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Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5 μM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 μM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 μM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 μM riluzole or 100 μM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 μM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca 2+ /high Mg 2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Breathing movements in mammals are driven by rhythmic neural activity automatically generated within spatially and functionally organized brainstem neural circuits comprising the respiratory central pattern generator (CPG). This chapter reviews up-to-date experimental information and theoretical studies of the cellular and circuit mechanisms of respiratory rhythm and pattern generation operating within critical components of this CPG in the lower brainstem. Over the past several decades, there have been substantial advances in delineating the spatial architecture of essential medullary regions and their regional cellular and circuit properties required to understand rhythm and pattern generation mechanisms. A fundamental concept is that the circuits in these regions have rhythm-generating capabilities at multiple cellular and circuit organization levels. The regional cellular properties, circuit organization, and control mechanisms allow flexible expression of neural activity patterns for a repertoire of respiratory behaviors under various physiologic conditions that are dictated by requirements for homeostatic regulation and behavioral integration. Many mechanistic insights have been provided by computational modeling studies driven by experimental results and have advanced understanding in the field. These conceptual and theoretical developments are discussed., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The perfused working heart brainstem preparation of rodents has become a widely used tool to study brainstem function. Here, we adapt this experimental technique for newborn guinea pigs (postnatal day 7-14) to develop a tool that enables investigation of airway defense mechanisms not observed in other rodents. The perfused guinea pig brainstem preparation generates a stable eupnea-like motor pattern recorded from the phrenic, recurrent laryngeal and intercostal nerves and basic cardio-respiratory reflexes, including the arterial chemoreceptor, the baroreceptor reflex. In addition a fictive laryngeal cough reflex can be reliably elicited after mechanical stimulation of the trachea. Single unit recordings within the ponto-medullary respiratory column show robust central respiratory neuronal activity. Additionally, as in other species ponto-medullary transection of the brainstem produces apneusis. The latter suggests that the preparation fully preserves ponto-medullary synaptic connectivity that is required for eupnea-like respiratory rhythm and pattern formation and the mediation of various cardio-respiratory reflexes. We conclude that this novel research tool provides an alternative to established rat and mouse preparations and may become a experimental tool for the investigation of central mechanisms that mediate laryngeal cough., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABA A antagonist bicuculline and glycine antagonist strychnine or GABA B antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na + current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO 2 -sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 μM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 μM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

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

In the clinical setting, the use of QX-314 (a quaternary derivative of lidocaine) has been proposed to achieve the selective inhibition of nociceptors that express transient receptor potential vanilloid 1 (TRPV1) channels with fewer motor deficits. However, it has been also reported that QX-314 may produce systemic CNS toxicities with relative potencies that are approximately twice as high as those of lidocaine. There are no reports concerning the effects of extracellular QX-314 on the rhythm-generating neurons in the respiratory center. In the present study, we examined the effects of QX-314 on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats. The extracellular application of QX-314 (200μM) decreased the C4 burst rate, amplitude and slope during the initial rising phase, and the effects slowly developed with a half-decay time of approximately 20min. The combined application of capsaicin (10 or 100μM) with QX-314 (100μM) showed no additional effect. The intracellular application of QX-314 (100μM) to respiratory neurons depressed the action potentials with a half-decay time of around 5min. Our findings could explain one of the mechanisms underlying the central toxicities that occur after the systemic application of QX-314., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The transient receptor potential (TRP) channels are widely distributed in the central nervous system (CNS) and peripheral nervous system. We examined the effects of TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and allyl isothiocyanate) on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats [postnatal days 0-3 (P0-P3)] and in in situ-perfused preparations from juvenile rats (P11-P13). Preparations were superfused with modified Krebs solution at 25-26°C, and activity of inspiratory C4 ventral root (or phrenic nerve) was monitored. In the newborn rat, an in vitro preparation of cinnamaldehyde (0.5 mM) induced typically biphasic responses in C4 rate: an initial short increase and subsequent decrease, then a gradual recovery of rhythm during 15 min of bath application. After washout, the respiratory rhythm rate further increased, remaining 200% of control for >120 min, indicating long-lasting facilitation. Allyl isothiocyanate induced effects similar to those of cinnamaldehyde. The long-lasting facilitation of respiratory rhythm was partially antagonized by the TRPA1 antagonist HC-030031 (10 μM). We obtained similar long-lasting facilitation in an in situ-perfused reparation from P11-P13 rats. On the basis of results from transection experiments of the rostral medulla and whole-cell recordings from preinspiratory neurons in the parafacial respiratory group (pFRG), we suggest that the rostral medulla, including the pFRG, is important to the induction of long-lasting facilitation. A histochemical analysis demonstrated a wide distribution of TRPA1 channel-positive cells in the reticular formation of the medulla, including the pFRG. Our findings suggest that TRPA1 channel activation could induce long-lasting facilitation of respiratory rhythm and provide grounds for future study on the roles of TRPA1 channels in the CNS., (Copyright © 2015 the American Physiological Society.)

