RESUMEN
Although a number of studies have considered the neural circuitry that regulates diaphragm activity, these pathways have not been adequately discerned, particularly in animals such as cats that utilize the respiratory muscles during a variety of different behaviors and movements. The present study employed the retrograde transneuronal transport of rabies virus to identify the extended neural pathways that control diaphragm function in felines. In all animals deemed to have successful rabies virus injections into the diaphragm, large, presumed motoneurons were infected in the C(4)-C(6) spinal segments. In addition, smaller presumed interneurons were labeled bilaterally throughout the cervical and upper thoracic spinal cord. While in short and intermediate survival cases, infected interneurons were concentrated in the vicinity of phrenic motoneurons, in late survival cases, the distribution of labeling was more expansive. Within the brain stem, the earliest infected neurons included those located in the classically defined pontine and medullary respiratory groups, the medial and lateral medullary reticular formation, the region immediately ventral to the spinal trigeminal nucleus, raphe pallidus and obscurus, and the vestibular nuclei. At longer survival times, infection appeared in the midbrain, which was concentrated in the lateral portion of the periaqueductal gray, the region of the tegmentum that contains the locomotion center, and the red nucleus. Considerable labeling was also present in the fastigial nucleus of the cerebellum, portions of the posterior and lateral hypothalamus and the adjacent fields of Forel known to contain hypocretin-containing neurons and the precruciate gyrus of cerebral cortex. These data raise the possibility that several parallel pathways participate in regulating the activity of the feline diaphragm, which underscores the multifunctional nature of the respiratory muscles in this species.
Asunto(s)
Encéfalo/patología , Diafragma/inervación , Interneuronas/patología , Neuronas Motoras/patología , Rabia/patología , Nervios Espinales/patología , Coloración y Etiquetado/métodos , Animales , Transporte Axonal , Encéfalo/virología , Gatos , Diafragma/patología , Diencéfalo/patología , Diencéfalo/virología , Modelos Animales de Enfermedad , Femenino , Interneuronas/virología , Bulbo Raquídeo/patología , Bulbo Raquídeo/virología , Mesencéfalo/patología , Mesencéfalo/virología , Neuronas Motoras/virología , Vías Nerviosas/patología , Vías Nerviosas/virología , Puente/patología , Puente/virología , Rabia/virología , Virus de la Rabia/aislamiento & purificación , Virus de la Rabia/metabolismo , Nervios Espinales/virología , Telencéfalo/patología , Telencéfalo/virologíaRESUMEN
The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization.
Asunto(s)
Proteínas Fluorescentes Verdes/sangre , Herpesvirus Suido 1/metabolismo , Interneuronas/metabolismo , Núcleo Accumbens/metabolismo , Somatostatina/metabolismo , Coloración y Etiquetado , Animales , Interneuronas/citología , Interneuronas/virología , Masculino , Ratones , Núcleo Accumbens/citología , Núcleo Accumbens/virologíaRESUMEN
The subventricular zone produces neuroblasts that migrate to the olfactory bulb (OB) and differentiate into interneurons throughout postnatal life (Altman and Das, 1966; Hinds, 1968; Altman, 1969; Kishi et al., 1990; Luskin, 1993; Lois and Alvarez-Buylla, 1994). Although such postnatally generated interneurons have been characterized morphologically, their physiological differentiation has not been thoroughly described. Combining retroviral-mediated labeling of newly generated neurons with patch-clamp electrophysiology, we demonstrated that soon after new cells enter the layers of the olfactory bulb, they display voltage-dependent currents typical of more mature neurons. We also show that these "newcomers" express functional GABA and glutamate receptor channels, respond synaptically to stimulation of the olfactory nerve, and may establish both axodendritic and dendrodendritic synaptic contacts within the olfactory bulb. These data provide a basic description of the physiology of newly generated cells in the OB and show that such new cells are functional neurons that synaptically integrate into olfactory bulb circuitry soon after their arrival.
