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We examined respiratory sinus arrhythmia (RSA) and possible interaction with respiratory frequency (f R ) and heart rate (HR) in spontaneously breathing, unanesthetized newborn Wistar rats (2- to 5-day-old; n = 54) and the adult rats (8-week-old; n = 34). Instantaneous heart rate (inst-HR) was calculated as the reciprocal of the inter-beat-interval. For each breath, RSA was determined as the difference between the maximum and minimum inst-HR value. The absolute RSA or RSA% (RSA per HR) were calculated as the average RSA of 10 consecutive breaths. RSA (or RSA%) in the newborn rats was significantly lower than that in the adult rats. Correlation coefficient between RSA (or RSA%) and 1/f R or HR/f R , but not HR, was significant in newborn rats, whereas only that between RSA (or RSA%) and HR was significant in adult rats. The power spectrum density of heartbeat fluctuation was detectable in both age groups. The present findings suggest that RSA exists and could be influenced by f R , rather than HR, in newborn rats., Competing Interests: Declaration of Competing Interest The authors report no competing financial interest or personal relationships that could inappropriately influence the work presented in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: We examined the cardiorespiratory effect of dexmedetomidine, an α2- adrenoceptor/imidazoline 1 (I1) receptor agonist, in spontaneously breathing adult rats., Methods: Male rats (226-301 g, n = 49) under isoflurane anesthesia had their tail vein cannulated for drug administration and their tail artery cannulated for analysis of mean arterial pressure (MAP), pulse rate (PR), and arterial blood gases (PaO2, PaCO2, pH). After recovery, one set of rats received normal saline for control recording and was then divided into three experimental groups, two receiving dexmedetomidine (5 or 50 μg·kg-1) and one receiving normal saline (n = 7 per group). Another set of rats was divided into four groups receiving dexmedetomidine (50 μg·kg-1) followed 5 min later by 0.5 or 1 mg∙kg-1 atipamezole (selective α2-adrenoceptor antagonist) or efaroxan (α2-adrenoceptor/I1 receptor antagonist) (n = 6 or 8 per group). Recordings were performed 15 min after normal saline or dexmedetomidine administration., Results: Compared with normal saline, dexmedetomidine (5 and 50 μg·kg-1) decreased respiratory frequency (fR, p = 0.04 and < 0.01, respectively), PR (both p < 0.01), and PaO2 (p = 0.04 and < 0.01), and increased tidal volume (both p = 0.049). Dexmedetomidine at 5 μg·kg-1 did not significantly change minute ventilation (V'E) (p = 0.87) or MAP (p = 0.24), whereas dexmedetomidine at 50 μg·kg-1 significantly decreased V'E (p = 0.03) and increased MAP (p < 0.01). Only dexmedetomidine at 50 μg·kg-1 increased PaCO2 (p < 0.01). Dexmedetomidine (5 and 50 μg·kg-1) significantly increased blood glucose (p < 0.01), and dexmedetomidine at 50 μg·kg-1 increased hemoglobin (p = 0.04). Supplemental atipamezole or efaroxan administration similarly prevented the 50 μg·kg-1 dexmedetomidine-related cardiorespiratory changes., Principal Conclusion: These results suggest that dexmedetomidine-related hypoventilation and hypertension are observed simultaneously and occur predominantly through activation of α2-adrenoceptors, but not I1 receptors, in spontaneously breathing adult rats., Competing Interests: The authors have declared that no competing interests exist.

The activation of imidazoline 1 (I 1 ) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I 1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3-5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I 1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aβ), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aβ removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H 2 O 2 )-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H 2 O 2 -treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H 2 O 2 -induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ 1-42 -treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aβ clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease., Competing Interests: None

