Silencing by raised extracellular Ca of pre-Bötzinger complex neurons in newborn rat brainstem slices without change of membrane potential or input resistance

Breathing is controlled by inspiratory pre-Bötzinger complex (preBötC) networks that remain active in transversal brainstem slices from perinatal rodents. In 600 μm thick preBötC slices, inspiratory-related bursting in physiological (3 mM) [K⁺] is depressed by <1 mM elevation of superfusate [Ca²⁺]. Here, we studied underlying cellular mechanisms in whole-cell-recorded neurons of 400 μm thin newborn rat slices with the <200 μm thin preBötC in the middle (“m-preBötC[400]” slices). Extracellular activity in the ventrolateral slice area in 3 mM K⁺ and a most common physiological Ca²⁺ range (1–1.2 mM) stopped spontaneously within 2 h (“in vitro apnea”). Contrary, rhythm was stable for >3 h at 6–8 bursts/min in 7 mM K⁺ and 1.2 mM Ca²⁺ solution. In non-pacemaker preBötC inspiratory cells and neighboring inspiratory or tonically active neurons, block or frequency depression by >90% of rhythm in the latter solution by 2–3 mM Ca²⁺ changed neither resting potential nor input resistance. High Ca²⁺ silenced inspiratory neurons and depressed tonic discharge of non-respiratory neurons. However, in both cell types current injection evoked normal action potentials with unchanged threshold potential. The findings show that m-preBötC[400] slices represent a good compromise between long term viability of rhythmogenic preBötC neurons and minimal modulation of these cells by adjacent tissue, but need to be studied in elevated K⁺. The lack of postsynaptic K⁺ channel-mediated hyperpolarization suggests that saturation of surface charges, presynaptic block of transmission and/or inhibition of postsynaptic burst-promoting conductances such as Ca²⁺ activated non-selective cation channels are involved in inspiratory depression by high Ca²⁺.     

K and Ca dependence of inspiratory-related rhythm in novel “calibrated” mouse brainstem slices

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

Embryonic emergence of the respiratory rhythm generator

Breathing is vital for life ex utero and therefore requires that the respiratory rhythm generator (RRG), the central neural network generating the continuous rhythmic motor command, be functional at birth. The RRG, located in the brainstem, appears to comprise two interacting respiratory oscillators: the parafacial respiratory group (pFRG), and the preBötzinger complex (preBötC). Data on the establishment of these respiratory oscillators during embryonic and foetal periods are beginning to be produced. The present paper provides a short review of the current knowledge regarding: (i) the emergence of activity in the two respiratory oscillators and (ii) their functional coupling during prenatal development in rodents.     

Reconfiguration of respiratory-related population activity in a rostrally tilted transversal slice preparation following blockade of inhibitory neurotransmission in neonatal rats

Recent studies showed that respiratory rhythm generation depends on oscillators located in the pre-Bötzinger complex (pre-BötC) and the parafacial respiratory group (pFRG). To study inhibitory synaptic interactions between these two oscillators, we developed a rostrally tilted transversal slice preparation, which preserves these regions. The onset of rhythmic mass activity in the retrotrapezoid nucleus (RTN)/pFRG preceded that of the pre-BötC. Blockade of glycinergic and gamma-aminobutyric acidic inhibition synchronized pre-BötC and RTN/pFRG activity and significantly increased pre-BötC burst frequency, amplitude, and duration. Population imaging revealed recruitment of inspiratory-like neurones, while expiratory-like neurones lost their phasic activity. The reconfiguration after disinhibition reveals: (1) synaptic inhibition of the pre-BötC arising from the RTN/pFRG, (2) excitatory drive from the RTN/pFRG that triggers the pre-BötC burst. Our findings support the view that these synaptic interactions in vitro relate to the initiation of the inspiratory phase or to the steering of the expiratory–inspiratory phase transition in vivo.     

