Many neuronal networks are multifunctional, producing different patterns of activity in different circumstances, but the mechanisms responsible for this reconfiguration are in many cases unresolved. The mammalian respiratory network is an example of such a system. Normal respiratory activity (eupnea) is periodically interrupted by distinct large-amplitude inspirations known as sighs. Both rhythms originate from a single multifunctional neural network, and both are preserved in the in vitro transverse medullary slice of mice. Here we show that the generation of fictive sighs were more sensitive than eupnea to reductions of excitatory synapse strength caused by either the P/Q-type (alpha1A-containing) calcium channel antagonist omega-agatoxin TK or the non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX). In contrast, the NMDA receptor antagonist MK-801, while also inhibiting eupnea, increased the occurrence of sighs. This suggests that among the glutamatergic synapses subserving eupneic rhythmogenesis, there is a specific subset-highly sensitive to agatoxin and insensitive to NMDA receptor blockade-that is essential for sighs. Blockade of N-type calcium channels with omega-conotoxin GVIA also had pattern-specific effects: eupneic activity was not affected, but sigh frequency was increased and postsigh apnea decreased. We hypothesize that N-type (alpha1B) calcium channels selectively coupled to calcium-activated potassium channels contribute to the generation of the postsigh apnea.
"For this purpose we used an AMPA/kainate-subtype glutamatergic receptor antagonist 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX, 30 µM) but not an NMDA receptor blocker. This is because we considered that the synchronizing excitatory interactions would only require AMPA/kainate-subtype glutamatergic receptors as demonstrated by previous pharmacological experiments (Funk et al., 1993; Koshiya and Smith, 1999), although Lieske and Ramirez (2006a) argued that NMDA receptor is involved in respiratory rhythm generation. Voltage-imaging video signals taken at "
[Show abstract][Hide abstract] ABSTRACT: The preBötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.
"Interestingly, the period of rodent embryonic development when the preBötC network normally acquires the ability to generate sighs corresponds closely to the time when chloride-mediated conductances switch from depolarizing (excitatory) to hyperpolarizing (inhibitory) synaptic actions (Ren & Greer, 2006). Thus, the capacity for generating different inspiratory-related rhythmic activities is likely to depend upon maturational changes in synaptic properties within the preBötC network itself, this process being most probably accompanied by a maturation of neuronal membrane properties required for the large-amplitude inspiratory burst generation (Lieske & Ramirez, 2006; Koch et al. 2013). The mechanism(s) by which glycinergic synapses achieve the phase-coupling between the two components of sigh bursts remains unclear. "
[Show abstract][Hide abstract] ABSTRACT: In mammals, eupneic breathing is periodically interrupted by spontaneous augmented breaths (sighs) that include a larger-amplitude inspiratory effort, typically followed by a post-sigh apnea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre-Bötzinger complex (preBötC) can generate the distinct inspiratory motor patterns for both eupnea- and sigh-related behaviour. During mouse embryonic development, the preBötC begins to generate eupneic rhythmicity at embryonic day (E) 15.5, but the network's ability to also generate sigh-like activity remains unexplored at prenatal stages. Using transverse brainstem slice preparations we monitored the neuronal population activity of the preBötC at different embryonic ages. Spontaneous sigh-like rhythmicity was found to emerge progressively, being expressed in 0/32 slices at E15.5, 7/30 at E16.5, 9/22 at E17.5 and 23/26 at E18.5. Calcium imaging showed that the preBötC cell population that participates in eupneic-like discharge was also active during fictive sighs. However, patch-clamp recordings revealed the existence of an additional small subset of neurons that fired exclusively during sigh activity. Changes in glycinergic inhibitory synaptic signalling, either by pharmacological blockade, functional perturbation or natural maturation of the chloride co-transporters KCC2 or NKCC1 selectively, and in an age-dependent manner, altered the bi-phasic nature of sigh bursts and their coordination with eupneic bursting, leading to the generation of an atypical monophasic sigh-related event. Together our results demonstrate that the developmental emergence of a sigh generating capability occurs after the onset of eupneic rhythmogenesis and requires the proper maturation of chloride-mediated glycinergic synaptic transmission.
The Journal of Physiology 03/2014; 592(10). DOI:10.1113/jphysiol.2013.268730 · 5.04 Impact Factor
"Loss of Ca v 2.1 reduced glutamatergic transmission within the preBötC. Our pharmacological experiments confirmed that Ca v 2.2 accounts for ~30% of excitatory synaptic transmission (Lieske and Ramirez, 2006). In KO slices, EPSP amplitudes were reduced, and further pharmacological blockade of Ca v 2.2 abolished EPSPs. "
[Show abstract][Hide abstract] ABSTRACT: P/Q-type voltage-gated calcium channels (Ca(v)2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Ca(v)2.1 pore-forming α(1A) subunit (α(1A)(-/-)) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolated in vitro brainstem slice preparation containing the pre-Bötzinger complex. The loss of Ca(v)2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Ca(v)2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α(1A)(-/-) mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α(1A)(-/-) mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α(1A)(-/-) mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α(1A)(-/-) mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2013; 33(8):3633-3645. DOI:10.1523/JNEUROSCI.6390-11.2013 · 6.34 Impact Factor
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