Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Respiratory responses evoked by blockades of ionotropic glutamate receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit

The respiratory role of excitatory amino acid (EAA) receptors within the Bötzinger complex (BötC) and the pre‐Bötzinger complex (pre‐BötC) was investigated in α‐chloralose–urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30–50 nL) of EAA receptor antagonists. Blockade of both N‐methyl‐d ‐aspartic acid (NMDA) and non‐NMDA receptors by 50 mm kynurenic acid (KYN) within the BötC induced a pattern of breathing characterized by low‐amplitude, high‐frequency irregular oscillations superimposed on tonic phrenic activity and successively the disappearance of respiratory rhythmicity in the presence of intense tonic inspiratory discharges (tonic apnea). KYN microinjections into the pre‐BötC caused similar respiratory responses that, however, never led to tonic apnea. Blockade of NMDA receptors by D(–)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; 1, 10 and 20 mm ) within the BötC induced increases in respiratory frequency and decreases in peak phrenic amplitude; the highest concentrations caused tonic apnea insensitive to chemical stimuli. Blockade of non‐NMDA receptors by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 1, 10 and 20 mm ) within the BötC produced only less pronounced increases in respiratory frequency. Responses to D‐AP5 in the pre‐BötC were similar, although less pronounced than those elicited in the BötC and never characterized by tonic apnea. In the same region, CNQX provoked increases in respiratory frequency similar to those elicited in the BötC, associated with slight reductions in peak phrenic activity. The results show that EAA receptors within the investigated medullary subregions mediate a potent control on both the intensity and frequency of inspiratory activity, with a major role played by NMDA receptors.     

AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitro

Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca²⁺-activated non-specific cation current (I_CAN). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca²⁺ channels to directly evoke I_CAN. Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca²⁺ release also evoked I_CAN. Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca²⁺ influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic I_CAN, which underlies inspiratory drive potentials. The coupling of AMPA receptors with I_CAN suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network.     

Optical imaging of medullary ventral respiratory network during eupnea and gasping in situ

In severe hypoxia, respiratory rhythm is shifted from an eupneic, ramp‐like motor pattern to gasping characterized by a decrementing pattern of phrenic motor activity. However, it is not known whether hypoxia reconfigures the spatiotemporal organization of the central respiratory rhythm generator. Using the in situ arterially perfused juvenile rat preparation, we investigated whether the shift from eupnea to gasping was associated with a reconfiguration of the spatiotemporal pattern of respiratory neuronal activity in the ventral medullary respiratory network. Optical images of medullary respiratory network activity were obtained from male rats (4–6 weeks of age). Part of the medullary network was stained with a voltage‐sensitive dye (di‐2 ANEPEQ) centred both within, and adjacent to, the pre‐Bötzinger complex (Pre‐BötC). During eupnea, optical signals initially increased prior to the onset of phrenic activity and progressively intensified during the inspiratory phase peaking at the end of inspiration. During early expiration, fluorescence was also detected and slowly declined throughout this phase. In contrast, hypoxia shifted the respiratory motor pattern from eupnea to gasping and optical signals were restricted to inspiration only. Areas active during gasping showed fluorescence that was more intensive and covered a larger region of the rostral ventrolateral medulla compared to eupnea. Regions exhibiting peak inspiratory fluorescence did not coincide spatially during eupnea and gasping. Moreover, there was a recruitment of additional medullary regions during gasping that were not active during eupnea. These results provide novel evidence that the shift in respiratory motor pattern from eupnea to gasping appears to be associated with a reconfiguration of the central respiratory rhythm generator characterized by changes in its spatiotemporal organization.     