Respiratory responses to somatostatin microinjections into the Bötzinger complex and the pre-Bötzinger complex of the rabbit

The respiratory responses to bilateral microinjections (30–50 nl) of 5 mM somatostatin (SOM) or 10 mM cyclosomatostatin (c-SOM, a SOM antagonist) into the Bötzinger complex (BötC), the pre-Bötzinger complex (preBötC) and the rostral inspiratory portion of the ventral respiratory group (iVRG) were investigated in urethane–chloralose anesthetized, vagotomized, paralysed and artificially ventilated rabbits. SOM microinjections into the BötC decreased respiratory frequency and the rate of rise of phrenic nerve activity without obvious changes in its peak amplitude. SOM microinjected into the preBötC caused increases in respiratory frequency and decreases in peak phrenic activity associated with increases in its rate of rise. No changes in respiration were induced by SOM microinjections into the iVRG. Microinjections of c-SOM into the preBötC caused decreases in respiratory frequency as well as in peak amplitude and rate of rise of phrenic nerve activity. The results show that endogenously released SOM within the preBötC contributes to shape the pattern of baseline respiratory activity and that SOM receptors within the BötC and the preBötC have a role in the modulation of respiration in the rabbit.     

Fluorescence imaging of active respiratory networks

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

Nociceptin/orphanin FQ causes non-quantal slowing of respiratory rhythm in brainstem-spinal cord preparation from newborn rat

Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the N/OFQ peptide receptor, an inhibitory G protein-coupled receptor. N/OFQ acts as a neuromodulator to depress respiratory rhythm in the brainstem. Although the mechanisms of respiratory rhythm generation remain poorly understood, the pre-inspiratory neuron (Pre-I) and the pre-Bötzinger complex (preBötC) inspiratory neuron (Insp) network in the rostral ventrolateral medulla (RVLM) have been proposed to be essential for respiratory rhythm generation. Opioids presumably cause quantal slowing via selective depression of preBötC Insps. However, it is unclear whether N/OFQ depresses respiratory rhythm via the same mechanism. In this study, using in vitro newborn rat en bloc preparations, we examined the slowing pattern of N/OFQ (quantal or non-quantal) and the effects of N/OFQ on the extracellularly recorded discharge of Pre-Is and Insps in the RVLM. N/OFQ caused non-quantal slowing with a synchronous decrease in burst rates of Insps and of C4 discharge whereas the intraburst spike number in Insps remained unchanged. It also caused a significant decrease in burst rates and intraburst spike numbers in Pre-Is, while the 1:1 coupling of Pre-Is bursts to C4 bursts was preserved. When superfusate K⁺ was elevated from 6.2 to 11.2 mM, Pre-I activity was increasingly uncoupled from C4 bursts. After the application of N/OFQ in a high [K⁺] superfusate, the 1:1 coupling of Pre-Is to C4 bursts was restored. We conclude that N/OFQ suppresses burst and spike generation of Pre-Is, and that suppression of Pre-Is activity with synchronous coupling to the Insps network contributes to N/OFQ-induced non-quantal slowing.     

The adrenergic modulation of firings of respiratory rhythm-generating neurons in medulla-spinal cord preparation from newborn rat

We analysed the modulation of respiratory neurons by adrenaline or noradrenaline (NA) in a newborn rat brainstem-spinal cord preparation. Adrenaline or NA caused a dose-dependent depression of the respiratory rhythm and induced C4 spinal tonic discharges. The inhibitory effect of adrenaline (ED₅₀=0.5 μM) on the respiratory rhythm was stronger than NA (ED₅₀=5 μM). The adrenaline respiratory rhythm depression was partially blocked by the α₁-antagonist prazosin or by the α₂-antagonist yohimbine. The C4 tonic discharge elicited by adrenaline was blocked by the α₁-antagonist prazosin. The direct effects of adrenaline on pre-inspiratory (Pre-I) neurons were examined in a synaptic blockade solution (low Ca), and fifty-six percent of Pre-I neurons were found to continue firing. In low-Ca solution, Pre-I neurons were excited (n=29 of 39) or depressed (n=5 of 39) by adrenaline, and excited by α₁-agonist phenylephrine or depressed by α₂-agonist clonidine. These results suggest that the respiratory rhythm depression under intact network conditions is mediated by some other inhibitory system. The inhibitory effect of adrenaline on the respiratory rhythm was partially blocked by the GABA_A-antagonists bicuculline or picrotoxin, but not by the GABA_B-antagonist phaclofen. The present results suggest that: (1) respiratory rhythm generation is more sensitive to adrenaline than NA through α-adrenergic action of adrenaline; (2) the activity of Pre-I neurons could be directly regulated by excitation via α₁-receptors and inhibition via α₂-receptors; and (3) the depression of the respiratory rhythm by adrenaline is partly mediated by GABA_Aergic neurons. Received: 8 April 1997 / Accepted: 6 October 1997     

Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven “type-I” Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The µ-opioid [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (~5 mV) and decreased their resistance (~30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.     

Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla

Two putative respiratory rhythm generators (RRGs), the para-facial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC), have been identified in the neonatal rodent brainstem. To elucidate their functional roles during the neonatal period, we evaluated developmental changes of these RRGs by optical imaging using a voltage-sensitive dye. Optical signals, recorded from the ventral medulla of brainstem–spinal cord preparations of neonatal (P0–P4) rats ( n = 44), were analysed by a cross correlation method. With development during the first few postnatal days, the respiratory-related activity in the pFRG reduced and shifted from a preinspiratory (P0–P1) to an inspiratory (P2–P4) pattern, whereas preBötC activity remained unchanged. The μ-opioid agonist [ d -Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) augmented preinspiratory activity in the pFRG, while the μ-opioid antagonist naloxone induced changes in spatiotemporal activation profiles that closely mimicked the developmental changes. These results are consistent with the recently proposed hypothesis by Janczewski and Feldman that the pFRG is activated to compensate for the depression of the preBötC by perinatal opiate surge. We conclude that significant reorganization of the respiratory neuronal network, characterized by a reduction of preinspiratory activity in the pFRG, occurs at P1–P2 in rats. The changes in spatiotemporal activation profiles of the pFRG neurones may reflect changes in the mode of coupling of the two respiratory rhythm generators.     

Isolation of the kernel for respiratory rhythm generation in a novel preparation: the pre-Bötzinger complex "island"

The pre-B?tzinger complex (pre-B?tC), a bilaterally distributed network of rhythmogenic neurons within the ventrolateral medulla, has been proposed to be the critical locus for respiratory rhythm generation in mammals. To date, thin transverse medullary slice preparations that capture the pre-B?tC have served as the optimal experimental model to study the region's inherent cellular and network properties. We have reduced the thin slices to isolated pre-B?tC "islands" to further establish whether the pre-B?tC has intrinsic rhythmicity and is the kernel for rhythmogenesis in the slice. We recorded neuron population activity locally in the pre-B?tC with macroelectrodes and fluorescent imaging of Ca(2+) activities with Calcium Green-1AM dye before and after excising the island. The isolated island remained rhythmically active with a population burst profile similar to the inspiratory burst in the slice. Rhythmic population activity persisted in islands after block of GABA(A)ergic and glycinergic synaptic inhibition. The loci of pre-B?tC Ca(2+) activity imaged in thin slices and islands were similar, and imaged pre-B?tC neurons exhibited synchronized flashing after blocking synaptic inhibition. Population burst frequency increased monotonically as extracellular potassium concentration was elevated, consistent with mathematical models consisting entirely of an excitatory network of synaptically coupled pacemaker neurons with heterogeneous, voltage-dependent bursting properties. Our results provide further evidence for a rhythmogenic kernel in the pre-B?tC in vitro and demonstrate that the islands are ideal preparations for studying the kernel's intrinsic properties.     

Energetics and oxygen transport mechanisms in embryos

Abrupt, bilateral destruction of the pre-Bötzinger Complex (preBötC) leads to terminal apnea in unanesthetized goats and rats. In contrast, respiratory rhythm and pattern and arterial blood gases in goats during wakefulness and sleep are normal after incremental (over a month) destruction of >90% of the preBötC. Here, we tested the hypothesis that the difference in effects between abrupt and incremental destruction of the preBötC are a result of time-dependent plasticity, which manifests as anatomic changes at sites within the respiratory network. Accordingly, we report data from histological analyses comparing the brainstems of control goats, and goats that had undergone bilateral, incremental, ibotenic acid (IA)-induced preBötC lesioning. A major focus was on the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pontine respiratory group (PRG), which are sites thought to contribute to respiratory rhythmogenesis. We also studied the facial (FN), rostral nucleus ambiguus (NA), medullary raphé (MRN), hypoglossal (HN), and the dorsal motor vagal (DMV) nuclei. Neuronal counts, count region area (mm²), and neuronal densities were calculated using computer-assisted analyses and/or manual microscopy to compare control and preBötC-lesioned animals. We found that within the ventral and lateral medulla 2 mm rostral to the caudal pole of the FN (presumed pFRG/RTN), there were 25% and 65% more (P < 0.001) neurons, respectively, in preBötC-lesioned compared to control goats. Lesioned goats also showed 14% and 13% more (P < 0.001) neurons in the HN and medial parabrachialis nucleus, but 46%, 28%, 7%, and 17% fewer (P < 0.001) neurons in the FN, NA, DMV, and Kölliker-Fuse nuclei, respectively. In the remaining sites analyzed, there were no differences between groups. We conclude that anatomic changes at multiple sites within the respiratory network may contribute to the time-dependent plasticity in breathing following incremental and near-complete destruction of the preBötC.     

