Studying rhythmogenesis of breathing comparison of in vivo and in vitro models trends in neurosciences brain anoxia

Fig. 1

Respiratory brainstem regions that are involved in generating a three-phased respiratory rhythm. (a) A dorsal view of the brainstem after removal of the cerebellum showing the localization of different components of the respiratory network. The dorsal group of respiratory neurons (DRG) is localized around the nucleus of the tractus solitarii (blue area). The ventral group of respiratory neurons (VRG) extends bilaterally from the compact division of the rostral nucleus ambiguus to the C1 level (orange color). A ‘noeud vital’ is the pre-bötzinger complex (PBC, red color) which is primarily involved in respiratory rhythm generation 6. The kölliger-fuse nucleus and the nucleus parabrachialis in the pons (blue area) are involved in reflex regulation and shaping of respiratory activity patterns. (b) A 300–700 mm thick coronal ‘rhythmic brainstem slice’ can be isolated at the level of the PBC, which retains rhythmic activity after in vitro isolation.Brain anoxia respiratory neurons can be patch-recorded under visual control. For functional identification, neuronal activity is temporally correlated with the inspiratory motor discharges recorded with a suction electrode from the rootlet of the hypoglossal nerve. Characteristic structures of this rhythmic brainstem slice are: PBC, pre-bötzinger complex; amb, nucleus ambiguus; XII, hypoglossal nucleus; RO, nucleus raphe obscurus; sol, nucleus of the solitary tract containing DRG neurons; mve, medial vestibular nucleus; spve, spinal vestibular nucleus; sp5i, spinal trigeminal nucleus; ICP, inferior cerebellar peduncle; IO, inferior olive; P, pyramidal tract. (c) in the mature cat in vivo, medullary respiratory neurons oscillate in a three-phased respiratory rhythm.Brain anoxia this is illustrated by a simultaneous triple-recording of the main populations of neurons, that is, extracellular recordings of the action potential discharges of (1) an inspiratory neuron (large spikes) and (2) a post-inspiratory neuron (small spikes). A parallel intracellular recording shows (3) an expiratory neuron. The sequences of discharges and correlated fluctuations of the membrane potential reveal a clear distinction between three respiratory cycles: inspiration, post-inspiration and expiration. Whereas the transitions from expiration to inspiration and from inspiration to post-inspiration occur within a few millisconds (vertical arrows), the transition from post-inspiration to expiration is quite variable (horizontal arrow; data from ref. 97 ).Brain anoxia

Fig. 3

Maturational changes in pre-inspiratory delays in bulbar to spinal transmission. (a) A medullary inspiratory neuron in the anesthetized mature cat shows an augmenting membrane depolarization, which parallels an augmenting pattern of phrenic nerve burst discharges. Onset of inspiratory membrane depolarization of medullary neurons precedes phrenic nerve bursts by no more than a few milliseconds (data from ref. 21 ). (b) on the first day after birth (P0), a medullary inspiratory neuron of the anesthetized neonatal cat already shows an augmenting membrane depolarization and an augmenting pattern of rapidly oscillating phrenic nerve burst discharges (compare with fig. 2 e). However, onset of inspiratory membrane depolarization of medullary neurons precedes phrenic bursts by more than half a second (data from ref. 26 ). (c) in the perfused brainstem of the juvenile mouse, medullary inspiratory neurons also reveal an augmenting membrane depolarization in parallel with an augmenting pattern of phrenic burst discharges.Brain anoxia in some neurons this is followed by a membrane hyperpolarization during post-inspiration. Onset of inspiratory membrane depolarization of medullary neurons precedes phrenic nerve bursts by more than 130 ms (data from ref. 27 ). (d) in the en bloc brainstem–spinal cord preparation of neonatal rat, a medullary inspiratory neuron typically shows a slowly augmenting membrane depolarization preceding inspiratory bursts in the phrenic nerve rootlet (C4) by about 400 ms. The inspiratory C4 burst consists of rapidly oscillating mini-bursts, the amplitudes of which decline (compare with fig. 2 e). The neuron repolarizes slowly after inspiratory bursting of C4 rootlets, which could indicate the existence of a post-inspiratory phase. (data from ref. 11 ). (e) in the en bloc brainstem spinal cord preparation of neonatal rat, a spinal inspiratory motoneuron of phrenic nerve typically shows a rapid membrane depolarization; there is only a short delay between depolarization of motoneurons and discharge in the phrenic rootlet (data from ref. 29 ). (f) the behavior of a medullary post-inspiratory neuron from the perfused brainstem of juvenile mouse: synaptic hyperpolarization typical for inspiratory inhibition precedes phrenic bursts by more than 150 ms (data from ref. 13 ).Brain anoxia

