RESUMEN
The fundamental role of calcium ions (Ca(2+)) in an excitable tissue, the frog heart, was first demonstrated in a series of classical reports by Sydney Ringer in the latter part of the nineteenth century (1882a, b; 1893a, b). Even so, nearly a century elapsed before it was proven that Ca(2+) regulated the excitability of primary sensory neurons. In this chapter we review the sites and mechanisms whereby internal and external Ca(2+) can directly or indirectly alter the excitability of primary sensory neurons: excitability changes being manifested typically by variations in shape of the action potential or the pattern of its discharge.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Células Receptoras Sensoriales/metabolismo , Potenciales de Acción , Animales , Transporte Biológico , Neuropatías Diabéticas/metabolismo , Humanos , Cinética , Neuralgia/metabolismo , Traumatismos del Sistema Nervioso/metabolismoRESUMEN
Adult rat sensory trigeminal ganglion neurons innervating the cornea (cTGNs) were isolated and identified following retrograde dye labeling with FM1-43. Using standard whole-cell patch clamp recording techniques, cTGNs could be subdivided by their action potential (AP) duration. Fast cTGNs had AP durations <1 ms (40%) while slow cTGNs had AP durations >1 ms and an inflection on the repolarization phase of the AP. With the exception of membrane input resistance, the passive membrane properties of fast cTGNs were different from those of slow cTGNs (capacitance: 61+/-4.5 pF vs. 42+/-2.6 pF, resting membrane potential: -59+/-0.7 mV vs. -53+/-0.9 mV, for fast and slow cTGNs respectively). Active membrane properties also differed between fast and slow cTGNs. Slow cTGNs had a higher AP threshold (-25+/-1.6 mV vs. -38+/-0.8 mV), a larger rheobase (14+/-1.9 pA/pF vs. 6.8+/-1.0 pA/pF), and a smaller AP undershoot (-56+/-1.7 mV vs. -67+/-2.5 mV). The AP overshoot, however was similar between the two types of neurons (46+/-3.1 mV vs. 48+/-4 mV). Slow cTGNs were depolarized by capsaicin (1 microM, 80%) and 60% of their APs were blocked by tetrodotoxin (TTX) (100 nM). Fast cTGNs were unaffected by capsaicin and 100% of their APs were blocked by TTX. Similarly, cTGNs were also heterogeneous with respect to their responses to exogenous ATP and 5-HT. The current work shows that cTGNs have distinctive electrophysiological properties and chemosensitivity profiles. These characteristics may mirror the distinct properties of corneal sensory nerve terminals. The availability of isolated identified cTGNs constitutes a tractable model system to investigate the biophysical and pharmacological properties of corneal sensory nerve terminals.
Asunto(s)
Potenciales de Acción/fisiología , Córnea/inervación , Neuronas Aferentes/fisiología , Nociceptores/fisiología , Nervio Oftálmico/fisiología , Ganglio del Trigémino/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Capsaicina/farmacología , Células Cultivadas , Córnea/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Aferentes/efectos de los fármacos , Nociceptores/efectos de los fármacos , Nervio Oftálmico/efectos de los fármacos , Dolor/fisiopatología , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Prostaglandin E(2) (PGE(2)) is a prototypical inflammatory mediator that excites and sensitizes cell bodies [Kwong K, Lee LY (2002) PGE(2) sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli. J Appl Physiol 93:1419-1428; Kwong K, Lee LY (2005) Prostaglandin E(2) potentiates a tetrodotoxin (TTX)-resistant sodium current in rat capsaicin-sensitive vagal pulmonary sensory neurons. J Physiol 56:437-450] and peripheral nerve terminals [Ho CY, Gu Q, Hong JL, Lee LY (2000) Prostaglandin E (2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat. Am J Respir Crit Care Med 162:528-533] of primary vagal sensory neurons. Nearly all central nerve terminals of vagal afferents are in the nucleus tractus solitarius (NTS), where they operate with a high probability of release [Doyle MW, Andresen MC (2001) Reliability of monosynaptic sensory transmission in brain stem neurons in vitro. J Neurophysiol 85:2213-2223]. We studied the effect of PGE(2) on synaptic transmission between tractus solitarius afferent nerve terminals and the second-order NTS neurons in brain stem slices of Sprague-Dawley rats. Whole-cell patch recording in voltage clamp mode was used to study evoked excitatory postsynaptic glutamatergic currents (evEPSCs) from NTS neurons elicited by electrical stimulation of the solitary tract (ST). In 34 neurons, bath-applied PGE(2) (200 nM) decreased the evEPSC amplitude by 49+/-5%. In 22 neurons, however, PGE(2) had no effect. We also tested 15 NTS neurons for capsaicin sensitivity. Seven neurons generated evEPSCs that were equally unaffected by PGE(2) and capsaicin. Conversely, evEPSCs of the other eight neurons, which were PGE(2)-responsive, were abolished by 200 nM capsaicin. Furthermore, the PGE(2-)induced depression of evEPSCs was associated with an increase in the paired pulse ratio and a decrease in both the frequency and amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) and TTX-independent spontaneous miniature excitatory postsynaptic currents (mEPSCs). These results suggest that PGE(2) acts both presynaptically on nerve terminals and postsynaptically on NTS neurons to reduce glutamatergic responses.
