Unstable breathing rhythms and quasiperiodicity in the pre-Bötzinger complex.
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Animals↗
Biomedical subjects
N Koshiya
Publications and source records attributed to N Koshiya.
Stationary organotypic culture of the pre-Bötzinger complex from the newborn rat.
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Animals↗
Isolation of the kernel for respiratory rhythm generation in a novel preparation: the pre-Bötzinger complex "island".
The pre-Bötzinger complex (pre-BötC), 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-BötC 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-BötC "islands" to further establish whether the pre-BötC has intrinsic rhythmicity and is the kernel for rhythmogenesis in the slice. We recorded neuron population activity locally in the pre-BötC 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-BötC Ca(2+) activity imaged in thin slices and islands were similar, and imaged pre-BötC 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-BötC in vitro and demonstrate that the islands are ideal preparations for studying the kernel's intrinsic properties.
Animals↗
Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker-network model.
We review a new unified model of respiratory rhythm generation - the hybrid pacemaker-network model. This model represents a comprehensive synthesis of cellular and network mechanisms that can theoretically account for rhythm generation in different functional states, from the most reduced states in the neonatal nervous system in vitro to the intact adult system in vivo. The model incorporates a critical neuronal kernel consisting of a network of excitatory neurons with state-dependent, oscillatory bursting or pacemaker properties. This kernel, located in the pre-Bötzinger complex of the ventrolateral medulla, provides a rudimentary pacemaker network mechanism for generating an inspiratory rhythm, revealed predominately in functionally reduced states in vitro. In vivo the kernel is embedded in a larger network that interacts with the kernel via inhibitory synaptic connections that provide the dynamic control required for the evolution of the complete pattern of inspiratory and expiratory network activity. The resulting hybrid of cellular pacemaker and network properties functionally endows the system with multiple mechanisms of rhythm generation. New biophysically realistic mathematical models of the hybrid pacemaker-network have been developed that illustrate these concepts and provide a computational framework for investigating interactions of cellular and network processes that must be analyzed to understand rhythm generation.
Animals↗
Neuronal pacemaker for breathing visualized in vitro.
Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-Bötzinger complex in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown. The central question is whether the rhythm is generated by a network of bursting pacemaker neurons coupled by excitatory synapses that synchronize pacemaker activity. Here we visualized the activity of inspiratory pacemaker neurons at single-cell and population levels with calcium-sensitive dye. We developed methods to label these neurons retrogradely with the dye in neonatal rodent brainstem slices that retain the rhythmically active respiratory network. We simultaneously used infrared structural imaging to allow patch-clamp recording from the identified neurons. After we pharmacologically blocked glutamatergic synaptic transmission, a subpopulation of inspiratory neurons continued to burst rhythmically but asynchronously. The intrinsic bursting frequency of these pacemaker neurons depended on the baseline membrane potential, providing a cellular mechanism for respiratory frequency control. These results provide evidence that the neuronal kernel for rhythm generation consists of a network of synaptically-coupled pacemaker neurons.
Calcium↗
Tonic sympathetic chemoreflex after blockade of respiratory rhythmogenesis in the rat.
1. We sought to determine whether the increase in sympathetic nerve discharge (SND) caused by carotid chemoreceptor stimulation requires the integrity of ventrolateral medullary structures involved in generating respiratory rhythm and pattern. Experiments were done in urethane-anaesthetized, vagotomized, aortic deafferented, ventilated rats except when indicated (see paragraph 3). 2. Brief hypoxia (N2 for 5-12 s) or I.V. NaCN (50-100 micrograms kg-1) activated SND in bursts synchronized with the phrenic nerve discharge (PND). No effect was produced in chemo-deafferented rats. 3. In unanaesthetized vagotomized decerebrated rats, ligation of the internal carotid arteries preserved peripheral chemoreceptor function but abolished baroreflexes. In this preparation, stimulation of peripheral chemoreceptors (N2 for 2-6 s) also activated SND in bursts synchronized with PND. 4. Bilateral microinjection of the GABAA receptor agonist muscimol into the caudal ventrolateral medulla (CVLM) instantly blocked the sympathetic baroreflex, eliminated PND at rest and during chemoreceptor stimulation but did not change the mean increase in SND produced by chemoreceptor stimulation. Sympathoactivation in response to chemoreceptor stimulation became tonic after 1-13 min and was still totally dependent on the integrity of the carotid sinus nerves. 5. Muscimol injection instantly eliminated the respiratory outflow of the Xth and XIIth cranial nerves, both at rest and during chemoreceptor stimulation. 6. Muscimol eliminated the on-off respiratory pattern of neurons in the rostral ventrolateral medulla (RVLM). During chemoreceptor stimulation, these cells became activated or inhibited tonically. 7. Muscimol injection raised the resting discharge rate of vasomotor presympathetic cells in RVLM, blocked their baroreceptor inputs but did not change the magnitude of their excitation by chemoreceptor stimulation. Muscimol injection eliminated their respiratory modulation. 8. In conclusion, the sympathetic response to chemoreceptor stimulation may be due to convergence and integration in RVLM of two processes: respiration-independent excitatory input to RVLM neurons and respiratory patterning of their activities via inputs from the pre-Bötzinger complex.