Metabotropic glutamate receptors (mGluR5) activate transient receptor potential canonical channels to improve the regularity of the respiratory rhythm generated by the pre-Bötzinger complex in mice

Metabotropic glutamate receptors (mGluRs) are hypothesized to play a key role in generating the central respiratory rhythm and other rhythmic activities driven by central pattern generators (e.g. locomotion). However, the functional role of mGluRs in rhythmic respiratory activity and many motor patterns is very poorly understood. Here, we used mouse respiratory brain‐slice preparations containing the pre‐Bötzinger complex (pre‐BötC) to identify the role of group I mGluRs (mGluR1 and mGluR5) in respiratory rhythm generation. We found that activation of mGluR1/5 is not required for the pre‐BötC to generate a respiratory rhythm. However, our data suggest that mGluR1 and mGluR5 differentially modulate the respiratory rhythm. Blocking endogenous mGluR5 activity with 2‐Methyl‐6‐(phenylethynyl)pyridine (MPEP) decreases the inspiratory burst duration, burst area and frequency, whereas it increases the irregularity of the fictive eupneic inspiratory rhythm generated by the pre‐BötC. In contrast, blocking mGluR1 reduces the frequency. Moreover, the mGluR1/5 agonist 3,5‐dihydroxyphenylglycine increases the frequency and decreases the irregularity of the respiratory rhythm. Based on previous studies, we hypothesized that mGluR signaling decreases the irregularity of the respiratory rhythm by activating transient receptor potential canonical (TRPC) channels, which carry a non‐specific cation current (ICAN). Indeed, 3,5‐dihydroxyphenylglycine (DHPG) application reduces cycle‐by‐cycle variability and subsequent application of the TRPC channel blocker 1‐[2‐(4‐methoxyphenyl)‐2‐[3‐(4‐methoxyphenyl)propoxy]ethyl]imidazole (SKF‐96365) hydrochloride reverses this effect. Our data suggest that mGluR5 activation of ICAN‐carrying TRPC channels plays an important role in governing the cycle‐by‐cycle variability of the respiratory rhythm. These data suggest that modulation of TRPC channels may correct irregular respiratory rhythms in some central neuronal diseases.     

Background sodium current underlying respiratory rhythm regularity

Rhythm-generating neural circuits underlying diverse behaviors such as locomotion, sleep states, digestion and respiration play critical roles in our lives. Irregularities in these rhythmic behaviors characterize disease states – thus, it is essential that we identify the ionic and/or cellular mechanisms that are necessary for triggering these rhythmic behaviors on a regular basis. Here, we examine which ionic conductances underlie regular or ‘stable’ respiratory activities, which are proposed to underlie eupnea, or normal quiet breathing. We used a mouse in vitro medullary slice preparation containing the rhythmogenic respiratory neural circuit, called the preBötzinger complex (preBötC), that underlies inspiratory respiratory activity. We varied either [K⁺]_o or [Na⁺]_o, or blocked voltage-gated calcium channels, while recording from synaptically isolated respiratory pacemakers, and examined which of these manipulations resulted in their endogenous bursting becoming more irregular. Of these, lowering [Na⁺]_o increased the irregularity of endogenous bursting by synaptically isolated pacemakers. Lowering [Na⁺]_o also decreased the regularity of fictive eupneic activity generated by the ventral respiratory group (VRG) population and hypoglossal motor output. Voltage clamp data indicate that lowering [Na⁺]_o, in a range that results in irregular population rhythm generation, decreased persistent sodium currents, but not transient sodium currents underlying action potentials. Our data suggest that background sodium currents play a major role in determining the regularity of the fictive eupneic respiratory rhythm.     

Endogenous rhythm generation in the pre-Bötzinger complex and ionic currents: modelling and in vitro studies

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.     

Isocitrate supplementation promotes breathing generation,gasping, and autoresuscitation in neonatal mice