Asunto(s)
Dinoprostona/farmacología , Inhibición Neural/efectos de los fármacos , Oxitócicos/farmacología , Núcleo Solitario/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Analgésicos no Narcóticos/farmacología , Animales , Animales Recién Nacidos , Capsaicina/farmacología , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Técnicas In Vitro , Masculino , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Whereas spontaneous point mutation operates on nucleotides individually, sexual recombination manipulates the set of nucleotides within an allele as an essentially particulate unit. In principle, these two different scales of variation enable selection to follow fitness gradients in two different spaces: in nucleotide sequence space and allele sequence space respectively. Epistasis for fitness at these two scales, between nucleotides and between genes, may be qualitatively different and may significantly influence the advantage of mutation-based and recombination-based evolutionary trajectories respectively. We examine scenarios where the genetic sequence within a gene strongly influences the fitness effect of a mutation in that gene, whereas epistatic interactions between sites in different genes are weak or absent. We find that, in cases where beneficial alleles of a gene differ from one another at several nucleotide sites, sexual populations can exhibit enormous benefit compared with asexual populations: not only discovering fit genotypes faster than asexual populations, but also discovering high-fitness genotypes that are effectively not evolvable in asexual populations.
Asunto(s)
Meiosis/genética , Recombinación Genética/genética , Animales , Genotipo , Modelos Genéticos , Mutación/genética , Reproducción/genéticaAsunto(s)
Toma de Decisiones , Diseño de Fármacos , Farmacogenética , Proyectos de Investigación , Animales , Ensayos Clínicos como Asunto/métodos , Ensayos Clínicos como Asunto/normas , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Humanos , Estudios ProspectivosRESUMEN
An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C-fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C-fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C-fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C-fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C-fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C-fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C-fibre population responded with action potential discharge to the P2X selective receptor agonist alpha,beta-methylene-ATP. Whole cell patch clamp recording of capsaicin-sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C-fibre neurones express functional P2X receptors, whereas lung specific jugular C-fibres do not. The data support the hypothesis that both neural crest-derived neurones (jugular ganglia) and placode-derived neurones (nodose ganglia) project C-fibres in the vagus, and that these two C-fibre populations represent distinct phenotypes.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Vías Aferentes/fisiología , Pulmón/inervación , Fibras Nerviosas Amielínicas/fisiología , Nervio Vago/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adenosina Trifosfato/farmacología , Animales , Bradiquinina/farmacología , Bronquios/inervación , Péptido Relacionado con Gen de Calcitonina/análisis , Capsaicina/farmacología , Electrofisiología , Ganglios/química , Ganglios/fisiología , Cobayas , Inmunohistoquímica , Técnicas In Vitro , Nervios Laríngeos/fisiología , Pulmón/efectos de los fármacos , Pulmón/fisiología , Masculino , Fibras Nerviosas Amielínicas/clasificación , Fibras Nerviosas Amielínicas/efectos de los fármacos , Proteínas de Neurofilamentos/análisis , Ganglio Nudoso/química , Ganglio Nudoso/fisiología , Técnicas de Placa-Clamp , Estimulación Física , Sustancia P/análisis , Tráquea/inervaciónRESUMEN
Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.
