Ceftriaxone preserves glutamate transporters and prevents intermittent hypoxia-induced vulnerability to brain excitotoxic injury.

Hypoxia alters cellular metabolism and although the effects of sustained hypoxia (SH) have been extensively studied, less is known about chronic intermittent hypoxia (IH), commonly associated with cardiovascular morbidity and stroke. We hypothesize that impaired glutamate homeostasis after chronic IH may underlie vulnerability to stroke-induced excitotoxicity. P16 organotypic hippocampal slices, cultured for 7 days were exposed for 7 days to IH (alternating 2 min 5% O2-15 min 21% O2), SH (5% O2) or RA (21% O2), then 3 glutamate challenges. The first and last exposures were intended as a metabolic stimulus (200 µM glutamate, 15 min); the second emulated excitotoxicity (10 mM glutamate, 10 min). GFAP, MAP2, and EAAT1, EAAT2 glutamate transporters expression were assessed after exposure to each hypoxic protocol. Additionally, cell viability was determined at baseline and after each glutamate challenge, in presence or absence of ceftriaxone that increases glutamate transporter expression. GFAP and MAP2 decreased after 7 days IH and SH. Long-term IH but not SH decreased EAAT1 and EAAT2. Excitotoxic glutamate challenge decreased cell viability and the following 200 µM exposure further increased cell death, particularly in IH-exposed slices. Ceftriaxone prevented glutamate transporter decrease and improved cell viability after IH and excitotoxicity. We conclude that IH is more detrimental to cell survival and glutamate homeostasis than SH. These findings suggest that impaired regulation of extracellular glutamate levels is implicated in the increased brain susceptibility to excitotoxic insult after long-term IH.

Respiratory circuits: development, function and models.

Breathing is a rhythmic motor behavior generated and controlled by hindbrain neuronal networks. Respiratory motor output arises from two distinct, but functionally interacting, rhythmogenic networks: the pre-Bötzinger complex (preBötC) and the retrotrapezoïd nucleus/parafacial respiratory group (RTN/pFRG). This review outlines recent advances in delineating the genetic specification of the neuronal constituents of these two rhythmogenic networks, their respective roles in respiratory function and how they interact to constitute a functional respiratory circuit ensemble. The often lethal consequences of disruption to these networks found in naturally occurring developmental disorders, transgenic animals, and highly specific lesion studies are described. In addition, we discuss how recent computational models enhance our understanding of how respiratory networks generate and regulate respiratory behavior.

Functional anatomical evidence for respiratory rhythmogenic function of endogenous bursters in rat medulla.

Endogenous burster neurons (EBs) have been found at the level of the facial nucleus (VIIn), and 500 mum caudally, within the pre-Bötzinger complex (preBötC). They have been proposed as either causal to or playing no role in respiratory rhythmogenesis. Little is known about their broader distribution in ventrolateral medulla. Here, a Ca(2+) indicator was used to record respiratory network activity in ventrolateral medulla, and, following synaptic blockade, to identify EBs active at perfusate K(+) concentrations ([K(+)](o)) of 3, 6, and 9 mm. Recordings were made along the respiratory column, extending 300 mum rostrally, and 1100 mum caudally from the caudal pole of VIIn (VIIc), in the in vitro tilted sagittal slab preparation, isolated from neonate male and female Sprague Dawley rats. Activity under matching [K(+)](o) in the intact respiratory network was subsequently investigated. Respiratory neurons (n = 401) formed statistically significant clusters at the VIIc, within the preBötC, and 100 mum caudal to the preBötC. EBs (n = 693) formed statistically significant clusters that overlapped with respiratory clusters at the VIIc and preBötC. EB activity increased significantly as [K(+)](o) was increased, as did neurons that remained coupled following synaptic blockade. The overlap between respiratory and EB clusters in regions of ventrolateral medulla identified as rhythmogenic supports the hypothesis that EBs are constituents of rhythmogenic networks. In addition, the observation of truncated inspiratory bursts and ectopic bursting in respiratory neurons when [K(+)](o) was elevated in the intact network is consistent with a causal role for EBs in respiratory rhythmogenesis.

Degeneracy as a substrate for respiratory regulation.

