RESUMEN
C1 catecholamine neurons reside within the rostroventrolateral medulla (RVLM), an area that plays an integral role in blood pressure regulation through reticulospinal projections to sympathetic preganglionic neurons in the thoracic spinal cord. In a previous investigation we mapped the efferent projections of C1 neurons, documenting supraspinal projections to cell groups in the preautonomic network that contribute to the control of cardiovascular function. Light microscopic study also revealed putative local circuit connections within RVLM. In this investigation we tested the hypothesis that RVLM C1 neurons elaborate a local circuit synaptic network that permits communication between C1 neurons giving rise to supraspinal and reticulospinal projections. A replication defective lentivirus vector that expresses enhanced green fluorescent protein (EGFP) under the control of a synthetic dopamine beta hydroxylase (DßH) promoter was used to label C1 neurons and their processes. Confocal fluorescence microscopy demonstrated thin varicose axons immunopositive for EGFP and tyrosine hydroxylase that formed close appositions to C1 somata and dendrites throughout the rostrocaudal extent of the C1 area. Dual-labeled electron microscopic analysis revealed axosomatic, axodendritic and axospinous synaptic contacts with C1 and non-C1 neurons with a distribution recapitulating that observed in the light microscopic analysis. Labeled boutons were large, contained light axoplasm, lucent spherical vesicles, and formed asymmetric synaptic contacts. Collectively these data demonstrate that C1 neurons form a synaptic network within the C1 area that may function to coordinate activity among projection-specific subpopulations of neurons. The data also suggest that the boundaries of RVLM should be defined on the basis of function criteria rather than the C1 phenotype of neurons.
Asunto(s)
Catecolaminas/metabolismo , Bulbo Raquídeo/citología , Red Nerviosa/fisiología , Neuronas/fisiología , Sinapsis/metabolismo , Animales , Mapeo Encefálico , Dopamina beta-Hidroxilasa/metabolismo , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microscopía Inmunoelectrónica , Red Nerviosa/ultraestructura , Neuronas/ultraestructura , Ratas , Ratas Sprague-Dawley , Sinapsis/ultraestructura , Transducción Genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the U(L)21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha U(L)21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.
Asunto(s)
Proteínas de la Cápside/genética , Herpesvirus Suido 1/genética , Neuronas/virología , Animales , Técnica del Anticuerpo Fluorescente , Herpesvirus Suido 1/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Bleomycin hydrolase (BLMH) is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer's disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the CNS were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/JxB6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the CNS.
Asunto(s)
Conducta Animal/fisiología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/fisiología , Gliosis/genética , Gliosis/psicología , Animales , Encéfalo/enzimología , Encéfalo/fisiología , Condicionamiento Operante/fisiología , Señales (Psicología) , ADN/genética , Conducta Exploratoria/fisiología , Miedo/psicología , Femenino , Genotipo , Hipocampo/fisiología , Inmunohistoquímica , Luz , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Equilibrio Postural/fisiologíaRESUMEN
The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.
Asunto(s)
Axones/metabolismo , Infecciones Virales del Ojo/metabolismo , Herpesvirus Suido 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Seudorrabia/metabolismo , Replicación Viral , Animales , Axones/patología , Axones/virología , Técnicas de Cocultivo , Ojo/inervación , Ojo/metabolismo , Ojo/patología , Ojo/virología , Infecciones Virales del Ojo/genética , Infecciones Virales del Ojo/patología , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/patogenicidad , Mutación , Células PC12 , Proteínas Serina-Treonina Quinasas/deficiencia , Seudorrabia/genética , Seudorrabia/patología , Ratas , Replicación Viral/genéticaRESUMEN
Prior experiments have shown that a region of the medial and inferior vestibular nuclei contributes to cardiovascular and respiratory regulation. In addition to labyrinthine inputs, the majority of neurons in this region of the vestibular nuclei receive signals from the skin, muscle, and viscera, although the pathways conveying these nonlabyrinthine inputs to the vestibular nucleus neurons are unknown. To gain further insight into the afferent pathways to this functionally distinct subdivision of the vestibular complex, we combined monosynaptic mapping with viral transneuronal tracing in the ferret. First order afferent projections were defined by retrograde transport of the beta-subunit of cholera toxin (CTbeta), and the extended polysynaptic circuitry was defined in the same animals by injection of a recombinant of pseudorabies virus Bartha (PRV) into the contralateral vestibular nuclei. Neurons containing CTbeta or infected by retrograde transneuronal transport and replication of PRV were distributed throughout the spinal cord, but were 10 times more prevalent in the cervical cord than the lumbar cord. The labeled spinal neurons were most commonly observed in Rexed's laminae IV-VI and the dorsal portions of laminae VII-VIII. Both the CTbeta and PRV injections also resulted in labeling of neurons in all four vestibular nuclei, the prepositus hypoglossi, the reticular formation, the inferior olivary nucleus, the medullary raphe nuclei, the spinal and principal trigeminal nuclei, the facial nucleus, and the lateral reticular nucleus. Following survival times >/=3 days, PRV-infected neurons were additionally present in nucleus solitarius and the gracile and cuneate nuclei. These data show that an anatomical substrate is present for somatosensory and visceral inputs to influence the activity of cells in the autonomic region of the vestibular nuclei and suggest that these signals are primarily transmitted through brainstem relay neurons.