Asunto(s)
Canales de Calcio/fisiología , Sustancia P/farmacología , Cationes , Línea Celular , Humanos , Canales Iónicos/fisiología , Receptores de Neuroquinina-2/fisiología , Canales Catiónicos TRPCRESUMEN
1. Nodose ganglion neurones (NGNs) become less excitable following section of the vagus nerve. To determine the role of sodium currents (I(Na)) in these changes, standard patch-clamp recording techniques were used to measure I(Na) in rat NGNs maintained in vivo for 5-6 days following vagotomy, and then in vitro for 2-9 h. 2. Total I(Na) and I(Na) density in vagotomized NGNs were similar to control values. However, steady-state I(Na) inactivation in vagotomized NGNs was shifted -9 mV relative to control values (V(1/2), -74 +/- 2 vs. -65 +/- 2 mV, P < 0.01) and I(Na) activation was shifted by -7 mV (V(1/2), -21 +/- 2 vs. -14 +/- 2 mV, P < 0.006). I(Na) recovery from inactivation was also slower in vagotomized NGNs (fast time constant, 2.8 +/- 0.4 vs. 1.6 +/- 0.3 ms, P < 0.02). 3. The fraction of I(Na) resistant to 1 microM tetrodotoxin (TTX-R) was halved in vagotomized NGNs (21 +/- 8 vs. 56 +/- 8 % of total I(Na), P < 0.05). This change from TTX-R I(Na) to TTX-sensitive (TTX-S) I(Na) may explain altered I(Na) activation, inactivation and repriming in vagotomized NGNs. 4. The contribution of alterations in I(Na) to NGN firing patterns was assessed by measuring I(Na) evoked by a series of action potential (AP) waveforms. In general, control NGNs produced large, repetitive TTX-R I(Na) while vagotomized NGNs produced smaller TTX-S I(Na) that rapidly inactivated during AP discharge. We conclude that TTX-R I(Na) is important for sustained AP discharge in NGNs, and that its diminution underlies the decreased AP discharge of vagotomized NGNs.
Asunto(s)
Neuronas Aferentes/metabolismo , Ganglio Nudoso/citología , Canales de Sodio/metabolismo , Sodio/metabolismo , Vagotomía , Potenciales de Acción/fisiología , Anestésicos Locales/farmacología , Animales , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Masculino , Ganglio Nudoso/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Tetrodotoxina/farmacologíaRESUMEN
1. The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low-threshold vagal afferent nerve terminals in the guinea-pig trachea/bronchus. 2. Using extracellular single-unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea-pig airway afferent fibres. 3. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. 4. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride approximately benzamil > amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil > amiloride >> dimethylamiloride). 5. As voltage-gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole-cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 microM), benzamil caused significant inhibition of voltage-gated sodium currents in neuronal cell bodies acutely isolated from guinea-pig nodose ganglia. 6. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage-gated sodium currents.
Asunto(s)
Amilorida/análogos & derivados , Amilorida/farmacología , Bronquios/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Bloqueadores de los Canales de Sodio , Canales de Sodio/metabolismo , Tráquea/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Bronquios/inervación , Bronquios/metabolismo , Electrofisiología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Gadolinio/farmacología , Cobayas , Activación del Canal Iónico/efectos de los fármacos , Mecanorreceptores/efectos de los fármacos , Mecanorreceptores/fisiología , Neuronas Aferentes/fisiología , Técnicas de Placa-Clamp , Tetrodotoxina/farmacología , Tráquea/inervación , Tráquea/metabolismo , Nervio Vago/efectos de los fármacos , Nervio Vago/fisiologíaRESUMEN
