Neurons in the cochlear nuclei controlling the tensor tympani muscle in the rat: a study using pseudorabies virus.

The middle ear muscle reflex has been implicated in modulation of auditory input and protection of the inner ear from acoustic trauma. However, the identification of neurons in the cochlear nuclei participating in this reflex has not been fully elucidated. In the present study, we injected the retrograde transynaptic tracer pseudorabies virus into single tensor tympani (TT) muscles, and identified transynaptically labeled cochlear nucleus neurons at multiple survival times. Motoneurons controlling TT were located ventral to the ipsilateral motor trigeminal nucleus and extended rostrally towards the medial aspect of the lateral lemniscus. Transynaptically labeled neurons were observed bilaterally in the dorsal and dorso-medial parts of ventral cochlear nuclei as early as 48 h after virus injection, and had morphological features of radiate multipolar cells. After >or=69 h, labeled cells of different types were observed in all cochlear nuclei. At those times, labeling was also detected bilaterally in the medial nucleus of the trapezoid body and periolivary cell groups in the superior olivary complex. Based on the temporal course of viral replication, our data strongly suggest the presence of a direct projection of neurons from the ventral cochlear nuclei bilaterally to the TT motoneuron pool in rats. The influence of neurons in the cochlear nuclei upon TT activity through direct and indirect pathways may account for multifunctional roles of this muscle in auditory functions.

A monosynaptic pathway links the vestibular nuclei and masseter muscle motoneurons in rats.

Physiological evidence indicates that vestibular signals modulate the activity of motoneurons innervating the masseter muscle. Recently, experiments using transynaptic retrograde transport of pseudorabies virus provided anatomical evidence that many neurons concentrated in the dorsomedial part of the parvicellular division of the medial vestibular nucleus (MVePC) and the caudal prepositus hypoglossi (PH) provide inputs to motoneurons innervating the lower third of the superficial layer of the masseter muscle. However, it was not clear whether this vestibulo-trigeminal projection was monosynaptic or polysynaptic. The present study sought to determine whether neurons in the MVePC or PH project directly to motoneurons controlling the masseter muscle in rats. For this purpose, an anterograde tracer (biotinylated dextran amine, BDA) was injected into vestibular nuclei (mainly MVePC) or PH and a retrograde tracer (the beta-subunit of cholera toxin, b-CT) was injected into the masseter muscle ipsilateral or contralateral to the BDA injection site. Following injections of BDA into the vestibular nuclei or PH, anterogradely labeled axon terminals were observed bilaterally in the motor trigeminal nucleus (Mo5), particularly in the ventral, medial, and lateral portions of the nucleus; projections to dorsal Mo5 were sparse. In addition, retrogradely labeled motoneurons were located in the ventral and lateral portions of the ipsilateral Mo5. Moreover, anterogradely labeled terminals were observed to be in close proximity to motoneurons in the Mo5 that were retrogradely labeled from b-CT injections into the masseter muscle. This study provides direct evidence that a monosynaptic pathway exists between the MVePC and PH and masseter motoneurons.

Transneuronal tracing of vestibulo-trigeminal pathways innervating the masseter muscle in the rat.

Previous studies reported that the activity of trigeminal motoneurons innervating masseter muscles is modulated by vestibular inputs. We performed the present study to provide an anatomical substrate for these physiological observations. The transynaptic retrograde tracer pseudorabies virus-Bartha was injected into multiple sites of the lower third of the superficial layer of the masseter muscle in rats, a subset of which underwent a sympathectomy prior to virus injections, and the animals were euthanized 24-120 h later. Labeled masseteric motoneurons were first found in the ipsilateral trigeminal motor nucleus following a 24-h postinoculation period; subsequent to 72-h survival times, the number of infected motoneurons increased, and at > or =96 h many of these cells showed signs of cytopathic changes. Following 72-h survival times, a few transynaptically labeled neurons appeared bilaterally in the medial vestibular nucleus (MVe) and the caudal prepositus hypoglossi (PH) and in the ipsilateral spinal vestibular nucleus (SpVe). At survival times of 96-120 h, labeled neurons were consistently observed bilaterally in all vestibular nuclei (VN), although the highest concentration of infected cells was located in the caudal part of the MVe, the SpVe, and the caudal portion of PH. The distribution and density of labeling in the VN and PH were similar in sympathectomized and nonsympathectomized rats. These anatomical data provide the first direct evidence that neurons in the VN and PH project bilaterally to populations of motoneurons innervating the lower third of the superficial layer of the masseter muscle. The MVe, PH, and SpVe appear to play a predominant integrative role in producing vestibulo-trigeminal responses.