Anesthesia↗
Role of medulla oblongata in generation of sympathetic and vagal outflows.
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Animals↗
NTS neurons with carotid chemoreceptor inputs arborize in the rostral ventrolateral medulla.
Neurons that were excited by hypoxic stimulation of carotid chemoreceptors were recorded in the caudal portion of the nucleus of the solitary tract (cNTS) of urethan-anesthetized, vagotomized, aortic-deafferented, artificially ventilated rats (n = 23). The focus of the study is on 26 chemosensitive neurons (classified as early- and late-response cells) that were tonically activated by chemoreceptor stimulation and never fired in bursts synchronized with the phrenic nerve discharge (PND) cycle. The discharge of early-response cells (n = 14) started up to 2.5 s before the onset of PND activation, whereas the discharge of late-response cells (n = 14) started 1.5-5 s after onset of PND response. Four early-response cells were antidromically activated from the rostral ventrolateral medulla (RVLM; latencies: 7-13 ms), and two had axenal collaterals in the region of the nucleus ambiguus. Four late-response neurons were antidromically activated from the RVLM (latencies: 6-12 ms), but no collateral was found in this area. The basal discharge of early- and late-response cells ranged from 0 to 10 and 0 to 30 spikes/s, respectively, but most of them had a very low spontaneous firing rate (median: 0.2 and 0.6 spikes/s, respectively). Neither type was excited by baroreceptor stimulation. The cNTS also contained neurons that were firing in bursts synchronized with the PND cycle. These cells were activated by chemoreceptor stimulation and were not antidromically activated from the RVLM. Chemosensitive neurons made up 33% of cNTS neurons antidromically activated from the RVLM (8/24). In conclusion, a population of cNTS chemosensitive neurons devoid of respiratory modulation projects through the RVLM and arborizes in this region.
Afferent Pathways↗
Sympatholytic effect of clonidine depends on the respiratory phase in rat splanchnic nerve.
Peri-event averaging of the sympathetic nerve discharge was done to measure the magnitude of the sympatholytic effect of the anti-hypertensive drug clonidine during three different phases of the respiratory cycle (inspiration, I; postinspiration, post-I; late expiration, pre-I). Arterial pressure (AP) and discharges of splanchnic sympathetic (SND) and phrenic nerves (PND, onset used for peri-event averaging) were recorded in urethane-anesthetized, vagotomized, aortic deafferentated, paralyzed and artificially ventilated Sprague-Dawley rats (n = 7). During control periods (mean AP 106 +/- 10 mmHg) SND was distributed equally throughout the three selected respiratory periods, though two brief peaks were noted during the I and post-I periods. Low doses of clonidine (15-30 micrograms/kg i.v.) produced brief hypertension (< 30 s, 150 +/- 9 mmHg at peak) followed by moderate hypotension (89 +/- 3 mmHg) and a reduction in mean SND (-63 +/- 11% from control value). High doses of clonidine (200-250 micrograms/kg i.v.) produced sustained hypertension (> 10 min, 173 +/- 3 mmHg) and silence of SND. During this sustained hypertension, lowering AP by i.v. nitroprusside retrieved a component of SND that was barosensitive but insensitive to clonidine. During those hypotensive periods (spontaneous after a low dose of clonidine, and induced by nitroprusside after a high dose of clonidine), SND was most attenuated during the pre-I period and least during the I period. The I component of SND was significantly less attenuated than the post-I component by clonidine and, in most cases (6 out of 7), SND showed a single inspiratory peak following clonidine administration. It is concluded that (i) the pre-I component of SND is the most sensitive to clonidine and (ii) the I component of SND is the most resistant to the drug.
Action Potentials↗
Role of the pons in the carotid sympathetic chemoreflex.