Oxidative stress causes severe tissue injury of the central nervous system in ischemic brain damage (IBD), traumatic brain injury (TBI) and neurodegenerative disorders. In this study, we used hydrogen peroxide (H₂O₂) to induce oxidative stress in organotypic brain slice cultures (OBSCs), and investigated the protective effects of oxidative stress-tolerant (OST) stem cells harvested from human exfoliated deciduous teeth (SHED) which were co-cultivated with OBSCs. Using presto blue assay and immunostaining, we demonstrated that both normal SHED and OST-SHED could prevent H₂O₂-induced cell death, and increase the numbers of mature neuron and neuronal progenitors in the hippocampus of OBSCs. During co-cultivation, OST-SHED, but not normal SHED, exhibited neuronal cell morphology and expressed neuronal markers. Results from ELISA showed that both normal SHED and OST-SHED significantly decreased oxidative DNA damage in H₂O₂-treated OBSCs. SHED could also produce neurotrophic factor BDNF (brain derived neurotrophic factor) and promoted the production of IL-6 in OBSCs. Although OST-SHED had lower cell viability, the neuronal protection of OST-SHED was significantly superior to that of normal SHED. Our findings suggest that SHED, especially OST-SHED, could prevent oxidative stress induced brain damage. OST-SHED can be explored as a new therapeutic tool for IBD, TBI and neurodegenerative disorders.

The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro., Competing Interests: The authors declare no conflict of interest.

Background: Dexmedetomidine is an alpha-2 (α 2 ) adrenoceptor and imidazoline 1 (I 1 ) receptor agonist that provides sedation without loss of respiratory drive., Aims: The aim of this study was to elucidate the involvement of α 2 -adrenoceptor and I 1 receptor in the cardiorespiratory changes induced by dexmedetomidine in spontaneously breathing newborn rats., Methods: An abdominal catheter to administer drugs and three subcutaneous electrodes to record electrocardiographic data were inserted into 2- to 5-day-old Wistar rats under isoflurane anesthesia. In individual chambers, each rat was intraperitoneally administered dexmedetomidine (50 μg·kg -1 ) followed 5 min later by normal saline or 1, 5, or 10 mg·kg -1 atipamezole (selective α 2 -adrenoceptor antagonist) or efaroxan (α 2 -adrenoceptor/I 1 receptor antagonist). Cardiorespiratory indices were recorded before and after drug administration., Results: The administration of dexmedetomidine alone resulted in significant changes to most of the cardiorespiratory indices examined. The addition of 5 or 10 mg·kg -1 atipamezole or 1 mg·kg -1 efaroxan completely ameliorated the dexmedetomidine-associated reduction in heart rate (HR). The addition of 1 mg·kg -1 atipamezole or 1 or 5 mg·kg -1 efaroxan completely ameliorated the dexmedetomidine-associated reduction in respiratory frequency. Mean inspiratory flow (V T /T I ; V T is tidal volume and T I is inspiratory time), which is an index of respiratory drive, was not significantly affected by the administration of dexmedetomidine alone (P = 0.273) or dexmedetomidine + atipamezole (P = 0.605, 0.153, 0.138 for 1, 5, 10 mg·kg -1 atipamezole, respectively); however, it was significantly decreased after the administration of dexmedetomidine + efaroxan (P = 0.029, <0.001, <0.001 for 1, 5, 10 mg·kg -1 efaroxan, respectively)., Conclusions: Our results suggest that in newborn rats undergoing dexmedetomidine sedation, the α 2 -adrenoceptor, but not I 1 receptor, is involved in the regulation of HR and respiratory frequency, and that activation of the I 1 receptor plays a major role in the maintenance of respiratory drive., (© 2017 John Wiley & Sons Ltd.)

