RESUMEN
The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium tuberculosis/genética , Sinorhizobium meliloti/fisiología , Simbiosis , Antiinfecciosos/farmacología , Proteínas Bacterianas/genética , Medicago sativa/microbiología , Medicago sativa/fisiología , Proteínas de Transporte de Membrana/genética , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/genética , beta-Defensinas/farmacologíaRESUMEN
The production of MG (methylglyoxal) in bacterial cells must be maintained in balance with the capacity for detoxification and protection against this electrophile. Excessive production of MG leads to cell death. Survival of exposure to MG is best understood in the Gram-negative bacteria. The major mechanism of protection is the spontaneous reaction of MG with GSH to form hemithiolacetal, followed by detoxification by the glyoxalase system leading to the production of D-lactate. The KefB and KefC glutathione-gated K(+) efflux systems are integrated with the activity of the glyoxalase system to regulate the cytoplasmic pH in response to exposure to electrophiles. Bacteria only produce MG when an imbalance occurs in metabolism. Operation of the MG bypass enables cells to adapt, such that balance is restored to metabolism. Excessive production of MG is an adaptive ploy, which, if it fails, has fatal consequences. On this basis one might define MG-induced loss of life as "death by misadventure" rather than suicide!
Asunto(s)
Bacterias/metabolismo , Piruvaldehído/metabolismoRESUMEN
The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.
Asunto(s)
Antiportadores/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Canales de Potasio/fisiología , Piruvaldehído/toxicidad , ADN Bacteriano , Escherichia coli/fisiología , Plásmidos , Antiportadores de Potasio-HidrógenoRESUMEN
Dicarbonyl-containing compounds such as methylglyoxal (MG) are toxic to cells since they can interact with the nucleophilic centers of macromolecules. MG has been found to accumulate during hyperglycemia, and it has been suggested that this reactive dicarbonyl may contribute to the tissue damage and long-term complications of diabetes. A sensitive bacterial assay for investigating the ability of nucleophilic agents to interact with and detoxify MG has been developed. This assay utilizes the sensitivity of exponential phase cells of an Escherichia coli double mutant lacking the KefB and KefC potassium channels toward MG. The bidentate nucleophile, phenylacylthiazolium bromide (PTB), was found to protect and allow the growth of E. coli cells in the presence of either externally added or endogenously produced MG. In the absence of PTB, growth was completely inhibited and rapid cell death occurred under these conditions. PTB protected E. coli against MG almost as well as aminoguanidine, a compound shown previously to be involved in detoxification. The level of protection by PTB against MG was much greater than for the endogenous nucleophile, glutathione. These data suggested that PTB could interact with and detoxify MG. The mechanism of this interaction was characterized by NMR and mass spectroscopy.
Asunto(s)
Proteínas de Escherichia coli , Piruvaldehído/metabolismo , Tiazoles/farmacología , Antiportadores/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Glutatión/metabolismo , Guanidinas/farmacología , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Canales de Potasio/genética , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/química , Piruvaldehído/farmacología , Tiazoles/químicaRESUMEN
Escherichia coli are exposed to toxic electrophiles from both endogenous and exogenous sources. To survive, E. coli have developed novel protective mechanisms that appear unique to bacteria. In the absence of one of these mechanisms, electrophiles induce rapid killing. Hence it appears that electrophile-protective mechanisms represent novel targets for antibacterial drug research.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/fisiología , Piruvaldehído/metabolismo , Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Glutatión/metabolismo , Concentración de Iones de Hidrógeno , Canales de Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/efectos adversosRESUMEN
Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.
Asunto(s)
Bacterias/metabolismo , Proteínas de Escherichia coli , Piruvaldehído/metabolismo , Antiportadores/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/fisiología , Glutatión/metabolismo , Glucólisis/fisiología , Canales de Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno , Factor sigma/metabolismoRESUMEN
The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to E. coli cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K+ pool and survival of exposure to methylglyoxal.
Asunto(s)
Escherichia coli/metabolismo , Glutatión/metabolismo , Potasio/metabolismo , Piruvaldehído/farmacología , División Celular/efectos de los fármacos , Citoplasma/metabolismo , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Piruvaldehído/metabolismoRESUMEN
The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Etilmaleimida/farmacología , Factor sigma/fisiología , Antiportadores/genética , Antiportadores/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Farmacorresistencia Microbiana , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Mutación , Canales de Potasio/genética , Canales de Potasio/fisiología , Antiportadores de Potasio-Hidrógeno , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
The glyoxalase I gene (gloA) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E. coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.
Asunto(s)
Antiportadores/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Lactoilglutatión Liasa/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Piruvaldehído/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glutatión/metabolismo , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/farmacologíaRESUMEN
The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH. However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation. No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.
