RESUMEN
During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation in vivo but is not well studied. As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization. We compared acutely isolated retinal ganglion cells (RGCs) at various developmental stages and RGCs differentiated in vitro from embryonic retinal progenitors for the effects of aging and, independently, of retinal environment age on their GABAA receptor (GABAAR) responses, elicited by muscimol. We found that neurons generated in vitro from progenitors exhibited depolarizing, immature GABA responses, like those of early postnatal RGCs. As progenitor-derived neurons aged from 1 to 3 weeks, their GABA responses matured. Interestingly, signals secreted by the early postnatal retina suppressed acquisition of mature GABA responses. This suppression was not associated with changes in expression of GABAAR or of the chloride co-transporter KCC2, but rather with inhibition of KCC2 dimerization in differentiating neurons. Taken together, these data indicate GABA response maturation depends on release of inhibition by developmentally regulated diffusible signals from the retina.
Asunto(s)
Receptores de GABA-A/metabolismo , Células Ganglionares de la Retina/fisiología , Neuronas Retinianas/fisiología , Células Madre/fisiología , Simportadores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Ratas , Células Ganglionares de la Retina/citología , Neuronas Retinianas/citología , Células Madre/citología , Cotransportadores de K ClRESUMEN
What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENT Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in â¼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy.
Asunto(s)
Envejecimiento/fisiología , Redes Reguladoras de Genes/fisiología , Células Ganglionares de la Retina/fisiología , Factores de Transcripción SOXC/metabolismo , Activación Transcripcional/fisiología , Vías Visuales/fisiología , Animales , Células Cultivadas , Retroalimentación Fisiológica/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ratones , Ratas Sprague-DawleyRESUMEN
Retinal ganglion cells (RGCs) degenerate in diseases like glaucoma and are not replaced in adult mammals. Here we investigate whether transplanted RGCs can integrate into the mature retina. We have transplanted GFP-labelled RGCs into uninjured rat retinas in vivo by intravitreal injection. Transplanted RGCs acquire the general morphology of endogenous RGCs, with axons orienting towards the optic nerve head of the host retina and dendrites growing into the inner plexiform layer. Preliminary data show in some cases GFP(+) axons extending within the host optic nerves and optic tract, reaching usual synaptic targets in the brain, including the lateral geniculate nucleus and superior colliculus. Electrophysiological recordings from transplanted RGCs demonstrate the cells' electrical excitability and light responses similar to host ON, ON-OFF and OFF RGCs, although less rapid and with greater adaptation. These data present a promising approach to develop cell replacement strategies in diseased retinas with degenerating RGCs.
Asunto(s)
Axones , Dendritas , Cuerpos Geniculados , Luz , Nervio Óptico , Células Ganglionares de la Retina/trasplante , Colículos Superiores , Animales , Recuento de Células , Femenino , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Inyecciones Intravítreas , Masculino , Ratones , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , RetinaRESUMEN
Innexins are bifunctional membrane proteins in invertebrates, forming gap junctions as well as non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Pannexins appear to form only non-junctional membrane channels (pannexons). The membrane channels formed by pannexins and innexins are similar in their biophysical and pharmacological properties. Innexons and pannexons are permeable to ATP, are present in glial cells, and are involved in activation of microglia by calcium waves in glia. Directional movement and accumulation of microglia following nerve injury, which has been studied in the leech which has unusually large glial cells, involves at least 3 signals: ATP is the "go" signal, NO is the "where" signal and arachidonic acid is a "stop" signal.
Asunto(s)
Señalización del Calcio , Conexinas/metabolismo , Potenciales de la Membrana , Adenosina Trifosfato/metabolismo , Animales , Conexinas/genética , Uniones Comunicantes/metabolismo , Uniones Comunicantes/fisiología , Invertebrados/química , Invertebrados/metabolismo , Neuroglía/metabolismo , Neuroglía/fisiologíaRESUMEN
Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin- and of pannexin-injected oocytes; ArA also blocked K(+) -induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye-release and, evidently, ATP-release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non-metabolizable analog eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions.
Asunto(s)
Ácido Araquidónico/farmacología , Movimiento Celular/efectos de los fármacos , Conexinas/metabolismo , Microglía/metabolismo , Animales , Calcio/metabolismo , Movimiento Celular/fisiología , Uniones Comunicantes/efectos de los fármacos , Sanguijuelas/metabolismo , Compresión Nerviosa/métodos , Oocitos/metabolismoRESUMEN
Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.