Recent studies in vivo and in vitro suggest that both respiratory rhythmogenesis and its central chemosensory modulation arise from multiple, mechanistically and/or anatomically distinct networks whose outputs are similar. These observations are consistent with degeneracy, defined as the ability of structurally distinct elements to generate similar function. This review argues that degeneracy is an essential feature of respiratory networks, ensuring the survival of the individual organism over the course of development, and accounting for the transformation of respiratory biomechanics over evolutionary time. At faster timescales, respiration must adapt continuously and rapidly to changes in metabolic demand and ambient conditions to maintain blood-gas homeostasis. Control theory, which formalizes homeostasis, states axiomatically that rapid responsiveness can only be achieved with high gain, but high gain comes at the cost of instability. Homeostatic systems displaying highly optimized tolerance (HOT) mitigate the instability accompanying high gain by incorporating regulatory mechanisms that provide protection against expected perturbations, yet these systems remain fragile to catastrophic failure in response to rare events. Because the multiple mechanisms that are conjectured to mediate respiratory rhythmogenesis and chemosensation have distinct ranges of activity and responses to modulatory input, they provide a richer substrate for respiratory regulation than those of any single mechanism. Respiration, though robust, remains fragile to rare perturbations, matching a key feature of HOT. These observations support the conclusion that degeneracy provides the substrate for respiratory regulation, and that the resulting regulatory system conforms to HOT.

Semi-automated region of interest generation for the analysis of optically recorded neuronal activity.

Bath-applied membrane-permeant Ca(2+) indicators offer access to network function with single-cell resolution. A barrier to wider and more efficient use of this technique is the difficulty of extracting fluorescence signals from the active constituents of the network under study. Here we present a method for semi-automatic region of interest (ROI) detection that exploits the spatially compact, slowly time-varying character of the somatic signals that these indicators typically produce. First, the image series is differenced to eliminate static and very slowly varying fluorescence values, and then the differenced image series undergoes low-pass filtering in the spatial domain, to eliminate temporally isolated fluctuations in brightness. This processed image series is then thresholded so that pixel regions of fluctuating brightness are set to white, while all other regions are set to black. Binary images are averaged, and then subjected to iterative thresholding to extract ROIs associated with both dim and bright cells. The original image series is then analyzed using the generated ROIs, after which the end-user rejects spurious signals. These methods are applied to respiratory networks in the neonate rat tilted sagittal slab preparation, and to simulations with signal-to-noise ratios ranging between 1.0-0.2. Simulations established that algorithm performance degraded gracefully with increasing noise. Because signal extraction is the necessary first step in the analysis of time-varying Ca(2+) signals, semi-automated ROI detection frees the researcher to focus on the next step: selecting traces of interest from the relatively complete set generated using these methods.

Belt-and-suspenders as a biological design principle.

Recent studies have shown both the pFRG and the preBötC are sufficient to generate respiratory rhythm, and are hypothesized to do so via distinct mechanisms (Onimaru and Homma 2003; Mellen, Janczewski, Bocchiaro and Feldman 2003). The coexistence of mechanistically distinct, functionally matching networks (defined as degeneracy, Edelman and Gally 2001) is a ubiquitous feature of motor networks in both invertebrates (Selverston and Miller 1980) and vertebrates (DiDomenico, Nissanov and Eaton 1988). In almost all cases, a consensus exists about which subsystem is the "primary" rhythm generator, yet consistently, the effect of modulators on the isolated primary rhythm generator is qualitatively different than their effect on the more intact network (Ayali and Harris-Warrick 1999) and, in the intact animal, all rhythmogenic networks are active during motor pattern generation. Thus, at best, ascribing primacy to a particular network has weak support (since the other networks can produce qualitatively similar patterns; Prinz, Bucher and Marder 2004) and little explanatory power (since effects of modulatory inputs on the isolated "primary" rhythm generator do not persist in more intact networks). The ubiquity of degenerate networks for motor pattern generation suggests that a more useful question is why such an organization exists. We propose that degeneracy is ubiquitous because it reduces the phenotype's sensitivity to genetic mutation and environmental perturbation, and broadens the adaptiveness of motor patterns.

A vibrating microtome attachment for cutting brain slice preparations at reproducible compound angles relative to the midline.