Background: Dexmedetomidine, a selective α2-adrenoceptor agonist, is a new sedative agent., Objective: To examine the dexmedetomidine-associated changes in cardiorespiratory indices in spontaneously breathing newborn rats., Methods: An abdominal catheter to administer drugs and subcutaneous electrodes to record electrocardiographic data were inserted into 2- to 4-day-old rats under isoflurane anesthesia; the rats were then placed in individual chambers. After recovery from the anesthesia, the rats received intraperitoneal administrations of normal saline (NS, vehicle), dexmedetomidine (50 μg·kg(-1)), or dexmedetomidine (50 μg·kg(-1)) followed 5 min later with NS or the selective α2-adrenoceptor antagonist atipamezole (1 mg·kg(-1)) (n = 10 in each group). Cardiorespiratory indices were recorded for each animal throughout the experiment., Results: Dexmedetomidine administration significantly decreased heart rate (HR) and minute ventilation (V'E) (P < 0.05) compared with control, whereas NS administration did not. The decrease in HR and V'E after dexmedetomidine administration was significantly less in rats that received atipamezole (P < 0.05) than in those that received NS after dexmedetomidine administration. The dexmedetomidine-associated V'E depression was attributed to a significant decrease in respiratory frequency (fR) but not tidal volume (VT ). The change in fR was reversed by atipamezole administration, which itself induced no significant changes in HR and fR., Conclusion: In spontaneously breathing immature rats, dexmedetomidine administration significantly reduced HR and V'E. Because atipamezole fully reversed decreases in fR and therefore V'E, dexmedetomidine-related respiratory suppression occurs predominantly through α2-adrenoceptor-related suppression of fR., (© 2014 John Wiley & Sons Ltd.)

Mammalian adult central nerve system (CNS) injuries are devastating because of the intrinsic difficulties for effective neuronal regeneration. The greatest problem to be overcome for CNS recovery is the poor regeneration of neurons and myelin-forming cells, oligodendrocytes. Endogenous neural progenitors and transplanted exogenous neuronal stem cells can be the source for neuronal regeneration. However, because of the harsh local microenvironment, they usually have very low efficacy for functional neural regeneration which cannot compensate for the loss of neurons and oligodendrocytes. Glial cells (including astrocytes, microglia, oligodendrocytes and NG2 glia) are the majority of cells in CNS that provide support and protection for neurons. Inside the local microenvironment, glial cells largely influence local and transplanted neural stem cells survival and fates. This review critically analyzes current finding of the roles of glial cells in CNS regeneration, and highlights strategies for regulating glial cells' behavior to create a permissive microenvironment for neuronal stem cells.

Abnormalities of the serotonin (5-hydroxytryptamine, 5-HT) system may induce respiratory disorders. We examined which regions in the rostral medulla are important for the effect of 5-HT on the frequency of respiratory-like nerve (fR-like) activity by transecting the preparations at different levels near the facial nucleus (nVII) in newborn rat brainstem-spinal cord preparations. The fR-like activity at the fourth cervical ventral root (C4) of the Pons-medulla-spinal cord preparations in 2-3-day-old rats (n=25) was monitored at 26°C, and the change in fR-like activity in response to application of 10μM 5-HT before and after transection was compared among three groups, in which nVII was retained (group A, n=10), partially retained (group B, n=7), or eliminated (group C, n=8) by the transection. Before transection, the resting fR-like activity (set to 100%) and stimulant effect of 5-HT (+101-143%) were similar among the groups. After transection, resting fR-like activity increased in all groups, but the facilitatory effects of 5-HT on the fR-like activity were abolished in groups A and C (fR-like activity of -4% and +7%, respectively). In group B, 5-HT became inhibitory (fR-like activity of -28%). In conclusion, a distinct part of the rostral medulla in the absence of pontine influences may mediate the inhibitory effects of 5-HT on the respiratory rhythm., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

