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![Criteria for identifying pacemakers. A, In 8 mM [K ] o aCSF, inspiratory pacemaker bursts are in phase with PBC bursts. B, Bath application of CNQX, CPP, bicuculline, and strychnine eliminates PBC bursts while pacemaker neurons continue bursting. Brief current injection reset the ongoing rhythm, the timing of which is demarcated by black bars above the neuron recording. C, Injecting depolarizing current into synaptically isolated pacemakers increased the bursting frequency (top neural trace), whereas hyperpolarizing current terminated bursting during current injection (bottom trace). The y-axis scale in A applies to B and C.](profile/Andrew-Tryba/publication/7402463/figure/fig2/AS:394497751044105@1471067001856/Criteria-for-identifying-pacemakers-A-In-8-mM-K-o-aCSF-inspiratory-pacemaker-bursts.png)
Criteria for identifying pacemakers. A, In 8 mM [K ] o aCSF, inspiratory pacemaker bursts are in phase with PBC bursts. B, Bath application of CNQX, CPP, bicuculline, and strychnine eliminates PBC bursts while pacemaker neurons continue bursting. Brief current injection reset the ongoing rhythm, the timing of which is demarcated by black bars above the neuron recording. C, Injecting depolarizing current into synaptically isolated pacemakers increased the bursting frequency (top neural trace), whereas hyperpolarizing current terminated bursting during current injection (bottom trace). The y-axis scale in A applies to B and C.
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![Figure 2. Criteria for identifying pacemakers. A, In 8 mM [K ] o aCSF,...](profile/Andrew-Tryba/publication/7402463/figure/fig2/AS:394497751044105@1471067001856/Criteria-for-identifying-pacemakers-A-In-8-mM-K-o-aCSF-inspiratory-pacemaker-bursts_Q320.jpg)
![Figure 4. Pacemaker activity in 3 mM [K ] o aCSF is restored during...](profile/Andrew-Tryba/publication/7402463/figure/fig4/AS:394497751044109@1471067001982/Pacemaker-activity-in-3-mM-K-o-aCSF-is-restored-during-depolarizing-current-injection_Q320.jpg)
![Figure 5. Pacemakers can burst in aCSF with 3 and 8 mM [K ] o without...](profile/Andrew-Tryba/publication/7402463/figure/fig5/AS:394497755238400@1471067002037/Pacemakers-can-burst-in-aCSF-with-3-and-8-mM-K-o-without-blocking-synaptic_Q320.jpg)
Synaptic and endogenous pacemaker properties have been hypothesized as principal cellular mechanisms for respiratory rhythm generation. This rhythmic activity is thought to originate in the pre-Bötzinger complex, an area that can generate fictive respiration when isolated in brainstem slice preparations of mice. In slice preparations, external pota...
Citations
... Spike Shape Regulates Intrinsic Bursting. Spike shapes vary widely, and in the preBötC spike amplitudes range from approximately 15 to 125 mV (20,24,25). Due to the voltagedependence of I NaP (in)activation (8,18,21), we wondered whether spike shape could impact intrinsic bursting. ...
... However, another artificial aspect of in vitro experiments is low temperature, which has an inverse relationship with spike amplitude and AHP (33); Fig. 7. Warmer temperatures in vivo are therefore expected to counteract the effects of low [K + ] e on intrinsic bursting. As a result, our model predicts that at physiological temperature and [K + ] e some neurons remain burst-capable, consistent with experiments that have identified intrinsic bursting preBötC neurons at physiological [K + ] e but at slightly warmer temperatures (30°C to 31°C) (24,54). ...
Breathing is a vital rhythmic process originating from the pre-Bötzinger complex. Since its discovery in 1991, there has been a spirited debate about whether respiratory rhythm generation emerges as a network property or is driven by a subset of specialized neurons with rhythmic bursting capabilities, endowed by intrinsic currents. Here, using computational modeling, we propose a unifying,data-driven model of respiratory rhythm generation, which bridges the gap between these competing theories. In this study, we demonstrate that the interaction of cellular (a persistent sodium current) and network properties (recurrent excitation) gives rise to multiple (i.e., degenerate) modes of rhythm generation that are consistent with both theories. Importantly, conditional factors impacting spike shape can promote one mode over the other.
