Yasumasa Okada

Preinspiratory calcium rise in putative pre-Bötzinger complex astrocytes

•u2002 Autonomic respiratory rhythm is essential to maintain lives and is generated in the lower brainstem. The ventrolateral medullary region, called the pre‐Bötzinger complex (preBötC), is the kernel for respiratory rhythm generation. Despite previous extensive studies focusing on neurons, the mechanism of how respiratory rhythm is generated has not been fully understood. •u2002 Here we show that non‐neuronal glial cells (a subset of putative astrocytes) in the preBötC are periodically activated preceding inspiratory neuronal activity, periodic activity of putative astrocytes persists during blockade of neuronal activity, and stimulation of astrocytes in the preBötC induces inspiratory neuronal firings. •u2002 These findings together with the previous report that blockade of astrocytic metabolism abolishes inspiratory neural output suggest that astrocytes are functionally involved in respiratory rhythm generation. •u2002 These results will help us better understand how respiratory rhythm is generated and how respiratory output is disturbed in various pathological conditions.

Anatomical and functional pathways of rhythmogenic inspiratory premotor information flow originating in the pre-Bötzinger complex in the rat medulla.

The pre-Bötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during a single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.

The hypoxic ventilatory response and TRPA1 antagonism in conscious mice.

Recently, TRPA1 channels, richly expressed in both peripheral and central neural systems, have been proposed as novel sensors of changes in oxygen concentration along the hypoxic–hyperoxic continuum. In this study, we investigated the hypothesis that TRPA1 channels blockade should profoundly affect the hypoxic ventilatory response (HVR).

The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology

Respiratory activities are produced by medullary respiratory rhythm generators and are modulated from various sites in the lower brainstem, and which are then output as motor activities through premotor efferent networks in the brainstem and spinal cord. Over the past few decades, new knowledge has been accumulated on the anatomical and physiological mechanisms underlying the generation and regulation of respiratory rhythm. In this review, we focus on the recent findings and attempt to elucidate the anatomical and functional mechanisms underlying respiratory control in the lower brainstem and spinal cord.

Effects of arundic acid, an astrocytic modulator, on the cerebral and respiratory functions in severe hypoxia.

Mild hypoxia increases ventilation, but severe hypoxia depresses it. The mechanism of hypoxic ventilatory depression, in particular, the functional role of the cerebrum, is not fully understood. Recent progress in glial physiology has provided evidence that astrocytes play active roles in information processing in various brain functions. We investigated the hypothesis that astrocytic activation is necessary to maintain the cerebral function and ventilation in hypoxia, by examining the responses of EEG and ventilation to severe hypoxia before and after administration of a modulator of astrocytic function, arundic acid, in unanesthetized mice. Ventilatory parameters were measured by whole body plethysmography. When hypoxic ventilatory depression occurred, gamma frequency band of EEG was suppressed. Arundic acid further suppressed ventilation, and the EEG power was suppressed in a dose-dependent manner. Arundic acid also suppressed hypoxia-induced c-Fos expression in the hypothalamus. We conclude that severe hypoxia suppresses the cerebral function which could reduce the stimulus to the brainstem resulting in ventilatory depression. Astrocytic activation in hypoxia may counteract both cerebral and ventilatory suppression.

Respiratory Toxicity of Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents for hydrophobic substances in biological experiments. In addition, the compound exhibits a plethora of bioactivities, which makes it of potential pharmacological use of its own. The influence on respiration, and thus on arterial blood oxygenation, of DMSO is unclear, contentious, and an area of limited study. Thus, in the present investigation we set out to determine the influence on lung ventilation of cumulated doses of DMSO in the amount of 0.5, 1.5, 3.5, 7.5, and 15.5 g/kg; each dose given intraperitoneally at 1 h interval in conscious mice. Ventilation and its responses to 7 % hypoxia (N(2) balanced) were recorded in a whole body plethsymograph. We demonstrate a dose-dependent inhibitory effect of DMSO on lung ventilation and its hypoxic responsiveness, driven mostly by changes in the tidal component. The maximum safe dose of DMSO devoid of meaningful consequences for respiratory function was 3.5 g/kg. The dose of 7.5 g/kg of DMSO significantly dampened respiration, with yet well preserved hyperventilatory response to hypoxia. The highest dose of 15.5 g/kg severely impaired ventilation and its responses. The study delineates the safety profile of DMSO regarding the respiratory function which is essential for maintaining proper tissue oxygenation. Caution should be exercised concerning dose concentration of DMSO.