Zonal organization of the vestibulo-cerebellar pathways controlling the horizontal eye muscles using two recombinant strains of pseudorabies virus.

Many studies have documented the influence of the flocculus upon vestibulo-ocular reflex eye movements. Electrical stimulation of Purkinje cells in a central longitudinal zone evoked slow ipsilateral eye movements in the horizontal plane. Recently, the organization of neurons in the vestibulo-cerebellar pathways controlling single lateral rectus and medial rectus muscles was identified in rats using the transynaptic transport of pseudorabies virus. Overlapping distributions of neurons innervating single muscles were located predominantly in a central longitudinal zone of ventral paraflocculi/dorsal flocculi, and the rostral half of ventral flocculi. This study used two isogenic pseudorabies virus recombinants to determine whether individual cells in those brain regions have collateralized projections to motoneuron pools innervating the right lateral rectus and the left medial rectus muscles using different survival times and dual injection paradigms. The infected neurons were detected using dual-labeling immunofluorescence. Three populations of labeled neurons were observed: two populations replicated only one reporter while a third contained both viruses (i.e. dual-labeled). Most dual-labeled cells were located in a central longitudinal zone of the ventral paraflocculus, ipsilateral to the injection into the medial rectus, whereas very few were in the flocculus. This finding suggests that the flocculus and ventral paraflocculus may exert influence upon distinct vestibulo-cerebellar pathways. Most Purkinje cells in the ventral paraflocculus may influence the vestibulo-ocular reflex pathways through collateralization, whereas those in the flocculus may instead provide a monocular control of eye movements.

Zonal organization of the vestibulo-cerebellum in the control of horizontal extraocular muscles using pseudorabies virus: I. Flocculus/ventral paraflocculus.

Much literature has studied the relationship between the organization of neurons in the flocculus/ventral paraflocculus and vestibulo-ocular reflex pathways. Although activation of a flocculus central zone produces ipsilateral horizontal eye movement, anatomical tracing evidence in rats suggests that there may not be a simple one-to-one correspondence between flocculus/ventral paraflocculus zones and control of single extraocular muscles or coplanar pairs of antagonistic extraocular muscles. This study used the retrograde transynaptic transport of pseudorabies virus to identify the topographical organization of Purkinje cells in the flocculus/ventral paraflocculus that control the lateral rectus (LR) and medial rectus (MR) muscles in rats. A survival time of 80 h and 84 h was necessary to observe consistent transynaptically labeled cells in the flocculus/ventral paraflocculus following injections of pseudorabies virus into the MR and LR, respectively. The organization of Purkinje cells in the dorsal flocculus and ventral paraflocculus abided by the traditional boundaries, whereas the labeling pattern in the ventral flocculus showed a more complex, interdigitated arrangement. In agreement with prior studies, transynaptically labeled neurons were also observed in specific vestibular nuclear regions within the medial and superior vestibular nuclei and dorsal Y group. The distribution of labeled neurons in ipsilateral and contralateral vestibular nuclei was associated with features of ipsilateral and contralateral retrograde labeling of Purkinje cells in flocculus/ventral paraflocculus. Importantly, this study provides the first evidence of vestibulo-cerebellar zones controlling individual extraocular muscles and also overlapping distribution of neurons in flocculo-vestibular zones that influence the LR and MR motoneuron pools. This suggests that some of these neurons may be responsible for controlling both muscles.

Transneuronal tracing of neural pathways influencing both diaphragm and genioglossal muscle activity in the ferret.