The mass discharges of the splanchnic sympathetic (SND) and phrenic nerves (PND) were recorded in urethananesthetized rats with resected vagal and aortic nerves. Carotid chemoreceptor (CC) stimulation with N2 inhalation (4-12 s) or cyanide (50-100 micrograms/kg iv) activated SND in bursts synchronized with the postinspiratory phase (mean SND increase: 105 +/- 8%), raised AP, and increased PND rate and amplitude (n = 40). Brain transection at superior collicular level produced no effect. The sympathetic (SChR) and respiratory chemoreflexes (RChR) were reduced after transections through the pons. Lesions of the dorsolateral pons (dl-pons) produced CO2-dependent apneusis and/or tachypnea at rest. After such lesions, CC stimulation produced expiratory apnea and a 30% increase in SChR due to tonic activation of SND. In contrast, bilateral lesions of the ventrolateral pons (vl-pons) reduced the SChR by 54-76%. Muscimol (Mus) injections (bilateral, 175 pmol/side) into vl-pons did not change resting SND, MAP, baroreflex, and RChR but reduced the SChR (54-82%). In conclusion, under anesthesia: 1) the pathway of the carotid chemoreflex is confined to the pons and medulla, 2) the dl-pons exerts indirect control over the SChR via its role in respiratory rhythmogenesis, and 3) neurons in the vl-pons contribute selectively to the SChR but not to PND activation during CC activation.
Animals↗
A5 noradrenergic neurons and the carotid sympathetic chemoreflex.
Inhibition of neural activity in the caudal ventrolateral pons (A5 area) by microinjection of muscimol (Mus) attenuates (-65%) the carotid sympathetic chemoreflex (SChR) without altering the concomitant activation of the phrenic nerve (PND). The present study, performed in urethan-anesthetized rats, explores the possibility that activation of the noradrenergic (NE) neurons of the A5 area is involved in the SChR. The NE neuron-selective toxin 6-hydroxydopamine (6-OHDA) was microinjected bilaterally into the spinal cord at T2 level (4 micrograms). This dose reduced the SChR by 55% (n = 5) 90 min after injection, while 0.4 microgram of 6-OHDA produced no effect (n = 5). In seven rats that had received 250 micrograms 6-OHDA intracisternally 2 wk before, Mus injections into the A5 area failed to attenuate the SChR. These rats also had a lower resting mean arterial pressure than controls (97 vs. 112 mmHg). Spinal intrathecal injection of alpha-adrenergic receptor antagonists (prazosin, 10 and 20 micrograms) or phentolamine (20 and 40 micrograms) attenuated resting sympathetic nerve discharge (SND) and SChR in a roughly proportional manner (25-40%); the beta-adrenergic antagonist nadolol (10 and 20 microgram(s) intrathecally) attenuated the SChR selectively but modestly (-10%). The results are generally compatible with the hypothesis that A5 NE neurons and particularly their spinal cord projection could play a facilitating role in the SChR. However, clear evidence that A5 cells contribute selectively to sympathoactivation during chemoreceptor stimulation by releasing NE in the spinal cord could not be obtained.
Animals↗
Ventrolateral medulla and sympathetic chemoreflex in the rat.
Splanchnic sympathetic nerve discharge (SND), phrenic nerve activity (PND) and putative sympathetic premotor neurons of the rostral ventrolateral medulla (RVL) were recorded in urethane-anesthetized vagotomized rats without aortic baroreceptor afferents. Carotid chemoreceptor stimulation with brief N2 inhalation increased SND by 101 +/- 7%, raised mean arterial pressure (MAP) and increased the discharge rate of RVL premotor neurons by 46 +/- 12% (N = 32). During chemoreceptor activation. SND and most RVL neurons displayed pronounced central respiratory rhythmicity with maximal firing probability immediately after cessation of the PND (postinspiratory phase) and lowest probability during PND (inspiratory phase). Bilateral microinjection of the breed spectrum glutamate receptor antagonist kynurenic acid (Kyn, 5 nmol in 100 nl) into RVL blocked the sympathetic chemoreflex but left the sympathetic baroreflex intact. In contrast, bilateral microinjection of the same dose of Kyn into the caudal ventrolateral medulla (at obex level CVL) blocked the baroreflex but left the sympathetic chemoreflex intact. Bilateral microinjection of the GABAA agonist muscimol (87.5 pmol in 50 nl) into CVL produced effects identical to those of Kyn. These results confirm that the caudal ventrolateral medulla contains an essential relay of the sympathetic baroreflex and demonstrate that the same area plays no role in the sympathetic chemoreflex. The data suggests that these two reflexes could have a largely independent course through the medulla oblongata and that integration between the baroreceptor and chemoreceptor information used for sympathetic vasomotor control may occur as late as the premotor neuronal stage in RVL.