... Spike Shape Regulates Intrinsic Bursting. Spike shapes vary widely, and in the preBötC spike amplitudes range from approximately 15 to 125 mV (20,24,25). Due to the voltagedependence of I NaP (in)activation (8,18,21), we wondered whether spike shape could impact intrinsic bursting. ...
... However, another artificial aspect of in vitro experiments is low temperature, which has an inverse relationship with spike amplitude and AHP (33); Fig. 7. Warmer temperatures in vivo are therefore expected to counteract the effects of low [K + ] e on intrinsic bursting. As a result, our model predicts that at physiological temperature and [K + ] e some neurons remain burst-capable, consistent with experiments that have identified intrinsic bursting preBötC neurons at physiological [K + ] e but at slightly warmer temperatures (30°C to 31°C) (24,54). ...
... Spike Shape Regulates Intrinsic Bursting. Spike shapes vary widely, and in the preBötC spike amplitudes range from approximately 15 to 125 mV (20,24,25). Due to the voltagedependence of I NaP (in)activation (8,18,21), we wondered whether spike shape could impact intrinsic bursting. ...
... However, another artificial aspect of in vitro experiments is low temperature, which has an inverse relationship with spike amplitude and AHP (33); Fig. 7. Warmer temperatures in vivo are therefore expected to counteract the effects of low [K + ] e on intrinsic bursting. As a result, our model predicts that at physiological temperature and [K + ] e some neurons remain burst-capable, consistent with experiments that have identified intrinsic bursting preBötC neurons at physiological [K + ] e but at slightly warmer temperatures (30°C to 31°C) (24,54). ...
... To determine whether activation of the motor pattern was specific to block of synaptic inhibition vs. a generalized response caused by enhanced excitability, we tested additional treatments that increase excitability in the thick slice. In the neonatal rodent slice preparation, elevated external [K + ] is often used to sustain rhythmic output Tryba et al., 2003;Kam et al., 2013). In 3 out of 5 preparations increasing extracellular potassium failed to initiate rhythmic vagal motor bursting ( Figure 4). ...
Breathing is generated by a rhythmic neural circuit in the brainstem, which contains conserved elements across vertebrate groups. In adult frogs, the "lung area" located in the reticularis parvocellularis is thought to represent the core rhythm generator for breathing. Although this region is necessary for breathing-related motor output, whether it functions as an endogenous oscillator when isolated from other brainstem centers is not clear. Therefore, we generated thick brainstem sections that encompass the lung area to determine if it can generate breathing-related motor output in a highly reduced preparation. Brainstem sections did not produce activity. However, subsaturating block of glycine receptors reliably led to the emergence of rhythmic motor output that was further enhanced by blockade of GABAA receptors. Output occurred in singlets and multi-burst episodes resembling the intact network. However, bursts were slower and had longer duration that those produced by the intact preparation. In addition, burst frequency was reduced by norepinephrine and µ opioids, and increased by serotonin, as observed in the intact network and in vivo. These results suggest that the "lung area" can be activated to produce rhythmic respiratory-related motor output in a reduced brainstem section and provide new insights into respiratory rhythm generation in adult amphibians. First, clustering breaths into episodes can occur within the rhythm generating network without long-range input from structures such as the pons. Second, local inhibition near, or within, the rhythmogenic center may need to be overridden to express the respiratory rhythm.
... When individuals become dehydrated during prolonged intense exercise, factors such as chemosensory discharge induced by increased blood osmolarity, detection of hyperosmolarity by brain osmoreceptors, hypovolaemia and/or increased release of ventilatory stimuli (Fujii et al., 2008b) could all modulate the breathing response. Likewise, the concomitant core hyperthermia could drive the rise in ventilation owing to high temperatures in the hypothalamus, medulla oblongata, spinal cord and/or skeletal muscles (Chai & Lin, 1972;Hertel et al., 1976;Holmes et al., 1960;Kumazawa & Mizumura, 1977;Tryba et al., 2003). However, the independent effects of dehydration-induced hyperosmolality and hypovolemia and the separate influence of internal body hyperthermia on the ventilatory response to prolonged exercise have not been documented. ...
