Rostral ventral medulla 5-HT1A receptors selectively inhibit the somatosympathetic reflex

The role of the 5-hydroxytryptamine (5-HT1A) receptors in the rostral ventrolateral medulla (RVLM) on somatosympathetic, baroreceptor, and chemoreceptor reflexes was examined in anesthetized rats. Microinjection of the selective 5-HT1A agonist 8-hydroxy-di-n-propylamino tetralin (8-OH-DPAT) decreased arterial blood pressure and splanchnic sympathetic nerve activity (SNA). Electrical stimulation of the hindlimb evoked early and late excitatory sympathetic responses. Bilateral microinjection in the RVLM of 8-OH-DPAT markedly attenuated both the early and late responses. This potent inhibition of the somatosympathetic reflex persisted even after SNA and arterial blood pressure returned to preinjection levels. Preinjection of the selective 5-HT1A antagonist NAN-190 in the RVLM blocked the sympathoinhibitory effect of 8-OH-DPAT and attenuated the inhibitory effect on the somatosympathetic reflex. 8-OH-DPAT injected in the RVLM did not affect baroreceptor or chemoreceptor reflexes. Our findings suggest that activation of 5-HT1A receptors in the RVLM exerts a potent, selective inhibition on the somatosympathetic reflex.     

Spinal GABA(A) receptors do not mediate the sympathetic baroreceptor reflex in the rat

Activation of baroreceptors causes efferent sympathetic nerve activity (SNA) to fall. Two mechanisms could account for this sympathoinhibition: disfacilitation of sympathetic preganglionic neurons (SPN) and/or direct inhibition of SPN. The roles that spinal GABA and glycine receptors play in the baroreceptor reflex were examined in anesthetized, paralyzed, and artificially ventilated rats. Spinal GABA(A) receptors were blocked by an intrathecal injection of bicuculline methiodide, whereas glycine receptors were blocked with strychnine. Baroreceptors were activated by stimulation of the aortic depressor nerve (ADN), and a somatosympathetic reflex was used as control. After an intrathecal injection of vehicle, there was no effect on any measured variable or evoked reflex. In contrast, bicuculline caused a dose-dependent increase in arterial pressure, SNA, phrenic nerve discharge, and it significantly facilitated the somatosympathetic reflex. However, bicuculline did not attenuate either the depressor response or sympathoinhibition evoked after ADN stimulation. Similarly, strychnine did not affect the baroreceptor-induced depressor response. Thus GABA(A) and glycine receptors in the spinal cord have no significant role in baroreceptor-mediated sympathoinhibition.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Estimating the Hidden Burden of Bovine Tuberculosis in Great Britain

The number of cattle herds placed under movement restrictions in Great Britain (GB) due to the suspected presence of bovine tuberculosis (bTB) has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown) within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC) to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI) where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR). Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26–4.9; 95% CI) in a herd of 30 cattle up to 4.9 (0.99–14.0) in a herd of 400. Under this model we estimate that 50% (33–67) of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11–42) of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence unless this extrinsic infectious pressure is simultaneously addressed.     

The site of action of pyrethrin I in the nervous system of the cockroach Periplaneta americana

Spontaneous nervous activity in the sixth abdominal ganglia of the cockroach is increased by much smaller concentrations of pyrethrin I than are needed to affect conduction in giant fibre axons, suggesting that the fatal lesions caused by pyrethrin I may be within the ganglia rather than associated with axonic conduction.Pyrethrin I is about 30 times more active than allethrin (Narahashi, 1962, a & b) against conduction in giant fibres.

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Zusammenfassung Neurophysiologische Methoden wurden angewendet, um die Wirkung von Pyrethrin I auf die Leitung von Aktionspotentialen in Riesenfasern und den Umfang der Spontanaktivität im 6. Abdominalganglion erwachsener männlicher Küchenschaben festzustellen. In Versuchen an isolierten Nervenpräparaten hatten Pyrethrin I-Konzentrationen von weniger als 10^-7 M wenig Wirkung auf die Riesenfasern, während so geringe Konzentrationen wie 10^-10 M im Ganglion die Höhe der Aktivität beeinflußten. Riesenfasern von Küchenschaben, die vorher örtlich mit DL95s von Pyrethrin I behandelt worden waren, wurden 1–4 Stunden nach der Vergiftung stark beeinflußt, wenn die Insekten stark angegriffen waren, doch war die Aktivität im 6. Abdominalganglion zu diesem Zeitpunkt stark erhöht. Einige intraganglionäre Neurone sind also gegenüber der Vergiftung mit Pyrethrinen wesentlich empfindlicher als die Riesenfasern, was darauf hindeutet, daß die gefährlichen Schädigungen durch Pyrethrine wahrscheinlich eher innerhalb der Ganglien als in peripheren Nerven auftreten.Die Wirkung von Pyrethrin I an Riesenfasern ähnelt den von Narahashi (1962 a+b) für Allethrin beschriebenen, doch war Pyrethrin I etwa 30mal aktiver.