Slice preparations isolate functional networks, permitting single unit recording under visual control, and the use of fluorescent indicators. Circuits of interest often lie at a tilt in both the rostrocaudal and ventrodorsal axis, thus exposing circuits of interest at the cut surface of a slice would require a device for tilting a preparation along two orthogonal axes relative to the blade. Such a device, designed to be used in conjunction with a vibrating microtome, permitting the isolation of slice preparations at reproducible angles, is described here. Because the two orthogonal axes of tilt can be independently and continuously adjusted, it is possible to use this device to successively refine tilt parameters from preparation to preparation for optimal exposure of circuits of interest, facilitating the development of new slice preparations. Its use in cutting a thick medullary slab preparation, isolated from the neonate rat, which exposes respiratory networks at the cut surface is described.

Functional imaging reveals respiratory network activity during hypoxic and opioid challenge in the neonate rat tilted sagittal slab preparation.

In mammals, respiration-modulated networks are distributed rostrocaudally in the ventrolateral quadrant of the medulla. Recent studies have established that in neonate rodents, two spatially separate networks along this column-the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC)-are hypothesized to be sufficient for respiratory rhythm generation, but little is known about the connectivity within or between these networks. To be able to observe how these networks interact, we have developed a neonate rat medullary tilted sagittal slab, which exposes one column of respiration-modulated neurons on its surface, permitting functional imaging with cellular resolution. Here we examined how respiratory networks responded to hypoxic challenge and opioid-induced depression. At the systems level, the sagittal slab was congruent with more intact preparations: hypoxic challenge led to a significant increase in respiratory period and inspiratory burst amplitude, consistent with gasping. At opioid concentrations sufficient to slow respiration, we observed periods at integer multiples of control, matching quantal slowing. Consistent with single-unit recordings in more intact preparations, respiratory networks were distributed bimodally along the rostrocaudal axis, with respiratory neurons concentrated at the caudal pole of the facial nucleus, and 350 microns caudally, at the level of the pFRG and the preBötC, respectively. Within these regions neurons active during hypoxia- and/or opioid-induced depression were ubiquitous and interdigitated. In particular, contrary to earlier reports, opiate-insensitive neurons were found at the level of the preBötC.

Afferent modulation of neonatal rat respiratory rhythm in vitro: cellular and synaptic mechanisms.

In mammals, expiration is lengthened by mid-expiratory lung inflation (Breuer-Hering Expiratory reflex; BHE). The central pathway mediating the BHE is paucisynaptic, converging on neurones in the rostral ventrolateral medulla. An in vitro neonatal rat brainstem-lung preparation in which mid-expiratory inflation lengthens expiration was used to study afferent modulation of respiratory neurone activity. Recordings were made from respiratory neurones in or near the pre-Bötzinger Complex (preBötC). Respiratory neurone membrane properties and BHE-induced changes in activity were characterized. Our findings suggest the following mechanisms for the BHE: (i) lung afferent signals strongly excite biphasic neurones that convey these signals to respiratory neurones in ventrolateral medulla; (ii) expiratory lengthening is mediated by inhibition of rhythmogenic and (pre)motoneuronal networks; and (iii) pre-inspiratory (Pre-I) neurones, some of which project to abdominal expiratory motoneurones, are excited during the BHE. These findings are qualitatively similar to studies of the BHE in vivo. Where there are differences, they can largely be accounted for by developmental changes and experimental conditions.

Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation.

Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-Bötzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preBötC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.

Hypothermia and recovery from respiratory arrest in a neonatal rat in vitro brain stem preparation.

This study was designed to examine the possibility that respiratory arrest during hypothermia occurs at the level of premotor or motor neurons rather than at the level of the central rhythm generator itself. Specifically, we sought to determine the consequences of hypothermic cooling until respiratory arrest, and subsequent rewarming, on neurons in the pre-Bötzinger Complex, as an indication of the output of the entire rhythmogenic network; and from cervical spinal (phrenic) ventral roots, as an indication of motor neuron output, in an in vitro neonatal rat brain stem-spinal cord preparation. We found that hypothermia led to a slowing of the respiratory rhythm with little or no decrease in the magnitude of phrenic motor output or the field potential of pre-Bötzinger Complex neurons. Ultimate arrest occurred abruptly and simultaneously in recordings from both sites, indicating that the arrest was due to failure of the central rhythm-generating network, primarily due to removal of a conditional excitation. On being rewarmed, the motor output recorded at both sites was usually fractionated, initially suggesting that changes occurred in network synchronization either during cooling or during reactivation following hypothermic arrest.