Asunto(s)
Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Dinitroclorobenceno/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Glutatión/metabolismo , Yodoacetatos/farmacología , Canales de Potasio/metabolismo , División Celular/efectos de los fármacos , Dinitroclorobenceno/toxicidad , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Yodoacetatos/toxicidad , Ácido Yodoacético , Activación del Canal Iónico , Transporte Iónico/efectos de los fármacos , Antiportadores de Potasio-HidrógenoRESUMEN
The role of the KefB and KefC potassium efflux systems in protecting Escherichia coli cells against the toxic effects of the electrophile N-ethylmaleimide has been investigated. Activation of KefB and KefC aids the survival of cells exposed to high concentrations (> 100 microM) of NEM. High potassium concentrations reduce the protection afforded by activation of KefB and KefC, but the possession of these systems is still important under these conditions. The Kdp system, which confers sensitivity to the electrophile methylglyoxal, did not affect the survival of cells exposed to NEM. Survival is correlated with the reduction of the cytoplasmic pH upon activation of the channels. In particular, the kinetics of the intracellular pH (pHi) change are crucial to the retention of viability of cells exposed to NEM; slow acidification does not protect cells as effectively as rapid lowering of pHi. Cells treated with low levels of NEM (10 microM) recover faster if they activate KefB and KefC, and this correlates with changes in pHi. The pHi does not significantly alter the rate of NEM metabolism. The possible mechanisms by which protection against the electrophile is mediated are discussed.
Asunto(s)
Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Etilmaleimida/farmacología , Canales de Potasio/metabolismo , Potasio/metabolismo , Transporte Biológico , Escherichia coli/metabolismo , Etilmaleimida/metabolismo , Antiportadores de Potasio-HidrógenoRESUMEN
Expression of the Kdp system sensitizes cells to methylglyoxal (MG) whether this electrophile is added externally or is synthesized endogenously. The basis of this enhanced sensitivity is the maintenance of a higher cytoplasmic pH (pHi) in cells expressing Kdp. In such cells, MG elicits rapid cytoplasmic acidification via KefB and KefC, but the steady-state pHi attained is still too high to confer protection Lowering pHi further by incubation with acetate increases the sensitivity of cells to MG.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Potasio/metabolismo , Piruvaldehído/farmacología , Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Canales de Potasio/metabolismo , Antiportadores de Potasio-HidrógenoRESUMEN
Escherichia coli possesses two glutathione-gated potassium channels, KefB and KefC, that are activated by glutathione-S-conjugates formed with methylglyoxal. We demonstrate that activation of the channels leads to cytoplasmic acidification and that this protects cells during electrophilic attack. Further, we demonstrate that mutants lacking the channels can be protected against the lethal effects of methylglyoxal by acidification of the cytoplasm with a weak acid. The degree of protection is determined by the absolute value of the pHi and the time at which acidification takes place. Alterations in the pHi do not accelerate the rate of detoxification of methylglyoxal. The mechanism by which methylglyoxal causes cell death and the implications for pHi-mediated resistance to methylglyoxal are discussed.
Asunto(s)
Antiportadores/efectos de los fármacos , Proteínas Bacterianas/efectos de los fármacos , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Glutatión/fisiología , Canales de Potasio/efectos de los fármacos , Potasio/metabolismo , Piruvaldehído/farmacología , Acetatos/farmacología , Ácido Acético , Antiportadores/genética , Antiportadores/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fenómenos Químicos , Química Física , Citoplasma/química , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Líquido Intracelular/química , Estrés Oxidativo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno , ProtonesRESUMEN
1. The role of the paired serotonergic cerebral giant cells (CGCs) in the feeding system of Lymnaea was examined by electrophysiological and pharmacological techniques. 2. The firing characteristics of the CGCs were recorded by fine wires attached to their cell bodies in freely moving intact snails (in vivo recording) and their "physiological" rates of firing determined during feeding and other behaviors. 3. The mean CGC firing rates recorded in vivo varied between 1 and 20 spikes/min but never reached the average rates seen in the isolated CNS (60-120 spikes/min). Maximum rates of firing were seen during bouts of radula biting/rasping movements characteristic of the consummatory phase of feeding (15 +/- 1.69 spikes/min, mean +/- SE, range 7-20 spikes/min), with lower rates seen during locomotion (6.7 +/- 0.75 spikes/min; range 5-9 spikes/min. The cells were rarely active when the animal was quiescent (1.45 +/- 0.91 spikes/min; range 0-2 spikes/min). 4. In vivo recorded CGC firing was phase locked to the feeding movements of the animal, with spikes occurring just before the opening of the mouth, during the protraction phase of the feeding cycle. 5. Evoking firing rates on the CGCs in the isolated preparation similar to those seen in vivo during rasping movements (7-20 spikes/min) did not elicit a fictive feeding pattern in an inactive preparation. Neither did bath application of 10(-9) M serotonin (5-HT; the transmitter of the CGCs). 6. To allow the modulatory role of the CGCs to be examined during patterned activity, the fictive feeding pattern was evoked in the isolated preparation by injecting depolarizing current into a modulatory neuron, the slow oscillator (SO). 