Araya Ruangkittisakul

High Sensitivity to Neuromodulator-Activated Signaling Pathways at Physiological [K+] of Confocally Imaged Respiratory Center Neurons in On-Line-Calibrated Newborn Rat Brainstem Slices

The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8–10 mm) superfusate [K+], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel “on-line histology” based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0–4. At physiological superfusate [K+] (3 mm), 500- and 600-μm-thick slices with the PBC in the center and the caudal boundary 0.70 and 0.76 mm caudal to the facial motonucleus generated rhythm for >2 and ∼4 h, respectively. Rhythm was abolished by low nanomolar concentrations of the μ-opiate receptor agonist DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin). After spontaneous arrest of bursting, rhythm was reactivated at clinically relevant or physiological concentrations by 3,5-dihydroxyphenylglycine, thyrotropin-releasing hormone, or rolipram, each affecting distinct second-messenger pathways. Two-photon/confocal Ca2+ imaging revealed that these agents reactivated the same PBC neurons initially active in 3 mm [K+]. The data show that “calibrated” PBC slices at physiological [K+] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous “in vitro apnea” is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure–function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K+].

Generation of Eupnea and Sighs by a Spatiochemically Organized Inspiratory Network

The discovery of the rhythmogenic pre-Bötzinger complex (preBötC) inspiratory network, which remains active in a transverse brainstem slice, greatly increased the understanding of neural respiratory control. However, basic questions remain unanswered such as (1) What are the necessary and sufficient slice boundaries for a functional preBötC? (2) Is the minimal preBötC capable of reconfiguring between inspiratory-related patterns (e.g., fictive eupnea and sighs)? (3) How is preBötC activity affected by surrounding structures? Using newborn rat slices with systematically varied dimensions in physiological [K+] (3 mm), we found that a 175 μm thickness is sufficient for generating inspiratory-related rhythms. In 700-μm-thick slices with unilaterally exposed preBötC, a kernel <100 μm thick, centered 0.5 mm caudal to the facial nucleus, is necessary for rhythm generation. Slices containing this kernel plus caudal structures produced eupneic bursts of regular amplitude, whereas this kernel plus rostral tissue generated sighs, intermingled with eupneic bursts of variable amplitude (“eupnea–sigh pattern”). After spontaneous arrest of rhythm, substance-P or neurokinin-1 (NK1) receptor agonist induced the eupnea–sigh burst pattern in ≥250-μm-thick slices, whereas thyrotropin-releasing hormone or phosphodiesterase-4 blockers evoked the eupnea burst pattern. Endogenous rhythm was depressed by NK1 receptor antagonism. Multineuronal Ca2+ imaging revealed that preBötC neurons reconfigure between eupnea and eupnea–sigh burst patterns. We hypothesize a (gradient-like) spatiochemical organization of regions adjacent to the preBötC, such that a small preBötC inspiratory-related oscillator generates eupnea under the dominant influence of caudal structures or thyrotropin-releasing hormone-like transmitters but eupnea–sigh activity when the influence of rostral structures or substance-P-like transmitters predominates.

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 Ca2+ 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.

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. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated optogenetic actuators. However, spatially distinct patterns of optogenetic activation and voltage imaging are required to avoid fluorescence artifacts due to photoactivation of the FlicR1 chromophore.

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

Background: Aβ and human amylin peptides share similar biophysical and neurotoxic properties. Results: Aβ directly activates amylin-3 receptor (AMY3) isoform and triggers multiple signaling pathways. Conclusion: Aβ actions are expressed via AMY3 receptors. Significance: AMY3 could serve as a therapeutic target for attenuating Aβ toxicity. The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ 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). Aβ1–42 and human amylin (hAmylin) increase cytosolic cAMP and Ca2+, trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1–42 and hAmylin also induce cell death during exposure for 24–48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1–42 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 Ca2+, activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1–42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem

The pre‐Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstem–spinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K+] and [Ca2+] (both of which determine neuronal excitability) is not clear. We report here that inspiratory‐related rhythms in newborn rat slices and brainstem–spinal cords with defined boundaries were basically similar in physiological Ca2+ (1.2 mm) and K+ (3 mm). Hypoglossal nerve rhythm was 1 : 1‐coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC‐related slice rhythms were inhibited in 1.5 mm Ca2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mm Ca2+. Ca2+ depression of slice rhythms was antagonized by raising superfusate K+ to 8–10 mm. This strong extracellular Ca2+/K+ antagonism of inspiratory (motor) rhythms was also revealed in brainstem–spinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K+, respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscles

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.

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

Cancer cells are critically dependent on ER–mitochondria Ca2+ flux that regulates their bioenergetics. Here, Raturi et al. identify the ER oxidoreductase TMX1 as a thiol-dependent regulator of this intracellular signaling mechanism within cancer cells.

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.

Activity and metabolism-related Ca2+ and mitochondrial dynamics in co-cultured human fetal cortical neurons and astrocytes.

Neurons and neighboring astrocytic glia are mostly studied in nervous tissues from rodents whereas less is known on their properties and interactions in the human brain. Here, confocal/multiphoton fluorescence imaging for several hours revealed that co-cultured fetal human cortical neurons and astrocytes show pronounced spontaneous rises of cytosolic Ca(2+) which last for up to several minutes without concomitant changes in either movements or membrane potential of mitochondria. Similar Ca(2+) rises were evoked mainly in neurons by bath-applied glutamate or γ-aminobutyric acid (GABA) acting via N-methyl-d-aspartate (NMDA)+AMPA/Kainate and GABAA receptors, respectively. Predominantly in astrocytes, Ca(2+) baseline was elevated by adenosine diphosphate (ADP) and adenosine triphosphate (ATP) acting via P2Y1 and P2X7 receptors, likely causing the release of glutamate and glutamine. Mainly astrocytes responded to histamine, whereas the activation of muscarinic acetylcholine (ACh) receptors raised Ca(2+) in both cell types. Evoked neuronal and astrocytic Ca(2+) rises could last for several minutes without affecting mitochondrial movements or membrane potential. In contrast, reversible depolarization of mitochondrial membrane potential accompanied neuronal Ca(2+) rises induced by cyanide-evoked chemical anoxia or the uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). During such metabolic perturbation, mitochondrial depolarization also occurred in astrocytes, whereas Ca(2+) was largely unaffected. In summary, fetal human cortical neurons and astrocytes show distinct patterns of neuro/glio-transmitter- and metabolically-evoked Ca(2+) rises and possess active mitochondria. One aspect of our discussion deals with the question of whether the functional mitochondria contribute to cellular Ca(2+) homeostasis that seems to be already well-developed in fetal human cortical brain cells.