Effect on breathing of surface ventrolateral medullary cooling in awake, anesthetized and asleep goats

In adult and neonatal goats, we chronically implanted thermodes on the ventrolateral (VLM) medullary surface to create reversible neuronal dysfunction and thereby gain insight into the role of superficial VLM neurons in control of breathing in anesthetized, awake and asleep states. Consistent with data of others, cooling caudal area M and rostral area S caused sustained apnea under anesthesia. However, in the awake and NREM sleep states, cooling at this site caused only a modest reduction in breathing, indicating that neurons at this site are not critical for respiratory rhythm in these states. Moreover, data in the awake state over multiple conditions suggest neurons at this site do not integrate all intracranial and carotid chemoreception. The data suggest though that neurons at this site have a facilitatory-like effect on breathing both unrelated and related to intracranial chemoreception. We believe that this facilitation serves a function similar to the facilitation provided by the carotid chemoreceptors and by sources associated with wakefulness. Accordingly, elimination/attenuation of any one of these three influences (caudal M rostral S VLM, wakefulness, carotid chemoreception) results in a slight decrease in breathing, removal of two of the three results in a greater decrease in breathing, and removal of all three results in sustained apnea.     

Classification of caudal ventrolateral pontine neurons with sympathetic nerve-related activity

This study was designed to answer three questions concerning caudal ventrolateral pontine (CVLP) neurons whose naturally occurring discharges are correlated to sympathetic nerve discharge (SND). 1) What are the proportions of CVLP neurons that have activity correlated to both the cardiac-related and 10-Hz rhythms in SND, to only the 10-Hz rhythm, and to only the cardiac-related rhythm? 2) Do CVLP neurons with activity correlated to the cardiac-related and/or 10-Hz rhythm in SND subserve a sympathoexcitatory or sympathoinhibitory function? 3) Do CVLP neurons with activity correlated to the cardiac-related and/or 10-Hz rhythm in SND project to the thoracic spinal cord? To address these issues we recorded from 476 CVLP neurons in 24 urethan-anesthetized cats. Spike-triggered averaging, arterial pulse-triggered analysis, and coherence analysis revealed that the discharges of 66 of these neurons were correlated to inferior cardiac postganglionic SND. For 39 of these neurons, we were able to determine whether their discharges were correlated to one or both rhythms. The results showed that the CVLP contained a heterogeneous population of neurons with sympathetic nerve-related activity. The discharges of 21 neurons were correlated to both the 10-Hz and cardiac-related rhythms in SND, 9 neurons had activity correlated to only the 10-Hz rhythm, and 9 neurons had activity correlated to only the cardiac-related rhythm. The firing rates of CVLP neurons with activity correlated to both rhythms or to only the 10-Hz rhythm were decreased during the inhibition of SND induced by baroreceptor reflex activation (rapid obstruction of the abdominal aorta). These neurons are presumed to exert sympathoexcitatory actions. The time-controlled collision test verified that 11 of 12 CVLP neurons with activity correlated to both rhythms were antidromically activated by stimulation of the first thoracic segment of the spinal cord. Antidromic mapping at this level showed that the site requiring the least stimulus current to elicit the longest latency response (nearest the terminal) was in the vicinity of the intermediolateral nucleus (IML). In contrast, only 1 of 13 CVLP neurons with activity correlated to only one of the rhythms in SND could be antidromically activated by spinal stimulation. These data demonstrate for the first time that there is a direct pathway from the CVLP to the IML that is comprised almost exclusively of sympathoexcitatory neurons whose discharges are correlated to both the 10-Hz and cardiac-related rhythms in SND.     

Innervation of serotonergic medullary raphe neurons from cells of the rostral ventrolateral medulla in rats

The rostral ventral medulla has been shown to consist of three distinct subregions: the midline or raphé region, the lateral paragigantocellular-gigantocellular region and the rostro-ventrolateral reticular nucleus. All three regions have been shown to contribute to central vaso-regulation and to project towards sympathetic preganglionic neurons of the thoracic spinal cord. Therefore it is of particular interest to describe the interconnections between the three regions and to see if local afferents reach cells which have been implicated in the regulation of descending inputs. Following injections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the lateral paragigantocellular nucleus or the rostroventrolateral reticular nucleus, labelled axons were traced into the medullary raphé nuclei and the contralateral rostral ventrolateral medulla. Efferents originating from both regions innervated the raphé pallidus, raphé obscurus and raphé magnus. However the distribution of terminals originating from the two regions was different in the contralateral ventrolateral medulla oblongata. The data indicate that the connection between the ipsi- and contralateral equivalents of both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus are stronger than the cross-connection between the ipsi- and contralateral parts of the two different regions. In the second part of the study, the existence of direct projections from the rostroventrolateral reticular nucleus and the lateral paragigantocellular-gigantocellular region onto serotonin-immunogold-labelled cells of the ventromedial medulla were investigated. The correlated light and electron microscopic analysis revealed direct synaptic contacts between axons originating from both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus, and serotonin-immunoreactive cells of the raphé obscurus and raphé pallidus. The results of the present light microscopic tract-tracing study revealed a different pattern of the intramedullary projection of the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus. These data are in support of the proposed parcellation of the two cytoarchitectonically different areas of the rostral ventrolateral medulla into two functionally distinct subdivisions. Furthermore, the direct anatomical connection revealed in the present study between cells of the rostral ventrolateral and ventromedial medulla oblongata indicates the possibility that vasoregulatory effects of some cells of the rostral ventrolateral medulla oblongata might be executed via direct projections onto serotonin-immunoreactive cells of the medullary raphé nuclei.     