Asunto(s)
Electrofisiología/métodos , Neuronas/fisiología , Bulbo Olfatorio/fisiología , Canales de Potasio con Entrada de Voltaje , Animales , Diferenciación Celular , Linaje de la Célula , Movimiento Celular/fisiología , Canales de Potasio de Tipo Rectificador Tardío , Femenino , Proteínas Fluorescentes Verdes , Interneuronas/fisiología , Interneuronas/virología , Ventrículos Laterales/citología , Ventrículos Laterales/fisiología , Proteínas Luminiscentes/biosíntesis , Masculino , Neuronas/clasificación , Neuronas/virología , Bulbo Olfatorio/citología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Ratas , Ratas Wistar , Receptores de GABA/efectos de los fármacos , Receptores de GABA/metabolismo , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Retroviridae/fisiología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Retrograde transneuronal tracing with rabies virus from the right orbicularis oculi muscle was used to identify neural networks underlying spontaneous, reflex, and learned blinks. The kinetics of viral transfer was studied at sequential 12 hr intervals between 3 and 5 d after inoculation. Rabies virus immunolabeling was combined with the immunohistochemical detection of choline acetyltransferase expression in brainstem motoneurons or Fluoro-Ruby injections in the rubrospinal tract. Virus uptake involved exclusively orbicularis oculi motoneurons in the dorsolateral division of the facial nucleus. At 3-3.5 d, transneuronal transfer involved premotor interneurons of trigeminal, auditory, and vestibular reflex pathways (in medullary and pontine reticular formation, trigeminal nuclei, periolivary and ventral cochlear nuclei, and medial vestibular nuclei), motor pathways (dorsolateral quadrant of contralateral red nucleus and pararubral area), deep cerebellar nuclei (lateral portion of interpositus nucleus and dorsolateral hump ipsilaterally), limbic relays (parabrachial and Kölliker-Fuse nuclei), and oculomotor structures involved in eye-eyelid coordination (oculomotor nucleus, supraoculomotor area, and interstitial nucleus of Cajal). At 4 d, higher order neurons were revealed in trigeminal, auditory, vestibular, and deep cerebellar nuclei (medial, interpositus, and lateral), oculomotor and visual-related structures (Darkschewitsch, nucleus of the posterior commissure, deep layers of superior colliculus, and pretectal area), lateral hypothalamus, and cerebral cortex (particularly in parietal areas). At 4.5 and 5 d the labeling of higher order neurons occurred in hypothalamus, cerebral cortex, and blink-related areas of cerebellar cortex. These results provide a comprehensive picture of the premotor networks mediating reflex, voluntary, and limbic-related eyelid responses and highlight potential sites of motor learning in eyelid classical conditioning.
Asunto(s)
Parpadeo/fisiología , Neuronas Motoras/fisiología , Red Nerviosa/anatomía & histología , Red Nerviosa/fisiología , Virus de la Rabia/fisiología , Animales , Transporte Axonal/fisiología , Tronco Encefálico/anatomía & histología , Tronco Encefálico/fisiología , Tronco Encefálico/virología , Corteza Cerebelosa/anatomía & histología , Corteza Cerebelosa/fisiología , Corteza Cerebelosa/virología , Núcleos Cerebelosos/anatomía & histología , Núcleos Cerebelosos/fisiología , Núcleos Cerebelosos/virología , Corteza Cerebral/anatomía & histología , Corteza Cerebral/fisiología , Corteza Cerebral/virología , Párpados/inervación , Párpados/fisiología , Técnica del Anticuerpo Fluorescente , Hipotálamo/anatomía & histología , Hipotálamo/fisiología , Hipotálamo/virología , Interneuronas/citología , Interneuronas/fisiología , Interneuronas/virología , Neuronas Motoras/citología , Neuronas Motoras/virología , Red Nerviosa/virología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.
Asunto(s)
Trasplante de Células/métodos , Neuronas GABAérgicas/trasplante , Interneuronas/fisiología , Interneuronas/virología , Eminencia Media/fisiología , Eminencia Media/virología , Animales , Femenino , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/fisiología , Neuronas GABAérgicas/virología , Células HEK293 , Humanos , Interneuronas/citología , Interneuronas/trasplante , Lentivirus/genética , Eminencia Media/citología , Eminencia Media/trasplante , Ratones , Células-Madre Neurales/citología , Embarazo , Transducción de Señal , Transducción GenéticaRESUMEN
Primary pain and touch sensory neurons not only detect internal and external sensory stimuli, but also receive inputs from other neurons. However, the neuronal derived inputs for primary neurons have not been systematically identified. Using a monosynaptic rabies viruses-based transneuronal tracing method combined with sensory-specific Cre-drivers, we found that sensory neurons receive intraganglion, intraspinal, and supraspinal inputs, the latter of which are mainly derived from the rostroventral medulla (RVM). The viral-traced central neurons were largely inhibitory but also consisted of some glutamatergic neurons in the spinal cord and serotonergic neurons in the RVM. The majority of RVM-derived descending inputs were dual GABAergic and enkephalinergic (opioidergic). These inputs projected through the dorsolateral funiculus and primarily innervated layers I, II, and V of the dorsal horn, where pain-sensory afferents terminate. Silencing or activation of the dual GABA/enkephalinergic RVM neurons in adult animals substantially increased or decreased behavioral sensitivity, respectively, to heat and mechanical stimuli. These results are consistent with the fact that both GABA and enkephalin can exert presynaptic inhibition of the sensory afferents. Taken together, this work provides a systematic view of and a set of tools for examining peri- and extrasynaptic regulations of pain-afferent transmission.