We examined in infant rats whether cardiac afferent neurons in the nodose ganglia (NG) express transient receptor potential vanilloid 1 (TRPV1) receptors. Anesthetized rats (9-11 days old) were injected with 2 μl of a fluorescent retrograde tracer (Fluoro-Gold, FG) into the ventricular wall through the anterior epicardial surface. After 2 days, the NG were excised under anesthesia for identification of FG-labeled neurons and immunohistochemical analysis. Of 3858 NG neurons, 5% (202 neurons) were labeled with FG. Among the FG-labeled neurons, 180 (89%) were TRPV1 positive, of which 123 co-expressed 200-kDa neurofilaments (NF200), which is specific for myelinated nerve fibers. Among the FG-labeled neurons that expressed both TRPV1 and NF200, 37 had relatively large cell diameters (>26 μm) (range: 12-38 μm). In conclusion, in infant rats, most cardiac afferent neurons (both myelinated and unmyelinated) in the NG may express TRPV1 receptors. Although functional properties such as those related to the arterial baroreflex may vary among the neurons, our results suggest that, in immature animals, TRPV1 receptors help convey cardiac sensations and control autonomic reflexes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The purpose of the present study was to determine the relationship between the responses of transient and sustained K(+) currents, and action potentials to ouabain, and to compare the immunoreactive expression of alpha Na(+)-K(+)-ATPase isoforms (α(1), α(2) and α(3)) in neonatal rat small-diameter nodose ganglion neurons. We used perforated patch-clamp techniques. We first confirmed that the neurons (n=20) were insensitive to 0.5 μM tetrodotoxin (TTX). Application of 1 μM ouabain 1) decreased the transient K(+) currents in 60% of neurons and the sustained K(+) currents in 20%, 2) increased voltage-gated transient and sustained K(+) currents in 20% of neurons, and 3) had no effect on transient K(+) currents in 20% of neurons and on sustained K(+) currents in 60%. Thirteen of the neurons were of a rapidly adapting type, and the remaining 7 were of a slowly adapting type. In 6 rapidly adapting type neurons (46%), their activity was not significantly altered by ouabain application, but in 4 rapidly adapting type neurons, the activity increased. In the remaining 3 rapidly adapting type neurons, ouabain application hyperpolarized the resting membrane potential. The slowly adapting type 7 neurons each showed increased activity after 1 μM ouabain application. The α(1) isoform of Na(+)-K(+)-ATPase was identified as the predominant immunoreactive isoforms in small-diameter nodose ganglion neurons. These results suggest that the increased activity of small-diameter nodose ganglion neurons seen after application of 1 μM ouabain is mediated by direct inhibition of the transient K(+) current., (Published by Elsevier B.V.)

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K(+) current (I(A) ) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4-AP attenuated or blocked the CO(2) -induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch-clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT-PCR of mRNA and the expression of voltage-gated K(+) (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I(A) by approximately 27% and the additional application of 4-AP (1 mM) further inhibited I(A) by 48%. The application of 0.1 μM α-dendrotoxin (α-DTX), a slow inactivating transient K(+) current (I(D) ) blocker, inhibited the baseline I(A) by approximately 27%, and the additional application of 1 mM AZ further decreased the I(A) by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage-gated K(+) channel proteins were present, and most (80-90%) of the four Kv channels immunoreactive neurons showed the co-expression of carbonic anhydrase-II (CA-II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I(A) via the inhibition of four voltage-gated K(+) channel (Kv) proteins without affecting I(D)., (© 2010 Blackwell Publishing Ltd.)

Aims: the purpose of the present study was to investigate (a) whether maintained inflations result in the inhibition of slowly adapting pulmonary stretch receptor (SAR) discharge to elicit an abrupt change in receptor activity and (b) whether pretreatment with veratridine, a Na(+) channel opener, and/or flecainide, a Na(+) channel blocker, alters the responses of SAR properties to maintained inflations., Main Methods: we measured the properties of SAR activity during maintained inflations at different pressures in 31 anesthetized, artificially ventilated rats with unilateral vagotomy., Key Findings: During maintained inflations (approximately 5, 10 and 15 cmH(2)O) for about 5s, the procedures did not produce the induction of inhibition of either 16 low-threshold SARs (firing during both inflation and deflation) or 15 high-threshold SARs (firing during inflation only). In these preparations, the excitatory responses of SARs to maintained inflations at three different pressures were markedly enhanced after administration of veratridine (50 μg/kg), but under such conditions, the inhibition of SAR discharges was not observed. In the same SAR preparations, after flecainide treatment (9 mg/kg) sufficient for the blockade of veratridine (50 μg/kg)-induced SAR stimulation, maintained inflations at higher pressures (10 and 15 cmH(2)O) greatly inhibited SAR discharges. Under these conditions, the inhibition of SAR discharges was not observed during maintained inflations at 5 cmH(2)O., Significance: These results suggest that neither low-threshold SARs nor high-threshold SARs in the rat lung are deactivated during maintained inflations at higher pressures., (2010 Elsevier Inc. All rights reserved.)