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5–5 μM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 μM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 μM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 μM riluzole or 100 μM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 μM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment. [ABSTRACT FROM AUTHOR]

Mammalian respiratory rhythm-generating circuits in the brainstem are subject to neuromodulation by multiple peptidergic afferent inputs controlling circuit behavior and outputs. Although functionally important, actions of neuropeptide modulators have not been fully characterized. We analyzed at cellular and circuit levels two inspiratory patterns intrinsically generated by the preBötzinger complex (preBötC) and their modulation by the neuropeptides bombesin and substance P (SP) in neonatal rat medullary slices in vitro. We found that, in recordings of hypoglossal nerve and preBötC neuron inspiratory activity, some inspiratory bursts occurring spontaneously under basal conditions have a biphasic shape with longer duration than normal inspiratory bursts and occur at a lower frequency. This biphasic burst pattern has been proposed to represent inspiratory activity underling periodic sighs. Bath-applied bombesin or SP decreased the period and increased the duration of both normal inspiratory and biphasic bursts and their underlying synaptic drives. The ratio of the biphasic long-duration burst period to the normal inspiratory burst period and the ratio of their burst durations remained the same before and after peptidergic modulation. Bombesin increased the frequency of the inspiratory rhythm in a Ca2+-independent manner and the frequency of long-duration bursts in a Ca2+-dependent manner. This finding suggests that period and burst duration coupling are due to intrinsic mechanisms controlling simultaneously timing and burst termination within the inspiratory rhythm-generating network. We propose a model in which signaling cascades activated by bombesin and SP modulate mechanisms controlling inspiratory burst frequency and duration to coordinate preBötC circuit behavioral outputs. [ABSTRACT FROM AUTHOR]

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABA antagonist bicuculline and glycine antagonist strychnine or GABA antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol. [ABSTRACT FROM AUTHOR]

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 μM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 μM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception. [ABSTRACT FROM AUTHOR]

The persistent sodium channel is an important pacemaker component in rhythm generation. In the present study, we examined the effects of a persistent sodium channel blocker, riluzole on pre-inspiratory (Pre-I) and inspiratory neurons in the rostral medulla as well as on 4th cervical ventral root (C4)-inspiratory activity in brainstem-spinal cord preparations. Preparations were isolated from postnatal day 0–3 Wistar rats and were superfused with artificial cerebrospinal fluid, equilibrated with 95% O 2 and 5% CO 2 , pH 7.4, at 25–26 °C. The C4 inspiratory burst rate decreased in a dose-dependent manner (50–200 μM) after 15 min application of riluzole. Riluzole caused a strong reduction in the drive potential of Pre-I neurons but not of inspiratory neurons. After washout, C4 inspiratory burst gradually changed into an episodic pattern, in which one burst consisted of 3–9 short separate bursts. Riluzole also depressed the induction of repetitive firing induced by depolarizing stimulation. Under voltage clamp conditions, riluzole suppressed the negative-slope component of Pre-I neurons. Riluzole also depressed the intrinsic burst generation of Pre-I neurons in low calcium and high magnesium solution. Our findings indicate that the burst generation of Pre-I neurons is more sensitive than inspiratory burst generation to riluzole and thus suggested that persistent sodium channels have an important role in the burst generation of Pre-I neurons and are involved in the primary respiratory rhythm generation. [ABSTRACT FROM AUTHOR]

