RESUMO
Many developing networks exhibit a transient period of spontaneous activity that is believed to be important developmentally. Here we investigate the initiation of spontaneous episodes of rhythmic activity in the embryonic chick spinal cord. These episodes recur regularly and are separated by quiescent intervals of many minutes. We examined the role of motoneurons and their intraspinal synaptic targets (R-interneurons) in the initiation of these episodes. During the latter part of the inter-episode interval, we recorded spontaneous, transient ventral root depolarizations that were accompanied by small, spatially diffuse fluorescent signals from interneurons retrogradely labeled with a calcium-sensitive dye. A transient often could be resolved at episode onset and was accompanied by an intense pre-episode (approximately 500 ms) motoneuronal discharge (particularly in adductor and sartorius) but not by interneuronal discharge monitored from the ventrolateral funiculus (VLF). An important role for this pre-episode motoneuron discharge was suggested by the finding that electrical stimulation of motor axons, sufficient to activate R-interneurons, could trigger episodes prematurely. This effect was mediated through activation of R-interneurons because it was prevented by pharmacological blockade of either the cholinergic motoneuronal inputs to R-interneurons or the GABAergic outputs from R-interneurons to other interneurons. Whole-cell recording from R-interneurons and imaging of calcium dye-labeled interneurons established that R-interneuron cell bodies were located dorsomedial to the lateral motor column (R-interneuron region). This region became active before other labeled interneurons when an episode was triggered by motor axon stimulation. At the beginning of a spontaneous episode, whole-cell recordings revealed that R-interneurons fired a high-frequency burst of spikes and optical recordings demonstrated that the R-interneuron region became active before other labeled interneurons. In the presence of cholinergic blockade, however, episode initiation slowed and the inter-episode interval lengthened. In addition, optical activity recorded from the R-interneuron region no longer led that of other labeled interneurons. Instead the initial activity occurred bilaterally in the region medial to the motor column and encompassing the central canal. These findings are consistent with the hypothesis that transient depolarizations and firing in motoneurons, originating from random fluctuations of interneuronal synaptic activity, activate R-interneurons, which then trigger the recruitment of the rest of the spinal interneuronal network. This unusual function for R-interneurons is likely to arise because the output of these interneurons is functionally excitatory during development.
Assuntos
Interneurônios/fisiologia , Neurônios Motores/fisiologia , Vias Neurais/fisiologia , Medula Espinal/citologia , Medula Espinal/embriologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Atropina/farmacologia , Bicuculina/farmacologia , Embrião de Galinha , Corantes Fluorescentes , Antagonistas GABAérgicos/farmacologia , Bloqueadores Ganglionares/farmacologia , Glicinérgicos/farmacologia , Interneurônios/citologia , Mecamilamina/farmacologia , Neurônios Motores/citologia , Antagonistas Muscarínicos/farmacologia , Compostos Orgânicos , Técnicas de Patch-Clamp , Raízes Nervosas Espinhais/fisiologia , Estricnina/farmacologiaRESUMO
Underwater mass spectrometry systems can be used for direct in situ detection of volatile organic compounds and dissolved gases in oceans, lakes, rivers and waste-water streams. In this work we describe the design and operation of (1) a linear quadrupole mass filter and (2) a quadrupole ion trap mass spectrometer interfaced, in each case, with a membrane introduction/fluid control system and packaged for underwater operation. These mass spectrometry systems can operate autonomously, or under user control via a wireless rf link. Detection limits for each system were determined in the laboratory using pure solutions. The quadrupole mass filter system provides detection limits in the 1-5 ppb range with an upper mass limit of 100 amu. Its power requirement is approximately 95 Watts. The ion trap system has detection limits well below 1 ppb, an upper mass limit of 650 amu and MS/MS capability. Its power consumption is on the order of 150 Watts. The present membrane limits analysis to non-polar compounds (<300 amu) with analysis cycles of 5-15 minutes. Deployments of both types of instruments are described, along with a discussion of the challenges associated with in-water mass spectrometry and descriptions of alternative in-water mass spectrometer configurations.
RESUMO
The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1beta induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1beta induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases' transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium. A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.
