Expiratory abdominal muscle nerve is active at flexor phase, while inspiratory phrenic nerve is not active during locomotion evoked by 5-HT and NMDA in the neonatal rat.

Abdominal muscles are involved in respiration and locomotion. In the isolated pons-spinal cord-rib attached preparation from neonatal rat, the phrenic nerve and abdominal muscles show inspiratory and expiratory activity, respectively. Using this preparation, we investigated whether the bath application of NMDA and 5-HT could evoke locomotor activities in the fourth cervical ventral root (C4VR), phrenic nerve, and abdominal muscle nerve (ilioinguinal nerve, IIG-n). We also observed rib and abdominal muscle movements visually. The phrenic nerve and C4VR showed inspiratory activity consistently under the control conditions, whereas IIG-n showed expiratory activity only at the beginning of the experiment. During the chemically-induced locomotion, both C4VR and IIG-n showed locomotor activity, and IIG-n in particular showed flexor activity. During the flexor activity, lateral bending of the rib cage to the recording site was observed. The phrenic nerve showed weak or no apparent locomotor activity. We concluded that the central pattern generator (CPG) for locomotion provides stronger excitatory synaptic inputs to C4 motoneurons innervating neck and shoulder muscles than the inputs to the phrenic motoneurons. Thus, the locomotor CPG provides a suitable amount of inputs to the functionally proper motoneurons. This preparation will be useful to explore how the respiratory and locomotor CPGs select proper motoneurons to give synaptic inputs and are coordinated with each other.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking.

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous 'spiking' might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 µm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 µM) and activating µ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 µM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

Genetically Encoded Glutamate Indicators with Altered Color and Topology.

Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and Rncp-iGluSnFR1 display glutamate affinities of 11 µM and 0.9 µM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that Gncp-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.

TMX1 determines cancer cell metabolism as a thiol-based modulator of ER-mitochondria Ca2+ flux.

The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.

A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.

The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.

Identification of the pre-Bötzinger complex inspiratory center in calibrated "sandwich" slices from newborn mice with fluorescent Dbx1 interneurons.

Inspiratory active pre-Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow-up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1-derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0-4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging-based studies, as well as the application of other Cre-dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory-related oscillations of preBötC Dbx1-derived neurons and thus breathing.

Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response.

We demonstrate a simple, low cost and disposable microfluidic fluorescence activated cell sorting system (µFACS) for directed evolution of fluorescent proteins (FP) and FP-based calcium ion (Ca(2+)) indicators. The system was employed to pre-screen libraries of up to 10(6) variants of a yellow FP-based Ca(2+) indicator (Y-GECO) with throughput up to 300 cells per s. Compared to traditional manual screening of FP libraries, this system accelerated the discovery of improved variants and saved considerable time and effort during the directed evolution of Y-GECO. Y-GECO1, the final product of the µFACS-aided directed evolution, has a unique fluorescence hue that places it in the middle of the spectral gap that separates the currently available green and orange FP-based Ca(2+) indicators, exhibits bright fluorescence in the resting (Ca(2+) free) state, and gives a large response to intracellular Ca(2+) fluctuations in live cells.

Anoxia response in physiological potassium of the isolated inspiratory center in calibrated newborn rat brainstem slices.

Using newborn rat brainstem-spinal cords, we were the first to show that medullary inspiratory networks can generate the neonatal biphasic (initial acceleration-secondary slowing) respiratory response to severe hypoxia causing tissue anoxia. Our findings also indicated that medullary inspiratory interneurons remain functional during sustained anoxia due to effective utilization of anaerobic metabolism. In that previous work by us and related studies by others on respiratory anoxia responses in the above en bloc model or brainstem slices, presumptive recording sites within the pre-Bötzinger complex (preBötC) inspiratory center were not histologically verified. Moreover, preBötC slices were studied in 7-9 mM K(+) to stabilize rhythm which can, however, affect respiratory neuromodulation. Here, we summarize our previous findings on respiratory anoxia responses in the en bloc model in physiological (3 mM) K(+). Using our recently developed 'calibrated' slices, we also exemplify anoxia effects in anatomically identified preBötC cells in physiological K(+) based on recording electrophysiological population activity in conjunction with either membrane potential or cytosolic Ca(2+).

Amyloid ß (Aß) peptide directly activates amylin-3 receptor subtype by triggering multiple intracellular signaling pathways.

The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid ß protein (Aß) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aß and amylin may express their effects through the amylin receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the amylin receptor family, amylin receptor-3 (AMY3). Aß1-42 and human amylin (hAmylin) increase cytosolic cAMP and Ca(2+), trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aß1-42 and hAmylin also induce cell death during exposure for 24-48 h at low micromolar concentrations. In the presence of hAmylin, Aß1-42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin receptor antagonist AC253 blocks increases in intracellular Ca(2+), activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aß1-42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aß and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

K(+) and Ca²(+) dependence of inspiratory-related rhythm in novel "calibrated" mouse brainstem slices.