In prior experiments that employed the transneuronal transport of isogenic recombinants of pseudorabies virus (PRV), we demonstrated that neurons located ventrally in the medial medullary reticular formation (MRF) of the ferret provide collateralized projections to both diaphragm and abdominal muscle motoneurons as well as to multiple abdominal muscle motoneuron pools. The goal of the present study was to determine whether single MRF neurons also furnish inputs to diaphragm motoneurons and those innervating an airway muscle with inspiratory-related activity: the tongue protruder genioglossus. For this purpose, PRV recombinants expressing unique reporters (beta-galactosidase or enhanced green fluorescent protein) were injected into either the diaphragm or the genioglossal muscle. The virus injections produced transneuronal infection of overlapping populations of MRF neurons. A small proportion of these neurons (<15%) was infected by both PRV recombinants, which indicated that they provide collateralized inputs to genioglossal and diaphragm motoneurons. These findings show that, whereas some MRF neurons simultaneously influence the activity of upper airway and respiratory pump muscles, other cells in this brain stem region independently contribute to diaphragm and genioglossal muscle contraction regulation.

Neurochemical phenotypes of MRF neurons influencing diaphragm and rectus abdominis activity.

In prior studies that used transneuronal transport of isogenic recombinants of pseudorabies virus, we established that medial medullary reticular formation (MRF) neurons sent collateralized projections to both diaphragm and abdominal muscle motoneurons. Furthermore, inactivation of MRF neurons in cats and ferrets increased the excitability of diaphragm and abdominal motoneurons, suggesting that MRF neurons controlling respiratory activity are inhibitory. To test this hypothesis, the present study determined the neurochemical phenotypes of MRF premotor respiratory neurons in the ferret by using immunohistochemical procedures. Dual-labeling immunohistochemistry combining pseudorabies virus injections into respiratory muscles with the detection of glutamic acid decarboxylase-like immunoreactive and glutamate-like immunoreactive cells showed that both GABAergic and glutamatergic MRF neurons project to respiratory motoneurons, although the latter are more common. These data suggest that the role of the MRF in respiratory regulation is multifaceted, as this region provides both inhibitory and excitatory influences on motoneuron activity.

Role of the vestibular system in regulating respiratory muscle activity during movement.

1. Changes in posture can affect the resting length of the diaphragm, which is corrected through increases in both diaphragm and abdominal muscle activity. Furthermore, postural alterations can diminish airway patency, which must be compensated for through increases in firing of particular upper airway muscles. 2. Recent evidence has shown that the vestibular system participates in adjusting the activity of both upper airway muscles and respiratory pump muscles during movement and changes in body position. 3. Vestibulo-respiratory responses do not appear to be mediated through the brainstem respiratory groups; labyrinthine influences on respiratory pump muscles may be relayed through neurons in the medial medullary reticular formation, which have recently been demonstrated to provide inputs to both abdominal and diaphragm motoneurons. 4. Three regions of the cerebellum that receive vestibular inputs, the fastigial nucleus, the nodulus/uvula and the anterior lobe, also influence respiratory muscle activity, although the physiological role of cerebellar regulation of respiratory activity is yet to be determined. 5. It is practical for the vestibular system to participate in the control of respiration, to provide for rapid adjustments in ventilation such that the oxygen demands of the body are continually matched during movement and exercise.

Plasma hormone levels and central c-Fos expression in ferrets after systemic administration of cholecystokinin.

Posterior pituitary hormone secretion and central neural expression of the immediate-early gene product c-Fos was examined in adult ferrets after intravenous administration of CCK octapeptide. Pharmacological doses of CCK (1, 5, 10, or 50 microg/kg) did not induce emesis, but elicited behavioral signs of nausea and dose-related increases in plasma vasopressin (AVP) levels without significant increases in plasma oxytocin (OT) levels. CCK activated neuronal c-Fos expression in several brain stem viscerosensory regions, including a dose-related activation of neurons in the dorsal vagal complex (DVC). Activated brain stem neurons included catecholaminergic and glucagon-like peptide-1-positive cells in the DVC and ventrolateral medulla. In the forebrain, activated neurons were prevalent in the paraventricular and supraoptic nuclei of the hypothalamus and also were observed in the central nucleus of the amygdala and bed nucleus of the stria terminalis. Activated hypothalamic neurons included cells that were immunoreactive for AVP, OT, and corticotropin-releasing factor. Comparable patterns of brain stem and forebrain c-Fos activation were observed in ferrets after intraperitoneal injection of lithium chloride (LiCl; 86 mg/kg), a classic emetic agent. However, LiCl activated more neurons in the area postrema and fewer neurons in the nucleus of the solitary tract compared with CCK. Together with results from previous studies in rodents, our findings support the view that nauseogenic treatments activate similar central neural circuits in emetic and nonemetic species, despite differences in treatment-induced emesis and pituitary hormone secretion.