Asunto(s)
Vías Aferentes/fisiología , Vías Eferentes/fisiología , Red Nerviosa/fisiología , Nocicepción/fisiología , Células Receptoras Sensoriales/fisiología , Asta Dorsal de la Médula Espinal/citología , Animales , Virus Defectuosos/fisiología , Encefalinas/fisiología , Miembro Anterior/inervación , Neuronas GABAérgicas/fisiología , Neuronas GABAérgicas/virología , Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Interneuronas/fisiología , Interneuronas/virología , Proteínas del Tejido Nervioso/análisis , Conducción Nerviosa , Neuronas Aferentes/fisiología , Neuronas Aferentes/virología , Neuronas Eferentes/fisiología , Neuronas Eferentes/virología , Nociceptores/fisiología , Células del Asta Posterior/fisiología , Células del Asta Posterior/virología , Terminales Presinápticos/fisiología , Virus de la Rabia/fisiología , Células Receptoras Sensoriales/clasificación , Células Receptoras Sensoriales/virología , Piel/inervación , Asta Dorsal de la Médula Espinal/fisiología , Asta Dorsal de la Médula Espinal/ultraestructura , Replicación Viral , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Male Sprague-Dawley rats, with their pelvic and hypogastric nerves transected, were infected with pseudorabies virus (PRV) injected into the external urethral sphincter. Animals were sacrificed at 2, 2.5, 3, and 4 days postinfection. Spinal cord and brain tissue were sectioned and processed by immunohistochemical techniques with antisera against PRV and choline acetyl transferase (CAT). At 2 days postinfection, virus-labeled neurons were found in the ventrolateral divisions of Onuf's nucleus and in the dorsal gray commissure (DGC). At progressively later incubation times, labeled neurons were found in the intermediolateral regions, the superficial layer of the dorsal horn, and the brainstem, in particular, the pontine micturition center. PRV/CAT-positive neurons were only found in Onuf's nucleus. Preganglionic neurons in the L6-S1 intermediolateral regions were CAT positive but PRV negative, thus suggesting that they are interneurons, not sacral parasympathetic preganglionic neurons. After 4 days, virus had spread to neurons in the paraventricular, preoptic, and even cortical regions. The distribution of these PRV-labeled brain neurons strongly resembled that obtained after the injection of PRV into the urinary bladder (Nadelhaft et al. [1992] Neurosci. Lett. 143:271-274). In both cases, neurons were labeled in the DGC in the spinal cord. The data therefore suggest that neurons in the DGC may be involved in the integrated control of the bladder and the external urethral sphincter.
Asunto(s)
Encéfalo/virología , Herpesvirus Suido 1/aislamiento & purificación , Neuronas/virología , Médula Espinal/virología , Uretra/virología , Animales , Encéfalo/citología , Mapeo Encefálico , Colina O-Acetiltransferasa/análisis , Ganglios Espinales/citología , Ganglios Espinales/virología , Inmunohistoquímica , Inyecciones , Interneuronas/virología , Masculino , Puente/virología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Uretra/inervaciónRESUMEN
Although indirect evidence suggests that the control of sympathetic preganglionic neurons is mediated to a great extent through interneurons, little is known about the location, morphology or neurotransmitter phenotype of such interneurons. This limitation seriously impedes our understanding of spinal synaptic circuits crucial to control of arterial pressure and other visceral functions. We used a highly neurotropic, minimally cytopathic recombinant herpes simplex virus type-1 to study spinal "sympathetic" interneurons labelled by trans-synaptic transport of the virus from the adrenal gland in rats. Approximately 120-320 infected neurons/rat were identified by immunocytochemical detection of the viral antigen. We distinguished between virus-infected preganglionic neurons and infected interneurons by (i) their location within the spinal laminae, (ii) their size and shape and (iii) the presence or absence of immunoreactivity for the acetylcholine-synthesizing enzyme, choline acetyltransferase, a marker of sympathetic preganglionic neurons. Virus-labelled sympathetic preganglionic neurons were found within the known spinal preganglionic nuclei. Non-cholinergic, virus-labelled neurons were located throughout lamina VII and in the ventral portion of lamina V. These putative interneurons were found in the major spinal preganglionic nuclei, usually intermingled with the preganglionic neurons. Sometimes, they were located in clusters separate from the preganglionic neurons. The interneurons were approximately 15 microm in diameter, smaller than the average preganglionic neuron (diameter=25 microm), and had a few fine processes emanating from them. These non-cholinergic interneurons constituted approximately one-half of the population of virus-infected neurons. In summary, with the use of a recombinant herpes simplex virus, we identified a large number of non-cholinergic interneurons close to, or intermingled with, adrenal sympathetic preganglionic neurons. The neurotransmitter phenotype of these neurons remains to be determined but they likely integrate much of the supraspinal and primary afferent inputs to spinal preganglionic neurons that control arterial pressure and other visceral functions.