Altered sympathetic nervous system activity has been implicated often in hypertension. We examined short-term potentiation [posttetanic potentiation (PTP)] and long-term potentiation (LTP) in the isolated superior cervical ganglia (SCG) from Sprague-Dawley (SD) rats given vehicle, digoxin, or ouabain by subcutaneous implants as well as in animals with ouabain-induced hypertension (OHR), and inbred Baltimore ouabain-resistant (BOR) and Baltimore ouabain-sensitive (BOS) strains of rats. Postganglionic compound action potentials (CAP) were used to determine PTP and LTP following a tetanic stimulus (20 Hz, 20 s). Baseline CAP magnitude was greater in ganglia from OHR than in vehicle-treated SD rats before tetanus, but the decay time constant of PTP was significantly decreased in OHR and in rats infused with digoxin that were normotensive. In hypertensive BOS and OHR, the time constants for the decay of both PTP and LTP (t(L)) were increased and correlated with blood pressure (slope = 0.15 min/mmHg, r = 0.52, P < 0.047 and 6.7 min/mmHg, r = 0.906, P < 0.0001, respectively). In BOS and OHR, t(L) (minutes) was 492 +/- 40 (n = 7) and 539 +/- 41 (n = 5), respectively, and differed (P < 0.05) from BOR (257 +/- 48, n = 4), SD vehicle rats (240 +/- 18, n = 4), and captopril-treated OHR (370 +/- 52, n = 5). After the tetanus, the CAP at 90 min in BOS and OHR SCG declined less rapidly vs. SD vehicle rats or BOR. Captopril normalized blood pressure and t(L) in OHR. We conclude that the duration of ganglionic LTP and blood pressure are tightly linked in ouabain-dependent hypertension. Our results favor the possibility that enhanced duration of LTP in sympathetic neurons contributes to the increase in sympathetic nerve activity in ouabain-dependent hypertension and suggest that a captopril-sensitive step mediates the link of ouabain with LTP.
Asunto(s)
Presión Sanguínea/fisiología , Hipertensión/fisiopatología , Plasticidad Neuronal/fisiología , Ouabaína/farmacología , Ganglio Cervical Superior/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Digoxina/farmacología , Modelos Animales de Enfermedad , Electrofisiología , Humanos , Hipertensión/inducido químicamente , Técnicas In Vitro , Potenciación a Largo Plazo , Masculino , Ratas , Ratas Endogámicas , Ratas Sprague-Dawley , Ganglio Cervical Superior/efectos de los fármacosRESUMEN
Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.
Asunto(s)
Cafeína/farmacología , Calcio/farmacocinética , Estimulantes del Sistema Nervioso Central/farmacología , Neuronas Aferentes/metabolismo , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Inhibidores Enzimáticos/farmacología , Femenino , Hidroquinonas/farmacología , Masculino , Mamíferos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Aferentes/efectos de los fármacos , Conejos , Rianodina/farmacologíaRESUMEN
Standard patch-clamp and intracellular recording techniques were used to monitor membrane excitability changes in adult inferior vagal ganglion neurons (nodose ganglion neurons, NGNs) 5 days following section of the vagus nerve (vagotomy). NGNs were maintained in vivo for 5 days following vagotomy, and then in vitro for 2-9 h prior to recording. Vagotomy increased action potential (AP) threshold by over 200% (264 +/- 19 pA, mean +/- SE, n = 66) compared with control values (81 +/- 20 pA, n = 68; P < 0.001). The number of APs evoked by a 3 times threshold 750-ms depolarizing current decreased by >70% (from 8.3 to 2.3 APs, P < 0.001) and the number of APs evoked by a standardized series of (0.1-0.9 nA, 750 ms) depolarizing current steps decreased by over 80% (from 16.9 APs to 2.6 APs, P < 0.001) in vagotomized NGNs. Similar decreases in excitability were observed in vagotomized NGNs in intact ganglia in vitro studied with "sharp" microelectrode techniques. Baseline electrophysiological properties and changes following vagotomy were similar in right and left NGNs. A "sham" vagotomy procedure had no effect on NGN properties at 5 days, indicating that changes were due to severing the vagus nerve itself, not surrounding tissue damage. NGNs isolated after being maintained 17 h in vivo following vagotomy revealed no differences in excitability, suggesting that vagotomy-induced changes occur some time from 1-5 days after injury. Decreased excitability was still observed in NGNs isolated after 20-21 days in vivo following vagotomy. These data indicate that, in contrast to many primary sensory neurons that are thought to become hyperexcitable following section of their axons, NGNs undergo a marked decrease in electrical excitability following vagotomy.