Transneuronal tracing of neural pathways controlling abdominal musculature in the ferret.

Abdominal musculature participates in generating a large number of behaviors and protective reflexes, although each abdominal muscle is frequently activated differentially during particular motor responses. For example, rectus abdominis has been reported to play less of a role in respiration than other abdominal muscles, such as transversus abdominis. In the present study, the inputs to transversus abdominis and rectus abdominis motoneurons were determined and compared using the transneuronal transport of two recombinant isogenic strains of pseudorabies virus. After a 5-day post-inoculation period, infected presumed motoneurons were observed principally in cord levels T10-T15 ipsilateral to the injections. The injection of a monosynaptic tracer, beta-cholera toxin, into transversus abdominis confirmed the distribution of motoneurons innervating this muscle. In the brainstem, neurons transneuronally infected following injection of pseudorabies virus into rectus abdominis or transversus abdominis were located in the same regions, which included the medial medullary reticular formation, the medullary raphe nuclei, and nucleus retroambiguus (the expiration region of the caudal ventral respiratory group). Double-labeled cells providing inputs to both rectus and transversus motoneurons were present in both the medial medullary reticular formation and nucleus retroambiguus. These data show that the medial medullary reticular formation contains neurons influencing the activity of multiple abdominal muscles, and support our hypothesis that this region globally affects the excitability of motoneurons involved in respiration.

Definition of neuronal circuitry controlling the activity of phrenic and abdominal motoneurons in the ferret using recombinant strains of pseudorabies virus.

During a number of behaviors, including vomiting and some postural adjustments, activity of both the diaphragm and abdominal muscles increases. Previous transneuronal tracing studies using injection of pseudorabies virus (PRV) into either the diaphragm or rectus abdominis (RA) of the ferret demonstrated that motoneurons innervating these muscles receive inputs from neurons in circumscribed regions of the spinal cord and brainstem, some of which have an overlapping distribution in the magnocellular part of the medullary reticular formation (MRF). This observation raises two possibilities: that two populations of MRF neurons provide independent inputs to inspiratory and expiratory motoneurons or that single MRF neurons have collateralized projections to both groups of motoneurons. The present study sought to distinguish between these prospects. For this purpose, recombinant isogenic strains of PRV were injected into these respiratory muscles in nine ferrets; the strain injected into the diaphragm expressed beta-galactosidase, whereas that injected into RA expressed green fluorescent protein. Immunofluorescence localization of the unique reporters of each virus revealed three populations of infected premotor neurons, two of which expressed only one virus and a third group that contained both viruses. Dual-infected neurons were predominantly located in the magnocellular part of the MRF, but were absent from both the dorsal and ventral respiratory cell groups. These data suggest that coactivation of inspiratory and expiratory muscles during behaviors such as emesis and some postural adjustments can be elicited through collateralized projections from a single group of brainstem neurons located in the MRF.

Transneuronal tracing of neural pathways controlling an abdominal muscle, rectus abdominis, in the ferret.

Abdominal muscles participate in generating a large number of behaviors and reflex responses, including expiration, coughing, sneezing, vomiting, postural control, production of speech, straining, facilitation of venous return to the heart, and reaction to vestibular stimulation. However, the only premotor neurons that have been conclusively shown to influence abdominal motoneurons are located in nucleus retroambiguus, the expiratory region of the caudal ventral respiratory group. In the present study, the neural circuitry controlling the activity of one abdominal muscle, rectus abdominis, was mapped using the transneuronal tracer pseudorabies virus (PRV) in the ferret. Injections of PRV into rectus abdominis labeled large presumed motoneurons in the ventral horn of T12-L4, and smaller presumed interneurons that were scattered in laminae VII, VIII, IX, and X of T4-L4. In addition, neurons in several areas of the medulla and caudal pons, including the retroambigual nucleus, medial and ventromedial reticular formation, nucleus prepositus hypoglossi, vestibular nuclei, and raphe nuclei, were infected by transynaptic passage of PRV from rectus abdominis motoneurons. Thus, the multifunctional roles of abdominal muscles appear to be coordinated by premotor neurons located in both the spinal cord and several regions of the brainstem.