The Kv currents are divided into three different K+ currents, such as slow inactivating transient K+ current (I(D)), fast inactivating transient K+ current (I(A)) and dominant sustained K+ current (I(K)), in small-diameter rat trigeminal ganglion (TG) neurons. The concentration of alpha-DTX (an I(D) blocker) to evoke the maximal inhibition of I(A) was 0.1 microM, and this concentration caused a 20 % inhibition of I(A) and increased the number of action potentials. Irrespective of the presence of 0.1 microM alpha-DTX, the application of 0.5 mM 4-AP (an IA blocker) caused a 51 % inhibition of I(A) and increased the number of action potentials. The responses were associated with the decreases in the resting membrane potential (RMP) and duration of depolarization phase of action potential (DDP). The application of 2 mM tetraethylammonium (TEA, an I(K) blocker) produced a 55 % inhibition of I(K). Irrespective of the presence of both I(D) and I(A) blockers, the I(K) was the predominant K+ current. The prolongation of duration of action potential was usually observed following TEA treatment, suggesting that I(A) and I(K) had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively. Furthermore, the response characteristics of action potentials in the presence of both 4-AP and TEA resemble those of TG neurons in rats following chronic constriction nerve injury of the infraorbital nerve as well as after inferior alveolar nerve section. Thus, reducing effects of both I(A) and I(K) may be useful to investigate the mechanism of allodynia.

Aims: We examined whether responses of respiratory frequency (fR) to noradrenaline (NA) were eliminated by mechanical lesions in the ventrolateral area at the ponto-medullary junction in preparations of newborn rat pons-medulla-spinal cord (PMS)., Main Methods: Preparations obtained from 2- to 4-day-old rats were superfused with artificial cerebrospinal fluid that was equilibrated with oxygenated (95% O2 plus 5% CO2 gas, and fR was monitored at the C4 ventral root at 24 degrees C. Bilateral lesions were made in the ventrolateral area between the VIth cranial nerve root and the anterior inferior cerebellar artery in PMS (n=11). The resting fR and response to exogenous NA (7 microM) were compared with those of medulla-spinal cord (MS) preparations (n=6). Immunohistochemistry of PMS preparations was performed to detect tyrosine hydroxylase (TH)-positive neurons at the ponto-medullary junction., Key Findings: PMS preparations with the lesions had (1) a significantly higher resting fR but 2 significantly less fR facilitation after NA application than those of intact PMS preparations, and (3) significantly lower resting fR and (4) significantly less fR reduction after NA application than those of MS preparations. TH-positive neurons were detected in the region from the rostral dorsolateral to the caudal ventrolateral pons (the A5 area), as well as in the ventral area near the facial nucleus., Significance: Results suggest that ventrolateral area at ponto-medullary junction plays a significant role in exogenous NA-induced fR changes under the influence of pons-induced tonic fR inhibition in newborn rat brainstem-spinal cord preparations.

To determine whether there is a difference between a transient K(+) current (I(A)) and a sustained K(+) current (I(K)) regarding the neuronal function in small-diameter adult rat trigeminal ganglion (TG) neurons, which were insensitive to tetrodotoxin (TTX, 1 microM), we performed two different types of experiments. Primary cultures of dissociated TG neurons were prepared, and electrophysiological recordings were performed with the whole-cell configuration using the patch-clamp technique. In the voltage-clamp mode, two distinct K(+) current components, (I(A)) and (I(K)), were identified, and two different components (59.5% and 96.3%) of I(K) to the total K(+) current were observed at a +50 mV step-pulse. The IC(50) value for 4-aminoprydine (4-AP, 0.05-50 mM), which inhibited the I(A) by 50%, was 0.7 mM. That for tetraethylammonium (TEA, 0.02-20 mM) to inhibit 50% of I(K) was 1.5 mM. In the current-clamp mode, we used 0.5 mM 4-AP and 2 mM TEA at each concentration nearly equal to the IC(50) value. Irrespective of the absence or presence of TEA (2 mM), 0.5 mM 4-AP application increased the number of action potentials due to the decreased duration of the depolarization phase (DDP). TEA in the presence and absence of 4-AP prolonged the duration of action potentials as well as the duration of repolarization phase (DRP). These results suggested that I(A) and I(K) had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively, in adult rat TTX-R TG neurons.