Asunto(s)
Fibras Nerviosas/fisiología , Células Ganglionares de la Retina/citología , Ingeniería de Tejidos/métodos , Animales , Axones/fisiología , Supervivencia Celular , Fenómenos Electrofisiológicos , Ratones , Ratas , Ratas Sprague-Dawley , Andamios del Tejido/químicaRESUMEN
Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells' ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.
Asunto(s)
Adenosina Trifosfato/metabolismo , Conexinas/metabolismo , Neuroglía/metabolismo , Neuroglía/fisiología , Animales , Señalización del Calcio , Movimiento Celular , Conexinas/genética , Sanguijuelas , Microglía , Compresión Nerviosa , Oocitos/metabolismo , Interferencia de ARN , Traumatismos del Sistema Nervioso/metabolismo , XenopusRESUMEN
Respiratory rhythms arise from neurons situated in the ventral medulla. We are investigating their spatial and functional relationships optically by measuring changes in intracellular calcium using the fluorescent, calcium-sensitive dye Oregon Green 488 BAPTA-1 AM while simultaneously recording the regular firing of motoneurons in the phrenic nerve in isolated brainstem/spinal cord preparations of E17 to E19 mice. Responses of identified cells are associated breath by breath with inspiratory and expiratory phases of respiration and depend on CO(2) and pH levels. Optical methods including two-photon microscopy are being developed together with computational analyses. Analysis of the spatial pattern of neuronal activity associated with respiratory rhythm, including cross-correlation analysis, reveals a network distributed in the ventral medulla with intermingling of neurons that are active during separate phases of the rhythm. Our experiments, aimed at testing whether initiation of the respiratory rhythm depends on pacemaker neurons, on networks or a combination of both, suggest an important role for networks.
Asunto(s)
Tronco Encefálico/fisiología , Calcio/metabolismo , Feto/fisiología , Microscopía Fluorescente/métodos , Red Nerviosa/fisiología , Mecánica Respiratoria/fisiología , Animales , Tronco Encefálico/anatomía & histología , Dióxido de Carbono/metabolismo , Colorantes Fluorescentes , Ratones , Red Nerviosa/anatomía & histología , Compuestos OrgánicosRESUMEN
Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP-PNP (adenyl-5'-yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 microM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC-specific inhibitor LY83583 applied at 50 microM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.
Asunto(s)
Adenosina Trifosfato/farmacología , Movimiento Celular/efectos de los fármacos , Microglía/efectos de los fármacos , Óxido Nítrico/metabolismo , Traumatismos del Sistema Nervioso/patología , Aminoquinolinas/farmacología , Análisis de Varianza , Animales , GMP Cíclico/metabolismo , Óxidos N-Cíclicos/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Imidazoles/farmacología , Técnicas In Vitro , Sanguijuelas , Microglía/fisiología , Nucleótidos/farmacología , Traumatismos del Sistema Nervioso/fisiopatología , TriazinasRESUMEN
Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.
Asunto(s)
Conexinas/fisiología , Animales , Señalización del Calcio , Citoplasma/metabolismo , Uniones Comunicantes/metabolismo , Sanguijuelas/metabolismo , Modelos Biológicos , Neuroglía/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Xenopus/metabolismoRESUMEN
Functional recovery following central nervous system (CNS) injury in adult animals may depend on the reestablishment of the precise pattern of connections made during development. When the nervous system is injured during embryonic development, functional recovery may involve the formation of atypical connections. Can such atypical synapses regenerate in adults, particularly in a nervous system known for its capacity for repair? When the S interneuron in one segmental ganglion of the leech Hirudo is killed during development, two neighboring S cells extend their axons into the ganglion and restore function by making electrical synapses with the usual synaptic targets of the killed S cell. Although adult S-cell axons reliably regenerated their usual synaptic connections, the novel synapses induced following embryonic injury failed to regenerate in adults. In these preparations severed S-cell axons did not reach the denervated ganglion but grew close to it, independent of the distance required to grow. Thus, the developmental changes that permit aberrant but functional connections in embryos do not lead to a similar change in the capacity for axon growth and subsequent synapse regeneration in adults.