... In this context, increases in T c could impact respiratory control mechanisms through changes in brain and spinal cord temperature. Animal studies have consistently shown that the spinal cord, hypothalamus and medulla oblongata are responsive to heat (Chai & Lin, 1972;Holmes et al., 1960;Tryba et al., 2003). In particular, increases in temperature within the physiological range (i.e., from ∼37 to 41 • C) have been shown to increase activity of the respiratory pacemaker neurons located within the ventral respiratory group in the medulla oblongata (Tryba et al., 2003), hence to accelerate breathing frequency. ...
... Animal studies have consistently shown that the spinal cord, hypothalamus and medulla oblongata are responsive to heat (Chai & Lin, 1972;Holmes et al., 1960;Tryba et al., 2003). In particular, increases in temperature within the physiological range (i.e., from ∼37 to 41 • C) have been shown to increase activity of the respiratory pacemaker neurons located within the ventral respiratory group in the medulla oblongata (Tryba et al., 2003), hence to accelerate breathing frequency. Unlike animals that use panting as an evaporative heatloss mechanism (Robertshaw, 2006), the modification of breathing pattern in our study participants (i.e., accelerated rate of breathing) did not lead to a simultaneous increase in dead space (V D /V T ratios remained constant at ∼0.13 from 20 min onwards in both experimental trials). ...
The mechanisms driving hyperthermic hyperventilation during exercise are unclear. In a series of retrospective analyses, we evaluated the impact of combined versus isolated dehydration and hyperthermia and the effects of sympathoadrenal discharge on ventilation and pulmonary gas exchange during prolonged intense exercise. In the first study, endurance‐trained males performed two submaximal cycling exercise trials in the heat. On day 1, participants cycled until volitional exhaustion (135 ± 11 min) while experiencing progressive dehydration and hyperthermia. On day 2, participants maintained euhydration and core temperature (Tc) during a time‐matched exercise (control). At rest and during the first 20 min of exercise, pulmonary ventilation (V̇E${\skew2\dot V_{\rm{E}}}$), arterial blood gases, CO2 output and O2 uptake were similar in both trials. At 135 ± 11 min, however, V̇E${\skew2\dot V_{\rm{E}}}$ was elevated with dehydration and hyperthermia, and this was accompanied by lower arterial partial pressure of CO2, higher breathing frequency, arterial partial pressure of O2, arteriovenous CO2 and O2 differences, and elevated CO2 output and unchanged O2 uptake despite a reduced pulmonary circulation. The increased V̇E${\skew2\dot V_{\rm{E}}}$ was closely related to the rise in Tc and circulating catecholamines (R² ≥ 0.818, P ≤ 0.034). In three additional studies in different participants, hyperthermia independently increased V̇E${\skew2\dot V_{\rm{E}}}$ to an extent similar to combined dehydration and hyperthermia, whereas prevention of hyperthermia in dehydrated individuals restored V̇E${\skew2\dot V_{\rm{E}}}$ to control levels. Furthermore, adrenaline infusion during exercise elevated both Tc and V̇E${\skew2\dot V_{\rm{E}}}$. These findings indicate that: (1) adjustments in pulmonary gas exchange limit homeostatic disturbances in the face of a blunted pulmonary circulation; (2) hyperthermia is the main stimulus increasing ventilation during prolonged intense exercise; and (3) sympathoadrenal activation might partly mediate the hyperthermic hyperventilation.