Effects of ACh on the electric activity of rostral ventrolateral medullary neurons of rat in vitro

Extracellular single-unit discharges were obtained from 165 spontaneously active neurons within the region of the rostral ventrolateral medulla (RVLM) by glass microelectrode from 89 brain slices of the Sprague-Dawley rats. The units could be divided into three types: regular (61.8%), irregular (24.2%) and silent (14%). Acetylcholine (ACh, 0.1, 0.3 mumol/L) showed four kinds of effects on spontaneous discharges of RVLM neurons: excitatory, inhibitory, biphasic and non-responsive, counting respectively 41.8%, 20%, 3% and 35.2% of the neurons tested. The excitatory effect of ACh was dose-dependent. The effects, either excitatory or inhibitory, of ACh (n = 49) were mostly blocked by atropine (0.3 mumol/L, n = 42). The excitatory effect of ACh (n = 14) could be blocked mainly by selective antagonist of M1 receptor, pirenzepine (PZ, 30 nmol/L, n = 9), but not by selective antagonist of M2 receptor, methoctramine (MT) and AFDX-116. The inhibitory effect of ACh (n = 10) could be blocked mostly by M2 receptor antagonist MT (30 nmol/L, n = 7); and this inhibitory effect (n = 9) could be blocked mostly by another M2 receptor antagonist AFDX-116 (30 nmol/L, n = 6), but not by M1 receptor antagonist PZ.     

Temporary leukocyte depletion reduces ventricular dysfunction during prolonged postischemic reperfusion

Leukocyte depletion improves early postischemic ventricular performance in neonatal models of global myocardial ischemia. However, the rate of leukocyte reaccumulation after cardiopulmonary bypass and its subsequent impact on myocardial function is not known. This laboratory study examined the effect of leukocyte depletion on myocardial performance during the initial 6-hour period after bypass in an in situ, in vivo porcine model of neonatal cardiac surgery. Fifteen 3- to 5-day-old piglets (eight control and seven leukocyte depleted animals) were instrumented by placement of left ventricular short-axis sonomicrometry crystals and an intraventricular micromanometer catheter. Mechanical leukocyte depletion was achieved with Pall RC100 filters (Pall Biomedical, Inc., Fajardo, Puerto Rico) in the cardiopulmonary bypass circuit. Neonatal hearts were subjected to 90 minutes of hypothermic ischemia after a single dose of cold crystalloid cardioplegia. Two control animals died after the operation and were excluded from data analysis. Leukocyte filtration reduced the granulocyte count during initial myocardial reperfusion to 0.8% of control values. However, circulating granulocyte counts increased in leukocyte depleted animals throughout the postoperative period, reaching 68% of control values by 6 hours. Despite this rapid return of circulating granulocytes, animals subjected to leukocyte depletion had significantly better preservation of left ventricular performance (measured by preload recruitable stroke work, p < or = 0.02), left ventricular systolic function (measured by end-systolic pressure-volume relationship, p < or = 0.05), and ventricular compliance (p < or = 0.04) during the experiment. These changes in ventricular function were associated with a significant increase in left ventricular water content (p < or = 0.02) and tissue myeloperoxidase activity (p < or = 0.005) in control animals compared with leukocyte depleted animals. This study demonstrates that leukocyte depletion during initial reperfusion results in sustained improvement in postischemic left ventricular function despite the rapid return of granulocytes to the circulation.     