Asunto(s)
Neuronas Aferentes/fisiología , Ganglio Nudoso/fisiología , Vagotomía , Nervio Vago/fisiología , Potenciales de Acción/fisiología , Animales , Membrana Celular/fisiología , Células Cultivadas , Masculino , Ganglio Nudoso/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/fisiología , Umbral Sensorial/fisiología , Nervio Vago/citología , Nervio Vago/cirugíaRESUMEN
A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA.
Asunto(s)
ADN Mitocondrial/genética , Evolución Molecular , Mutación/genética , Filogenia , Primates/genética , Roedores/genética , Animales , Grupo Citocromo b/genética , Complejo IV de Transporte de Electrones/genética , Modelos GenéticosRESUMEN
We report that patterns of nonneutral DNA sequence evolution among published nuclear and mitochondrially encoded protein-coding loci differ significantly in animals. Whereas an apparent excess of amino acid polymorphism is seen in most (25/31) mitochondrial genes, this pattern is seen in fewer than half (15/36) of the nuclear data sets. This differentiation is even greater among data sets with significant departures from neutrality (14/15 vs. 1/6). Using forward simulations, we examined patterns of nonneutral evolution using parameters chosen to mimic the differences between mitochondrial and nuclear genetics (we varied recombination rate, population size, mutation rate, selective dominance, and intensity of germ line bottleneck). Patterns of evolution were correlated only with effective population size and strength of selection, and no single genetic factor explains the empirical contrast in patterns. We further report that in Arabidopsis thaliana, a highly self-fertilizing plant with effectively low recombination, five of six published nuclear data sets also exhibit an excess of amino acid polymorphism. We suggest that the contrast between nuclear and mitochondrial nonneutrality in animals stems from differences in rates of recombination in conjunction with a distribution of selective effects. If the majority of mutations segregating in populations are deleterious, high linkage may hinder the spread of the occasional beneficial mutation.
Asunto(s)
ADN Mitocondrial/genética , ADN/genética , Evolución Molecular , Genoma , Proteínas/genética , Animales , Núcleo Celular/genética , Simulación por Computador , Variación Genética , Humanos , Modelos Genéticos , Polimorfismo GenéticoRESUMEN
Neurons can display sexual dimorphism in receptor expression, neurotransmitter release, and synaptic plasticity. We have detected sexual dimorphism in functional tachykinin receptors in vagal afferents (nodose ganglion neurons, NGNs) by studying the effects of hormonal variation on the depolarizing actions of substance P (SP) in female guinea pig NGNs. Using conventional "sharp" microelectrode recording plus measurement of serum 17beta-estradiol values, we examined SP responses in NGNs isolated from 1) ovariectomized females (OVX), 2) OVX females treated with 17beta-estradiol (OVX + E2), 3) pregnant females, and 4) males. Depending on various manipulations, 19-41% female NGNs were depolarized (16 +/- 1.1 mV, mean +/- SE) by 100 nM SP acting through NK-1 receptors. The NGNs of OVX + E2 females (41%, 15/37; 17 +/- 2.1 mV) and pregnant females (41%, 32/79; 16 +/- 1.7 mV) were more likely to respond to SP than those of control males (P < 0.001). The percentage of SP-sensitive NGNs from OVX females (19%, 21/109; 15 +/- 1.9 mV) was not significantly different (P = 0.361) from that of control males (13%, 11/83; 13 +/- 2.0 mV). The serum 17beta-estradiol values for OVX + E2, pregnant, and OVX females were 23.9 +/- 3.3 pg/ml (n = 8), 16.0 +/- 2.4 pg/ml (n = 4), and 3.9 +/- 0.3 pg/ml (n = 8), respectively. These data indicate that there is a gender difference in NK-1 receptor expression in guinea pig nodose neurons, and they suggest that estrogen may modulate SP responsiveness in these neurons.