Breathing is a vital function generated and controlled by a brainstem neural network, which is able to adjust its function to fit different metabolic demands. For instance, the pre‐Bötzinger complex (preBötC) can respond to low oxygen availability (hypoxia) by an initial increase in rhythm frequency followed by a decrease in respiratory efforts that leads to gasping generation. Gasping is essential for autoresuscitation, which has motivated studies of the cellular mechanisms involved in these processes. Hypoxia has different effects on enzymes that participate in the Krebs cycle. In particular, aconitase is downregulated, whereas isocitrate dehydrogenase is unaffected or upregulated under hypoxic conditions. We hypothesized that the application of isocitrate, the product of aconitase and the substrate of isocitrate dehydrogenase as well as an alternative metabolic substrate, might enhance breathing and render it more resistant to hypoxic insult. We tested the effects of isocitrate applied on brainstem slices containing the preBötC as well as its central effects in vivo using plethysmography. Our results show that isocitrate increases the frequency of fictive eupnea and fictive gasping produced by the preBötC in vitro. Moreover, isocitrate increases the amplitude of ventilation in vivo in normoxia, increases ventilation during gasping, and favors autoresuscitation when animals were subjected to asphyxiation. In conclusion, we have found that isocitrate improves ventilation under both normoxic and hypoxic conditions through a mechanism that involves the preBötC and possibly other respiratory neural networks. Thus, isocitrate would be useful to avoid the failure of gasping generation and autoresuscitation in pathological conditions. © 2013 Wiley Periodicals, Inc.     

Respiratory and metabolic acidosis differentially affect the respiratory neuronal network in the ventral medulla of neonatal rats

Two respiratory‐related areas, the para‐facial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pre‐Bötzinger complex/ventral respiratory group (preBötC/VRG), are thought to play key roles in respiratory rhythm. Because respiratory output patterns in response to respiratory and metabolic acidosis differ, we hypothesized that the responses of the medullary respiratory neuronal network to respiratory and metabolic acidosis are different. To test these hypotheses, we analysed respiratory‐related activity in the pFRG/RTN and preBötC/VRG of the neonatal rat brainstem–spinal cord in vitro by optical imaging using a voltage‐sensitive dye, and compared the effects of respiratory and metabolic acidosis on these two populations. We found that the spatiotemporal responses of respiratory‐related regional activities to respiratory and metabolic acidosis are fundamentally different, although both acidosis similarly augmented respiratory output by increasing respiratory frequency. PreBötC/VRG activity, which is mainly inspiratory, was augmented by respiratory acidosis. Respiratory‐modulated pixels increased in the preBötC/VRG area in response to respiratory acidosis. Metabolic acidosis shifted the respiratory phase in the pFRG/RTN; the pre‐inspiratory dominant pattern shifted to inspiratory dominant. The responses of the pFRG/RTN activity to respiratory and metabolic acidosis are complex, and involve either augmentation or reduction in the size of respiratory‐related areas. Furthermore, the activation pattern in the pFRG/RTN switched bi‐directionally between pre‐inspiratory/inspiratory and post‐inspiratory. Electrophysiological study supported the results of our optical imaging study. We conclude that respiratory and metabolic acidosis differentially affect activities of the pFRG/RTN and preBötC/VRG, inducing switching and shifts of the respiratory phase. We suggest that they differently influence the coupling states between the pFRG/RTN and preBötC/VRG.     

Somatostatin 2a receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column of the rat

Microinjection of somatostatin (SST) causes site‐specific effects on respiratory phase transition, frequency, and amplitude when microinjected into the ventrolateral medulla (VLM) of the anesthetized rat, suggesting selective expression of SST receptors on different functional classes of respiratory neurons. Of the six subtypes of SST receptor, somatostatin 2a (sst_2a) is the most prevalent in the VLM, and other investigators have suggested that glutamatergic neurons in the preBötzinger Complex (preBötC) that coexpress neurokinin‐1 receptor (NK1R), SST, and sst_2a are critical for the generation of respiratory rhythm. However, quantitative data describing the distribution of sst_2a in respiratory compartments other than preBötC, or on functionally identified respiratory neurons, is absent. Here we examine the medullary expression of sst_2a with particular reference to glycinergic/expiratory neurons in the Bötzinger Complex (BötC) and NK1R‐immunoreactive/inspiratory neurons in the preBötC. We found robust sst_2a expression at all rostrocaudal levels of the VLM, including a large proportion of catecholaminergic neurons, but no colocalization of sst_2a and glycine transporter 2 mRNA in the BötC. In the preBötC 54% of sst_2a‐immunoreactive neurons were also positive for NK1R. sst_2a was not observed in any of 52 dye‐labeled respiratory interneurons, including seven BötC expiratory‐decrementing and 11 preBötC preinspiratory neurons. We conclude that sst_2a is not expressed on BötC respiratory neurons and that phasic respiratory activity is a poor predictor of sst_2a expression in the preBötC. Therefore, sst_2a is unlikely to underlie responses to BötC SST injection, and is sparse or absent on respiratory neurons identified by classical functional criteria. J. Comp. Neurol. 524:1384–1398, 2016. © 2015 Wiley Periodicals, Inc.     