Assuntos
Artrite Reumatoide/enzimologia , Metaloproteinases da Matriz/metabolismo , Proteína Amiloide A Sérica/biossíntese , Transcrição Gênica , Regulação para Cima , Sequência de Aminoácidos , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem Articular/metabolismo , Movimento Celular/genética , Condrócitos/enzimologia , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/isolamento & purificação , Proteína Amiloide A Sérica/fisiologia , Membrana Sinovial/enzimologia , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Transcrição Gênica/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
A number of homeodomain transcription factors have been implicated in controlling the differentiation of various types of neurons including spinal motoneurons. Some of these proteins are also expressed in spinal interneurons, but their function is unknown. Progress in understanding the role of transcription factors in interneuronal development has been slow because the synaptic connections of interneurons, which in part define their identity, are difficult to establish. Using whole cell recording in the isolated spinal cord of chick embryos, we assessed the synaptic connections of lumbosacral interneurons expressing the Engrailed-1 (En1) transcription factor. Specifically we established whether En1-expressing interneurons made direct connections with motoneurons and whether they constitute a single interneuron class. Cells were labeled with biocytin and subsequently processed for En1 immunoreactivity. Our findings indicate that the connections of En1-expressing cells with motoneurons and with sensory afferents were diverse, suggesting that the population was heterogeneous. In addition, the synaptic connections we tested were similar in interneurons that expressed the En1 protein and in many that did not. The majority of sampled En1 cells did, however, exhibit a direct synaptic connection to motoneurons that is likely to be GABAergic. Because our physiological methods underestimate the number of direct connections with motoneurons, it is possible that the great majority, perhaps all, En1-expressing cells make direct synaptic connections with motoneurons. Our results raise the possibility that En1 could be involved in interneuron-motoneuron connectivity but that its expression is not restricted to a distinct functional subclass of ventral interneuron. These findings constrain hypotheses about the role of En-1 in interneuron development and function.
Assuntos
Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/biossíntese , Interneurônios/química , Interneurônios/fisiologia , Lisina/análogos & derivados , Medula Espinal/citologia , Fatores de Transcrição , Potenciais de Ação/fisiologia , Animais , Embrião de Galinha , Imuno-Histoquímica , Neurônios Motores/citologia , Técnicas de Patch-Clamp , Medula Espinal/embriologia , Raízes Nervosas Espinhais/citologia , Sinapses/fisiologiaRESUMO
Studies on the development of synaptic specificity, embryonic activity, and neuronal specification in the spinal cord have all been limited by the absence of a functionally identified interneuron class (defined by its unique set of connections). Here, we identify an interneuron population in the embryonic chick spinal cord that appears to be the avian equivalent of the mammalian Renshaw cell (R-interneurons). These cells receive monosynaptic nicotinic, cholinergic input from motoneuron recurrent collaterals. They make predominately GABAergic connections back onto motoneurons and to other R-interneurons but project rarely to other spinal interneurons. The similarity between the connections of the developing R-interneuron, shortly after circuit formation, and the mature mammalian Renshaw cell raises the possibility that R-interneuronal connections are formed precisely from the onset. Using a newly developed optical approach, we identified the location of R-interneurons in a column, dorsomedial to the motor nucleus. Functional characterization of the R-interneuron population provides the basis for analyses that have so far only been possible for motoneurons.
Assuntos
Interneurônios/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/embriologia , Sinapses/fisiologia , Animais , Axônios/fisiologia , Bicuculina/farmacologia , Comunicação Celular , Embrião de Galinha , Estimulação Elétrica , Potenciais Evocados , Interneurônios/classificação , Interneurônios/citologia , Mamíferos , Mecamilamina/farmacologia , Morfogênese , Neurônios Motores/citologia , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/fisiologia , Receptores Colinérgicos/fisiologia , Receptores Nicotínicos/fisiologia , Especificidade da Espécie , Medula Espinal/citologia , Estricnina/farmacologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
To investigate the origin of spontaneous activity in developing spinal networks, we examined the activity patterns and synaptic organization of ventrally located lumbosacral interneurons, including those whose axons project into the ventrolateral funiculus (VLF), in embryonic day 9 (E9)-E12 chick embryos. During spontaneous episodes, rhythmic synaptic potentials were recorded from the VLF and from spinal interneurons that were synchronized, cycle by cycle, with rhythmic ventral root potentials. At the beginning of an episode, ventral root potentials started before the VLF discharge and the firing of individual interneurons. However, pharmacological blockade of recurrent motoneuron collaterals did not prevent or substantially delay interneuron recruitment during spontaneous episodes. The synaptic connections of interneurons were examined by stimulating the VLF and recording the potentials evoked in the ventral roots, in the VLF, or in individual interneurons. Low-intensity stimulation of the VLF evoked a short-latency depolarizing potential in the ventral roots, or in interneurons, that was probably mediated mono- or disynaptically. At higher intensities, long-latency responses were recruited in a highly nonlinear manner, eventually culminating in the activation of an episode. VLF-evoked potentials were reversibly blocked by extracellular Co2+, indicating that they were mediated by chemical synaptic transmission. Collectively, these findings indicate that ventral interneurons are rhythmically active, project to motoneurons, and are likely to be interconnected by recurrent excitatory synaptic connections. This pattern of organization may explain the synchronous activation of spinal neurons and the regenerative activation of spinal networks when provided with a suprathreshold stimulus.