Functional imaging reveals respiratory network activity during hypoxic and opioid challenge in the neonate rat tilted sagittal slab preparation

In mammals, respiration-modulated networks are distributed rostrocaudally in the ventrolateral quadrant of the medulla. Recent studies have established that in neonate rodents, two spatially separate networks along this column-the parafacial respiratory group (pFRG) and the pre-B?tzinger complex (preB?tC)-are hypothesized to be sufficient for respiratory rhythm generation, but little is known about the connectivity within or between these networks. To be able to observe how these networks interact, we have developed a neonate rat medullary tilted sagittal slab, which exposes one column of respiration-modulated neurons on its surface, permitting functional imaging with cellular resolution. Here we examined how respiratory networks responded to hypoxic challenge and opioid-induced depression. At the systems level, the sagittal slab was congruent with more intact preparations: hypoxic challenge led to a significant increase in respiratory period and inspiratory burst amplitude, consistent with gasping. At opioid concentrations sufficient to slow respiration, we observed periods at integer multiples of control, matching quantal slowing. Consistent with single-unit recordings in more intact preparations, respiratory networks were distributed bimodally along the rostrocaudal axis, with respiratory neurons concentrated at the caudal pole of the facial nucleus, and 350 microns caudally, at the level of the pFRG and the preB?tC, respectively. Within these regions neurons active during hypoxia- and/or opioid-induced depression were ubiquitous and interdigitated. In particular, contrary to earlier reports, opiate-insensitive neurons were found at the level of the preB?tC.     

Late-expiratory activity: emergence and interactions with the respiratory CpG

The respiratory rhythm and motor pattern are hypothesized to be generated by a brain stem respiratory network with a rhythmogenic core consisting of neural populations interacting within and between the pre-B?tzinger (pre-B?tC) and B?tzinger (B?tC) complexes and controlled by drives from other brain stem compartments. Our previous large-scale computational model reproduced the behavior of this network under many different conditions but did not consider neural oscillations that were proposed to emerge within the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) and drive preinspiratory (or late-expiratory, late-E) discharges in the abdominal motor output. Here we extend the analysis of our previously published data and consider new data on the generation of abdominal late-E activity as the basis for extending our computational model. The extended model incorporates an additional late-E population in RTN/pFRG, representing a source of late-E oscillatory activity. In the proposed model, under normal metabolic conditions, this RTN/pFRG oscillator is inhibited by B?tC/pre-B?tC circuits, and the late-E oscillations can be released by either hypercapnia-evoked activation of RTN/pFRG or by hypoxia-dependent suppression of RTN/pFRG inhibition by B?tC/pre-B?tC. The proposed interactions between B?tC/pre-B?tC and RTN/pFRG allow the model to reproduce several experimentally observed behaviors, including quantal acceleration of abdominal late-E oscillations with progressive hypercapnia and quantal slowing of phrenic activity with progressive suppression of pre-B?tC excitability, as well as to predict a release of late-E oscillations by disinhibition of RTN/pFRG under normal conditions. The extended model proposes mechanistic explanations for the emergence of RTN/pFRG oscillations and their interaction with the brain stem respiratory network.     