Abstract: The anuran brainstem is segmentally organized into rhombomeres (r) and this segmental organization is uniquely preserved throughout development. We hypothesized that rhombomeres associated with cranial nerves (CN) also contain oscillators that are capable of producing rhythmic motor output (bursts) in isolation. We used in vitro brainstem preparations from pre- and post-metamorphic bullfrogs (Lithobates catesbeianus) to determine if rhombomeric organization of oscillators is present throughout development. Brainstems were transected into segments containing one or more rhombomeres and motor output was measured with suction electrodes attached to CN V, X and XII. Rhythmic motor output was observed in 85% of tadpoles and 91% of frogs in an anterior segment (r0–r5), 27% of tadpoles and 18% of frogs in the middle segment (r6–r7), and 77% of tadpoles and 55% of frogs in the caudal segment (r8). There were significant reductions in burst frequency and whole nerve amplitude following transections. These data support the hypothesis that brainstem oscillators associated with specific groups of rhombomeres are present throughout development in anurans. [Copyright &y& Elsevier]

Dopamine receptor 1 (DR) plays an essential role in regulating respiratory activity in mammals, however, little is known about how this receptor acts to modulate the basic respiratory rhythmogenesis. Here, by simultaneously recording the discharge activities of biphasic expiratory (biphasic E) neurons/inspiratory (I) neurons and the XII nerve rootlets from brainstem slices, we found that the application of DR agonist cis-(±)-1-(aminomethyl)-3,4-dihydro-3-phenyl-1 H-2-benzopyran-5,6-diolhydrochloride (A68930, 5 μM), or forskolin, an intracellular cAMP-increasing agent, substantially decreased respiratory cycle and expiratory time of both types of neurons, and elevated the integral amplitude and frequency of XII nerve rootlets discharge. These changes were reversed by subsequent application of their antagonists SCH-23390 and Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), respectively. Importantly, after pretreatment with Rp-cAMPS, the effects of A68930 in both types of neurons were blocked, suggestive of a cAMP-dependent action of A68930. Thus, the current study indicates that DR may modulate basic breathing rhythmogenesis via cAMP-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: Our previous study has shown that activating peripheral μ-receptors is necessary for switching the bronchopulmonary C-fibers (PCFs)-mediated rapid shallow breathing (RSB) into an apnea by systemic administration of fentanyl. The brainstem nuclei, such as the medial nucleus tractus solitarius (mNTS) and the pre-Botzinger complex (PBC), are required for completing the PCF-mediated respiratory reflexes. Moreover, these areas contain abundant μ-receptors and their activation prolongs expiratory duration (TE). Thus, we asked if central μ-receptors, especially those in the mNTS and PBC, are involved in fully expressing this RSB-apnea switch by fentanyl. In anesthetized rats, the cardiorespiratory responses to right atrial injection of phenylbiguanide (PBG, 3–6μg/kg) were repeated after: (1) fentanyl (iv), a μ-receptor agonist, alone (8μg/kg, iv); (2) fentanyl following microinjection of naloxone methiodide (NXM, an opioid receptor antagonist) into the cisterna magna (10μg/4μl); (3) the bilateral mNTS (10mM, 20nl); or (4) PBC (10mM, 20nl). Our results showed that PBG shortened T E by 37±6% (RSB, from 0.41±0.05 to 0.26±0.03s, P <0.01), but it markedly prolonged TE by 5.8-fold (an apnea, from 0.50±0.04s to 2.9±0.57s, P <0.01) after fentanyl (iv). Pretreatment with NXM injected into the cisterna magna or the PBC, but not the mNTS, prevented the fentanyl-induced switch. This study, along with our previous results mentioned above, suggests that although peripheral μ-receptors are essential for triggering the fentanyl-induced switch, central μ-receptors, especially those in the PBC, are required to fully exhibit such switch. Summary statement: Our results suggest that the activation of central μ-receptors, especially those in the pre-Botzinger complex, is required for switching the pulmonary C-fiber-mediated rapid shallow breathing into an apnea by systemic administration of fentanyl. [Copyright &y& Elsevier]