Asunto(s)
Glándulas Suprarrenales/citología , Fibras Adrenérgicas/química , Interneuronas/química , Receptores Presinapticos/análisis , Simplexvirus , Fibras Adrenérgicas/enzimología , Animales , Colina O-Acetiltransferasa/análisis , Técnica del Anticuerpo Fluorescente , Ganglios Simpáticos/química , Ganglios Simpáticos/citología , Ganglios Simpáticos/enzimología , Ácido Glutámico/análisis , Herpes Simple , Interneuronas/enzimología , Interneuronas/virología , Masculino , Ratas , Ratas Wistar , Proteínas Recombinantes de FusiónRESUMEN
Mechanisms by which perinatal viral infections can disrupt hippocampal development and cause selective neuronal death may have implications for temporal lobe epilepsy and schizophrenia. Despite abnormalities of inhibitory interneurons in these diseases, the causal relationships between such neurotransmitter changes and viral infections remain unclear. This relationship was examined in a model in which rats, infected with lymphocytic choriomeningitis virus (LCMV) as neonates, manifest a gradual loss of hippocampal dentate granule cells and neuronal hyperexcitability. The current data demonstrate that GABAergic interneurons are dual immunostained for LCMV antigens prior to the loss of dentate granule cells, supporting the hypothesis that LCMV may disrupt developing inhibitory circuits causing unbalanced excitatory neurotransmission and the eventual death of dentate granule cells due to excitotoxicity.
Asunto(s)
Epilepsia del Lóbulo Temporal/virología , Hipocampo/crecimiento & desarrollo , Hipocampo/virología , Interneuronas/virología , Inhibición Neural/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Enfermedades Virales del Sistema Nervioso Central/patología , Enfermedades Virales del Sistema Nervioso Central/fisiopatología , Enfermedades Virales del Sistema Nervioso Central/virología , Giro Dentado/patología , Giro Dentado/fisiopatología , Giro Dentado/virología , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Hipocampo/patología , Hipocampo/fisiopatología , Interneuronas/metabolismo , Interneuronas/patología , Coriomeningitis Linfocítica/patología , Coriomeningitis Linfocítica/fisiopatología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/virología , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Endogámicas Lew , Esquizofrenia/patología , Esquizofrenia/fisiopatología , Esquizofrenia/virologíaRESUMEN
The potency of spinal sympathetic reflexes is increased after spinal injury, and these reflexes may result in life-threatening hypertensive crises in humans. Few, if any, primary afferents project directly to sympathetic preganglionic neurons (SPN). Therefore, spinal sympathetic interneurons (IN) must play a major role in generating dysfunctional sympathetic activity after spinal cord injury. Furthermore, these IN are potentially aberrant targets, either for ascending and descending axons that may sprout after spinal cord injury or for axons that regenerate after spinal cord injury. We identified IN via the transsynaptic retrograde transport of pseudorabies virus (PRV) injected into the kidneys of rats. The proportion of infected IN ranged from approximately 1/3 to approximately 2/3 of the number of infected SPN. IN were heavily concentrated among the SPN in spinal lamina VII. However, IN were located in all lamina of the dorsal horn. The longitudinal distribution of infected IN was closely correlated with the longitudinal distribution of infected SPN. Few infected IN were found rostral or caudal to the longitudinal range of infected SPN. Infected IN were heterogeneous in both their sizes and the extent of their dendritic trees. The strong correlation between longitudinal distributions of infected IN and SPN supports physiological data demonstrating a segmental organization of spinal sympathetic reflexes. The paucity of infected IN in segments distant from SPN suggests that multisegmental sympathetic reflexes are mediated by projections onto IN rather than onto SPN themselves. The morphological heterogeneity of IN probably manifests the variety of systems that affect spinal sympathetic regulation.