... Moreover, experiments show that the bursting capability of pre-BötC neurons and networks depends on the extracellular ion concentrations to which they are exposed. Slices of 250-350 m thickness prepared from the pre-BötC are nonrhythmic at physiological [K + ] ext , but some individual pre-BötC neurons do burst in these conditions (Del Negro et al., 2001;Tryba et al., 2003), especially if depolarized by a tonic input (Smith et al., 1991), and pharmacological blockade of GABA A and glycinergic inhibition also allows pre-BötC neurons to burst in these conditions (Tryba et al., 2003). In contrast to these results, modeling that explains how different extracellular potassium concentrations can produce corresponding forms of pre-BötC activity has led to the conclusion that individual pre-BötC neurons should not be able to burst at physiologically relevant extracellular potassium concentrations (Bacak et al., 2016b). ...
... Moreover, experiments show that the bursting capability of pre-BötC neurons and networks depends on the extracellular ion concentrations to which they are exposed. Slices of 250-350 m thickness prepared from the pre-BötC are nonrhythmic at physiological [K + ] ext , but some individual pre-BötC neurons do burst in these conditions (Del Negro et al., 2001;Tryba et al., 2003), especially if depolarized by a tonic input (Smith et al., 1991), and pharmacological blockade of GABA A and glycinergic inhibition also allows pre-BötC neurons to burst in these conditions (Tryba et al., 2003). In contrast to these results, modeling that explains how different extracellular potassium concentrations can produce corresponding forms of pre-BötC activity has led to the conclusion that individual pre-BötC neurons should not be able to burst at physiologically relevant extracellular potassium concentrations (Bacak et al., 2016b). ...
Intensive computational and theoretical work has led to the development of multiple mathematical models for bursting in respiratory neurons in the pre-Bötzinger Complex (pre-BötC) of the mammalian brainstem. Nonetheless, these previous models have not captured the pre-inspiratory ramping aspects of these neurons’ activity patterns, in which relatively slow tonic spiking gradually progresses to faster spiking and a full-blown burst, with a corresponding gradual development of an underlying plateau potential. In this work, we show that the incorporation of the dynamics of the extracellular potassium ion concentration into an existing model for pre-BötC neuron bursting, along with some parameter adjustments, suffices to induce this ramping behavior. Using fast-slow decomposition, we show that this activity can be considered as a form of parabolic bursting, but with burst termination at a homoclinic bifurcation rather than as a SNIC bifurcation. We also investigate the parameter-dependence of these solutions and show that the proposed model yields a greater dynamic range of burst frequencies, durations, and duty cycles than those produced by other models in the literature.
... Figure 4H highlights the relative shape of burstlets and bursts as well as the delay between burstlet generation and recruitment of the pattern-generating population and simulated hypoglossal output, which agrees qualitatively with experimental observations (Kallurkar et al., 2020). Experimentally, it is likely that postsynaptic Ca 2+ transients will increase with increasing K bath due to the change in the resting Vm in nonrhythmic preBötC neurons (Tryba et al., 2003) the voltage-gated activation dynamics of postsynaptic calcium channels (Elsen and Ramirez, 1998); see 'Discussion' for a full analysis of this point. Interestingly, in our simulations, increasing P SynCa (i.e., the amplitude of the postsynaptic calcium transients) with K bath (Figure 4 traces G1-G4) generated K bath -dependent changes in the burstlet fraction that are consistent with experimental observations (Kallurkar et al., 2020; see Figure 4I). ...
... Consistent with this idea, Cd 2+ -sensitive Ca 2+ channels in preBötC neurons appear to be primarily localized in distal dendritic compartments . Voltage-gated calcium channels in the preBötC start to activate at approximately −65 mV (Elsen and Ramirez, 1998), and importantly, the mean somatic resting membrane potential of non-rhythmogenic preBötC neurons increases from −67.034 mV to −61.78 mV when extracellular potassium concentration is elevated from 3 mM to 8 mM (Tryba et al., 2003). Moreover, at K bath = 9 mM , EPSPs in the preBötC are on the order of 2-5 mV (Kottick and Del Negro, 2015;Morgado-Valle et al., 2015;Baertsch et al., 2021) and the amplitude of EPSCs has been shown to decrease as K bath is lowered (Okada et al., 2005). ...