Abstract: Rhythmic ventilation is an automatic process controlled by the central nervous system. Groups of cells in the brainstem, predominantly the ventral and dorsal respiratory groups, are responsible for generating basic respiratory rhythm. This basic rhythm is subject to modulation by both conscious and reflex actions. In normal individuals the respiratory minute volume is set to closely regulate arterial carbon dioxide tension (PaCO2) at approximately 5.3 kPa, predominantly via a negative feedback reflex involving the central chemoreceptors. A separate group of chemoreceptors, the arterial chemoreceptors, are responsible for initiating the increased ventilatory response to counter arterial hypoxia, but a brisk response is not seen until PaO2 levels fall to approximately 8.0 kPa from the normal 13.3 kPa. Combined hypercarbia and hypoxia (asphyxia) is a very powerful stimulus to breathe as the two inputs interact in a synergistic manner. The chemoreceptor reflexes can be modified when the need arises (e.g. blockade of the respiratory part of the arterial chemoreflex by the trigeminal reflex as part of the diving response). Other reflexes such as the Hering–Breuer reflex contribute to setting the balance between tidal volume and respiratory rate to attain a given minute volume, although this reflex does not appear to play a major role in humans at resting tidal volumes. Superimposed on this ‘tonic’ control, additional protective reflexes (e.g. from receptors in the upper airways) are recruited to protect the lungs and airways with responses such as coughs and sneezes when required. [Copyright &y& Elsevier]

Abstract: Recently developed transversal newborn rat brainstem slices with “calibrated” rostrocaudal margins unraveled novel features of rhythmogenic inspiratory active pre-Bötzinger complex (preBötC) neural networks (). For example, slice rhythm in physiological (3mM) 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 400–600μm thickness with likely centered preBötC kernel (“m-preBötC slices”) generated rhythm in 3mM K+ and 1mM Ca2+ for several hours although its rate decreased to <5bursts/min after >1h. Rhythm was stable at 8–12bursts/min in 6–7mM K+, depressed by 2mM Ca2+, and restored by 9mM 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Breathing is controlled by inspiratory pre-Bötzinger complex (preBötC) networks that remain active in transversal brainstem slices from perinatal rodents. In 600μm thick preBötC slices, inspiratory-related bursting in physiological (3mM) [K+] is depressed by <1mM elevation of superfusate [Ca2+]. Here, we studied underlying cellular mechanisms in whole-cell-recorded neurons of 400μm thin newborn rat slices with the <200μm thin preBötC in the middle (“m-preBötC[400]” slices). Extracellular activity in the ventrolateral slice area in 3mM K+ and a most common physiological Ca2+ range (1–1.2mM) stopped spontaneously within 2h (“in vitro apnea”). Contrary, rhythm was stable for >3h at 6–8 bursts/min in 7mM K+ and 1.2mM 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 2–3mM 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+. [Copyright &y& Elsevier]

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

Abstract: The amygdala is important for the formation of emotions that are affected by olfactory information. The piriform cortex is involved in information processing related to olfaction. To investigate functional interactions between the piriform cortex and amygdala and their relation to medullary respiratory activity, we developed a novel in vitro preparation including the limbic system, brainstem, and spinal cord of newborn rats. With the use of optical and electrophysiologic recordings, we analyzed spontaneous neuronal activity in the piriform–amygdala complex in limbic–brainstem–spinal cord preparations from 0- to 1-day-old rats. For optical recordings, the preparation was stained with a voltage-sensitive dye, and inspiratory activity was monitored from the fourth cervical (C4) ventral root. Spontaneous oscillatory burst activity (up to 10/min) was detected from the rostral cut surface of limbic and para-limbic regions including the piriform cortex and amygdala. The burst activity initially appeared in the piriform cortex and then propagated to the amygdala. We averaged the imaging data in the limbic area with the use of C4 inspiratory activity as a trigger signal. The results suggest functional coupling of the rhythmic burst activity in the piriform–amygdala complex to medullary inspiratory activity, which was confirmed electrophysiologically by cross-correlation analysis of these signals. This rhythmic burst activity may be involved in the development of neuronal circuits that process information related to olfaction, emotion, and respiration. [Copyright &y& Elsevier]

Abstract: Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation. [Copyright &y& Elsevier]