Effects of inhibitory neurotransmitters on the mudpuppy (Necturus maculatus) locomotor pattern in vitro

Effects of inhibitory neurotransmitters on the locomotor rhythm and pattern generation were investigated using an in vitro preparation isolated from the mudpuppy (Necturus maculatus). The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial nerves. During N-methyl-d-aspartate (NMDA)-induced locomotion, the rhythmic motor output (EMG) was recorded unilaterally from elbow flexor and extensor muscles. While neither glycine nor γ-aminobutyric acid (GABA)-related substances induced locomotion in the absence of NMDA, they modulated NMDA-induced locomotion. Bath application of glycine and GABA suppressed the rhythmic motor pattern induced by NMDA. Addition of glycine receptor antagonist strychnine or GABA_A receptor antagonist bicuculline disrupted the phase relationship between antagonistic motor pools during ongoing locomotion, thereby changing the normal alternating pattern into synchronous EMG bursts. Both the GABA_A receptor agonist muscimol and GABA_B receptor agonist baclofen mimicked the effects of GABA as they either slowed down or stopped locomotion. Nipecotic acid, a GABA uptake blocker, had a similar effect. This suggested that an endogenous release of GABA modulated the locomotor rhythm. The endogenous release was antagonized by the GABA_A and GABA_B receptor antagonists bicuculline and CGP-35348, respectively. Immunocytochemistry revealed that glycine and GABA-positive neurons and fibers were present in mudpuppy spinal cord. Although the GABAergic neurons were more numerous than glycinergic neurons, both cell types contributed processes directed towards the white matter and occasionally towards the ependymal lining of the central canal. Our results suggest that inhibitory neurotransmitters exert powerful actions upon the neuronal network governing forelimb locomotion in the mudpuppy. The effects we observed may be mediated by a network of segmentally distributed glycinergic and GABAergic spinal neurons. Received: 1 December 1998 / Accepted: 26 April 1999     

Dynamics of neuromodulatory feedback determines frequency modulation in a reduced respiratory network: A computational study

Neuromodulators, such as amines and neuropeptides, alter the activity of neurons and neuronal networks. In this work, we investigate how neuromodulators, which activate G_q-protein second messenger systems, can modulate the bursting frequency of neurons in a critical portion of the respiratory neural network, the pre-Bötzinger complex (preBötC). These neurons are a vital part of the ponto-medullary neuronal network, which generates a stable respiratory rhythm whose frequency is regulated by neuromodulator release from the nearby Raphe nucleus. Using a simulated 50-cell network of excitatory preBötC neurons with a heterogeneous distribution of persistent sodium conductance and Ca²⁺, we determined conditions for frequency modulation in such a network by simulating interaction between Raphe and preBötC nuclei. We found that the positive feedback between the Raphe excitability and preBötC activity induces frequency modulation in the preBötC neurons. In addition, the frequency of the respiratory rhythm can be regulated via phasic release of excitatory neuromodulators from the Raphe nucleus. We predict that the application of a G_q antagonist will eliminate this frequency modulation by the Raphe and keep the network frequency constant and low. In contrast, application of a G_q agonist will result in a high frequency for all levels of Raphe stimulation. Our modeling results also suggest that high [K⁺] requirement in respiratory brain slice experiments may serve as a compensatory mechanism for low neuromodulatory tone.     

Expressions of 5-HT/5-HT2A receptors and phospho-protein kinase C theta in the pre-Bötzinger complex in normal and chronic intermittent hypoxic rats

