scholarly journals KCNQ Current Contributes to Inspiratory Burst Termination in the Pre-Bötzinger Complex of Neonatal Rats in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Ann L. Revill ◽  
Alexis Katzell ◽  
Christopher A. Del Negro ◽  
William K. Milsom ◽  
Gregory D. Funk
Keyword(s):  
Burst Duration ◽  
Neonatal Rat ◽  
Synaptic Inhibition ◽  
Neonatal Rats ◽  
Kcnq Channels ◽  
Ventral Medulla ◽  
Inspiratory Neurons ◽  
Bötzinger Complex ◽  
Synaptic Mechanisms

The pre-Bötzinger complex (preBötC) of the ventral medulla generates the mammalian inspiratory breathing rhythm. When isolated in explants and deprived of synaptic inhibition, the preBötC continues to generate inspiratory-related rhythm. Mechanisms underlying burst generation have been investigated for decades, but cellular and synaptic mechanisms responsible for burst termination have received less attention. KCNQ-mediated K+ currents contribute to burst termination in other systems, and their transcripts are expressed in preBötC neurons. Therefore, we tested the hypothesis that KCNQ channels also contribute to burst termination in the preBötC. We recorded KCNQ-like currents in preBötC inspiratory neurons in neonatal rat slices that retain respiratory rhythmicity. Blocking KCNQ channels with XE991 or linopirdine (applied via superfusion or locally) increased inspiratory burst duration by 2- to 3-fold. By contrast, activation of KCNQ with retigabine decreased inspiratory burst duration by ~35%. These data from reduced preparations suggest that the KCNQ current in preBötC neurons contributes to inspiratory burst termination.

Persistent Sodium Current, Membrane Properties and Bursting Behavior of Pre-Bötzinger Complex Inspiratory Neurons In Vitro

2002 ◽  
Vol 88 (5) ◽  
pp. 2242-2250 ◽  
Author(s):  
Christopher A. Del Negro ◽  
Naohiro Koshiya ◽  
Robert J. Butera ◽  
Jeffrey C. Smith
Keyword(s):  
Sodium Current ◽  
Neonatal Rat ◽  
Membrane Properties ◽  
Negative Slope ◽  
Inactivation Kinetics ◽  
Pacemaker Neurons ◽  
Inspiratory Neurons ◽  
Voltage Ramp ◽  
Bötzinger Complex

We measured persistent Na+current and membrane properties of bursting-pacemaker and nonbursting inspiratory neurons of the neonatal rat pre-Bötzinger complex (pre-BötC) in brain stem slice preparations with a rhythmically active respiratory network in vitro. In whole-cell recordings, slow voltage ramps (≤100 mV/s) inactivated the fast, spike-generating Na+ current and yielded N-shaped current-voltage relationships with nonmonotonic, negative-slope regions between −60 and −35 mV when the voltage-sensitive component was isolated. The underlying current was a TTX-sensitive persistent Na+ current ( I NaP) since the inward current was present at slow voltage ramp speeds (3.3–100 mV/s) and the current was blocked by 1 μM TTX. We measured the biophysical properties of I NaP after subtracting the voltage-insensitive “leak” current ( I Leak) in the presence of Cd2+ and in some cases tetraethylammonium (TEA). Peak I NaP ranged from −50 to −200 pA at a membrane potential of −30 mV. Decreasing the speed of the voltage ramp caused time-dependent I NaPinactivation, but this current was present at ramp speeds as low as 3.3 mV/s. I NaP activated at −60 mV and obtained half-maximal activation near −40 mV. The subthreshold voltage dependence and slow inactivation kinetics of I NaP, which closely resemble those of I NaP mathematically modeled as a burst-generation mechanism in pacemaker neurons of the pre-BötC, suggest that I NaP predominantly influences bursting dynamics of pre-BötC inspiratory pacemaker neurons in vitro. We also found that the ratio of persistent Na+conductance to leak conductance ( g NaP/ g Leak) can distinguish the phenotypic subpopulations of bursting pacemaker and nonbursting inspiratory neurons: pacemaker neurons showed g NaP/ g Leak> g NaP/ g Leakin nonpacemaker cells ( P < 0.0002). We conclude that I NaP is ubiquitously expressed by pre-BötC inspiratory neurons and that bursting pacemaker behavior within the heterogeneous population of inspiratory neurons is achieved with specific ratios of these two conductances, g NaP and g Leak.