Activity of medullary respiratory neurons during ventilator-induced apnea in sleep and wakefulness

Mechanical ventilation of cats in sleep and wakefulness causes apnea, often within two to three cycles of the ventilator. We recorded 137 medullary respiratory neurons in four adult cats during eupnea and during apnea caused by mechanical ventilation. We hypothesized that the residual activity of respiratory neurons during apnea might reveal its cause(s). The results showed that residual activity depended on 1) the amount of nonrespiratory inputs to the cell (cells with more nonrespiratory inputs had greater amounts of residual activity); 2) the cell type (expiratory cells had more residual activity than inspiratory cells); and 3) the state of consciousness (more residual activity in wakefulness and rapid-eye-movement sleep than in non-rapid-eye-movement sleep). None of the cells showed an activation during ventilation that could explain the apnea. Residual activity of approximately one-half of the cells was modulated in phase with the ventilator. The strength of this modulation was quantified by using an effect-size statistic and was found to be weak. The patterns of modulation did not support the idea that mechanoreceptors excite some respiratory cells that, in turn, inhibit others. Indeed, most cells, inspiratory and expiratory, discharged during the deflation-inflation transition of ventilation. Residual activity failed to reveal the cause of apnea but showed that during apnea respiratory neurons act as if they were disinhibited and disfacilitated.     

Changes in the impulse activity of neurons of the human thalamic ventrolateral nucleus during voluntary movement

Functional differences were revealed in evoked activity of two types (A and B) of units of the human thalamic ventro-lateral nucleus (VL). Collective activities of these polyfunctional neurons were selectively related to triggering and execution phases of movement. Common character of dynamics of the responses seems to be due to similar polyfunctional nature as well as to the functional role of these two complementary elements in the motor signal transmission. The collective activities reflect in the VL the integrative processes related to processing and programming of generalised parameters of motor signals, but unrelated to performance of a concrete motor act.     

Activation of NMDA and non-NMDA receptors in the caudal ventrolateral medulla dilates the airways

Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.     

Influence of preload and afterload on genioglossus muscle length in awake goats

The genioglossus is an upper airway dilator muscle, the length of which is directly related to patency in the oropharyngeal region. We hypothesized that genioglossal length (Lgg) is dynamically influenced by the afterload exerted by negative upper airway pressure during inspiration and by the intrinsic length-tension characteristics of the muscle (preload). Seven awake goats were chronically instrumented with electrodes for EMGgg and sonomicrometry for Lgg. We examined the Lgg-EMGgg relationship during hypercapnia and inspiratory resistive loading (18 cm H2O/L/s). The goats breathed through the upper airway (TC) or airflow was diverted through a tracheostomy (TO). We found that: (1) passive inspiratory lengthening was observed with negative upper airway pressure (UAP) but not when UAP = 0 (TO breathing), (2) Lgg shortening for a given EMGgg was significantly decreased with negative inspiratory UAP, and (3) phasic Lgg shortening per unit EMGgg was greatest when Lgg was near optimal length (Lo). We conclude that genioglossal length is substantially influenced by afterload exerted by negative UAP and that genioglossal active shortening may be limited if the muscle operates at a length significantly greater or less than the optimal length.     

Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2

Prostaglandin D2 (PGD2) is an extensively studied sleep-promoting substance, but the neuroanatomical basis of PGD2-induced sleep is only partially understood. To determine potential regions involved in this response, we used Fos immunohistochemistry to identify neurons activated by infusion of PGD2 into the subarachnoid space below the rostral basal forebrain. PGD2 increased nonrapid eye movement sleep and induced striking expression of Fos in the ventrolateral preoptic area (VLPO), a cluster of neurons that may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD2 also increased Fos immunoreactivity in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO.     

Adrenergic reactivity after inhibition of nitric oxide synthesis in the cerebral circulation of awake goats

The interaction between nitric oxide (NO) and adrenergic reactivity in the cerebral circulation was studied using in vivo and in vitro preparations. Blood flow to one brain hemisphere (cerebral blood flow) was electromagnetically measured in conscious goats, and the effects of norepinephrine, tyramine and cervical sympathetic nerve stimulation were recorded before (control) and after inhibition of NO formation with Nw-nitro-l-arginine methyl ester (l-NAME). The responses to norepinephrine, tyramine and electrical field stimulation were also recorded in segments, 4 mm in length, from the goat's middle cerebral artery under control conditions and after l-NAME. In vivo, l-NAME (10 goats, 47 mg kg-1 administered i.v.) reduced resting cerebral blood flow by 37+/-2%, increased mean systemic arterial pressure by 24+/-3%, reduced heart rate by 35+/-2%, and decreased cerebrovascular conductance by 52+/-2% (all P<0.01). Norepinephrine (0.3-9 microgram), tyramine (50-500 microgram), and supramaximal electrical sympathetic cervical nerve stimulation (1. 5-6 Hz) decreased cerebrovascular conductance, and these decreases were significantly higher after l-NAME than under control conditions, remaining higher for about 48 h after this treatment. Norepinephrine (10-8-10-3 M), tyramine (10-6-10-3 M) and electrical field stimulation (1.5-6 Hz) contracted isolated cerebral arteries, and the maximal contraction, but not the sensitivity, was significantly higher in the arteries treated than in non-treated with l-NAME (10-4 M). Therefore, the reactivity of cerebral vasculature to exogenous and endogenous norepinephrine may be increased after inhibition of NO synthesis. This increase might be related, at least in part, to changes at postjunctional level in the adrenergic innervation of the vessel wall, and it might contribute to the observed decreases in resting cerebral blood flow after inhibition of NO synthesis.     