Assuntos
Interneurônios/fisiologia , Medula Espinal/embriologia , Sinapses/fisiologia , Sinapses/ultraestrutura , Ciclos de Atividade , Animais , Embrião de Galinha , Estimulação Elétrica , Potenciais Evocados , Lateralidade Funcional , Técnicas In Vitro , Interneurônios/citologia , Potenciais da Membrana , Músculo Esquelético/inervação , Tempo de Reação , Medula Espinal/citologia , Raízes Nervosas Espinhais/embriologia , Transmissão SinápticaRESUMO
We examined the effects of spontaneous or evoked episodes of rhythmic activity on synaptic transmission in several spinal pathways of embryonic day 9-12 chick embryos. We compared the amplitude of synaptic potentials evoked by stimulation of the ventrolateral funiculus (VLF), the dorsal or ventral roots, before and after episodes of activity. With the exception of the short-latency responses evoked by dorsal root stimulation, the potentials were briefly potentiated and then reduced for several minutes after an episode of rhythmic activity. Their amplitude progressively recovered in the interval between successive episodes. The lack of post-episode depression in the short-latency component of the dorsal root evoked responses is probably attributable to the absence of firing in cut muscle afferents during an episode of activity. The post-episode depression of VLF-evoked potentials was mimicked by prolonged stimulation of the VLF, subthreshold for an episode of activity. By contrast, antidromically induced motoneuron firing and the accompanying calcium entry did not depress VLF-evoked potentials recorded from the stimulated ventral root. In addition, post-episode depression of VLF-evoked synaptic currents was observed in voltage-clamped spinal neurons. Collectively, these findings suggest that somatic postsynaptic activity and calcium entry are not required for the depression. We propose instead that the mechanism may involve a form of long-lasting activity-induced synaptic depression, possibly a combination of transmitter depletion and ligand-induced changes in the postsynaptic current accompanying transmitter release. This activity-dependent depression appears to be an important mechanism underlying the occurrence of spontaneous activity in developing spinal networks.
Assuntos
Rede Nervosa/embriologia , Medula Espinal/embriologia , Transmissão Sináptica/fisiologia , Animais , Embrião de Galinha , Embrião não Mamífero/fisiologia , Potenciais Evocados/fisiologia , Neurônios Motores/fisiologia , Raízes Nervosas Espinhais/embriologia , Sinapses/fisiologiaRESUMO
Developing networks of the chick spinal cord become spontaneously active early in development and remain so until hatching. Experiments using an isolated preparation of the spinal cord have begun to reveal the mechanisms responsible for this activity. Whole-cell and optical recordings have shown that spinal neurons receive a rhythmic, depolarizing synaptic drive and experience rhythmic elevations of intracellular calcium during spontaneous episodes. Activity is expressed throughout the neuraxis and can be produced by different parts of the cord and by the isolated brain stem, suggesting that it does not depend upon the details of network architecture. Two factors appear to be particularly important for the production of endogenous activity. The first is the predominantly excitatory nature of developing synaptic connections, and the second is the presence of prolonged activity-dependent depression of network excitability. The interaction between high excitability and depression results in an equilibrium in which episodes are expressed periodically by the network. The mechanism of the rhythmic bursting within an episode is not understood, but it may be due to a "fast" form of network depression. Spontaneous embryonic activity has been shown to play a role in neuron and muscle development, but is probably not involved in the initial formation of connections between spinal neurons. It may be important in refining the initial connections, but this possibility remains to be explored.