scholarly journals Developmental Expression of the Peripheral-Type Benzodiazepine Receptor and the Advent of Steroidogenesis in Rat Adrenal Glands*

Endocrinology ◽  
1999 ◽  
Vol 140 (2) ◽  
pp. 859-864 ◽  
Author(s):  
Alexandra Zilz ◽  
Hua Li ◽  
Rosa Castello ◽  
Vassilios Papadopoulos ◽  
Eric P. Widmaier
Keyword(s):  
Ligand Binding ◽  
Binding Capacity ◽  
Benzodiazepine Receptor ◽  
Neonatal Rat ◽  
Developmental Expression ◽  
Neonatal Rats ◽  
Adrenal Cells ◽  
Star Protein ◽  
Peripheral Type

Abstract Although the precise mechanism whereby cholesterol is transported across the outer mitochondrial membrane is uncertain, a multimeric receptor complex termed the peripheral-type benzodiazepine receptor (PBR) appears essential for this process. We therefore predicted that adrenal cells at different developmental stages would express PBR coincidentally with the advent of steroidogenesis. Adrenals of neonatal rats demonstrate greatly reduced sensitivity to ACTH that gradually increases after the first 2 weeks of life. Thus, neonates have lower circulating corticosterone levels following exposure to stress. We examined mitochondrial PBR ligand binding activity, immunoreactive (ir) PBR content, and adrenal sensitivity to ACTH in vivo and in vitro. Ontogeny of both mitochondrial PBR ligand binding capacity and irPBR directly paralleled that of ACTH-inducible steroidogenesis in isolated rat adrenal cells and in rats injected with ACTH. In addition, neonatal PBR had approximately 2-fold higher affinity for PK11195, a synthetic ligand that binds with high affinity to PBR. No correlation was observed during neonatal life between ir-steroidogenic acute regulatory (StAR) protein content and steroidogenesis. These results are consistent with the hypothesis that PBR is an absolute prerequisite for adrenocortical steroidogenesis, and suggest that the stress hyporesponsive period of neonatal rats may result from decreased PBR expression. In addition, the higher affinity of neonatal PBR and the relatively high basal expression of StAR protein in neonatal adrenals may partly explain the high constitutive steroidogenesis characteristic of neonatal rat adrenal cells.

Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO2

1992 ◽  
Vol 72 (2) ◽  
pp. 439-446 ◽  
Author(s):  
F. G. Issa ◽  
J. E. Remmers
Keyword(s):  
Spinal Cord ◽  
Neural Activity ◽  
Ventral Surface ◽  
Sudden Increase ◽  
Neonatal Rats ◽  
Central Chemoreceptors ◽  
Ventral Medulla ◽  
Ventromedial Medulla ◽  
Stimulation Of

The exact location of the central respiratory chemoreceptors sensitive to changes in PCO2 has not yet been determined. To avoid the confounding effects of the cerebral circulation, we used the in vitro brain stem-spinal cord of neonatal rats (1–5 days old) to identify areas within 500 microns of the ventral surface of the medulla where changes in PCO2 evoked a sudden increase in the rate of respiratory neural activity. The preparation was superfused with mock cerebrospinal fluid (CSF) while maintained at constant temperature (26 +/- 1 degrees C) and pH (7.34). Respiratory frequency increased linearly with decreases in superfusate pH (r2 = 0.92, P less than 0.001), indicating that the respiratory circuitry for the detection of CO2 and stimulation of breathing was intact in this preparation. The search for central chemoreceptors was performed with a specially designed micropipette that allowed microejection of 2–10 nl of mock CSF equilibrated with different CO2-O2 gas mixtures. The pipette was advanced in 50- to 100-microns steps by use of a microdrive to a maximum depth of 500 microns from the surface of the ventral medulla. Depending on the location of the micropipette, ejection of CO2-acidified mock CSF at depths of 100–350 microns below the ventral surface of the medulla stimulated neural respiratory output. Using this response as an indication of the location of central respiratory chemoreceptors, we found that chemoreceptive elements were located in a column in the ventromedial medulla extending from the hypoglossal rootlets caudally to an area 0.75 mm caudal to VI nerve in the rostral medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Afferent Inputs Modulate the Activity of a Rhythmic Burst Generator in the Rat Disinhibited Spinal Cord In Vitro

