminovestibular and trigeminospinal neurons were localized by injection of retrograde tracers. Immunohistochemical processing revealed gamma-aminobutyric acid (GABA)- and glutamate-containing neurons in these two populations. Trigeminovestibular neurons projecting to the MVN and the IVN were in the caudal principal nucleus (5P), pars oralis (5o), interpolaris (5i), and caudalis (5c) and scattered throughout the rostral 5P. Projections were bilateral to the IVN, with an ipsilateral dominance to the MVN, except from the rostral 5P, which was contralateral. Neurons projecting to the LVN were numerous in the ventral caudal 5P and the 5o and less abundant in the rostral 5P, 5i, and 5c. Our results suggested that only 5P and 5o project to the dorsal LVN. Neurons projecting to the SVN were in the dorsal 5P, 5o, and 5i but not in 5c. Trigeminospinal neurons were mainly in the ventral 5o and 5i and in the lateral 5c, rarely or never in 5P. Among trigeminovestibular neurons, most of the somas were immunoreactive for glutamate, but some reacted for GABA. Among trigeminospinal neurons, the number of somas immunoreactive for each of the two amino acids was similar. Trigeminal terminals were observed in contact with vestibulospinal neurons in the IVN and LVN, giving evidence of a trigeminovestibulospinal pathway. Therefore, inhibitory and excitatory facial inputs may contribute through trigeminospinal or trigeminovestibulospinal pathways to the control of head/neck movements.

Reticulo-collicular projections: a neuronal tracing study in the rat.

Neuroanatomical tract-tracing methods were used to study the topography of the reticulocollicular projections. Injections of gold-HRP or BDA tracers into the medial and/or central portions of the superior colliculus resulted in labelled neurones mainly in the medial reticular formation, whereas injections into the lateral portion of the superior colliculus showed labelling in the medial and lateral reticular formation. When tracer was injected into the lateral portion of the caudal superior colliculus, extensive lateral labelling was observed in the contralateral parvocellular reticular nucleus and the contralateral dorsal medullary reticular nucleus, two areas involved in reflex blinking. The present study shows that these reticular areas project to the lateral superior colliculus, which is known to be involved in the coordination of eye and eyelid movements.

Trigemino-solitarii-facial pathway in rats.

This study was undertaken to identify premotor neurons in the nucleus tractus solitarii (NTS) serving as relay neurons between the sensory trigeminal complex (STC) and the facial motor nucleus in rats. Trigemino-solitarii connections were first investigated following injections of anterograde and/or retrograde (biotinylated dextran amine, biocytin, or gold-HRP) tracers in STC or NTS. Trigemino-solitarii neurons were abundant in the ventral and dorsal parts of the STC and of moderate density in its intermediate part. They project throughout the entire rostrocaudal extent of the NTS with a strong lateral preponderance. Solitarii-trigeminal neurons were observed mostly in the rostral and rostrolateral NTS. They mainly project to the ventral and dorsal parts of the spinal trigeminal nucleus but not to the principal nucleus. Additional neurons located in the middle NTS were found to project exclusively to the spinal trigeminal nucleus pars caudalis. No solitarii-trigeminal cells were observed in the caudal NTS. In addition, evidence was obtained of NTS retrogradely labeled neurons contacted by anterogradely labeled trigeminal terminals. Second, solitarii-facial projections were analyzed following injections of anterograde and retrograde tracers into the NTS and the facial nucleus, respectively. NTS neurons, except those of the rostrolateral part, reached the dorsal aspect of the facial nucleus. Finally, simultaneous injections of anterograde tracer in the STC and retrograde tracer in the facial nucleus gave retrogradely labeled neurons in the NTS receiving contacts from anterogradely labeled trigeminal boutons. Thus, the present data demonstrate for the first time the existence of a trigemino-solitarii-facial pathway. This could account for the involvement of the NTS in the control of orofacial motor behaviors.

Projections from the superior colliculus to the trigeminal system and facial nucleus in the rat.

To determine the influence of the superior colliculus (SC) in orienting behaviors, we examined SC projections to the sensory trigeminal complex, the juxtatrigeminal region, and the facial motor nucleus in rats. Anterograde tracer experiments in the SC demonstrated predominantly contralateral colliculotrigeminal projections. Microinjections in the deep layers of the lateral portion showed labeled nerve fibers and terminals in the ventromedial parts of the caudal principal nucleus and of the rostral oral subnucleus and in the medial part of the interpolar subnucleus. Some terminals were also observed in the juxtatrigeminal region and in the dorsolateral part of the facial motor nucleus contralaterally, overlying the orbicularis oculi motoneuronal region. Verification by retrograde tracer injections into the trigeminal target regions showed labeled SC neurons mostly in lateral portions of layers 4-7. When the juxtatrigeminal region was involved, a remarkable increase of labeled neurons was observed, having a patch-like arrangement with a decreasing gradient from lateral to medial SC portions. Retrograde tracer injections in the dorsolateral VII nucleus showed bilateral labeled neurons mainly in the deep lateral SC portion. Retrograde BDA microinjections into the same trigeminal or juxtatrigeminal regions, followed by gold-HRP into the dorsolateral VII nucleus, demonstrated a significant number of SC neurons in deep layers 6-7 projecting to both structures by axon collaterals. These neurons are mediolaterally grouped in patches along the rostrocaudal SC extent; a subset of them are immunoreactive for glutamic acid decarboxylase (GAD). They could be involved in the coordination of facial movements. Simultaneous anterograde and retrograde tracer injections into the lateral SC portion and the VII nucleus respectively localized trigeminofacial neurons receiving collicular input in the trigeminal principal nucleus and pars oralis. Therefore the SC should play a crucial role in regulating motor programs of both eye and eyelid movements.