Assuntos
Interneurônios/fisiologia , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Medula Espinal/embriologia , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Animais , Embrião de Galinha , Rede Nervosa/citologia , Medula Espinal/citologia , Medula Espinal/fisiologiaRESUMO
Immunohistochemical and in vitro electrophysiological techniques were utilized to examine the distribution and possible role of calcitonin gene-related peptide (CGRP) in the spinal cord of the developing chick. CGRP-like immunoreactivity first appeared in the lateral motor column of the lumbosacral spinal cord at embryonic day 6 followed by the emergence of fiber immunoreactivity in the dorsal horn at embryonic day 11. A rostrocaudal survey of the cervical to lumbosacral spinal cord in embryonic day 18 chick demonstrated robust CGRP-like immunoreactivity at all levels in both putative motor neurons and dorsal horn fibers. Additionally, small immunoreactive lamina VII neurons were observed in sections of lumbosacral cord. In the embryonic day 10 (E10) in vitro reduced spinal cord preparation, bath application of the calcitonin gene-related peptide antagonist human alpha-CGRP fragment 8-37 decreased the frequency and increased the duration of episodes of spontaneously occurring rhythmic activity. Conversely, application of alpha or beta forms of calcitonin gene-related peptide increased the frequency of the rhythmic episodes. The electrophysiological results suggest there is a constitutive release of calcitonin gene-related peptide contributing to the spontaneous rhythmic activity. Immunohistochemical results from E10 animals suggest that CGRP-like immunoreactive putative motoneurons may be the source of the released CGRP.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/análise , Periodicidade , Medula Espinal/química , Análise de Variância , Animais , Embrião de Galinha , Imuno-Histoquímica , Região Lombossacral , Neurônios Motores/química , Medula Espinal/embriologiaRESUMO
The isolated lumbosacral cord of the chick embryo generates spontaneous episodes of rhythmic activity. Muscle nerve recordings show that the discharge of sartorius (flexor) and femorotibialis (extensor) motoneurons alternates even though the motoneurons are depolarized simultaneously during each cycle. The alternation occurs because sartorius motoneuron firing is shunted or voltage-clamped by its synaptic drive at the time of peak femorotibialis discharge. Ablation experiments have identified a region dorsomedial to the lateral motor column that may be required for the alternation of sartorius and femorotibialis motoneurons. This region overlaps the location of interneurons activated by ventral root stimulation. Wholecell recordings from interneurons receiving short latency ventral root input indicate that they fire at an appropriate time to contribute to the cyclical pause in firing of sartorius motoneurons. Spontaneous activity was modeled by the interaction of three variables: network activity and two activity-dependent forms of network depression. A "slow" depression which regulates the occurrence of episodes and a "fast" depression that controls cycling during an episode. The model successfully predicts several aspects of spinal network behavior including spontaneous rhythmic activity and the recovery of network activity following blockade of excitatory synaptic transmission.
Assuntos
Locomoção/fisiologia , Neurônios Motores/fisiologia , Medula Espinal , Animais , Embrião de Galinha , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/fisiologia , Transmissão Sináptica/fisiologiaAssuntos
Interneurônios/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Potenciais de Ação/fisiologia , Animais , Embrião de Galinha , Estimulação Elétrica , Eletrofisiologia , Lisina/análogos & derivados , Neurônios Motores/química , Neurônios Motores/fisiologia , Periodicidade , Medula Espinal/embriologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.
Assuntos
Embrião de Galinha/fisiologia , Neurônios Motores/citologia , Mutação , Junção Neuromuscular/fisiologia , Animais , Especificidade de Anticorpos , Canais de Cálcio/análise , Canais de Cálcio/imunologia , Proteínas de Ligação a Calmodulina/análise , Contagem de Células , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Eletrofisiologia , Heterozigoto , Microscopia Eletrônica , Neurônios Motores/química , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/análise , Proteínas Musculares/imunologia , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Doenças do Sistema Nervoso/fisiopatologia , Junção Neuromuscular/ultraestrutura , Paralisia/genética , Canal de Liberação de Cálcio do Receptor de Rianodina , Medula Espinal/embriologia , Medula Espinal/fisiopatologiaRESUMO
Different assays are employed to monitor the concentration of immunosuppressive drugs in biological fluids. None of these methods gives direct and precise information on the actual level of immunosuppression in the patient. Here we describe the use of an interleukin-2 (IL-2) reporter-gene assay (IL-2 RGA) to monitor the concentrations of immunosuppressants in body fluids. This assay is based on a chimeric gene construct in which the human IL-2 promoter drives the expression of a reporter gene. Upon mitogenic stimulation the reporter gene is expressed and can be easily quantified. The assay is very sensitive and selective for immunosuppressive compounds inhibiting IL-2 gene expression such as cyclosporine (CsA) and FK506, their active metabolites and derivatives, but not for others such as rapamycin. High reproducibility, fast performance time, and high capacity are additional characteristics of the assay. The assay was developed to monitor immunosuppressive drug levels in human volunteers or in animals receiving CsA analogues as the only immunosuppressive drugs. This assay is sensitive to CsA or ascomycin/FK506 analogues and metabolites, for which there are presently no specific monoclonal antibodies available. The IL-2 reporter-gene assay may be more suitable than other in vitro systems such as MLR or mitogen stimulated PBMC which were previously used to study the immunosuppressive activity of drugs in body fluids.