Identification of nerve endings in cat extraocular muscles.

BACKGROUND: The aim of the present study was to identify the varieties of sensory and motor nerve endings in cat extraocular muscles. METHODS: Sensory terminals were identify by injecting neuronal tracers (fast blue, biocytin, or peroxidase) into the trigeminal ganglion, which contains the sensory cells innervating the eye muscles. Motor terminals were identified by injections of horseradish peroxidase or DiI, a fluorescent carbocyanin dye, into either the oculomotor nerve or the IIIrd nuclei. RESULTS: Injections into the trigeminal ganglion anterogradely labelled three types of sensory nerve endings for each neuronal tracer used: (1) the well-known "palisade" endings at the myotendinous junction of each extraocular muscle; (2) "compact" endings consisting of a dense terminal arborization extending up to 60 microm in length on striated muscle fibres 10-15 microm in diameter; and (3) "complex" endings on muscle fibres 15-20 microm in diameter. The complex ending issued from multiple collateral branches of the parent nerve fibre, which stretched and turned around the muscle fibre and gave off numerous terminal varicosities over a distance of about 140 microm. The sensory complex and compact endings presented strong similarities with some "atypical muscle spindles" previously described. In addition to the classic motor "plate" and "grape," we found evidence for the existence of motor "spiral" endings with each tracer. CONCLUSIONS: The sensory nature of the palisade endings was demonstrated, and two other types of sensory terminals were identified and described. The spiral nerve terminals were demonstrated to be motor in nature, and a possible function in the microsaccadic movements associated with fixation is suggested.

Trigeminocerebellar and trigemino-olivary projections in rats.

Retrograde and anterograde neuronal tracers (HRP, biocytin, biotinylated dextran-amine) were used to study the organisation of trigeminocerebellar and trigemino-olivary connections, focusing on the connectivity between trigeminal nuclear regions and the sagittal zones of the rat cerebellar cortex. Trigeminocerebellar projections were bilateral, but mostly ipsilateral. Direct trigeminocerebellar fibres originated mostly in the principal trigeminal nucleus (VP) and pars oralis (Vo), pars interpolaris (Vi), and to a lesser extent in pars caudalis (Vc) of the spinal trigeminal nucleus. Consistent projections were found from the Vc to cerebellar lobules IX and X. The trigeminal fibres terminated in the cerebellum in an organised fashion. The ventral part of the VP, Vo and Vi projected to the medial A zone and the C3 and D2 subzones, whereas the dorsal part of the nuclei projected to the lateral A zone and the C2, D0 and D1 subzones. In lobules IX and X, the organisation was different. The medial half of the VP, Vo, Vi and Vc projected to the lateral aspects of these lobules whereas their lateral part projected to their medial aspects. Trigeminal projections to the deep cerebellar nuclei were also present. Projections to a given sagittal zone concomitantly reached its corresponding nuclear target. Trigemino-olivary projections were principally contralateral. The Vo, Vi and Vc projected to the rostromedial dorsal accessory olive, the adjacent dorsal leaf and the dorsomedial part of the ventral leaf of the principal olive, which are known to project subzones C3, D0 and D1 of the rat cerebellar cortex.

Cerebellar afferences from the mesencephalic trigeminal nucleus in the rat.

The aim of the present study was to test for and characterize the organization of a direct projection from neurones of the trigeminal mesencephalic nucleus (Vme) to the cerebellum. WGA-HRP was used as a retrograde tracer following injections in the cerebellar cortex. The extent of each injection site within the sagittal zones was determined according to corticonuclear and olivocortical connections. Retrogradely labelled neurons were observed in the caudal part of the ipsilateral Vme only following vermal injection. The Vme projections reached exclusively the ipsilateral sagittal zone X in the anterior lobe, lobule VI and lobule IX. This identification was confirmed by anterograde labelling of mossy fibre terminals following a biocytin injection restricted to the Vme.