Asunto(s)
Interneuronas/metabolismo , Sanguijuelas/embriología , Regeneración Nerviosa/fisiología , Sinapsis/fisiología , Animales , Axotomía , Electrofisiología , Ganglios de Invertebrados/lesiones , Ganglios de Invertebrados/fisiología , Interneuronas/patologíaAsunto(s)
Relojes Biológicos/fisiología , Bulbo Raquídeo/fisiología , Neuronas/fisiología , Insuficiencia Respiratoria/fisiopatología , Mecánica Respiratoria/fisiología , Animales , Antiinflamatorios/farmacología , Anticonvulsivantes/farmacología , Relojes Biológicos/efectos de los fármacos , Ácido Flufenámico/farmacología , Canales Iónicos/fisiología , Mamíferos/fisiología , Neurotransmisores/fisiología , Periodicidad , Centro Respiratorio/fisiología , Fenómenos Fisiológicos Respiratorios , Riluzol/farmacologíaRESUMEN
The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced. Pressure (P) sensory neurons were injected with biocytin to reveal the extent of their sprouting 24 hours after lesioning. To reduce microglia accumulation at lesions, cords were treated for 3.5 hours with 3 mM ATP or 2 mM N(omega)-nitro-L-arginine methyl ester (L-NAME) or 50 microM Reactive blue-2 (RB2) beginning 30 minutes before injury. Lesioned controls were either not treated with drug or treated 3 hours later with one of the drugs, after the migration and subsequent accumulation of most microglia had occurred, but before the onset of axon sprouting, for a total of seven separate conditions. There was a significant reduction in total sprout lengths compared with controls when microglial accumulation was reduced. The results suggest that microglial cells are necessary for the usual sprouting of injured axons.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/citología , Sanguijuelas/fisiología , Microglía/metabolismo , Regeneración Nerviosa/fisiología , Adenosina Trifosfato/fisiología , Animales , Sistema Nervioso Central/lesiones , Sistema Nervioso Central/fisiología , Sanguijuelas/citología , Microglía/citología , Compresión Nerviosa , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Óxido Nítrico/fisiología , Factores de Tiempo , Cicatrización de Heridas/fisiologíaRESUMEN
Sensitization of reflexive shortening in the leech has been linked to serotonin (5-HT)-induced changes in the excitability of a single interneuron, the S cell. This neuron is necessary for sensitization and complete dishabituation of reflexive shortening, during which it contributes to the sensory-motor reflex. The S cell does not contain 5-HT, which is released primarily from the Retzius (R) cells, whose firing enhances S-cell excitability. Here, we show that the S cell excites the R cells, mainly via a fast disynaptic pathway in which the first synapse is the electrical junction between the S cell and the coupling interneurons, and the second synapse is a glutamatergic synapse of the coupling interneurons onto the R cells. The S cell-triggered excitatory postsynaptic potential in the R cell diminishes and nearly disappears in elevated concentrations of divalent cations because the coupling interneurons become inexcitable under these conditions. Serotonin released from the R cells feeds back on the S cell and increases its excitability by activating a 5-HT7-like receptor; 5-methoxytryptamine (5-MeOT; 10 microM) mimics the effects of 5-HT on S cell excitability, and effects of both 5-HT and 5-MeOT are blocked by pimozide (10 microM) and SB-269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol] (5 microM). This feedback loop may be critical for the full expression of sensitization of reflexive shortening.
Asunto(s)
Umbral Diferencial/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Interneuronas/fisiología , Sanguijuelas/fisiología , Red Nerviosa/fisiología , Reflejo/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Retroalimentación/fisiología , Plasticidad Neuronal/fisiologíaRESUMEN
A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development. It cannot be assumed that once axons regenerate specific synapses, function will be restored. Recent work on the leech has shown following regeneration of the synapse between S-interneurons, which are required for sensitization of reflexive shortening, a form of non-associative learning, the capacity for sensitization is delayed. The steps in repair of synaptic connections in the leech are reviewed, with the aim of understanding general mechanisms that promote successful repair. New results are presented regarding the signals that regulate microglial migration to lesions, a first step in the repair process. In particular, microglia up to 900 microm from the lesion respond within minutes by moving rapidly toward the injury, controlled in part by nitric oxide (NO), which is generated immediately at the lesion and acts via a soluble guanylate cyclase (sGC). The cGMP produced remains elevated for hours after injury. The relationship of microglial migration to axon outgrowth is discussed.