This study was designed to examine how systemic administration of an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, altered respiratory timing in unanesthetized rats under normoxia and hypoxia. To detect fine changes in inspiratory time (TI) and expiratory time (TE), and cycle duration (TTOT), we prepared a diaphragmatic electromyogram (EMGdia). Diaphragm electrodes and arterial and venous catheters were inserted into Wistar rats (n = 8) under pentobarbital anesthesia. The next day, EMGdia was recorded before and after intravenous administration of MK-801 (3 mg/kg) under normoxia and hypoxia (12% O2) without anesthesia, and the respiratory timing (TI, TE, TTOT), respiratory frequency (fR), and amplitude of the integrated EMGdia were measured. Arterial blood gases (ABGs), mean arterial pressure (MAP), and heart rate (fH) were also measured with the EMGdia. Under normoxia, MK-801 increased fR owing to a significant decrease in TE, and elevated both MAP and fH. Under hypoxia, MK-801 suppressed an increase in fR owing to a significant increase in TI, and did not accelerate fH. In both gaseous conditions, on ABGs, MK-801 did not alter partial pressure of O2 (PaO2) or CO2 (PaCO2), and slightly decreased pH (but not less than 7.4). MK-801 significantly decreased hypoxic response (%change from normoxia) in fR, and increased that in EMGdia amplitude, and did not alter a total ventilatory index (fRxEMGdia amplitude). The results suggest that an NMDA receptor-mediated mechanism partially determines fR through significant alterations in respiratory timing, particularly in which the hypoxic ventilatory response was obtained in unanesthetized rats.

We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.

We tested whether depression of respiratory frequency (fR) under non-oxygenated artificial cerebrospinal fluid (aCSF) in pons-medulla-spinal cord (PMS) and medulla-spinal cord (MS) preparations is significantly influenced by the mitochondrial uncoupler 2,4-dinitrophenol (2,4-DNP) in newborn rats. Preparations were obtained from 0- to 4-day-old rats, and fR was monitored at the C4 ventral root in environmental temperature (24 degrees C). 2,4-DNP was dissolved in aCSF (1, 10 or 30 microM; pH 7.4), and we measured fR in PMS (n=19) and MS (n=16), both of which were superfused with aCSF equilibrated with oxygenated (95% O2-5% CO2) or non-oxygenated (10% O2-5% CO2, balanced with pure N2) gas. Our results showed that: (1) fR was significantly lower in PMS than MS, (2) fR was significantly decreased under non-oxygenated aCSF in both PMS and MS and (3) fR under non-oxygenated aCSF was significantly increased by 2,4-DNP applications at 10 and 30 microM in PMS but not in MS. Our results suggest that depression in fR under non-oxygenated aCSF in PMS and MS may not be due simply to O2 limitation, and 2,4-DNP has a stimulant effect on the medullary respiratory rhythm generator (RRG) through pontine RRG regulatory mechanisms under non-oxygenated aCSF.