Localization of trigeminal, spinal, and reticular neurons involved in the rat blink reflex.

Electrical stimulation of the supraorbital nerve (SO) induces eyelid closure by activation of orbicularis oculi muscle motoneurons located in the facial motor nucleus (VII). Neurons involved in brainstem central pathways implicated in rat blink reflex were localized by analyzing c-Fos protein expression after SO stimulation in conjunction with tracing experiments. A retrograde tracer (gold-horseradish peroxidase [HRP]) was injected into the VII. The distribution patterns of activated c-Fos-immunoreactive neurons and of neurons exhibiting both c-Fos immunoreactivity and gold-HRP labeling were determined in the sensory trigeminal complex (STC), the cervical spinal cord (C1), and the pontomedullary reticular formation. Within the STC, c-Fos immunoreactivity labeled neurons in the ipsilateral ventral part of the principal nucleus, the pars oralis and interpolaris, and bilaterally in the pars caudalis. Colocalization of gold-HRP and c-Fos immunoreactivity was observed in neurons of ventral pars caudalis layers I-IV and ventral pars interpolaris. In C1, SO stimulation revealed c-Fos neurons in laminae I-V. After additional injections in VII, the double-labeled c-Fos/gold-HRP neurons were concentrated in laminae IV and V. Although c-Fos neurons were found throughout the pontomedullary reticular formation, most appeared rostrally around the motor trigeminal nucleus and in the ventral parvocellular reticular nucleus medial to the fiber bundles of the seventh nerve. Caudally, c-Fos neurons were in the lateral portion of the dorsal medullary reticular field. In addition, these reticular areas contained double-labeled neurons in electrically stimulated rats that had received gold-HRP injections in the VII. The presence of double-labeled neurons in the STC, C1, and the reticular formation implies that these neurons receive sensory information from eyelids and project to the VII. These double-labeled neurons could then be involved in di- or trisynaptic pathways contributing to the blink reflex.

Haemophilus influenzae carriage in children attending French day care centers: a molecular epidemiological study.

The nasopharyngeal Haemophilus influenzae flora of healthy children under the age of 3 years attending day care centers in three distinct French geographic areas was analyzed by sampling during two periods, spring 1999 (May and June) and fall 1999 (November and December). The average carrier rate among 1,683 children was 40.9%. The prevalence of capsulated H. influenzae carriers was 0.4% for type f and 0.6% for type e. No type b strains were found among these children, of whom 98.5% had received one or more doses of anti-Haemophilus b vaccine. Among the strains, 44.5% were TEM-type beta-lactamase producers and nine (1.3%) were beta-lactamase-negative ampicillin-resistant strains. Pulsed-field gel electrophoresis restriction patterns showed a large diversity with 366 SmaI patterns from 663 strains. Among the strains isolated during a given period, 33% were isolated simultaneously in more than one area. In each area, depending on the sampling period, 68 to 72% of the strains had new pulsotypes and persistence of 28 to 32% of the strains was noted. For the 297 beta-lactamase-producing strains, 194 patterns were found. The genomic diversity of these strains was comparable to that of the whole set of strains and does not suggest a clonal diffusion. Among the beta-lactamase-producing strains isolated in November and December, depending on the area, 66 to 73% had new pulsotypes with persistence of only 27 to 33% of the strains. In any given geographic area, colonization by H. influenzae appears to be a dynamic process involving a high degree of genomic heterogeneity among the noncapsulated colonizing strains.

Vestibulotrigeminal and vestibulospinal projections in rats: retrograde tracing coupled to glutamic acid decarboxylase immunoreactivity.

Immunohistochemical experiments were performed using glutamic acid decarboxylase (GAD) to identify gamma-aminobutyric acid (GABA)ergic neurons in the vestibular nuclei (VN). VN neurons projecting to the sensory trigeminal complex (STC) or to the C1-C2 segments of the spinal cord were identified by injection of wheat germ agglutinin-apo-horseradish peroxidase coupled to colloidal gold (gold-HRP), a retrogradely transported tracer, in these structures. The experiments combining injection of gold-HRP in spinal cord and GAD immunohistochemistry revealed the existence in the medial, inferior and lateral VN of GAD immunoreactive neurons projecting to the spinal C1-C2 level. Experiments combining injection of gold-HRP in the STC and GAD immunohistochemistry demonstrated that, at least, 30-50% of the vestibulo-trigeminal neurons also contained GAD. Injections of two different retrograde tracers (gold-HRP and Biotinylated dextran amine) in the STC and the spinal cord demonstrated that some VN neurons project by axon collaterals to both structures. Because of the GABAergic spinal and STC vestibular projections we assume that these VN neurons with collateral projection are GABAergic. Therefore primary afferents from the face, neck or hindlimb could be modulated by inhibitory influences from GABAergic vestibular neurons.