7. The tonic firing activity of the CGCs was accurately maintained by current injection in the isolated preparation at rates equivalent to that occurring during feeding, locomotion, and quiescence in the intact snail. This was possible where the CGCs became silent after 1-2 h. Only when the CGCs activity was maintained at a rate (approximately 15 spikes/min) similar to that occurring during rasping, was the SO able to drive a full, high-frequency fictive feeding pattern (15-20 cycles/min). At lower rates of CGC firing, the SO-driven rhythm was either of lower frequency or no rhythm occurred at all (CGCs silent). 8. In many isolated preparations (80%) the CGCs remained active, and it was difficult to maintain specific levels of tonic activity by current injection.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Conducta Alimentaria/fisiología , Ganglios de Invertebrados/fisiología , Lymnaea/fisiología , Serotonina/fisiología , Transmisión Sináptica/fisiología , Animales , Nivel de Alerta/efectos de los fármacos , Nivel de Alerta/fisiología , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Conducta Alimentaria/efectos de los fármacos , Ganglios de Invertebrados/efectos de los fármacos , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram-negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation-translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram-negative bacteria, but not in the glutathione-deficient species of Gram-positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal-induced cell death. This establishes an essential physiological function for the KefB system.
Asunto(s)
Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Muerte Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Glutatión/metabolismo , Inactivación Metabólica , Cinética , Potasio/farmacología , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/farmacología , Factores de TiempoRESUMEN
This study reports the presence of high concentrations of free D-aspartic acid (D-Asp) in the circumoesophageal ganglia of the opisthobranch mollusc Aplysia fasciata. D-Asp was discovered using specific methods that employ Octopus D-aspartate oxidase and hog kidney D-amino acid oxidase to measure D-Asp levels. The concentration of D-Asp was 0.281 mumol/g wet tissue weight, which was 8.3% of the total free aspartic acid (D and L forms) present. No other free D-amino acids that were oxidised by D-amino acid oxidase were detected. To our knowledge the only molluscs that have previously been shown to have D-Asp within their nervous tissue are the cephalopods Octopus vulgaris, Loligo vulgaris and Sepia officinalis. In these, as in A. fasciata, no other D-amino acids were detected within the nervous tissue. The fact that free D-Asp occurs specifically in the nervous tissue of the above molluscs suggests that it may have a neurological function that is yet to be described.
Asunto(s)
Ácido Aspártico/metabolismo , Ganglios/metabolismo , Animales , Aplysia , Esófago/inervación , EstereoisomerismoRESUMEN
The neuronal activity of the cerebrobuccal connective (CBC) of Aplysia was recorded, using 2 implanted electrodes, under three conditions; 1) in the absence of feeding behaviour, 2) during appetitive feeding behaviour and 3) during consummatory feeding behaviour. Cross-correlation analysis of the recordings was then performed to subdivide spikes on the basis of their direction and speed of propagation. This revealed differences in the neuronal activity during the 3 conditions. There was little activity in the CBC when animals were not feeding. During appetitive and consummatory feeding behaviour the activity in the CBC increased. Units travelling in each direction were present, but with differential activity during the 2 behavioural patterns.
Asunto(s)
Aplysia/fisiología , Conducta Alimentaria/fisiología , Animales , Encéfalo/fisiología , Mejilla/inervación , Electrofisiología , Vías Nerviosas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Two muscle systems mediated the whole-body withdrawal response of Lymnaea stagnalis: the columellar muscle (CM) and the dorsal longitudinal muscle (DLM). The CM was innervated by the columellar nerves and contracted longitudinally to shorten the ventral head-foot complex and to pull the shell forward and down over the body. The DLM was innervated by the superior and inferior cervical nerves and the left and right parietal nerves. During whole-body withdrawal, the DLM contracted synchronously with the CM and shortened the dorsal head-foot longitudinally. The CM and the DLM were innervated by a network of motoneurones. The somata of these cells were located in seven ganglia of the central nervous system (CNS), but were especially concentrated in the bilaterally symmetrical A clusters of the cerebral ganglia. The CM was innervated by cells in the cerebral and pedal ganglia and the DLM by cells in the cerebral, pedal, pleural and left parietal ganglia. Individual motoneurones innervated large, but discrete, areas of muscle, which often overlapped with those innervated by other motoneurones. Motoneuronal action potentials evoked one-for-one non-facilitating excitatory junction potentials within muscle fibres. No all-or-nothing action potentials were recorded in the CM or DLM, and they did not appear to be innervated by inhibitory motoneurones. The whole network of motoneurones was electrotonically coupled, with most cells on one side of the CNS strongly coupled to each other but weakly coupled to cells on the contralateral side of the CNS. This electrotonic coupling between motoneurones is probably important in producing synchronous contraction of the CM and DLM when the animal retracts its head-foot complex during whole-body withdrawal.