Asunto(s)
Vías Aferentes/fisiología , Fenómenos Fisiológicos Cardiovasculares , Neuronas/fisiología , Respiración , Núcleos Vestibulares/citología , Núcleos Vestibulares/fisiología , Análisis de Varianza , Animales , Presión Sanguínea/fisiología , Recuento de Células , Toxina del Cólera/metabolismo , Hurones , Proteína Ácida Fibrilar de la Glía/metabolismo , Frecuencia Cardíaca/fisiología , Herpesvirus Suido 1/fisiología , Masculino , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
The sympathetic innervation of lumbar dorsal root ganglia (DRGs) and the possible presence of spinal cord circuits connecting primary sensory afferents to the sympathetic outflow to DRGs were investigated. We used simultaneous tracing of the sympathetic input to and sensory output from DRGs. Adult male rats received unilateral microinjections of the Bartha strain of pseudorabies virus into four lumbar DRGs. At 24 h post-inoculation, productive infection was detected in both DRG neurons and sympathetic postganglionic neurons. Infection of spinal cord neurons was first observed in sympathetic preganglionic neurons of the intermediolateral column. Subsequently, the infection spread to the contralateral intermediolateral column, the area around the central canal and the superficial dorsal horn layers. To investigate the relationship between infected spinal cord neurons and primary afferents from the corresponding DRGs, we injected pseudorabies virus for retrograde tracing together with cholera toxin B for anterograde tracing. We found that infected LIV/LV and LX neurons were in close apposition to cholera toxin B labeled afferents. Importantly, immunohistochemical detection of bassoon, a pre-synaptic zone protein, identified such contacts as synapses. Together, this suggests synaptic contacts between primary sensory afferents and neurons regulating sympathetic outflow to corresponding DRGs.
Asunto(s)
Vías Aferentes/citología , Ganglios Espinales/citología , Terminales Presinápticos/ultraestructura , Médula Espinal/citología , Raíces Nerviosas Espinales/citología , Sistema Nervioso Simpático/citología , Vías Aferentes/fisiología , Animales , Transporte Axonal/fisiología , Toxina del Cólera , Ganglios Espinales/irrigación sanguínea , Ganglios Espinales/fisiología , Herpesvirus Suido 1/metabolismo , Región Lumbosacra , Masculino , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/fisiología , Médula Espinal/fisiología , Médula Espinal/virología , Raíces Nerviosas Espinales/fisiología , Raíces Nerviosas Espinales/virología , Coloración y Etiquetado/métodos , Fibras Simpáticas Posganglionares/citología , Fibras Simpáticas Posganglionares/metabolismo , Sistema Nervioso Simpático/fisiología , Sistema Nervioso Simpático/virología , Transmisión Sináptica/fisiologíaRESUMEN
The activity of some neurons in the lateral mammillary nucleus (LMN) of the rat corresponds with the animal's current head direction (HD). HD cells have been studied extensively but the circuitry responsible for the generation and maintenance of the HD signal has not been established. The present study tested the hypothesis that a polysynaptic pathway connects the vestibular nuclei with the LMN via one or more relay nuclei. This circuitry could provide a substrate for the integration of sensory input necessary for HD cell activity. This hypothesis is based upon the prior demonstration that labyrinthectomy abolishes HD selectivity in thalamic neurons. Viral transneuronal tracing with pseudorabies virus (PRV) was used to test this hypothesis. We injected recombinants of PRV into the LMN and surrounding nuclei of adult male rats and defined the patterns of retrograde transneuronal infection at survival intervals of 60 and 72 h. Infected medial vestibular neurons (MVN) were only observed at the longest postinoculation interval in animals in which the injection site was localized largely to the LMN. Robust infection of the dorsal tegmental nucleus (DTN) and nucleus prepositus hypoglossi (PH) in these cases, but not in controls, at both survival intervals identified these nuclei as potential relays of vestibular input to the LMN. These data are consistent with the conclusion that vestibular information that contributes to the LMN HD cell activity is relayed to this caudal hypothalamic cell group via a polysynaptic brainstem circuit.