The pre-Bötzinger complex (pre-BötC), a functionally defined subregion in the ventrolateral medulla oblongata, is a presumed kernel of normal respiratory rhythmogenesis. However, less is known about the pre-BötC's contribution to respiratory neuroplasticity. The most frequently studied model for respiratory neuroplasticity is episodic hypoxia-induced phrenic long-term facilitation, which is 5-HT_2A receptors (5-HT_2AR)-dependent. We hypothesized that preconditioning with chronic intermittent hypoxic (CIH) would activate the 5-HT/5-HT_2AR system and the downstream protein kinase C (PKC) pathway in the pre-BötC. Animals were exposed to alternating 5 min of hypobaric hypoxia and 5 min of normoxia for 10 h/day for 7 days. Hypobaric hypoxia was achieved by continuous air evacuation to reach a pressure of 210–220 mm Hg, corresponding to an altitude of 9000–10000 m. In contrast to the CIH model, a group of animals were pretreated with chronic sustaining hypoxia (CSH), a protocol of continuous hypobaric hypoxia at 360 mm Hg, corresponding to an altitude of about 6000 m, for 10 h/day for 7 days. Immunoreactivity of 5-HT and 5-HT_2AR was examined in the pre-BötC, identified by the presence of neurokinin-1 receptor (NK1R). We found that 15.5% of 5-HT-immunoreactive (ir) terminals were in contact with NK1R-ir neurons. Asymmetric synapses could be identified between them. 38.7% of NK1R-ir dendrites were also immunoreactive for 5-HT_2AR, which was distributed along the inner surface of the plasma membrane in control animals. CIH challenge increased the expressions of 5-HT and 5-HT_2AR in the pre-BötC, an increase in the expressed 5-HT_2AR that was not detected in this region in CSH animals. Specifically, 5-HT_2AR was distributed not only along the inner surface, but also along the outer surface, or directly on the plasma membrane, a pattern not detectable in control animals. 5-HT_2AR was also detectable in the invaginations of the plasma membrane, where receptor endocytosis or exocytosis might occur, indicating CIH-induced higher trafficking of 5-HT_2AR. Concurrently, there was an up-regulation of phospho-PKC theta (P-PKCθ) in the pre-BötC, suggesting a 5-HT/5-HT_2AR-activated PKC mechanism that may contribute to hypoxia-induced respiratory neuroplasticity in the pre-BötC. The close association of P-PKCθ with the postsynaptic density implicates a postsynaptic mechanism mediating respiratory neuroplasticity in the pre-BötC.     

The role of GABAergic neuron on NMDA- and SP-induced phase delays in the suprachiasmatic nucleus neuronal activity rhythm in vitro

Gamma-aminobutyric acid (GABA), and its biosynthetic enzyme, glutamic decarboxylase, are widely distributed in the suprachiasmatic nucleus (SCN). In the present study, we examined the role of the GABA_A receptor on in vitro SCN responses to photic-like signals. We found that 100 μM GABA_A receptor antagonist bicuculline partially blocked field potentials evoked by optic nerve stimulation. NMDA- and SP-induced phase shifts of SCN neuronal activity rhythms, were blocked with 10 μM bicuculline. Application of 100 μM bicuculline alone induced phase advance of SCN neuronal activity rhythm. These results show that NMDA- and SP-induced phase shifts are blocked by bicuculline and suggest GABA has an important role as neurotransmitter in the neuronal network regulating phase shifts of the circadian clock.     

Respiration-related rhythmic activity in the rostral medulla of newborn rats

There are at least two respiration-related rhythm generators in the medulla: the pre-B?tzinger complex, which produces inspiratory (Insp) neuron bursts, and the parafacial respiratory group (pFRG), which produces predominantly preinspiratory (Pre-I) neuron bursts. The pFRG Pre-I neuron activity has not been correlated with motor neuron activity in slice or block preparations of rostral medulla. In this study, we attempted to detect pFRG Pre-I activity as motor output in the rostral medulla. We recorded respiratory activity of the facial nerve in the brain stem-spinal cord preparation of 0- to 2-day-old rats. Facial nerve activity consisted of preinspiratory, Insp, and postinspiratory activity. Pre- and postinspiratory activity corresponded well with membrane potential trajectories of Pre-I neurons in the rostral ventrolateral medulla. In response to perfusion of 1 microM DAMGO (a mu-opiate agonist), fourth cervical ventral root (C4) Insp activity was depressed and facial nerve activity continued to synchronize with Pre-I neuron bursts. After transverse sectioning between the levels of the pre-B?tzinger complex and the pFRG, C4 Insp activity recovered within 15 min, but facial nerve activity was inhibited. When DAMGO was applied, C4 Insp activity was inhibited, and rhythmic facial nerve activity recovered. Subsequent elevation of K+ concentration reinduced C4 activity, but facial nerve activity was inhibited. Whole cell recordings in the rostral block revealed the presence of putative Pre-I neurons, the activity of which was synchronized with facial nerve activity. These results show that the rostral medulla, not including the pre-B?tzinger complex, produces Pre-I-like rhythmic activity that can be monitored as facial nerve motor output in newborn rat in vitro preparations.