Asunto(s)
Herpesvirus Suido 1/química , Interneuronas/citología , Riñón/citología , Médula Espinal/citología , Animales , Interneuronas/química , Interneuronas/virología , Riñón/química , Riñón/virología , Masculino , Ratas , Ratas Sprague-Dawley , Médula Espinal/química , Médula Espinal/virologíaRESUMEN
We labeled interneurons in the L1-L2 and L6-S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus (PRV) in normal animals and in animals with selected nerve transections. Preganglionic neurons were identified using antisera against choline acetyltransferase (ChAT). In some experiments we labelled parasympathetic preganglionic neurons (PPNs) in the L6-S1 spinal cord by retrograde transport of Fluorogold from the major pelvic ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information from the bladder connects, via interneurons in L6-S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6-S1 ganglionectomies or L6-S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons (but not PPNs) with PRV in the L6-S1 spinal cord in a location very similar to those described in the intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1-L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve (presumably excitatory to the detrusor muscle); and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric (presumably inhibitory to the detrusor muscle).
Asunto(s)
Interneuronas/fisiología , Neuronas Aferentes/fisiología , Neuronas Eferentes/fisiología , Médula Espinal/fisiología , Estilbamidinas , Micción/fisiología , Animales , Toxina del Cólera , Colina O-Acetiltransferasa/metabolismo , Femenino , Colorantes Fluorescentes , Herpesvirus Suido 1/fisiología , Interneuronas/virología , Vértebras Lumbares , Fenómenos Fisiológicos del Sistema Nervioso , Vías Nerviosas/fisiología , Neuronas/enzimología , Pelvis/inervación , Ratas , Ratas Sprague-Dawley , Reflejo/fisiología , Médula Espinal/citología , Vejiga Urinaria/inervaciónRESUMEN
We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V-VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles.
Asunto(s)
Interneuronas/química , Neuronas Motoras/química , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Virus de la Rabia , Médula Espinal/química , Animales , Animales Recién Nacidos , Interneuronas/virología , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/fisiología , Neuronas Motoras/virología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/virologíaRESUMEN
BACKGROUND: The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells. CONCLUSIONS/SIGNIFICANCE: The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.
Asunto(s)
Herpesvirus Suido 1/crecimiento & desarrollo , Neuronas Motoras/virología , Médula Espinal/citología , Animales , Animales Recién Nacidos , Inyecciones Intramusculares , Interneuronas/virología , RatonesRESUMEN
Although monosynaptic bulbospinal projections to phrenic motoneurons have been extensively described, little is known about the organization of phrenic premotor neurons in the adult rat spinal cord. Because interneurons may play an important role in normal breathing and recovery following spinal cord injury, the present study has used anterograde and transneuronal retrograde tracing to study their distribution and synaptic relations. Exclusive unilateral, first-order labeling of the phrenic motoneuron pool with pseudorabies virus demonstrated a substantial number of second-order, bilaterally distributed cervical interneurons predominantly in the dorsal horn and around the central canal. Combined transneuronal and anterograde tracing revealed ventral respiratory column projections to prephrenic interneurons, suggesting that some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual-labeling studies with pseudorabies virus recombinants also showed prephrenic interneurons integrated with either contralateral phrenic or intercostal motoneuron pools. The stability of interneuronal pseudorabies virus labeling patterns following lateral cervical hemisection was then addressed. Except for fewer infected contralateral interneurons at the level of the central canal, the number and distribution of phrenic-associated interneurons was not significantly altered 2 weeks posthemisection (i.e., the point at which the earliest postinjury recovery of phrenic activity has been reported). These results demonstrate a heterogeneous population of phrenic-related interneurons. Their connectivity and relative stability after cervical hemisection raise speculation for potentially diverse roles in modulating phrenic function normally and postinjury.