Asunto(s)
Movimientos de la Cabeza/fisiología , Tubérculos Mamilares/fisiología , Sinapsis/fisiología , Núcleos Vestibulares/fisiología , Animales , Masculino , Tubérculos Mamilares/química , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/química , Núcleos Vestibulares/químicaRESUMEN
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.
Asunto(s)
Músculos Abdominales/inervación , Músculos Abdominales/fisiología , Bulbo Raquídeo/fisiología , Formación Reticular/fisiología , Lengua/inervación , Lengua/fisiología , Animales , Mapeo Encefálico/métodos , Tronco Encefálico/fisiología , Diafragma/inervación , Diafragma/fisiología , Vías Eferentes/fisiología , Hurones , Herpesvirus Suido 1 , Nervio Hipogloso/fisiología , Inyecciones , Neuronas Motoras/fisiología , Músculos/inervación , Músculos/fisiología , Vías Nerviosas/fisiología , Neuronas Eferentes/fisiología , Médula Espinal/fisiologíaRESUMEN
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.
Asunto(s)
Diafragma/inervación , Ácido Glutámico/metabolismo , Bulbo Raquídeo , Neuronas/fisiología , Recto del Abdomen/inervación , Formación Reticular/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Hurones , Glutamato Descarboxilasa/metabolismo , Inmunohistoquímica , Isoenzimas/metabolismo , Masculino , Neuronas Aferentes/fisiología , Fenotipo , Músculos Respiratorios/inervaciónRESUMEN
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.
Asunto(s)
Movimiento/fisiología , Músculos Respiratorios/fisiología , Pruebas de Función Vestibular/estadística & datos numéricos , Animales , Humanos , Vías Nerviosas/fisiología , Pruebas de Función Vestibular/métodosRESUMEN
Splenic immune function is modulated by sympathetic innervation, which in turn is controlled by inputs from supraspinal regions. In the present study, the characterization of central circuits involved in the control of splenic function was accomplished by injecting pseudorabies virus (PRV), a retrograde transynaptic tracer, into the spleen and conducting a temporal analysis of the progression of the infection from 60 hours to 110 hours postinoculation. In addition, central noradrenergic cell groups involved in splenic innervation were characterized by dual immunohistochemical detection of dopamine-beta-hydroxylase and PRV. Infection in the CNS first appeared in the spinal cord. Splenic sympathetic preganglionic neurons, identified in rats injected with Fluoro-Gold i.p. prior to PRV inoculation of the spleen, were located in T(3)-T(12) bilaterally; numerous infected interneurons were also found in the thoracic spinal cord (T(1)-T(13)). Infected neurons in the brain were first observed in the A5 region, ventromedial medulla, rostral ventrolateral medulla, paraventricular hypothalamic nucleus, Barrington's nucleus, and caudal raphe. At intermediate survival times, the number of infected cells increased in previously infected areas, and infected neurons also appeared in lateral hypothalamus, A7 region, locus coeruleus, subcoeruleus region, nucleus of the solitary tract, and C3 cell group. At longer postinoculation intervals, infected neurons were found in additional hypothalamic areas, Edinger-Westphal nucleus, periaqueductal gray, pedunculopontine tegmental nucleus, caudal ventrolateral medulla, and area postrema. These results demonstrate that the sympathetic outflow to the spleen is controlled by a complex multisynaptic pathway that involves several brainstem and forebrain nuclei.
Asunto(s)
Sistema Nervioso Central/fisiología , Ratas/fisiología , Bazo/inervación , Estilbamidinas , Animales , Dopamina beta-Hidroxilasa/metabolismo , Colorantes Fluorescentes , Herpesvirus Suido 1 , Inyecciones , Masculino , Vías Nerviosas/fisiología , Neuronas/fisiología , Neuronas/virología , Ratas Sprague-Dawley , Sistema Nervioso Simpático/fisiología , Sinapsis/fisiología , Factores de TiempoRESUMEN
Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.