Asunto(s)
Sanguijuelas/fisiología , Regeneración Nerviosa/fisiología , Vías Nerviosas/fisiología , Sinapsis/fisiología , AnimalesRESUMEN
Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.
Asunto(s)
Movimiento Celular/efectos de los fármacos , GMP Cíclico/biosíntesis , Inhibidores Enzimáticos/farmacología , Sanguijuelas/fisiología , Azul de Metileno/farmacología , Microglía/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Axones/fisiología , Movimiento Celular/fisiología , Sistema Nervioso Central/lesiones , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Microglía/fisiología , Compresión Nerviosa , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Óxido Nítrico/efectos adversos , Óxido Nítrico Sintasa , Óxido Nítrico Sintasa de Tipo IIIRESUMEN
The leech escape reflex-shortening of the body-can change with nonassociative conditioning, including sensitization, habituation, and dishabituation. Capacity for sensitization, which is an enhancement of the reflex, is lost when a single S-interneuron is ablated, but the reflex response itself remains. In the present experiments, the S-interneuron's axon in the living leech was filled with 6-carboxyfluorescein (6-CF) dye and cut with an argon laser microbeam (lambda = 488 nm). In contrast to sham-operated animals, axotomized preparations did not sensitize, reflecting the key role of the S-cell. By 2 weeks or more, S-cell axons had regenerated and reestablished synapses at their usual locations with neighboring S-cells. By 4 weeks, this restored the ability to sensitize to a level indistinguishable from that of controls, but an intermediate state of recovery was seen from 2-3 weeks after injury-a period not previously examined. The small capacity for sensitization among newly regenerated preparations was significantly lower than in sham controls but appeared higher than in animals whose cut S-cell axon had not regenerated its synapse. The results confirm the crucial role of the S-cell in sensitization. Moreover, full sensitization does not occur immediately upon synapse regeneration.
Asunto(s)
Interneuronas/fisiología , Aprendizaje/fisiología , Sanguijuelas/fisiología , Regeneración Nerviosa/fisiología , Sinapsis/fisiología , Animales , Axotomía , Electrofisiología , Conducción Nerviosa/fisiologíaRESUMEN
A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system.
Asunto(s)
3,3'-Diaminobencidina , Axones/efectos de los fármacos , Axotomía/métodos , Carbocianinas , Colorantes Fluorescentes , Estimulación Luminosa/efectos adversos , Fotoquímica/métodos , Médula Espinal/efectos de los fármacos , 3,3'-Diaminobencidina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Axones/efectos de la radiación , Axones/ultraestructura , Carbocianinas/farmacología , Femenino , Colorantes Fluorescentes/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/efectos de la radiación , Rayos Láser/efectos adversos , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa/fisiología , Zarigüeyas , Técnicas de Cultivo de Órganos , Estimulación Luminosa/instrumentación , Estimulación Luminosa/métodos , Fotoquímica/instrumentación , Médula Espinal/crecimiento & desarrollo , Médula Espinal/efectos de la radiación , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/fisiología , Raíces Nerviosas Espinales/cirugíaRESUMEN
Networks of electrically coupled neurons play an important role in coordinating activity among widely distributed neurons in the CNS. Such networks are sensitive to neuromodulation; but how modulation of individual cells affects activity of the entire network is not well understood. In the CNS of the medicinal leech, the S interneuron (S-cell) forms a network of electrically coupled neurons where each S-cell is linked to its two neighboring S-cells by electrical synapses. An action potential initiated in one cell is carried the length of the animal along this S-cell chain. The S-cell network is of interest because it is crucial for sensitization and dishabituation of the whole-body shortening reflex, although it is not necessary for reflexive shortening itself. Mechanosensory stimuli that produce shortening will directly elicit a train of action potentials by the S-cell network. This activity reflects the sum of action potential initiations in several S interneurons within the chain. The activity was enhanced by serotonin (5HT) in terms of both the total number of action potentials initiated and the average frequency of these initiations. Increases in evoked activity were accompanied by differential changes in the rates of action potential initiation in individual S-cells. 5HT only weakly enhanced initiations in S-cells that made a large contribution to the network-level response, while initiations in other, less active, S-cells were strongly enhanced by 5HT. This neurotransmitter also modulated the pattern of how activity was distributed throughout the network. 5HT-induced changes in activity patterns of the S-cell network may represent an important component of learning-related neuroplasticity in the leech shortening reflex.