Relationship between two types of vesicular glutamate transporters and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies

Our previous study demonstrated GABAergic and glycinergic synapses onto neurokinin‐1 receptor (NK1R)‐immunoreactive (ir) neurons in the pre‐Bötzinger complex (pre‐BötC), the hypothesized kernel of normal respiratory rhythmogenesis. In the present study, we aimed to identify glutamatergic synapses onto NK1R‐ir pre‐BötC neurons, as excitatory synaptic transmission is a prerequisite to normal respiratory rhythmogenesis. Two types of vesicular glutamate transporters (VGLUT), VGLUT1 and VGLUT2, have been recently implicated in glutamate‐mediated transmission. The present study used immunofluorescence and immunogold‐silver staining to determine the relationship between the transporters and NK1R‐ir neurons in the pre‐BötC of adult rats. Under the confocal laser‐scanning microscope, VGLUT2‐ir boutons were found to be widely distributed in the pre‐BötC, some of which were in close apposition to NK1R‐ir somas and dendrites. VGLUT1‐ir boutons were relatively rare and only a few were found to be in close apposition to NK1R‐ir somas and dendrites. Electron microscopic observation revealed that approximately 41% of VGLUT2‐ir terminals were in close apposition to, or made asymmetric synapses with NK1R‐ir somas and dendrites in the pre‐BötC. On the other hand, 50.5% of NK1R‐ir dendrites were closely apposed to, or synapsed with VGLUT2‐ir terminals. Occasionally, VGLUT1‐ir terminals were found in close apposition to NK1R‐ir somas or dendrites, but we were unable to identify synapses between them. The present findings provide the morphological basis for excitatory synaptic inputs onto NK1R‐ir neurons in the pre‐BötC. VGLUT2 may be involved in a dominant excitatory synaptic pathway for normal respiratory rhythmogenesis.     

Inhibition of protein kinase A activity depresses phrenic drive and glycinergic signalling,but not rhythmogenesis in anaesthetized rat

The cAMP–protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane‐permeable PKA inhibitor Rp‐adenosine 3′,5′‐cyclic monophosphothioate (Rp‐cAMPS) in urethane‐anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end‐tidal CO₂ and arterial pressure were recorded. Rp‐cAMPS in the preBötzinger complex (preBötC) caused powerful, dose‐dependent depression of phrenic burst amplitude and inspiratory period. Rp‐cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp‐cAMPS‐evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP–PKA signalling.     

Electrophysiological and morphological characteristics of GABAergic respiratory neurons in the mouse pre-Bötzinger complex

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre‐Bötzinger complex. We used 67‐kDa isoform of glutamic acid decarboxylase‐green fluorescence protein (GAD67‐GFP) (Δneo) knock‐in (GAD67^GFP/+) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre‐Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre‐Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP‐positive neurons in the pre‐Bötzinger complex were recorded in a perforated whole‐cell patch‐clamp mode. Six of 32 GFP‐positive neurons were respiratory and the remaining 26 neurons were non‐respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post‐synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP‐negative neurons were recorded in the pre‐Bötzinger complex. GFP‐positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP‐positive inspiratory neurons had bipolar, triangular or crescent‐shaped somata and GFP‐negative inspiratory neurons had multipolar‐shaped somata. The somata of GFP‐positive inspiratory neurons were smaller than those of GFP‐negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre‐Bötzinger complex.     