Asunto(s)
Genes fos/genética , Genes fos/efectos de la radiación , Hipotálamo/fisiología , Neuropéptidos/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de la radiación , Animales , Enucleación del Ojo , Técnica del Anticuerpo Fluorescente , Herpesvirus Suido 1 , Inmunohistoquímica , Luz , Masculino , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Glutamato de Sodio/farmacología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efectos de la radiación , Vías Visuales/citología , Vías Visuales/metabolismo , Vías Visuales/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Músculos Abdominales/inervación , Células del Asta Anterior/citología , Vías Eferentes/citología , Herpesvirus Suido 1/fisiología , Bulbo Raquídeo/citología , Centro Respiratorio/citología , Fenómenos Fisiológicos Respiratorios , Músculos Abdominales/fisiología , Músculos Abdominales/virología , Animales , Células del Asta Anterior/fisiología , Células del Asta Anterior/virología , Transporte Axonal/fisiología , Toxina del Cólera/farmacocinética , Vías Eferentes/fisiología , Vías Eferentes/virología , Hurones , Lateralidad Funcional/fisiología , Masculino , Bulbo Raquídeo/fisiología , Bulbo Raquídeo/virología , Sondas Moleculares/farmacocinética , Proteínas Recombinantes/metabolismo , Centro Respiratorio/fisiología , Centro Respiratorio/virologíaAsunto(s)
Encéfalo/fisiología , Encéfalo/virología , Herpesvirus Suido 1/fisiología , Herpesvirus Suido 1/patogenicidad , Seudorrabia/virología , Animales , Encéfalo/patología , Herpesvirus Suido 1/clasificación , Herpesvirus Suido 1/genética , Neuronas/citología , Neuronas/patología , Neuronas/virología , Virulencia , Replicación ViralRESUMEN
1. Despite the emerging framework that central neural pathways controlling the activity of the sympathetic nervous system are capable of producing highly selective responses, the specific neural pathways governing different sympathetic outflows are poorly understood. 2. Anatomical studies suggest that five brain areas, namely the rostral ventrolateral medulla, the rostral ventromedial medulla, the caudal raphe nuclei, the region containing the A5 noradrenergic neurons and the paraventricular hypothalamic nucleus, provide dominant supraspinal innervation of sympathetic preganglionic neurons. 3. The anatomical parcellation of different functions within and among these cell groups is uncertain. However, recent studies using transynaptic retrograde labelling of neural pathways connected to various sympathetic targets suggest that the circuits controlling these different targets may be partially distinct. Similarly, anatomical studies relying on stimulus-evoked expression of immediate early genes, such as c-fos, suggest that different sympathetic responses may be controlled by distinct, neural circuits. 4. Thus, although many similarities exist in the anatomical circuits innervating different sympathetic targets, possibly supporting the orchestration of global sympathetic responses, differences are also discernible.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Fibras Adrenérgicas/fisiología , Genes fos/fisiología , Bulbo Raquídeo/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Animales , Humanos , Riñón/fisiología , Bulbo Raquídeo/anatomía & histología , Vías Nerviosas/fisiología , Núcleo Hipotalámico Paraventricular/anatomía & histología , Glándula Pineal/fisiologíaRESUMEN
Over the past decade there has been a dramatic increase in the use of viruses as transneuronal tracers of neuronal circuitry. The method exploits the propensity of neurotropic viruses to invade neurons and then produce infectious progeny that cross synapses to infect other neurons within a circuit. The protocols and commentaries included in this unit focus upon the use of the swine alpha herpesvirus known as pseudorabies virus (PRV) for polysynaptic analysis. Here, the aspects of experimental design that have the greatest import for successful use of viruses in circuit definition are presented. Accordingly, the protocols included in this unit can be applied in concert with methods in which the use of classical tract tracers has been detailed. A procedure for retrograde infection of CNS circuits in the rat CNS by peripheral injection of virus is detailed, while transneuronal analysis by intracerebral injection is also described. A variant of these procedures, transneuronal analysis with multiple recombinant strains, is also described along with methods for growing and titering viral stocks, and procedures for single and dual immunohistochemical localization of viral antigens in fixed brain tissue.
Asunto(s)
Herpesvirus Suido 1/química , Inmunohistoquímica/métodos , Red Nerviosa/química , Red Nerviosa/virología , Neuronas/química , Neuronas/virología , Animales , Transporte Biológico/genética , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/metabolismo , Red Nerviosa/citología , Red Nerviosa/metabolismo , Neuronas/metabolismo , RatasRESUMEN
Transneuronal tracing of neuronal circuitry with neurotropic viruses has provided valuable insights in the way in which the nervous system imposes temporal organization on physiological processes and behavior. The swine alpha herpes virus known as pseudorabies virus, or PRV, has been particularly useful in this regard. Early studies identified attenuated mutants with selective tropism for visual circuitry involved in circadian regulation, and subsequent experiments employing this virus have provided considerable insight into the polysynaptic organization of the suprachiasmatic nuclei and associated circuitry. This literature, which has emerged during the past decade, is the subject of this mini review.