In decerebrate newborn rats, serotonin (5-HT) is a respiratory depressant via activation of 5-HT2 receptors, whereas it evokes respiratory stimulant effects when applied to the isolated brainstem obtained from the newborn rat. This discrepancy could be due to deafferentation in the in vitro preparation. The aim of our study was to analyse the role of vagal afferents in the modulation of central respiratory effects of 5-HT. In decerebrate cervically or abdominally bivagotomized newborn rats aged between 0 and 3 days, we recorded electrical activity from the diaphragm and from a hypoglossally innervated tongue muscle, as well as cardiac frequency ( Fc), before and after application of 5-HT to the floor of the IVth ventricle. The effects of related agents (a 5-HT1A agonist, 8-OH DPAT, and a 5-HT2 agonist, DOI) were studied in cervically bivagotomized animals. For comparison, and to assess the spontaneous variability in inspiratory frequency ( Fi) and Fc, sham groups were studied. Each group comprised ten newborn rats. In cervically bivagotomized newborn rats, 5-HT induces a significant increase in Fi, which is the opposite to that observed in decerebrate newborn rats with intact vagi. This respiratory effect is mediated in particular, via activation of 5-HT1A. By contrast, in abdominally bivagotomized newborn rats, a decrease in Fi was observed in response to 5-HT (as previously described in decerebrate animals with intact vagi). We conclude that pulmonary vagal afferents modulate the central respiratory action of 5-HT in decerebrate newborn rats, explaining the conflicting results between in vivo and in vitro experiments. [ABSTRACT FROM AUTHOR]

Abstract: Frogs produce two distinct yet highly coordinated ventilatory behaviors, buccal and lung. Lung ventilation occurs in short episodes, interspersed with periods of buccal ventilation. Recent data suggests that two brainstem oscillators are involved in generating these behaviors, one primarily responsible for buccal ventilation, the other for lung. Here we use a modeling approach to demonstrate that the episodic pattern of lung ventilation might be an emergent property of the coupling between the oscillators, and may not require a perturbing input from another, as yet unidentified but previously postulated, neuronal oscillator. [Copyright &y& Elsevier]

Abstract: We investigated how the spatio-temporal pattern of respiratory neuron network activity in the ventral medulla changes during the late fetal period of rat. Brainstem-spinal cord preparations isolated from rat fetuses on embryonic days 17–21 (E17–E21) were stained with a voltage-sensitive dye for optical image analysis of neuronal activity of the ventral medulla. The spatio-temporal pattern of respiratory neuron activity in the preparation from E20 to E21 was basically identical to that of neonatal rat; pre-inspiratory activity in a limited region of the rostral ventrolateral medulla, the para-facial region, preceded by several hundred milliseconds the onset of inspiratory activity in the more caudal ventrolateral medulla, the pre-Bötzinger complex level. In contrast, in E17–E18 specimens, pre-inspiratory activity could not be detected in the rostral medulla at the level of the facial nucleus. Neuronal activity appeared to begin at the pre-Bötzinger complex level shortly before onset of the inspiratory burst. Strong activity then developed in the facial nucleus and peaked in the post-inspiratory phase. The transition of these patterns of respiratory activity occurred at E19. We conclude that the changes in the spatio-temporal pattern of neuronal activity reflect developmental changes in the cellular elements underlying rhythm generation in the fetal respiratory neuron network. We suggest that the pre-inspiratory neuron network of the para-facial region in the rostral ventrolateral medulla functions as the rhythm generator after E19/20. [Copyright &y& Elsevier]

Abstract: This study investigated whether adenosine A1 receptors could modulate respiratory rhythm in mammals. Experiments were performed in in vitro brainstem slice preparations from neonatal rats. These preparations included the medial region of Nucleus Retrofacialis (mNRF) with the hypoglossal nerve (XII nerve) rootlets retained. The rhythmical discharges of the biphasic expiratory (biphasic E) neurons/inspiratory neurons (I neurons) and activities of the XII nerve rootlets were simultaneously recorded by using extracellular microelectrodes and suction electrodes, respectively. Roles of adenosine A1 receptors in modulation of respiratory rhythm were investigated by administration of the adenosine A1 receptor agonist R-phenylisopropyl-adenosine (R-PIA, 10μM) and its specific antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2μM). DPCPX decreased the respiratory period (27.19%) and expiratory duration (28.27%) of biphasic E neurons and at the same time increased the peak discharge frequency (48.13%). By contrast, R-PIA produced opposite effects. On the other hand, the effects of DPCPX and R-PIA on the I neurons were similar to that on the biphasic E neurons except that R-PIA shortened the discharge duration of I neurons (34.12%) and decreased the peak discharge frequency (37.75%) in the middle phase of inspiration, but not in the initial and terminal phases. These results suggest that adenosine A1 receptors are involved in the phase-switching between expiration and inspiration by affecting biphasic E neurons. Activation of adenosine A1 receptors may modulate the inhibitory synaptic inputs from I neurons to biphasic E neurons. [Copyright &y& Elsevier]