Asunto(s)
Neuronas Aferentes/fisiología , Ganglio Nudoso/citología , Receptores de Neuroquinina-1/fisiología , Caracteres Sexuales , Animales , Benzamidas/farmacología , Electrofisiología , Estradiol/sangre , Femenino , Hormonas Esteroides Gonadales/farmacología , Cobayas , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Aferentes/química , Ganglio Nudoso/fisiología , Orquiectomía , Ovariectomía , Piperidinas/farmacología , Embarazo , Quinolinas/farmacología , Serotonina/farmacología , Sustancia P/farmacología , Testosterona/farmacologíaRESUMEN
The majority of airway sensory innervation originates from afferent neurons whose somata reside in vagal (nodose and jugular) ganglia. Using guinea pigs immunized with chick ovalbumin, we have discovered that airway inflammation provokes phenotypic changes in the tachykinin responsiveness of nodose neurons. Bath application of substance P (SP; 0.1 to 10 microM) to nodose neurons isolated from guinea pigs with normal uninflamed airways did not elicit measurable changes in resting electrophysiological properties. In sharp contrast, 80% of nodose neurons isolated 24 h after in vivo aerosolized antigen challenge of the airway were depolarized by 100 nM SP. Inhalation of a nonantigenic protein did not evoke the expression of SP responses. Pharmacological analysis revealed that SP responses unmasked by airway inflammation were mediated by neurokinin-2 (NK-2) tachykinin receptors. There are several potential mechanisms for transduction of an "unmasking signal" from the inflamed airway to vagal afferent somata. The vagus nerve may relay the signal, either through anterograde transport and/or nerve impulse activity. Alternatively, a signal generated by airway inflammation may be carried by the circulation to the nodose ganglia. Unilateral vagotomy significantly reduced the percentage of SP-responsive neurons compared with intact controls, suggesting that the vagus nerve is required for unmasking of NK-2 responses.
Asunto(s)
Antígenos/administración & dosificación , Ovalbúmina/administración & dosificación , Receptores de Neuroquinina-2/metabolismo , Tráquea/inervación , Nervio Vago/metabolismo , Animales , Benzamidas/farmacología , Modelos Animales de Enfermedad , Cobayas , Masculino , Potenciales de la Membrana , Microscopía Electrónica de Rastreo , Antagonistas del Receptor de Neuroquinina-1 , Técnicas de Placa-Clamp , Piperidinas/farmacología , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/antagonistas & inhibidores , Sustancia P/farmacología , Tráquea/efectos de los fármacos , Tráquea/ultraestructura , Traqueítis/inducido químicamente , Traqueítis/patología , Traqueítis/fisiopatología , Vagotomía , Nervio Vago/efectos de los fármacos , Nervio Vago/cirugíaRESUMEN
The neutralist-selectionist debate should not be viewed as a dichotomy but as a continuum. While the strictly neutral model suggests a neutralist-selectionist dichotomy, the nearly neutral model is a continuous model spanning strict neutrality through weak selection (Ns approximately 1) to deterministic selection (Ns>3). We illustrate these points with polymorphism and divergence data from a sample of 73 genes (31 mitochondrial, 36 nuclear genes from Drosophila, and six Arabidopsis data sets). In an earlier study we used the McDonald-Kreitman (MK) test to show that amino acid replacement polymorphism in animal mitochondrial genes and Arabidopsis genes show a consistent trend toward negative selection, whereas nuclear genes from Drosophila span a range from negative selection, through neutrality, to positive selection. Here we analyze a subset of these genes (13 Drosophila nuclear, ten mitochondrial, and six Arabidopsis nuclear) for polymorphism and divergence of conservative and radical amino acid replacements (a protein-based conservative-radical MK, or pMK, test). The distinct patterns of selection between the different genomes is not apparent with the pMK test. Different definitions of conservative and radical (based on amino acid polarity, volume or charge) give inconsistent results across genes. We suggest that segregating fitness difference between silent and replacement mutations are more visible to selection than are segregating fitness differences between conservative and radical amino acid mutations. New data on the variation among genes with different opportunities for positive and negative selection are as important to the continuum view of the neutralist-selectionist debate as is the distribution of selection coefficients within individual genes.