Tooth pulp neurons in the caudal medulla oblongata of the cat

The medulla oblongata caudal to the obex was explored for neurons responsive to tooth pulp (TP) stimulation in cats. Four different subclasses of TP neurons were found. The latter included TP specific (TPS) neurons, trigeminal wide dynamic range (trigeminal WDR) neurons with TP input, trigeminal subnucleus reticularis ventralis (trigeminal SRV) neurons with TP input and convergent reticular formation (convergent RF) neurons with TP input. TPS neurons were located in the dorsal marginal rim of the trigeminal subnucleus caudalis, i.e., in the marginal layer or the outer zone of substantia gelatinosa. WDR neurons with TP input were found in the neck region of medullary dorsal horn which corresponds to the lateral part of subnucleus reticularis dorsalis (SRD). Trigeminal SRV neurons with TP input were located in the lateral part of SRV. Convergent RF neurons with TP input were found in the middle third of the caudal bulbar RF consisting of SRD and SRV. Both TPS neurons and WDR neurons with TP input included trigeminothalamic neurons as evidenced by the antidromic activation from the nucleus ventralis posteromedialis of the contralateral thalamus. A significant proportion of both trigeminal SRV and convergent RF neurons with TP input were antidromically activated by stimulation of the nucleus centralis lateralis of the contralateral thalamus. The former two subclasses may subserve the sensory-discriminative aspect of toothache, while the latter two subclasses, the emotional-motivational aspect.     

Role of the nucleus raphe obscurus in the inhibition of rostral ventrolateral medullary neurones induced by stimulation in the ventrolateral periaqueductal grey matter of the rabbit

The uptake, partitioning, and release of ingredients such as water, oil, surfactant, and ions are important factors to understand and control in the design and manufacture of detergent and personal products. Although conventional pulse NMR (PNMR) spectroscopy continues to be used to analyse bulk molecular mobility and phase composition, more recently MR imaging techniques have created unique opportunities for gaining spatial information about these processes in ways that are noninvasive and potentially quantitative. This paper describes the evaluation of MRI and associated PNMR techniques to study transport in three relevant cases: ion diffusion (e.g., fluoride) in concentrated dispersions, oil transport through powders, and water ingress into porous powders (zeolite). Results are presented to illustrate the potential of multiple pulse and gradient echo MRI methods for dealing with the short T2 scenarios that represent a common problem in quantitative imaging of water in solid-containing composites involving, for instance, zeolite, or silica. Pore-size characterisation results are also presented.     

Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices

Intracellular Ca2+ ([Ca2+]i) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5-5 Hz. [Ca2+]i increased by about 30 nm upon rising spike frequency by more than 200% due to 20-50 pA current pulses or 10 micrometer serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni2+ and also during hyperpolarization due to gamma-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K+ (KATP) channels with diazoxide. KATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca2+]i decrease which reversed into a secondary Ca2+ rise by less than 100 nm. Similar moderate rises of [Ca2+]i were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca2+]i transients did not correlate with the amplitude of the KATP channel-mediated outward current. [Ca2+]i did not change during diazoxide-induced or spontaneous activation of KATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca2+]i with cyclopiazonic acid did not activate KATP channels. [Ca2+]i was not affected upon block of outward current with sulphonylureas, but these KATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca2+]i fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced KATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca2+]i fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca2+]i to more than 1 micrometer. The results indicate that KATP channel activity in DVN is not affected by physiological changes of intracellular Ca2+ and the lack of a major perturbance of Ca2+ homeostasis contributes to their high tolerance to anoxia.     

The characteristics of the spontaneous activity of single neurons in the human ventrolateral thalamus during changes in the functional state of the brain

In the thalamic ventro-lateral nucleus of parkinsonian patients, two main types of convergent cells were shown to exist: 1) units with irregular discharges (A-cells, 74%) and 2) units with bursts of unstable rhythmic discharges (B-cells, 26%). The functional brain changes were accompanied by modifications of A-cells activity into the transient rhythmic burst-like pattern, characterized by two different types of intrinsic structure burst discharges being in some cases similar to the structure of B-cells. A correlation between activities of these units and the type of parkinsonian pathology was revealed.