Asunto(s)
Reacción de Fuga/fisiología , Neuronas Motoras/fisiología , Músculos/inervación , Animales , Encéfalo/citología , Cobalto , Electrofisiología , Ganglios/citología , Isoquinolinas , Lymnaea/fisiología , Neuronas Motoras/citología , Músculos/fisiologíaRESUMEN
The role of centrally located motoneurones in producing the whole-body withdrawal response of Lymnaea stagnalis (L.) was investigated. The motoneurones innervating the muscles used during whole-body withdrawal, the columellar muscle (CM) and the dorsal longitudinal muscle (DLM) were cells with a high resting potential (-60 to -70 mV) and thus a high threshold for spike initiation. In both semi-intact and isolated brain preparations these motoneurones showed very little spontaneous spike activity. When spontaneous firing was seen it could be correlated with the occurrence of two types of spontaneous excitatory postsynaptic potential (EPSP). One was a unitary EPSP that occasionally caused the initiation of single action potentials. The second was a larger-amplitude, long-duration (presumably compound) EPSP that caused the motoneurones to fire a burst of high-frequency action potentials. This second type of EPSP activity was associated with spontaneous longitudinal contractions of the body in semi-intact preparations. Tactile stimulation of the skin of Lymnaea evoked EPSPs in the CM and DLM motoneurones and in some other identified cells. These EPSPs summated and usually caused the motoneurone to fire action potentials, thus activating the withdrawal response muscles and causing longitudinal contraction of the semi-intact animal. Stimulating different areas of the body wall demonstrated that there was considerable sensory convergence on the side of the body ipsilateral to stimulation, but less on the contralateral side. Photic (light off) stimulation of the skin of Lymnaea also initiated EPSPs in CM and DLM motoneurones and in some other identified cells in the central nervous system (CNS). Cutting central nerves demonstrated that the reception of this sensory input was mediated by dermal photoreceptors distributed throughout the epidermis. The activation of the CM and DLM motoneurones by sensory input of the modalities that normally cause the whole-body withdrawal of the intact animal demonstrates that these motoneurones have the appropriate electrophysiological properties for the role of mediating whole-body withdrawal.
Asunto(s)
Reacción de Fuga/fisiología , Neuronas Motoras/fisiología , Músculos/inervación , Animales , Electrofisiología , Potenciales Evocados Somatosensoriales , Luz , Lymnaea/fisiología , Potenciales de la Membrana , Movimiento/fisiologíaRESUMEN
Aplysia egg laying is a complex sequence of head and neck movements initiated by the release of ovulatory and neuroactive hormones from the neurosecretory bag cells. This behavioral pattern is difficult to study in reduced preparations, because they do not show ovulation or egg laying behaviors. This paper describes the use of chronically implanted electrodes to elicit normal neurosecretory activity and provides an analysis of egg laying behaviors and the underlying muscle activity in intact, freely behaving A. californica and A. brasiliana. 1. Bag cell discharges elicited with a fine wire electrode implanted in the connective tissue sheath above the cell bodies were typically without noxious behavioral side effects. 2. Following selectively elicited bag cell discharges, egg laying consisted of four rhythmic head and neck movements that were separated functionally into appetitive behaviors ('waves' and 'undulations') used to explore and prepare the substrate and consummatory behaviors ('weaves' and 'tamps') used to distribute and attach the egg string. The amount of time an animal performed consummatory behaviors was positively related to the amount of eggs deposited. By contrast, the appetitive phase of egg laying was independent of the size of the egg mass. 3. The individual behaviors and their temporal sequence were similar following selectively elicited bag cell discharges, spontaneous discharges of animals with implanted electrodes and during normal egg laying of unoperated animals. 4. Three longitudinal muscle systems occurred within the head and neck. Following a selectively elicited bag cell discharge, spatially and temporally coordinated patterns of EJP bursts of different durations were recorded chronically from each muscle group. These EJP patterns were characteristic for specific head and neck movements used in appetitive and consummatory egg laying behaviors.