... For example, the calcium concentration at which the IP3 receptor is activated is dynamically regulated by IP3 (Kaftan et al., 1997), and therefore, activity-or neuromodulatory-dependent changes in the cytoplasmic Ca 2+ and/or IP3 concentration may impact ER Ca 2+ uptake and release dynamics. Storeoperated Ca 2+ dynamics are also affected by the transient receptor potential canonical 3 (TRPC3) channels (Salido et al., 2009), which are expressed in the preBötC, and manipulation of TRPC3 has been shown to impact burst amplitude and regularity (Tryba et al., 2003;Koizumi et al., 2018) as would be predicted by this model. It is also possible that calcium release is mediated by the ryanodine receptor, an additional calcium-activated channel located in the ER membrane (Lanner et al., 2010), since bath application of CPA (100 µM) and ryanodine (10 µM) removed large-amplitude oscillations in recordings of preBötC population activity (Toporikova et al., 2015). ...
Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBötzinger complex (preBötC). In in vitro slice preparations containing the preBötC, extracellular potassium must be elevated above physiological levels (to 7 - 9 mM ) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBötC projects. Reexamination of how extracellular K ⁺ affects preBötC neuronal activity has revealed that low amplitude oscillations persist at physiological levels. These oscillatory events are sub-threshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBötC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBötC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBötC population requires intracellular Ca ²⁺ dynamics and activation of a calcium-activated non-selective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca ²⁺ influx as well as Ca ²⁺ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated non-selective cationic current can reproduce and offer an explanation for many of the key properties associated with the burstlet theory of respiratory rhythm generation. Altogether, our modeling work provides a mechanistic basis that can unify a wide range of experimental findings on rhythm generation and motor output recruitment in the preBötC.
... Recently, chemogenetic activation of adult dorsal root ganglion neurons increased MT dynamics through tubulin acetylation, which resulted in axonal growth after nerve injury in vitro [170]. KCl-induced depolarization not only increases neuronal firing [171] but also releases neurotransmitters [172,173], including glutamate, GABA, and glycine [172,173], which modulate MT density [52, [174][175][176][177][178]. For instance, activation of glutamate receptors modulates MT function by regulating the expression of MAP2 [52, [175][176][177][178] and the upregulation of Tau translation and its accumulation in the somatodendritic compartments [179]. As will be reviewed later, NMDA receptor-dependent synaptic activation increases the proportion of dendritic spines containing dynamic MTs, contributing to spine morphological changes [53, 168,[180][181][182]. ...
Neuronal microtubules (MTs) are complex cytoskeletal protein arrays that undergo activity-dependent changes in their structure and function as a response to physiological demands throughout the lifespan of neurons. Many factors shape the allostatic dynamics of MTs and tubulin dimers in the cytosolic microenvironment, such as protein–protein interactions and activity-dependent shifts in these interactions that are responsible for their plastic capabilities. Recently, several findings have reinforced the role of MTs in behavioral and cognitive processes in normal and pathological conditions. In this review, we summarize the bidirectional relationships between MTs dynamics, neuronal processes, and brain and behavioral states. The outcomes of manipulating the dynamicity of MTs by genetic or pharmacological approaches on neuronal morphology, intrinsic and synaptic excitability, the state of the network, and behaviors are heterogeneous. We discuss the critical position of MTs as responders and adaptative elements of basic neuronal function whose impact on brain function is not fully understood, and we highlight the dilemma of artificially modulating MT dynamics for therapeutic purposes.
... Moreover, experiments show that the bursting capability of pre-BötC neurons and networks depends on the extracellular ion concentrations to which they are exposed. Slices of 250-350 µm thickness prepared from the pre-BötC are nonrhythmic at physiological [K + ] ext , but some individual pre-BötC neurons do burst in these conditions (Del Negro et al., 2001;Tryba et al., 2003), especially if depolarized by a tonic input (Smith et al., 1991), and pharmacological blockade of GABA A and glycinergic inhibition also allows pre-BötC neurons to burst in these conditions (Tryba et al., 2003). In contrast to these results, however, modeling that explains how different extracellular potassium concentrations can produce corresponding forms of pre-BötC activity has led to the conclusion that, according to existing modeling frameworks, individual pre-BötC neurons should not be able to burst at physiologically relevant extracellular potassium concentrations (Bacak et al., 2016b). ...