1997 ◽  
Vol 77 (6) ◽  
pp. 3157-3167 ◽  
Author(s):  
E. Bracci ◽  
M. Beato ◽  
A. Nistri
Keyword(s):  
Spinal Cord ◽  
Burst Duration ◽  
Neonatal Rat ◽  
Rhythmic Pattern ◽  
Synaptic Inputs ◽  
Electrical Pulses ◽  
Afferent Inputs ◽  
Spontaneous Bursts ◽  
Rhythmic Burst

Bracci, E., M. Beato, and A. Nistri. Afferent inputs modulate the activity of a rhythmic burst generator in the rat disinhibited spinal cord in vitro. J. Neurophysiol. 77: 3157–3167, 1997. Application of strychnine and bicuculline to the isolated spinal cord of the neonatal rat induces spontaneous bursting of regular rhythmicity (cycle period ∼30 s). This phenomenon is important because it shows that a spinal network, made up by excitatory connections only, generates a very reliable rhythmic pattern. To find out how signals from the periphery or higher centres might influence the operation of the rhythmogenic network, the present experiments examined whether synaptic inputs from dorsal root (DR) or ventrolateral (VL) afferent fibers could modulate this spontaneous rhythmicity. This issue was addressed with intracellular recording from motoneurons or extracellular recording from ventral roots after eliciting bursting with strychnine plus bicuculline. Single electrical shocks (0.1 ms; intensity 1–4 times threshold) applied to one DR reset spontaneous bursting without altering its period or duration. Repetitive stimulations at periods ranging from 20 to 2 s entrained bursts on a 1:1 basis. Burst duration was shorter at lower stimulation periods whereas burst amplitude was unchanged. The lowest stimulation period compatible with burst entrainment depended on stimulus strength. At stimulation periods <2-s entrainment was always lost and spontaneous bursts unexpectedly returned even if electrical pulses still elicited ventral root reflexes. Such spontaneous bursts had similar properties as those recorded in the absence of electrical pulses. Analogous results were obtained with VL stimulations. It is concluded that the spinal rhythmogenic network was highly susceptible to external synaptic inputs, which paced burst generation whereas burst duration was adapted to interstimulus interval. A scheme is provided to explain the modulatory role of synaptic inputs as well as the escape of bursting from fast stimulus entrainment in terms of a rhythmogenic network functionally separated from reflex pathways activated by DR or VL tracts.

scholarly journals CORM-2 can Attenuate Bleeding-mediated Inflammation by Increasing Phagocytic Capacity of Cerebral Microglial Cells in Neonatal Rat in Vitro

2021 ◽  
Author(s):  
Zhiying Chen ◽  
Huiyan Zhang ◽  
Jun Zhou ◽  
Xiaoqin Wu ◽  
Moxin Wu ◽  
...  
Keyword(s):  
Treated Group ◽  
Neonatal Rat ◽  
Neonatal Rats ◽  
Phagocytic Capacity ◽  
Inflammatory Reactions ◽  
Microglial Phagocytosis ◽  
Protein Expressions ◽  
Cell Groups ◽  
Cell Supernatant