Recently developed transversal newborn rat brainstem slices with "calibrated" rostrocaudal margins unraveled novel features of rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) neural networks (Ballanyi and Ruangkittisakul, 2009). For example, slice rhythm in physiological (3 mM) superfusate K(+) is depressed by modestly raised Ca²(+) and restored by raised K(+). Correspondingly, we generated here calibrated preBötC slices from commonly used newborn C57BL/6 mice in which rostrocaudal extents of respiratory marker structures, e.g., the inferior olive, turned out to be smaller than in newborn rats. Slices of 400-600 µm thickness with likely centered preBötC kernel ("m-preBötC slices") generated rhythm in 3 mM K(+) and 1mM Ca(2+) for several hours although its rate decreased to < 5 bursts/min after >1 h. Rhythm was stable at 8-12 bursts/min in 6-7 mM K(+), depressed by 2 mM Ca²(+), and restored by 9 mM K(+). Our findings provide the basis for future structure-function analyses of the mouse preBötC, whose activity depends critically on a "Ca(+)/K(+) antagonism" as in rats.

Control of breathing by "nerve glue".

Long regarded as mere structural support for neurons, neuroglial cells are now considered pivotal for brain metabolism, the blood-brain barrier, cerebral hemodynamics, and neuronal function. Multitasking by glia involves numerous signaling and effector pathways that control various processes, including neurotransmitter uptake and release of gliotransmitters, such as glutamate or adenosine 5'-triphosphate (ATP). Acidosis of cerebrospinal fluid causes ATP release from astrocytic glia at the ventral brainstem surface, which excites neighboring brainstem neurons that stimulate neurons in the pre-Bötzinger complex (preBötC), which controls inspiratory breathing movements. New insights into glial regulation of complex behavior, and particularly into respiratory circuit function, are evolving from application of genetically engineered optical stimulation and Ca(2+) imaging tools, combined with other molecular and electrophysiological approaches. These advances in technology will enable direct analyses of respiratory-related neuron-glia interactions not only at the ventral brainstem surface but also within the preBötC, which generates a vital brain rhythm.

Methylxanthine reversal of opioid-evoked inspiratory depression via phosphodiesterase-4 blockade.

Hypothetic mechanisms for respirogenic methylxanthine actions include blockade of adenosine receptors or phosphodiesterase-4 (PDE4) in inspiratory pre-Bötzinger complex (preBötC) networks. Here, we studied this by analyzing stimulating caffeine and theophylline actions on mu-opioid-depressed inspiratory-related rhythm in the ventrolateral aspect of rat brainstem slices. The methylxanthines restored DAMGO (0.5-1 microM) depressed rhythm only at >1mM, which is approximately 10-fold higher than selective for adenosine receptors. Adenosine receptor blockers did neither counter DAMGO inhibition nor change control rhythm, similar to adenosine (0.1-2.5 mM). The specific PDE4 blocker rolipram (5 microM) restored DAMGO-depressed rhythm incompletely, but effectively reversed similar inhibition by clinical mu-agonist (fentanyl, 0.1 microM). At 0.25 microM, rolipram boosted incomplete recovery by 1 mM theophylline of DAMGO-depressed rhythm. Findings indicate that methylxanthines excite rhythmogenic preBötC networks via blockade of cAMP dependent PDE4 and suggest that specific PDE4 inhibitors (plus low methylxanthine doses) stimulate breathing effectively. We discuss why methylxanthine doses for preBötC stimulation need to be higher than those for respirogenic effects in vivo.

Glia contribute to the purinergic modulation of inspiratory rhythm-generating networks.

Glia modulate neuronal activity by releasing transmitters in a process called gliotransmission. The role of this process in controlling the activity of neuronal networks underlying motor behavior is unknown. ATP features prominently in gliotransmission; it also contributes to the homeostatic ventilatory response evoked by low oxygen through mechanisms that likely include excitation of preBötzinger complex (preBötC) neural networks, brainstem centers critical for breathing. We therefore inhibited glial function in rhythmically active inspiratory networks in vitro to determine whether glia contribute to preBötC ATP sensitivity. Glial toxins markedly reduced preBötC responses to ATP, but not other modulators. Furthermore, since preBötC glia responded to ATP with increased intracellular Ca(2+) and glutamate release, we conclude that glia contribute to the ATP sensitivity of preBötC networks, and possibly the hypoxic ventilatory response. Data reveal a role for glia in signal processing within brainstem motor networks that may be relevant to similar networks throughout the neuraxis.

Indirect opioid actions on inspiratory pre-Bötzinger complex neurons in newborn rat brainstem slices.

Findings in newborn mouse brainstem slices led to the hypothesis that depression of breathing by opioids is caused by postsynaptic K(+) channel-mediated hyperpolarization of rhythmogenic inspiratory neurons of the pre-Bötzinger complex (preBötC). Subsequent observations in newborn en bloc medullas and juvenile rats in vivo indicated that excitatory drive from retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) neurons partly counters opioid-evoked inspiratory inhibition. Our recent study in newborn rat en bloc medullas supports the latter hypothesis, whereas we found in that report that inspiratory preBötC neurons constituting the interface with the RTN/pFRG are not hyperpolarized by opioids. Here, we show that opioids also do not hyperpolarize preBötC neurons in "calibrated" newborn rat slices. This supports the previous hypothesis by us and others that opioids rather target inspiratory networks indirectly, likely primarily via presynaptic mechanisms.