In anesthetized, artificially ventilated rats with one vagus nerve section, the purposes of the present study were to investigate whether release from phasic consecutive hyperinflations (inflation volume=3 tidal volumes) results in the afterhyperpolarization (AHP) of the slowly adapting pulmonary stretch receptor (SAR) activity and whether the effect of ouabain, a Na+-K+ ATPase inhibitor, alters AHP of the SAR activity seen after release from maintained inflations. Release from 10 consecutive phasic hyperinflations did not cause any significant inhibition of SAR activity. Release from maintained inflations (for approximately 10 and 15 cmH2O) for 5 s produced the induction of disappearance of SAR activity, corresponding with the AHP. Intravenous administration of ouabain (20 and 40 microg/kg) had no significant effects on the responses of SAR activity and SAR adaptation index (AI) to maintained inflations, but ouabain treatment with at 40 microg/kg resulted in a significant increase in the SAR activity after stopping the respirator and significantly attenuated the AHP of the SAR activity. In the immunohistochemical study, we found Na+-K+ ATPase alpha3-subunit-isoforms-like immunoreactivity in SAR terminals, forming leaflike extensions in the intrapulmonary bronchioles at different diameters, and those terminals were buried in the smooth muscle. In the same sections, the alpha1 subunit immunoreactivity of SAR terminals was not found. These results suggest that the mechanism of generating the AHP of SARs is mainly mediated by the activation of Na+-K+ ATPase alpha3 subunit isoform.

We hypothesized that facilitation of respiratory rhythm by noradrenaline (NA) in rat pons-medulla-spinal cord preparations is mediated through alpha1-adrenergic receptors. In 0- to 4-day-old rats, the respiratory frequency (fR) was monitored at the C4 ventral root and trigeminal motor (VMO) outputs. fR at temperature (Te)=23 degrees C was lower than that at a higher Te (27 degrees C) and was increased by NA. At 23 degrees C, lower concentrations of NA were needed to produce the same increases in fR seen at 27 degrees C. With highest NA concentration we tested (50 microM), activity at C4 was maintained in all preparations at both Te, whereas that at VMO was maintained in 50% (27 degrees C) or 88% (23 degrees C) of the preparations. Particularly, tonic activity at C4 appeared in all preparations at both Te, but that at the VMO occurred in 0% (27 degrees C) or 18% (23 degrees C) of the preparations. Based on these results, we used the lower Te (23 degrees C) and applied a low concentration of NA (3 microM) to the preparations. We found that: (1) with the addition of NA, fR was increased without the occurrence of tonic activity and (2) NA-related fR facilitation was inhibited by pre-treatment with the alpha1-adrenergic receptor antagonist prazosin (2 microM). fR was increased by application of the alpha1-adrenergic receptor agonist phenylephrine (4 microM), and this response was inhibited by prazosin (4 microM). At Te=23 degrees C, fR facilitation by NA in newborn rat pons-medulla-spinal cord preparations was obtained by activation of alpha1-adrenergic receptors.

The combined effects of ouabain (Na(+)-K(+) ATPase inhibitor) and hyperinflation (inflation volume=three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca(2+) channel blocker) and KB-R7943 (a reverse-mode Na(+)-Ca(2+) exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 microg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 microg/kg ouabain (total dose=90 microg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 microg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na(+) channel opener, 40 microg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 microg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na(+) overload, but not to a Ca(2+) influx via activation of L-type Ca(2+) channels, in the SAR endings.

The digastric muscle acts for both feeding (including mastication and swallowing) and respiration. In this study, we examined whether or not the muscle activity is detectable during anoxia in developing rats. Rats at 4 different ages, days 5, 10, 16, and 24, were exposed to 100% N2 under pentobarbital or ketamine-xylazine anesthesia, and the electromyograms of digastric muscles (dEMG) and the diaphragm (diaEMG) were examined simultaneously. Prior to the anoxic exposure, at all ages, the dEMG was similar to or less apparent than the diaEMG, which was detected at each inspiratory movement. In anoxia, we first observed dEMG activity, mostly sporadic (days 5 and 10) or mostly tonic (days 16 and 24), when diaEMG activity was temporarily suppressed (we termed it Phase 1). Second, synchronous phasic or tonic dEMG and phasic diaEMG were recorded temporarily before terminal apnea (we termed it Phase 2). These phenomena were also obtained in vagotomized rats (all ages) or in rats injected with the N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (days 16 and 24). In conclusion, our results suggest that in anoxia, dEMG activity is detectable during diaEMG suppression in early anoxia, irrespective of the developmental age, the anesthetic (pentobarbital or ketamine-xylazine), vagotomy, or MK-801 injections.