Abstract Objective: This study aimed to explore the mechanism of CORM-2 on attenuating bleeding-related inflammation. Methods: Microglia were isolated from the neonatal rats (1-2days old) and identified by the CD11b/c anti-body, and some microglia were co-cultured with RBCs marked with PKH26 fluorescent dye, and then treated with CORM-2. That is, the microglia cells were divided into the microglia, microglia+ PKH26+RBCs and microglia + PKH26+ RBCs+CORM-2 cell-groups. Microglial phagocytosis to RBCs PKH26+ was observed under an inverted fluorescence microscope; moreover, the fluorescence intensity of microglia that phagocytized PKH26+RBCs was detected through immunofluorescence. HO-1, NF-κB p65, and IL-1β expressions were detected using RT-qPCR, western blotting, and immunofluorescence, respectively. The levels of carbon monoxide hemoglobin (HbCO) in the cell supernatant in each group were detected with ELISA.Results. After 1- day of co-culturing, the number of residual PKH26+RBCs in the Microglia+ PKH26+RBCs+CORM-2 group decreased remarkably than that in the Microglia+ PKH26+RBCs groups (18 × 106 vs. 14 × 106, p=0.02), which revealed that microglia phagocytosis was stronger in CORM-2 treated group. More over, compared with microglia + PKH26+RBCs group, the microglia+ PKH26+RBCs +CORM-2 group showed higher levels of HO-1 mRNA and protein expressions at the 3rd day and the 5th day after co-culturing. Further more, CORM-2 significantly inhibited the expressions of mRNA and proteins of NF‐κB p65 and IL-1 after 3 days of co-culturing, meanwhile, CORM-2 did not increase the level of HbCO in the cell supernatant.Conclusions CORM-2 can inhibit inflammatory reactions in bleeding setting in vitro by promoting microglial phagocytosis to RBCs and decrease IL-1β and NF-κB; the mechanism may involve HO-1/CO system.

scholarly journals Chlorobenzoxime inhibits respiratory syncytial virus infection in neonatal rats via up-regulation of IFN-γ in dendritic cells

2020 ◽  
Vol 19 (2) ◽  
pp. 239-246
Author(s):  
Junzhao Li ◽  
Yonghai Zhang ◽  
Hongmei Qiao ◽  
Yingji Jin ◽  
Jianmin Wang ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Respiratory Syncytial Virus ◽  
Neonatal Rat ◽  
Interleukin 4 ◽  
Neonatal Rats ◽  
Alveolar Cells ◽  
Rsv Infection ◽  
Ifn Γ ◽  
Syncytial Virus

Purpose: To investigate the effect of chlorobenzoxime on respiratory syncytial virus (RSV) infection in vitro in lung alveolar cells and in vivo in neonatal rats, as well as the mechanism of action involved. Methods: RSV infection in neonatal rats was induced via intranasal administration of 2 x 106PFU viral particles. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used for determination of changes in interleukin expression. Results: RSV infection in BEAS-2B cells caused significant reduction in viability and marked alteration in morphological appearance (p < 0.05). Exposure of RSV-infected BEAS-2B cells to chlorobenzoxime prevented viability reduction and changes in morphology, and led to reductions in RSV-mediated increases in levels of interleukin-6 and interleukin-8. Moreover, RSV infection significantly enhanced ROS levels in BEAS-2B cells, when compared to control cells (p < 0.05). Chlorobenzoxime at a concentration of 30 μM completely suppressed RSV-mediated generation of ROS in BEAS-2B cells. In neonatal rats, RSV-induced upregulation of interleukin-4, interleukin-13 and TNF-α, were suppressed in bronchoalveolar lavage fluid (BALF) and lung tissues by chlorobenzoxime. Moreover, the RSVmediated reduction in IFN-γ was maximally blocked by chlorobenzoxime at a dose of 10 mg/mL. Chlorobenzoxime enhanced the proportion of IFN-γ -producing cells in neonatal rat BALF. Conclusion: Chlorobenzoxime exhibits antiviral against RSV infection in neonatal rats via increase in dendritic cell population, leading to inhibition of cytokine production. Therefore, chlorobenzoxime is a potential therapeutic agent for RSV infection. Keywords: Respiratory syncytial virus, Cytokines, Dendritic cells, Lung aveolar cells, Morphology, Interleukins

Thyrotropin-releasing hormone stimulates perinatal rat respiration in vitro

1996 ◽  
Vol 271 (5) ◽  
pp. R1160-R1164 ◽  
Author(s):  
J. J. Greer ◽  
Z. al-Zubaidy ◽  
J. E. Carter
Keyword(s):  
Spinal Cord ◽  
Brain Stem ◽  
Thyrotropin Releasing Hormone ◽  
Neonatal Rat ◽  
Discharge Frequency ◽  
Releasing Hormone ◽  
Respiratory Rhythmogenesis ◽  
Fetal Rat ◽  
Bötzinger Complex