Animals↗
Central respiratory modulation of facial motoneurons in rats.
Facial motoneurons (FMN) were recorded intracellularly in Sprague-Dawley rats anesthetized with halothane. The animals were vagotomized, paralyzed, and artificially ventilated. The average membrane potential of the cells was 62.6 +/- 1.9 mV and their input impedance ranged from 5 to 30 M omega (9.8 +/- 1.1 M omega, n = 38). The membrane potential of most FMNs varied throughout the central respiratory cycle and four distinct patterns were detected. Type I (post-inspiratory) cells (21/44) showed a two-phase Cl(-)-mediated hyperpolarization during the respiratory cycle, one during central inspiration and the second during late expiration. Type II cells (early inspiratory, n = 10) showed early inspiratory depolarization. Type III (n = 2, stage-2 expiratory) cells displayed late expiratory depolarization and one cell (type IV or throughout inspiratory) exhibited expiratory Cl(-)-mediated hyperpolarization. The remaining 10 cells showed no detectable respiratory modulation. The results reflect the heterogeneity of the central respiratory modulation of FMNs and suggest that these cells receive both excitatory and inhibitory inputs from elements of the central respiratory pattern generating network.
Animals↗
Central respiratory control of A5 and A6 pontine noradrenergic neurons.
Sympathetic nerve discharge (SND), phrenic nerve discharge (PND), and unit activity of locus ceruleus (LC) and of putative A5 noradrenergic cells were recorded in vagotomized rats anesthetized with urethan. SND was activated by stimulation of carotid chemoreceptors with hypoxia (N2 inhalation, 5-15 s or 12% O2 inhalation, 2-5 min) and displayed a prominent central respiratory modulation during the hypoxic challenge (postinspiratory pattern). LC cells were also activated by peripheral chemoreceptor stimulation. The discharge of most LC units (28 of 31) exhibited central respiratory modulation. 15 LC units had a postinspiratory pattern and 11 had an inspiratory one. Putative A5 cells were also excited by hypoxia and also displayed a clear central respiratory modulation (mostly postinspiratory pattern). These experiments indicate that 1) the firing rate of most pontine noradrenergic cells is increased by peripheral chemoreceptor stimulation, and 2) pontine noradrenergic neurons receive afferent information of a respiratory nature, possibly from their ventrolateral medullary inputs.
Animals↗
Neurons in the caudal ventrolateral medulla mediate the arterial baroreceptor reflex by inhibiting barosensitive reticulospinal neurons in the rostral ventrolateral medulla in rabbits.
Participation of the caudal ventrolateral medulla in the arterial baroreceptor reflex was examined in urethane-anesthetized, vagotomized and immobilized rabbits whose aortic nerve was cut bilaterally. The extent of the caudal ventrolateral medulla was mapped by decreases in the renal sympathetic nerve activity and arterial pressure following a local microinjection of a neuroexcitatory amino acid, sodium glutamate (0.075-1.5 nmol). It extended between the levels 1.3 mm rostral and 3.0 mm caudal to the obex. An injection of sodium glutamate into the caudal ventrolateral medulla also diminished spontaneous activity of barosensitive reticulospinal neurons in the rostral ventrolateral medulla. In the 'split medulla preparation' in which the medulla was split along the midsagittal plane to disrupt fiber connections associating both sides, a neurotoxic agent, kainic acid, was injected unilaterally into the rostral ventrolateral medulla. This treatment markedly attenuated responses of renal sympathetic nerve activity and arterial pressure induced by a sodium glutamate injection into the ipsilateral caudal ventrolateral medulla, whereas responses to an injection into the contralateral caudal ventrolateral medulla were totally preserved. In four separate experiments, three to five injections of kainic acid were made unilaterally to cover the whole extent of the caudal ventrolateral medulla. The sympathoinhibitory and depressor responses to stimulation of the ipsilateral aortic nerve were then totally abolished. Simultaneously, the cardiac cycle-related rhythmic fluctuation of renal sympathetic nerve activity, which represented activity of the carotid sinus baroreceptor reflex, was attenuated to the noise level. These results, together with our previous electrophysiological demonstration of barosensitive caudal ventrolateral medulla neurons with axonal projections to the rostral ventrolateral medulla, strongly support the hypothesis that neurons in the caudal ventrolateral medulla mediate the arterial baroreceptor-vasomotor reflex through inhibition of barosensitive reticulospinal neurons in the rostral ventrolateral medulla.
Animals↗
A5 noradrenergic unit activity and sympathetic nerve discharge in rats.