     

The spread of topically-applied pyrethrin I from the cuticle to the central nervous system of the cockroach Periplaneta americana

Penetration of topically-applied pyrethrin I into adult male American cockroaches (Periplaneta americana L.) and its subsequent distribution within the insects was studied microchemically and biologically. After applying an LD95 (0.5 g per insect), pyrethrin I was lost from the surface of the insects at a rate diminishing with time; 20% of the dose applied penetrated during the first hour, but elimination limited the amount found inside the insects to a maximum of 13%. The amount inside the insects increased for an hour after dosing, when symptoms of poisoning had become severe, but then remained steady until the end of the test, 48 hrs after dosing.Pyrethrin I sorbs strongly but reversibly on insect solids from aqueous solution, and at equilibrium is distributed between solids and solution in the ratio 3 × 10⁴: 1. This ratio would give a concentration of only 4 × 10^–11 M of pyrethrin I in the haemolymph of insects poisoned with an LD95 of the insecticide, but solids in the haemolymph would increase its pyrethrin I content and speed the spread of the insecticide from cuticle to nervous system. Chemical tests able to detect pyrethrin I at concentrations of 2×10^–8 M failed to show its presence in the haemolymph of poisoned cockroaches, but the haemolymph caused symptoms resembling those of pyrethroid poisoning when applied to nerve preparations from normal cockroaches.Although pyrethrin I at 4 × 10^–11 M in the haemolymph of cockroaches treated topically with LD95s of the insecticide seems too dilute to have produced the symptoms observed in their nervous systems, the results of additional tests did not support the alternative suggestion that the insecticide reached the nervous system by spreading over the cuticle and through the tracheal system. Larger concentrations of pyrethrin I occurring locally in the haemolymph near the nervous system during the early stages of poisoning may explain the anomaly.

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Résumé La pénétration d'une application topique de pyrethrine I chez le mâle adulte de Periplaneta americana et sa distribution ultérieure dans l'organisme est étudiée microchimiquement et biologiquement. Après application d'une dose égale à la DL 95 (0,5 g par insecte), la pyrethrine I est absorbée à la surface de l'insecte à un taux décroissant avec le temps; 20% de la dose appliquée pénètre durant la 1ère heure, mais l'élimination limite la quantité trouvée dans l'organisme à un maximum de 13%. La quantité trouvée dans l'organisme s'accroît pendant la 1ère heure après l'intoxication, quand les symptômes de l'intoxication deviennent importants, puis reste stationnaire jusqu'à la fin du test, soit 48 heures après l'intoxication.La pyrethrine I s'absorbe fortement mais réversiblement sur la phase solide de l'insecte, à partir de la solution aqueuse et le coefficient de partition est de 3×10⁴: 1. Ce coefficient donnarait une concentration de pyrethrine I de seulement 4 × 10^–11 M dans l'hémolymphe des insectes intoxiqués à la DL 95, mais la phase solide de l'hémolymphe augmenterait sa teneur en pyrethrine I et accélérerait la vitesse de pénétration de l'insecticide, de la cuticule vers le système nerveux. Des tests chimiques capables de déceler la pyrethrine I à la concentration de 2×10^–8 M n'ont pu établir sa présence dans l'hémolymphe des blattes intoxiquées, mais cette hémolymphe provoque des symptômes analogues à ceux de l'intoxication par les pyrethrines, quand elle est appliquée à des préparations nerveuses de blattes nonintoxiquées.Quoique la pyrethrine I à la concentration de 4 × 10^–11 M dans l'hémolymphe de blattes traitées localement à la DL 95 de cet insecticide, semble trop diluée pour produire les symptômes observés dans le système nerveux, des résultats supplémentaires n'ont pas autorisé une autre hypothèse, à savoir que l'insecticide atteindrait le système nerveux via la cuticule et le système trachéen. Des concentrations plus fortes de pyrethrine I, localisées près du système nerveux pendant les premiers stades de l'intoxication pourraient expliquer cette anomalie.