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Interneuronas/citología , Bulbo Raquídeo/citología , Nervio Frénico/citología , Centro Respiratorio/citología , Fenómenos Fisiológicos Respiratorios , Médula Espinal/citología , Animales , Biomarcadores , Mapeo Encefálico , Diafragma/inervación , Diafragma/fisiología , Estimulación Eléctrica , Femenino , Lateralidad Funcional/fisiología , Herpesvirus Suido 1 , Interneuronas/fisiología , Interneuronas/virología , Bulbo Raquídeo/fisiología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neuronas Motoras/virología , Red Nerviosa/citología , Red Nerviosa/fisiología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Nervio Frénico/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Centro Respiratorio/fisiología , Médula Espinal/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Coloración y EtiquetadoRESUMEN
In central nervous system (CNS) tissue preparations, wild-type Semliki Forest virus (SFV) mainly infects neurons, and in vivo it causes lethal encephalitis in neonatal and adult rodents. The SFV strain A7(74), by contrast, is avirulent in adult rodents, triggering only limited CNS infection. To examine A7(74) infection in hippocampal tissue, the authors constructed a replicon, termed SFV(A774nsP)-GFP, expressing green fluorescent protein. The results were compared to replication-proficient recombinant A7(74) encoding GFP, named VA7-EGFP. As nonstructural gene mutations can confer temperature sensitivity, the authors also tested whether infection was temperature-dependent. Indeed, at 31 degrees C both viral recombinants transduced significantly more baby hamster kidney cells than at 37 degrees C. When rat hippocampal slices and dissociated cells were incubated at 37 degrees C, SFV(A774nsP)-GFP transduced glial cells but virtually no neurons-the opposite of conventional SFV. For VA7-EGFP at 37 degrees C, the preferred GFP-positive cells in hippocampal slices were also non-neuronal cells. At 31 degrees C, however, a more wild-type phenotype was found, with 33% and 94% of the GFP-positive cells being neurons for SFV(A774nsP)-GFP in slices and dissociated cells, respectively, and 94% neurons for VA7-EGFP in slices. Immunochemical and electrophysiological analyses confirmed that at 37 degrees C virtually all cells transduced by SFV(A774nsP)-GFP in slices were astrocytes, while at 31 degrees C they also contained neurons. These results show that in addition to the developmental age, the temperature determines which cell type becomes infected by A7(74). Our data suggest that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurons at body temperature, whereas it still does so at lower temperatures.
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Astrocitos/virología , Vectores Genéticos , Hipocampo/virología , Células Piramidales/virología , Virus de los Bosques Semliki/genética , Transducción Genética , Animales , Astrocitos/citología , Células Cultivadas , Cricetinae , Genoma Viral , Proteínas Fluorescentes Verdes , Hipocampo/citología , Indicadores y Reactivos/metabolismo , Interneuronas/citología , Interneuronas/virología , Riñón/citología , Proteínas Luminiscentes/genética , Potenciales de la Membrana , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Fenotipo , Células Piramidales/citología , Células Piramidales/fisiología , Virus de los Bosques Semliki/crecimiento & desarrollo , Virus de los Bosques Semliki/patogenicidad , Temperatura , Virulencia , Replicación ViralRESUMEN
The transneuronal herpesvirus tracer, pseudorabies virus (PRV) was used to determine the dendritic architecture of cardiac-related neurons. We constructed a derivative of the Bartha strain of PRV called PRV-BaBlu, that carries the lacZ gene of E. coli. Expression of beta-galactosidase by this recombinant virus enabled us to define the dendritic morphology of motoneurons and interneurons that innervate the heart. beta-galactosidase antigen filled dendritic processes that were clearly revealed by antibodies to beta-galactosidase. In contrast, the standard enzymatic reaction for detection of beta-galactosidase activity stained the cell soma well, but was inferior for labeling dendrites. Following PRV-BaBlu cardiac injection, infected neurons were clearly defined and labeled dendrites could be traced for long distances, sometimes greater than 800 microns from the cell body. Labeled dendrites of cardiomotor neurons primarily located in the nucleus ambiguus (NA) were extensive and sometimes intertwined with dendrites from other labeled motoneurons. Dendrites of labeled neurons in the dorsal motor nucleus of the vagus (DMV) typically extended in the mediolateral direction in the transverse plane. Transynaptically labeled interneurons interposed between the cardiorespiratory region of the nucleus tractus solitarius (NTS) and the NA were primarily located in the NA region and the reticular arc, the area between the DMV and NA. These interneurons had long dendrites extending along the reticular arc in the transverse plane. The dendritic arborizations of infected cardiac-related neurons in the NTS were variable in extent. We conclude that antibody detection of beta-galactosidase expressed by PRV-BaBlu after infection of neural cardiac circuits provides a superior method to define the dendrites and dendritic fields of cardiac-related motoneurons and interneurons.