Unit recording experiments were designed to determine whether A5 noradrenergic neurons contribute to the generation of the splanchnic sympathetic nerve discharge (SSND) of halothane-anesthetized rats. Neurons (presumed A5 cells) were selected on the following bases: location in the ventrolateral tegmentum rostrolateral to facial nucleus (FN), antidromic (AD) activation from thoracic spinal cord, and complete inhibition by clonidine (10-15 micrograms/kg iv). These cells (n = 59) had low rates of spontaneous firing (1.4 +/- 0.2 spikes/s) and slow conduction velocities (2.6 +/- 0.2 m/s). The AD activation of seven of eight neurons was abolished within 1 h after intraspinal microinjection of 6-hydroxydopamine (4 micrograms), but the drug failed to affect the AD responses of eight sympathoexcitatory cells located caudal to the FN (control cells). The terminal fields of 16 A5 area neurons were found in the intermediolateral cell column of the spinal cord. Most neurons (63%, 37/59) were inhibited by raising arterial pressure and by train stimulation of the aortic depressor nerve (ADN, 47%, 9/20). A few cells responded to ADN stimulation but not to arterial pressure elevation or vice versa. The discharge of the cells was correlated to the SSND and preceded a peak of SSND by 69 +/- 6 ms (12/29 in intact and 3/9 in debuffered rats). We conclude that 40% of A5 cells may have a visceral vasomotor sympathoexcitatory function.
Afferent Pathways↗
Modulatory effects of endothelin-1 on central cardiovascular control in rats.
In urethane-anesthetized and immobilized rats, modulatory effects of endothelin-1 (ET-1) on central cardiovascular control were examined. An injection of 0.1 pmol of ET-1 into the cisterna magna caused immediate increases in arterial pressure (AP), renal sympathetic nerve activity (RSNA), and heart rate (HR) that lasted for 5-45 min. At doses of 1 and 10 pmol, intracisternal ET-1 elicited initial increases (phase I) followed by decreases in these variables below the pre-injection level (phase II). At the dose of 1 or 10 pmol, the arterial baroreceptor reflex was suppressed during the latter part of phase I and during phase II. The three variables subsequently returned to, or often exceeded, pre-injection levels in 30 to 60 min and reflex activity recovered (phase III). However, AP often remained below control throughout the 2-h observation period. Essentially identical responses to intracisternal ET-1 were observed in unanesthetized precollicular decerebrated or urethane-anesthetized rats. Application of a piece of filter paper soaked with 1 pmol of ET-1 to the ventral surface of the medulla (VSM) caused the pattern of changes similar to the following intracisternal injection. A microinjection of 4 pmol of ET-1 into the nucleus tractus solitarius (NTS) caused a moderate increase in RSNA with a minute fall in AP. Intrathecal administration of ET-1 resulted in moderate changes in AP and RSNA at the dose as high as 100 pmol. We conclude that intracisternally administered ET-1 modulates tonic and reflex control of AP and sympathetic vasomotor activity and that the VSM appears to be involved critically in this modulation.
Animals↗
Modulatory effects of rat endothelin on central cardiovascular control in rats.
To characterize the modulatory action of rat endothelin (endothelin-3 or ET-3) on the cardiovascular control by the central nervous system (CNS), ET-3 was injected into the cisterna magna of urethane-anesthetized and immobilized rats. An injection of 100 pmol of ET-3 caused immediate rises in arterial pressure (AP), renal nerve activity (RNA), and heart rate (HR). These variables subsequently decreased and, in 5-20 min, fell below the pre-injection level. Simultaneously, the arterial baroreceptor reflex was almost totally suppressed. Although RNA and HR subsequently returned to, or often exceeded, pre-injection levels in 20 to 60 min and reflex activity recovered, AP sometimes remained below control for at least 2 h. A similar pattern of changes was elicited in unanesthetized precollicular decerebrated rats. The responses to ET-3 were abolished by hexamethonium chloride, but were not conspicuously altered by arginine vasopressin antagonist or angiotensin II antagonist. The CNS sites responsible for ET-induced changes were subsequently searched. Topical application of ET-3 to the ventral surface of the medulla (VSM) caused the pattern of changes in AP, RNA, and HR similar to that following intracisternal injection. Microinjection of ET-3 into the nucl. tractus solitarius (NTS) increased AP and RNA, whereas intrathecal administration of it decreased them. We conclude that intracisternally administered ET-3 centrally modulates both tonic and reflex control of AP by the sympathetic nervous system and that the VSM appears to be primarily responsible for the modulation, although NTS and spinal cord may also be involved in it.
Angiotensin II↗