Inhibitory responses of slowly adapting pulmonary stretch receptor (SAR) activity to CO(2) inhalation (maximal tracheal CO(2) concentration ranging from 9.5 to 12.5%) for approximately 60 s were examined before and after administration of acetazolamide (a carbonic anhydrase inhibitor) or 4-aminopyridine (4-AP, a K(+) channel blocker). The experiments were performed in 35 anesthetized, artificially ventilated rats after unilateral vagotomy. Sixty-eight of eighty-four SARs were inhibited by CO(2) inhalation. The SAR inhibition was attenuated by pretreatment with either acetazolamide (20 mg/kg, n = 10) or 4-AP (0.7 and 2.0 mg/kg, n = 10). In other series of experiments, stainings to show the existence of carbonic anhydrase (CA) enzymatic reaction were not found in the smooth muscle of either extrapulmonary or intrapulmonary bronchi. Protein gene product 9.5 (PGP 9.5)-immunoreactive SAR terminals to form leaflike extensions were found in the bronchioles at different diameters and were smooth-muscle-related receptors. But in the same sections, CA isozyme II-like (erythrocyte CA) immunoreactive SAR terminals were not identified. These results suggest that CO(2)-induced inhibition of SARs may be involved in the CA-dependent CO(2) hydration in addition to the activation of 4-AP sensitive K(+) currents.

Many anesthetics are known to decrease ventilation (V(E)) and metabolic rate (MR). Because MR is known to contribute to the V(E) level, one would expect some parallelism between the changes in V(E) and MR during anesthesia. We tested this hypothesis in normoxia and hypoxia (12% O2) on male Wistar rats (n = 10; 221-288 g) by using a short-acting intravenous anesthetic, propofol. Propofol anesthesia was induced with a 7-7.5 mg kg(-1) (60-70 s) dose and maintained with a 20-22 mg kg(-1) h(-1) (<40 min) dose. In normoxia, propofol significantly decreased V(E) and MR and maintained the V(E)/MR ratio. In hypoxia, propofol decreased MR without a significant decrease in V(E), and the V(E)/MR ratio tended to increase. As a result, both in normoxia and hypoxia, propofol did not significantly increase the partial pressure of CO2 in arterial blood (PaCO2). Propofol was also associated with decreased body temperature and mean arterial pressure. The results suggest that during anesthesia, a large part of the drop in V(E) can be accounted for by the drop in MR, and that in both normoxia and hypoxia the V(E)/MR ratios and PaCO2values are maintained close to the levels of the conscious state.

The excitatory responses of deflationary slowly adapting pulmonary stretch receptor (SAR) activity to lung deflation ranging from approximately -15 to -25 cm of H(2)O for approximately 5 s were examined before and after administration of flecainide, a Na(+) channel blocker, and K(+) channel blockers, such as 4-aminopyridine (4-AP) and tetraethylammonium (TEA). The experiments were performed in anesthetized, artificially ventilated rats after unilateral vagotomy. The deflationary SARs increased their activity during lung deflation and its effect became more pronounced by increasing the degree of negative pressure. During lung deflation the average values for the deflationary SAR adaptation index (AI) were below 40%. Intravenous administration of veratridine (50 microg/kg), an Na(+) channel opener, stimulated deflationary SAR activity: one maintained excitatory activity mainly during deflation and the other receptors showed a tonic discharge during both deflation and inflation. Despite the difference in deflationary SAR firing patterns after veratridine administration, flecainide treatment (6.0 mg/kg) blocked veratridine-induced deflationary SAR stimulation and also caused strong inhibition of the excitatory responses of deflationary SARs to lung deflation. Under these conditions, the average values for deflationary SAR AI were over 90%. The responses of deflationary SARs and deflationary SAR AI to lung deflation were not significantly altered by pretreatment with either 4-AP (0.7 and 2.0 mg/kg) or TEA (2.0 and 6.0 mg/kg). These results suggest that the excitatory effect of lung deflation on deflationary SAR activity is mediated by the activation of flecainide-sensitive Na(+) channels on the nerve terminals of deflationary SARs.