Calcium/calmodulin-dependent protein kinases in the carotid body: an immunohistochemical study

We determined the presence of Ca2+/calmodulin-dependent protein kinases (CaMKs), a family of multifunctional proteins engaged in Ca2+-linked signaling, in carotid body chemoreceptor cells which are critical for the hypoxia-sensing. Carotid bodies were dissected from anesthetized normoxic adult Wistar rats and were double stained for individual CaMKs and for tyrosine hydroxylase (TH), a marker of chemoreceptor cells. Immunofluorescence was examined by confocal laser scanning microscopy. We found that CaMKI and CaMKII were expressed in chemoreceptor cells, but their distribution and intensity varied. CaMKI immunoreactivity was distributed throughout the cytoplasm, whereas that of CaMKII was localized in the cytoplasmic periphery of chemoreceptor cells. An overlap of CaMKI or CaMKII fluorescent probes with TH affirmed the presence of either protein in the chemoreceptor cells. CaMKIV could not be conclusively visualized by the used method. The study shows the expressions of CaMKI and CaMKII in chemoreceptor cells, which raises the plausibility of CaMKs` role in carotid body function.

Disharmony between wake- and respiration-promoting activities: effects of modafinil on ventilatory control in rodents

BackgroundModafinil is a wake-promoting drug and has been widely used for daytime sleepiness in patients with narcolepsy and other sleep disorders. A recent case series reported that daily oral modafinil alleviated hypercapnic respiratory failure in patients with COPD. However, the precise action of modafinil on respiration such as hypercapnic and/or hypoxic ventilatory responses remains unclear. The aim of this study is to clarify the effect of modafinil on the ventilatory control.MethodsWe investigated the hypothesis that modafinil enhances resting ventilation as well as the stimulatory ventilatory responses to hypercapnia and hypoxia. We addressed the issue by examining minute ventilation, respiratory rate and volume components using plethysmography, combined with a concurrent EEG monitoring of the level of wakefulness before and after administration of modafinil in two doses of 100xa0mg/kg and 200xa0mg/kg in unanesthetized mice. In addition, we monitored the effect of the lower dose of modafinil on mice locomotor activity in a freely moving condition by video-recording.ResultsWakefulness, locomotor activity and variability of the breathing pattern in tidal volume were promoted by both doses of modafinil. Neither dose of modafinil increased the absolute values of resting ventilation or promoted the ventilatory responses to hypercapnia and hypoxia. Rather, higher dose of modafinil slightly suppressed respiratory rate in room air condition.ConclusionsModafinil is conducive to the state of wakefulness but does not augment resting ventilation or the hyperventilatory responses to chemical stimuli in unanesthetized rodents.

Thermal Sensitivity and Dimethyl Sulfoxide (DMSO)

Dimethyl sulfoxide (DMSO) is commonly used as a solvent for hydrophobic substances, but the compounds innate bioactivity is an area of limited understanding. In this investigation we seek to determine the analgesic potential of DMSO. We addressed the issue by assessing the perception of thermal pain stimulus, using a 55xa0°C hotplate design, in conscious mice. The latency of withdrawal behaviors over a range of incremental accumulative intraperitoneal DMSO doses (0.5-15.5xa0g/kg) in the same mouse was taken as a measure of thermal endurance. The findings were that the latency, on average, amounted to 15-30xa0s and it differed inappreciably between the sequential DMSO conditions. Nor was it different from the pre-DMSO control conditions. Thus, DMSO did not influence the cutaneous thermal pain perception. The findings do not lend support to those literature reports that point to the plausible antinociceptive potential of DMSO as one of a plethora of its innate bioactivities. However, the findings concern the mouses footpad nociceptors which have specific morphology and stimulus transduction pathways, which cannot exclude DMSOs antinociceptive influence on other types of pain or in other types of skin. Complex and as yet unresolved neural mechanisms of perception of cutaneous noxious heat stimulus should be further explored with alternative experimental designs.

Standardization of Size, Shape and Internal Structure of Spinal Cord Images: Comparison of Three Transformation Methods

Functional fluorescence imaging has been widely applied to analyze spatio-temporal patterns of cellular dynamics in the brain and spinal cord. However, it is difficult to integrate spatial information obtained from imaging data in specific regions of interest across multiple samples, due to large variability in the size, shape and internal structure of samples. To solve this problem, we attempted to standardize transversely sectioned spinal cord images focusing on the laminar structure in the gray matter. We employed three standardization methods, the affine transformation (AT), the angle-dependent transformation (ADT) and the combination of these two methods (AT+ADT). The ADT is a novel non-linear transformation method developed in this study to adjust an individual image onto the template image in the polar coordinate system. We next compared the accuracy of these three standardization methods. We evaluated two indices, i.e., the spatial distribution of pixels that are not categorized to any layer and the error ratio by the leave-one-out cross validation method. In this study, we used neuron-specific marker (NeuN)-stained histological images of transversely sectioned cervical spinal cord slices (21 images obtained from 4 rats) to create the standard atlas and also to serve for benchmark tests. We found that the AT+ADT outperformed other two methods, though the accuracy of each method varied depending on the layer. This novel image standardization technique would be applicable to optical recording such as voltage-sensitive dye imaging, and will enable statistical evaluations of neural activation across multiple samples.