Asunto(s)
Aminoácidos/genética , ADN Mitocondrial/genética , Proteínas Nucleares/genética , Proteínas/genética , Animales , Arabidopsis/genética , Composición de Base , Codón/genética , Secuencia Conservada , Drosophila/genética , Evolución Molecular , Frecuencia de los Genes , Genes/genética , Modelos Genéticos , Mutación , Polimorfismo Genético , Selección GenéticaRESUMEN
McDonald/Kreitman tests performed on animal mtDNA consistently reveal significant deviations from strict neutrality in the direction of an excess number of polymorphic nonsynonymous sites, which is consistent with purifying selection acting on nonsynonymous sites. We show that under models of recurrent neutral and deleterious mutations, the mean age of segregating neutral mutations is greater than the mean age of segregating selected mutations, even in the absence of recombination. We develop a test of the hypothesis that the mean age of segregating synonymous mutations equals the mean age of segregating nonsynonymous mutations in a sample of DNA sequences. The power of this age-of-mutation test and the power of the McDonald/Kreitman test are explored by computer simulations. We apply the new test to 25 previously published mitochondrial data sets and find weak evidence for selection against nonsynonymous mutations.
Asunto(s)
ADN Mitocondrial/genética , Modelos Genéticos , Mutación/genética , Selección Genética , Alelos , Animales , Segregación Cromosómica/genética , Simulación por Computador , Ligamiento Genético/genética , Haplotipos/genética , Fenotipo , Polimorfismo Genético/genética , Distribuciones Estadísticas , Factores de TiempoRESUMEN
Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3-0.5 s) and a long duration (3-15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Calcio/fisiología , Neuronas Aferentes/fisiología , Ganglio Nudoso/fisiología , Nervio Vago/fisiología , Potenciales de Acción , Animales , Bradiquinina/farmacología , Canales de Calcio/efectos de los fármacos , Dinoprostona/farmacología , Potenciales Evocados , Cobayas , Histamina/farmacología , Leucotrieno C4/farmacología , Prostaglandina D2/farmacología , Conejos , Serotonina/farmacologíaRESUMEN
1. The regulation of substance P (SP) responsiveness in acutely isolated nodose neurones from adult guinea-pigs was investigated using standard intracellular recording techniques. 2. In control neurones, SP produced no measurable electrophysiological effects. However, following incubation with serotonin (5-HT, 10 microM), 64% of neurones were depolarized by 10 +/- 0.6 mV (n = 84 of 132 neurones) by SP (100 nM). 5-HT-induced SP responses were inhibited by SR48968 (100 nM, n = 6), a neurokinin 2 (NK-2) receptor antagonist, but were unaffected by CP99,994 and SR142801, NK-1 and NK-3 receptor antagonists (n = 3 each), respectively. 3. 5-HT-induced unmasking of SP responses was maximal within 5 min. Increasing the 5-HT incubation time up to 120 min did not increase the mean response amplitude or the percentage of SP responsive neurones (P = 0.611 and 0.867, respectively). 4. 5-HT-induced unmasking of SP responses was dose dependent (EC50 = 14 nM). A 5-HT3 receptor agonist CPBG (1 microM), mimicked the unmasking effects of 5-HT (n = 10 of 19 neurones), while 5-CT (10 microM), a non-selective 5-HT agonist devoid of action at 5-HT3 receptors, did not (n = 18). ICS205-930 (1 microM), a 5-HT3 receptor antagonist, completely blocked the 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 5. In 68% of the neurones tested, bath-applied 5-HT (10 microM) evoked a 178 +/- 29.5 nM increase in [Ca2+]i (n = 16), which was blocked by nominally zero [Ca2+]o (n = 4) or by ICS205-930 (1 microM, n = 4). Nodose neurones incubated with 5-HT in the presence of nominally zero [Ca2+]o did not respond to SP (n = 12 of 13 neurones) in Locke solution containing normal [Ca2+]o, indicating that the 5-HT-mediated elevation of [Ca2+]i is required for unmasking of SP responses. Calmidazolium (100 nM), a calmodulin inhibitor, inhibited the unmasking effects of 5-HT (n = 5 of 5 neurones). 6. Incubating neurones with the nitric oxide (NO) donors papaNONOate (1 mM, 15-30 min) or SNAP (50 microM, 30-60 min) unmasked depolarizing SP responses in 71% and 45% of the neurones studied, respectively. L-NMMA (30 microM), a NO synthase inhibitor, blocked 5-HT-induced unmasking of SP responses (n = 10 of 10 neurones). 7. In sum, these results suggest that stimulation of 5-HT3 receptors activates an intracellular signalling cascade that couples calcium-calmodulin and NO activation to NK-2 receptor unmasking in sensory neurones.