Asunto(s)
Encéfalo/virología , Ritmo Circadiano , Herpesvirus Suido 1/fisiología , Seudorrabia/fisiopatología , Animales , Herpesvirus Suido 1/genética , Neuronas/patología , Neuronas/fisiología , Seudorrabia/patología , PorcinosRESUMEN
Transsynaptic tracing with live virus is a powerful tool that has been used extensively to analyze central efferents that regulate peripheral targets. More recently, investigators have begun to use this new methodology with central injections to identify circuit anatomy within the brain. Although transsynaptic tracing with peripheral injection of pseudorabies virus has been extensively characterized, several methodological issues related to central application of this tracer have not been addressed. Here, we review the following issues relevant to the use of pseudorabies virus (PRV; Bartha strain) in experiments involving injection of virus into rat brain: (i) factors that determine the zone of viral uptake; (ii) uptake of pseudorabies virus by fibers of passage; (iii) viral invasion of the brain after leakage of virus into the brain ventricles; (iv) considerations for double labeling for PRV with peptides and neurotransmitters; (v) use of PRV with conventional retrograde tracers to anatomically identify relays in a multisynaptic pathway; and (vi) transport of PRV throughout the dendritic tree as a means of identifying inputs to distal dendrites. Collectively, the data demonstrate that PRV provides a powerful means of dissecting the synaptology of CNS circuitry when appropriate controls are incorporated into the experimental design. A set of recipes for various procedures are included at the end of this article.
Asunto(s)
Encéfalo/citología , Herpesvirus Suido 1/metabolismo , Red Nerviosa/citología , Vías Nerviosas/citología , Neuronas/citología , Sinapsis/ultraestructura , Animales , Axones/fisiología , Axones/ultraestructura , Axones/virología , Encéfalo/fisiología , Encéfalo/virología , Líquido Cefalorraquídeo/virología , Colorantes , Red Nerviosa/fisiología , Red Nerviosa/virología , Vías Nerviosas/fisiología , Vías Nerviosas/virología , Neuronas/fisiología , Neuronas/virología , Seudorrabia/metabolismo , Sinapsis/patología , Sinapsis/virologíaRESUMEN
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.
Asunto(s)
Vías Eferentes/fisiología , Herpesvirus Suido 1/metabolismo , Neuronas Motoras/fisiología , Nervio Frénico/fisiología , Recto del Abdomen/inervación , Animales , Diafragma/inervación , Vías Eferentes/citología , Vías Eferentes/virología , Hurones , Técnica del Anticuerpo Fluorescente , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Herpesvirus Suido 1/genética , Proteínas Luminiscentes/genética , Masculino , Neuronas Motoras/citología , Neuronas Motoras/virología , Nervio Frénico/citología , Nervio Frénico/virología , Núcleos del Rafe/citología , Núcleos del Rafe/fisiología , Respiración , Formación Reticular/citología , Formación Reticular/fisiología , Vómitos/fisiopatología , beta-Galactosidasa/genéticaRESUMEN
The development of neuronal projections to a target and the establishment of synaptic connections with that target can be temporally distinct events, which typically are distinguished by functional assessments. We have applied a novel neuroanatomical approach to characterize the development of limbic forebrain synaptic inputs to autonomic neurons in neonatal rats. Transneuronal labeling of preautonomic forebrain neurons was achieved by inoculating the ventral stomach wall with pseudorabies virus (PRV) on postnatal day 1 (P1), P4, or P8. In each age group, PRV-positive neurons were present in autonomic and preautonomic regions of the spinal cord and brainstem 62-64 hr after inoculation. Transneuronal forebrain labeling in rats injected on P8 was similar to the transneuronal labeling reported previously in adult rats and included neurons in the medial and lateral hypothalamus, amygdala, bed nucleus of the stria terminalis, and visceral cortices. However, no cortex labeling and only modest amygdala and bed nucleus labeling were observed in rats injected with PRV on P4, and only medial hypothalamic labeling was observed in rats injected on P1. Additional tracing experiments involving central injections of PRV or cholera toxin beta indicated that lateral hypothalamic and telencephalic regions projected to the medullary dorsal vagal complex several days before establishing synaptic connections with gastric-related autonomic neurons. These results demonstrate a novel strategy for evaluating synaptic connectivity in developing neural circuits and show a temporally segregated postnatal emergence of medial hypothalamic, lateral hypothalamic, and telencephalic synaptic inputs to central autonomic neurons.