The sensory trigeminal complex projects contralaterally to the facial motor and the accessory abducens nuclei in the rat.

Anterograde tracer injections in the rat sensory trigeminal complex are shown here to demonstrate projections to the contralateral facial motor (VII) and accessory abducens (VIacc) nuclei. Most of the trigeminal fibres originated within the pars oralis (5o) and contacted neurones in the medial and intermediate VII. Moderate projections from the pars caudalis (5c) and interpolaris (5i) reached the lateral and dorsolateral VII. Rare projections from the principal nucleus (5P) were found. Trigeminal projections to the contralateral VIacc originated mainly from the 5P and 5o. Few projections from the 5i and 5c to the contralateral VIacc were found. Retrograde tracer injections in the VII showed premotor neurones to the contralateral VII scattered throughout the 5o and in the ventromedial portion of the caudal 5i and the 5c. Double retrograde tracing experiments provide evidence that neurones in the 5o and 5c project to both the ipsi- and contralateral VII. Such collateralization would play a significant role in the co-ordination of the musculature of the face.

Organization of trigeminocollicular connections and their relations to the sensory innervation of the eyelids in the rat.

Relationships between the trigeminal component of blinking and the superior colliculus (SC) were studied in rats. To localize primary afferent eyelid projections in the sensory trigeminal complex, neuronal tracing experiments were performed as well as analysis of c-Fos protein expression after supraorbital (SO) nerve stimulation. Labelled nerve fibers were found to enter ventrally within the ipsilateral sensory trigeminal complex. Labelled boutons were observed at the junction of the principal nucleus (5P) and the pars oralis (5o) and in the pars caudalis (5c). The c-Fos immunoreactivity was observed in neurons located in the ipsilateral ventral parts of 5P, 5o, and the pars interpolaris (5i) and bilaterally in 5c. Injections in 5P, 5o, 5i, and 5c resulted in anterogradely labelled fibers, with a contralateral preponderance, within the intermediate and deeper SC layers. Injections in 5P or 5o showed anterogradely labelled nerve fibers, profusely terminating in small patches in the medial and central portions of SC layer 4. Subsequently, dense labelling was found in the lateral portion of SC layers 4-7, without patch-like organization. Injections in SC showed retrogradely labelled neurons predominantly within the contralateral part of the sensory trigeminal complex (28% in 5P, 20% in 5o, 50% in 5i, and 2% in 5c). Colocalization of the retrograde tracer after SC injections and c-Fos immunoreactivity in neurons demonstrated that some 5P, 5o, and 5i neurons receive SO nerve inputs and project to SC. This implies that intermediate and deeper SC layers receive sensory information from the eyelids and may be directly involved in the regulation of eye-eyelid coordination.

Diversity of beta-lactam resistance-conferring amino acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae.

The sequences of the ftsI gene, encoding the transpeptidase domain of penicillin binding protein (PBP) 3A and/or PBP 3B, which are involved in septal peptidoglycan synthesis, were determined for 108 clinical strains of Haemophilus influenzae with reduced susceptibility to beta-lactam antibiotics with or without beta-lactamase production and were compared to those of the ampicillin-susceptible Rd strain and ampicillin-susceptible clinical isolates. The sequences have 18 different mutation patterns and were classified into two groups on the basis of amino acid substitutions deduced from the nucleotide sequences located between bp 960 and 1618 of the ftsI gene. In group I strains (n = 7), His-517 was substituted for Arg-517. In group II strains (n = 101), Lys-526 was substituted for Asn-526. In subgroup IIa (n = 5; H. influenzae ATCC 49247), the only observed substitution was Lys-526 for Asn-526; in subgroup IIb (n = 56), Val-502 was substituted for Ala-502 (n = 13), along with several other substitutions: Asn-350 for Asp-350 (n = 15), Asn-350 for Asp-350 and Glu-490 for Gly-490 (n = 14), and Asn-350 for Asp-350 and Ser-437 for Ala-437 (n = 5). In subgroup IIc (n = 25), Thr-502 was substituted for Ala-502. In subgroup IId, Val-449 was substituted for Ile-449 (n = 15). The MICs of beta-lactam antibiotics for the 108 strains were to 8 to 16 times the MICs for susceptible strains. The strains, isolated from both adults and children, were analyzed for genetic relationship by pulsed-field gel electrophoresis and by determination of ftsI sequence phylogeny. Both analyses revealed the lack of clonality and the heterogeneity of the strains, but some clusters suggest the spread and/or persistence of a limited number of strains of the same pulsotype and pattern of amino acid substitutions. Reduced susceptibility to beta-lactam, brought about by mutations of the ftsI gene, is becoming a frequent phenomenon, affecting both strains that produce beta-lactamase and those that do not. The level of resistance remains low but opens the way to greater resistance in the future.