Multiphoton/confocal Ca2+-imaging of inspiratory pre-Bötzinger complex neurons at the rostral or caudal surface of newborn rat brainstem slices.

Breathing is controled by neural networks of the pre-Bötzinger complex (preBötC). We have previously established that multiphoton/confocal Ca(2+) imaging is a potent tool for studying preBötC functions in transversal newborn rat brainstem slices. Here, we used such imaging to show that only 3 often dispersedly located preBötC neurons are typically inspiratory active per transversal imaging plane in slices with rostrally-exposed preBötC contrary to rhythmic optical activity in 11 densely aggregated neurons in slices with the preBötC at the caudal margin. In both slice types, glutamate raises Ca(2+) in >30 cells (both neurons and glia). Factors are discussed that may be involved in the spatial inhomogeneity of superficially located active inspiratory preBötC neurons in both slice types.

Depression by Ca2+ and stimulation by K+ of fictive inspiratory rhythm in newborn rat brainstem slices.

Rhythmogenic pre-Bötzinger complex (preBötC) networks are inspiratory active in brainstem slices that are typically studied in 7-9 mM K(+)instead of physiological extracellular (3 mM) K(+). Also superfusate Ca(2+) is often high (up to 2.4 mM) compared to the most common physiological value (1.2 mM). Because both cations are potent neuromodulators, it is desirable to keep them as close as possible to normal levels for minimizing modulation of the isolated preBötC. We found that modestly raised Ca(2+) depresses preBötC slice rhythm while K(+) elevation counters this inhibition and low Ca(2+) accelerates inspiratory bursting. A Ca(2+) range of 0.75-1 mM and K(+) levels between 4-6 mM may be optimal for long term stable slice rhythm.

Caffeine reversal of opioid-evoked and endogenous inspiratory depression in perinatal rat en bloc medullas and slices.

Caffeine counters endogenous or drug-evoked depression of breathing in (preterm) infants. Despite its common clinical use, little is known on central nervous mechanisms of its stimulatory respiratory action. We show that millimolar concentrations of caffeine are needed in perinatal rat en bloc medullas and medullary slices for stimulation of fictive inspiratory rhythms that were either endogenously slow in fetuses or depressed by prostagandins or opioids. Findings suggests that caffeine blocks phospodiesterase-4 thus raising cAMP in rhythmogenic pre-Bötzinger complex (preBötC) networks and/or cells driving the inspiratory preBötC.

Fluorescence imaging of active respiratory networks.

Breathing in mammals is controlled by neural networks in the brainstem such as the pre-Bötzinger complex (preBötC) and the parafacial respiratory group (pFRG). Exploring these rhythmogenic networks and their interactions is greatly facilitated by live fluorescence imaging that enables analysis of (i) spatiotemporal patterns of respiratory (population) activities, (ii) (sub)cellular signaling in identified respiratory neurons, and (iii) membrane properties of respiratory neurons that are fluorescence-tagged for characteristic markers. Transversal medullary slices containing the preBötC and "en bloc" brainstem-spinal cord preparations with a functional preBötC/pFRG dual respiratory center which interacts, e.g., with pontine structures, are used for respiratory imaging in perinatal rodents. Imaging of less reduced (mature) respiratory networks is feasible in arterially-perfused "working-heart-brainstem" preparations from rodents. In these in situ models, imaging with voltage and Ca2+ sensitive dyes is established for assessment of respiratory (population) activities. Here, we summarize findings from diverse live imaging approaches in these models and point out potential pitfalls and future perspectives of respiratory-related optical recording.

Structure-function analysis of rhythmogenic inspiratory pre-Bötzinger complex networks in "calibrated" newborn rat brainstem slices.

Inspiratory pre-Bötzinger complex (preBötC) networks remain active in perinatal rodent brainstem slices. Our analysis of (crescendo-like) inspiratory-related population and cellular bursting in novel histologically identified transversal preBötC slices in physiological (3 mM) superfusate [K(+)] revealed: (i) the preBötC extent sufficient for rhythm in thin slices is at most 175 microm. (ii) In 700 microm thick slices with unilaterally exposed preBötC, a <100 microm kernel generates a eupnea-like inspiratory pattern under predominant influence of caudally adjacent structures or thyrotropin-releasing hormone-like transmitters, but a mixed eupnea-sigh-like pattern when influence of rostral structures or substance-P-like transmitters dominates. (iii) Primarily presynaptic processes may underlie inhibition of rhythm by opioids or raising superfusate [Ca(2+)] from lower to upper physiological limits (1-1.5 mM). (iv) High K(+) reverses depression of rhythm by raised Ca(2+), opioids and anoxia. In summary, distinct activity patterns of spatiochemically organized isolated inspiratory networks depend on both an extracellular "Ca(2+)-K(+) antagonism" and slice dimensions. This explains some discrepant findings between studies and suggests use of "calibrated" slices and more uniform experimental conditions.