Assuntos
Ciclosporina/sangue , Genes Reporter , Isomerases de Aminoácido/metabolismo , Bioensaio , Proteínas de Transporte/metabolismo , Linhagem Celular , Ciclosporina/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Imunossupressão , Imunossupressores/análise , Interleucina-2/genética , Peptidilprolil Isomerase , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
We describe a novel method for retrogradely labeling specific neuronal populations using voltage-sensitive dyes. Styryl dyes were injected into the ventral roots of the isolated embryonic chick spinal cord. After waiting several hours, the dye labeled motoneurons and autonomic preganglionic neurons. Neuronal cell bodies, dendrites and axons were labeled; we presume that the dye traveled either by retrograde transport or by diffusion within the membrane of the axon to which the dyes were initially applied. Using either a photodiode array or a photomultiplier, fluorescence changes could be recorded from motoneurons following antidromic or synaptic activation. Several characteristics of the fluorescence changes were measured indicating that the signals did indeed reflect changes in the motoneuron membrane potential. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. In the great majority of cases these dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic or antidromic depolarization of the motoneurons. We anticipate that these techniques should be useful in the mapping of activity patterns and connectivity in neural networks within a defined population of neurons.
Assuntos
Eletrofisiologia/métodos , Corantes Fluorescentes/farmacocinética , Medula Espinal/fisiologia , Potenciais de Ação/fisiologia , Animais , Embrião de Galinha , Ativação do Canal Iônico/fisiologia , Microeletrodos , Neurônios Motores/fisiologia , Óptica e Fotônica , Fotoquímica , Medula Espinal/citologia , Coloração e Rotulagem/métodosRESUMO
Using a novel method for retrogradely labeling specific neuronal populations, we tested different styryl dyes in attempt to find dyes whose staining would be specific, rapid, and lead to large activity dependent signals. The dyes were injected into the ventral roots of the isolated chick spinal cord from embryos at days E9-E12. The voltage-sensitive dye signals were recorded from synaptically activated motoneurons using a 464 element photodiode array. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. Over the 24 h period we examined, these dyes bound specifically to the cells with axons in the ventral roots. The dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic depolarization of the motoneurons. The signal-to-noise ratio obtained in a single trial from a detector that received light from a 14 x 14 microns2 area of the motoneuron population was about 10:1. Nonetheless, signals on neighboring diodes were similar, suggesting that we were not detecting the activity of individual neurons. Retrograde labeling and optical recording with voltage-sensitive dyes provides a means for monitoring the activity of identified neurons in situations where microelectrode recordings are not feasible.
Assuntos
Eletrofisiologia/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Neurônios Motores/fisiologia , Animais , Artefatos , Embrião de Galinha , Ativação do Canal Iônico/fisiologia , Óptica e Fotônica , Fotoquímica , Medula Espinal/citologia , Medula Espinal/fisiologia , Coloração e Rotulagem/métodosRESUMO
The source of environmental cues determining the central connections of muscle sensory neurons was investigated by manipulating chick embryos so that sensory neurons supplied a duplicate set of dorsal thigh muscles. These neurons projected out ventral nerve pathways and along motor axons that normally project to ventral muscles but their ultimate target tissue was the duplicate set of dorsal muscles. The central connections of these sensory neurons to motoneurons supplying normal dorsal muscles were then determined with intracellular recordings in isolated spinal cord preparations. Sensory neurons supplying individual duplicate dorsal muscles made the same connections as those supplying the corresponding normal dorsal muscles; the pattern of these connections was different than that made by afferents supplying ventral muscles. Sensory neurons thus made synaptic connections appropriate for their target muscle rather than for their more proximal ventral environment. These findings suggest that the target muscle is the source of cues that determine the central connections of the sensory neurons projecting to it. Motoneurons forced to innervate novel muscle received many of the same sensory inputs they would normally receive, suggesting that motoneurons are less influenced by their target tissue than sensory neurons.
Assuntos
Neurônios Motores/fisiologia , Fusos Musculares/fisiologia , Neurônios Aferentes/fisiologia , Medula Espinal/fisiologia , Sinapses/fisiologia , Animais , Embrião de Galinha , Músculo Esquelético/inervação , Vias Neurais/fisiologia , Medula Espinal/citologia , Coxa da PernaRESUMO
Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.