Fig. 2

Respiratory phases and inspiratory burst patterns. (a) in the en bloc brainstem-spinal cord preparation of neonatal rat, the pattern of inspiratory discharges peaks rapidly and then declines gradually. The function of the gradual decline of inspiratory activity might actually be passive expiration (data from ref. 11 ). (b) at higher time resolution, inspiratory bursts often reveal rapidly oscillating intra-inspiratory mini-bursts. Recordings are from a phrenic nerve rootlet (C4) in the en bloc brainstem-spinal cord preparation of neonatal rat (data from ref. 38 ). (c) the weak inspiratory bursts of the neonatal brainstem slice peak rapidly and then decline gradually. The inspiratory discharge also shows intra-burst oscillations.Brain anoxia recordings are from hypoglossal nerve rootlet (XII) in the brainstem slice from a neonatal mouse at postnatal day P1 (data from ref. 28 ). (d) the same type of rapidly oscillating intra-inspiratory mini-bursts are also seen in phrenic nerve recordings (PN) of the perfused brainstem-spinal cord preparation of neonatal mouse when glycine receptors are blocked by small doses (100 nm) of strychnine (data from ref. 27 ). (e) in the perfused brainstem preparation of juvenile mouse, phrenic nerves (PN) show an augmenting inspiratory burst pattern (I) and a declining post-inspiratory after-discharge (PI). This pattern is profoundly changed when glycine receptors are blocked selectively with a low dose (70 nm) of strychnine (middle).Brain anoxia now the inspiratory burst peaks much faster and is followed by a pronounced after-discharge, which might indicate enhancement and prolongation of post-inspiration. When strychnine is applied at higher concentrations (above 1 mm) strychnine is no longer selective for glycine receptors, but also blocks GABA A receptors 78. The consequence is a regular occurrence of rhythmic epileptifom discharges in phrenic and also non-respiratory nerves, for example, the plexus brachialis. Although the epileptiform rhythm alternates only initially with the respiratory rhythm, it finally suppresses the respiratory rhythm and is the only rhythm that persists. Such seizure can be blocked by transsection of the spinal cord (data from ref. 27 ). (f) comparable rapid intra-burst oscillations are seen during late inspiration in the anaesthetized cat, when synaptic interaction between neurons is partially blocked during acute hypoxia (data from ref. 39 ).Brain anoxia

Fig. 4

The different operational modes of the maturational network-burster model. (a) according to the hybrid-oscillator-network model, which has been formulated with regard to immature neonatal situations, respiratory rhythm generation operates through activation of inspiratory pacemaker neurons. Because the membrane potential is low in these early neonatal neurons, oscillations depend on a limited set of voltage-dependent ion conductances. Fast na f-channels are partly inactivated as evident from the negative peak of action potentials (data from refs 50 , 73 ). The assumed conductances and their activation thresholds (θ) are listed in the inset. (data from ref. 73 ). The membrane potential hyperpolarizes as the neurons mature (data from refs 6 , 31 , 50 ). (b) during maturation, the membrane potential of respiratory and pacemaker neurons becomes significantly more negative (by more than 20 mv) indicated by unbroken red arrow in part (a), which decreases neuronal excitability and stops endogenous pacemaker bursting.Brain anoxia synaptic interactions between neurons become more intense with the same time-course, as more complex internal and external interconnections between respiratory neurons are formed, pre- and postsynaptic processes mature and the processes of synaptic integration in the expanded dendritic tree are reinforced. All respiratory neurons have a rich repertoire of voltage-regulated ion conductances, which can be activated when the neurons are temporarily released from a functional synaptic voltage clamp. The consequence is that the respiratory rhythm oscillates in at least two antagonistic oscillatory phases: inspiration and post-inspiration. Specification of conductances and their activation thresholds (θ) are given in the inset.Brain anoxia action potentials with a clear positive overshoot are truncated in the figure (data are from several papers listed in the text).