Neurokinin-1 receptors (NK1) are present within the respiratory medullary network and in the phrenic nucleus, which controls the diaphragm. We compared the efficacy of substance P (SP) at inducing changes in respiratory frequency or the amplitude of the respiratory motor output between NK1 knockout (NK1–/–) and wild-type mice, using the in vitro brainstem-spinal cord preparation. The in vitro respiratory frequency, as well as the variability of the rhythm and the amplitude of the motor output were similar in both lines. In wild-type mice, application of exogenous SP induced either an increase in respiratory frequency (superfusion of the medulla) or an increase of the inspiratory motor output, as defined by the integral of C4 cervical ventral root activity (superfusion of the spinal cord). These two effects were not apparent in NK1–/– mice. In conclusion, NK1 receptors mediate the respiratory responses to SP but the lack of NK1 receptors in newborn NK1–/– mice does not change the respiratory activity. [ABSTRACT FROM AUTHOR]

Intracellular calcium signals are critical for modulation of neuronal function, and also for pathophysiological states during human neurodegenerative disease, such as Morbus Alzheimer and amyotrophic lateral sclerosis (ALS). We investigated intracellular calcium signals in motoneurones of the nucleus hypoglossus from the mouse, which were maintained in a functionally intact state of rhythmic, respiratory-related activity. Simultaneous patch-clamp recordings and calcium imaging demonstrated that rhythmic inspiratory-related clusters of action potential (AP) discharges are paralleled by calcium oscillations both in somatic and dendritic compartments. Calcium oscillations resulted primarily from the AP-induced opening of voltage-dependent calcium channels in the soma and dendrites. Dendritic calcium transients differed from somatic responses in their kinetics, amplitude, voltage dependence and regulation of basal calcium levels. Based on a combination of infrared differential interference contrast optics, microfluorimetric calcium imaging and electrophysiological patch-clamp recordings, our results demonstrate that the brainstem slice preparation is an attractive model system to study the integration and superposition of calcium signals in a functionally intact neuronal net. [ABSTRACT FROM AUTHOR]

Abstract: This special issue of Respiratory Physiology & Neurobiology summarizes the current standing of research concerned with synaptic mechanisms, membrane properties, plasticity, pre- and postnatal development and evolutionary origin of neurones involved in respiratory rhythm generation and central chemosensitivity. Moreover, a variety of articles link pathophysiological alterations of synaptic function in rhythmogenesis and chemosensitivity with breathing disorders in neurodevelopmental diseases. [Copyright &y& Elsevier]

Abstract The pre-Bötzinger complex is a small region in the mammalian brainstem involved in generation of the respiratory rhythm. As shown in vitro , this region, under certain conditions, can generate endogenous rhythmic bursting activity. Our investigation focused on the conditions that may induce this bursting behaviour. A computational model of a population of pacemaker neurons in the pre-Bötzinger complex was developed and analysed. Each neuron was modelled in the Hodgkin–Huxley style and included persistent sodium and delayed-rectifier potassium currents. We found that the firing behaviour of the model strongly depended on the expression of these currents. Specifically, bursting in the model could be induced by a suppression of delayed-rectifier potassium current (either directly or via an increase in extracellular potassium concentration, [K+ ]o ) or by an augmentation of persistent sodium current. To test our modelling predictions, we recorded endogenous population activity of the pre-Bötzinger complex and activity of the hypoglossal (XII) nerve from in vitro transverse brainstem slices (700 µm) of neonatal rats (P0–P4). Rhythmic activity was absent at 3 mm[K+ ]o but could be triggered by either the elevation of [K+ ]o to 5–7 mm or application of potassium current blockers (4-AP, 50–200 µm, or TEA, 2 or 4 mm), or by blocking aerobic metabolism with NaCN (2 mm). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50 µm). These findings are discussed in the context of the role of endogenous bursting activity in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping. [ABSTRACT FROM AUTHOR]