... Moreover, experiments show that the bursting capability of pre-BötC neurons and networks depends on the extracellular ion concentrations to which they are exposed. Slices of 250-350 µm thickness prepared from the pre-BötC are nonrhythmic at physiological [K + ] ext , but some individual pre-BötC neurons do burst in these conditions (Del Negro et al., 2001;Tryba et al., 2003), especially if depolarized by a tonic input (Smith et al., 1991), and pharmacological blockade of GABA A and glycinergic inhibition also allows pre-BötC neurons to burst in these conditions (Tryba et al., 2003). In contrast to these results, however, modeling that explains how different extracellular potassium concentrations can produce corresponding forms of pre-BötC activity has led to the conclusion that, according to existing modeling frameworks, individual pre-BötC neurons should not be able to burst at physiologically relevant extracellular potassium concentrations (Bacak et al., 2016b). ...
Intensive computational and theoretical work has led to the development of mutliple mathematical models for bursting in respiratory neurons in the pre-B\"otzinger Complex (pre-B\"otC) of the mammalian brainstem. Nonetheless, these previous models have not captured the pre-inspiratory ramping aspects of these neurons' activity patterns, in which relatively slow tonic spiking gradually progresses to faster spiking and a full blown burst, with a corresponding gradual development of an underlying plateau potential. In this work, we show that the incorporation of the dynamics of the extracellular potassium ion concentration into an existing model for pre-B\"otC neuron bursting, along with some parameter updates, suffices to induce this ramping behavior. Using fast-slow decomposition, we show that this activity can be considered as a form of parabolic bursting, but with burst termination at a homoclinic bifurcation rather than as a SNIC bifurcation. We also investigate the parameter-dependence of these solutions and show that the proposed model yields a greater dynamic range of burst frequencies, durations, and duty cycles than those produced by other models in the literature.
... Each slice was perfused by recirculating 200 mL of ACSF bubbled with carbogen at a flow rate of 10 mL/min (7). A temperature controller (Multi-Channel Systems; Reutlingen, Germany) was maintained at 30 C ± 1 C. Extracellular KCl was elevated from 3 mM to 8 mM over a span of 30 min before starting the recordings (55). Hypoxic conditions were induced by removing carbogen and bubbling the ACSF with a nitrogenous gas mixture (95% N 2 and 5% CO 2 ) for 15 min (8,32). ...
The preBötzinger complex (preBötC), located within the ventral respiratory column, produces inspiratory bursts in varying degrees of synchronization/amplitude. This wide range of population burst patterns reflects the flexibility of the preBötC neurons, which is expressed in variations in the onset/offset times of their activations and their activity during the population bursts, with respiratory neurons exhibiting a large cycle-to-cycle timing jitter both at the population activity onset and at the population activity peak; suggesting that respiratory neurons are stochastically activated before and during the inspiratory bursts. However, it is still unknown whether this stochasticity is maintained while evaluating the coactivity of respiratory neuronal ensembles. Moreover, the preBötC topology also remains unknown. Here, by simultaneously recording tens of preBötC neurons and using coactivation analysis during the inspiratory periods, we found that the preBötC has a scale-free configuration (mixture of not many highly connected nodes -hubs- with abundant poorly connected elements) exhibiting the rich-club phenomenon (hubs more likely interconnected with each other). PreBötC neurons also produce multineuronal activity patterns (MAPs) that are highly stable and change during the hypoxia-induced reconfiguration. Moreover, preBötC contains a coactivating core network shared by all its MAPs. Finally, we found a distinctive pattern of sequential coactivation of core network neurons at the beginning of the inspiratory periods, indicating that, when evaluated at the multicellular level, the coactivation of respiratory neurons seems not to be stochastic.