In the present study, we test whether thyrotropin-releasing hormone (TRH) stimulates respiratory frequency in perinatal rats by acting at regions of the medulla responsible for respiratory rhythmogenesis, the pre-Botzinger complex. We also test whether TRH stimulates respiration in the fetal rat at a time shortly after the inception of respiratory rhythmogenesis [embryonic days (E) 17-18]. Two in vitro experimental models were utilized: the isolated brain stem-spinal cord preparation from fetal (E17-E18) and neonatal [postnatal days (P) 0-2] rats and the medullary slice preparation isolated from neonatal rats (P1-P2). Bath application of TRH caused a dose-dependent, reversible increase (maximum increase approximately 60%) in the frequency of respiratory rhythmic neural discharge generated by brain stem-spinal cord [half-maximal effective concentration (EC50) approximately 9 nM] and medullary slice (EC50 approximately 2.5 nM) neonatal rat preparations. Pressure injection of TRH unilaterally into the region of the pre-Botzinger complex of the neonatal medullary slice caused an approximately 28% increase in the frequency of respiratory discharge. Application of TRH to the medium bathing fetal rat brain stem-spinal cord preparations caused an approximately threefold increase in respiratory discharge frequency. We conclude that TRH stimulates respiratory discharge frequency from the time near inception of respiratory motor discharge and acts directly at the pre-Botzinger complex.

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

2002 ◽  
Vol 282 (2) ◽  
pp. R484-R491 ◽  
Author(s):  
Nicholas M. Mellen ◽  
William K. Milsom ◽  
Jack L. Feldman
Keyword(s):  
Brain Stem ◽  
Motor Neurons ◽  
Respiratory Rhythm ◽  
Field Potential ◽  
Respiratory Arrest ◽  
Motor Output ◽  
Neonatal Rat ◽  
Bötzinger Complex ◽  
Neonatal Rat Brain

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

Modulation of respiratory rhythm in vitro: role of Gi/o protein-mediated mechanisms

1996 ◽  
Vol 80 (6) ◽  
pp. 2120-2133 ◽  
Author(s):  
S. M. Johnson ◽  
J. C. Smith ◽  
J. L. Feldman
Keyword(s):  
Respiratory Rhythm ◽  
Drug Effects ◽  
Receptor Activation ◽  
Respiratory Frequency ◽  
Neonatal Rat ◽  
Rhythm Generation ◽  
Gamma Aminobutyric Acid ◽  
Mu Opioid ◽  
Bötzinger Complex

Slice preparations from neonatal rat medulla that generate respiratory rhythm in vitro were used to test for Gi/o protein-mediated mechanisms affecting breathing rhythm in mammals. The frequency of inspiratory motor discharge recorded from hypoglossal (XII) nerve roots decreased with bath application of gamma-aminobutyric acid (GABA) and norepinephrine, as well as agonists specific for GABAB, alpha 2-adrenergic, and mu-opioid receptors; 5-hydroxytryptamine had little effect on frequency. Microinjection of these specific agonists into the pre-Botzinger complex, the site of respiratory rhythm generation in vitro, also decreased frequency. In contrast, substance P (SP) increased frequency when it was bath applied or microinjected into the pre-Botzinger complex. To test for involvement of Gi/o proteins, pertussis toxin (PTX) was injected into the cerebrospinal fluid of newborn rats, and slices from these animals were tested 48 h later for block of drug effects on rhythm. In PTX-treated slices the frequency decrease due to GABAB, mu-opioid, and alpha 2-adrenergic receptor activation was attenuated (P < or = 0.05), whereas the SP receptor-mediated response was unaltered. To test for involvement of K+ conductances linked to Gi/o proteins Ba2+ (0.2 mM) was added to the bath before application of drugs. Ba2+ attenuated the decrease in frequency associated with GABAB (P < or = 0.05) and mu-opioid (0.10 < or = P < or = 0.05) receptor activation, whereas the alpha 2-adrenergic and SP responses were unaltered. We conclude that GABAB and mu-opioid, but not alpha 2-adrenergic and SP, receptor activation modulates respiratory frequency via a Gi/o protein-dependent Ba(2+)-sensitive ionic conductance mechanism on neurons within the medullary locus for rhythm generation. This mechanism may be a convergent pathway for control of respiratory frequency.

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