GABAergic and glycinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies

The pre‐Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin‐1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre‐inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride‐mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold‐silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)‐ or glycine transporter 2 (GlyT2)‐immunoreactive (ir) boutons and NK1R‐ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD‐ and GlyT2‐ir boutons were in close apposition to NK1R‐ir somas and dendrites in the preBötC. Under the electron microscope, GAD‐ and GlyT2‐ir terminals were in close apposition to NK1R‐ir somas and dendrites. Symmetric synapses were identified between GAD‐ or GlyT2‐ir terminals and NK1R‐ir neurons. A total of 51.6% GAD‐ir and 38.2% GlyT2‐ir terminals were found to contact or make synapses with NK1R‐ir profiles, respectively. GAD‐ and GlyT2‐ir terminals synapsed not only upon NK1R‐ir neurons but also upon NK1R immuno‐negative neurons. NK1R‐ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R‐ir neurons in the preBötC, consistent with the suggestion that chloride‐mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.     

Efferent projections of excitatory and inhibitory preBötzinger Complex neurons

The preBötzinger Complex (preBötC), a compact medullary region essential for generating normal breathing rhythm and pattern, is the kernel of the breathing central pattern generator (CPG). Excitatory preBötC neurons in rats project to major breathing‐related brainstem regions. Here, we provide a brainstem connectivity map in mice for both excitatory and inhibitory preBötC neurons. Using a genetic strategy to label preBötC neurons, we confirmed extensive projections of preBötC excitatory neurons within the brainstem breathing CPG including the contralateral preBötC, Bötzinger Complex (BötC), ventral respiratory group, nucleus of the solitary tract, parahypoglossal nucleus, parafacial region (RTN/pFRG or alternatively, pF_L/pF_V), parabrachial and Kölliker‐Füse nuclei, as well as major projections to the midbrain periaqueductal gray. Interestingly, preBötC inhibitory projections paralleled the excitatory projections. Moreover, we examined overlapping projections in the pons in detail and found that they targeted the same neurons. We further explored the direct anatomical link between the preBötC and suprapontine brain regions that may govern emotion and other complex behaviors that can affect or be affected by breathing. Forebrain efferent projections were sparse and restricted to specific nuclei within the thalamus and hypothalamus, with processes rarely observed in cortex, basal ganglia, or other limbic regions, e.g., amygdala or hippocampus. We conclude that the preBötC sends direct, presumably inspiratory‐modulated, excitatory and inhibitory projections in parallel to distinct targets throughout the brain that generate and modulate breathing pattern and/or coordinate breathing with other behaviors, physiology, cognition, or emotional state.     

Computational modelling of 5-HT receptor-mediated reorganization of the brainstem respiratory network

Brainstem respiratory neurons express the glycine α₃ receptor (Glyα₃R), which is a target of modulation by several serotonin (5‐HT) receptor agonists. Application of the 5‐HT_1A receptor (5‐HT_1AR) agonist 8‐OH‐DPAT was shown (i) to depress cellular cAMP, leading to dephosphorylation of Glyα₃R and augmentation of postsynaptic inhibition of neurons expressing Glyα₃R ( Manzke et al., 2010 ) and (ii) to hyperpolarize respiratory neurons through 5‐HT‐activated potassium channels. These processes counteract opioid‐induced depression and restore breathing from apnoeas often accompanying pharmacotherapy of pain. The effect is postulated to rely on the enhanced Glyα₃R‐mediated inhibition of inhibitory neurons causing disinhibition of their target neurons. To evaluate this proposal and investigate the neural mechanisms involved, an established computational model of the brainstem respiratory network ( Smith et al., 2007 ), was extended by (i) incorporating distinct subpopulations of inhibitory neurons (glycinergic and GABAergic) and their synaptic interconnections within the Bötzinger and pre‐Bötzinger complexes and (ii) assigning the 5‐HT_1AR‐Glyα₃R complex to some of these inhibitory neuron types in the network. The modified model was used to simulate the effects of 8‐OH‐DPAT on the respiratory pattern and was able to realistically reproduce a number of experimentally observed responses, including the shift in the onset of post‐inspiratory activity to inspiration and conversion of the eupnoeic three‐phase rhythmic pattern into a two‐phase pattern lacking the post‐inspiratory phase. The model shows how 5‐HT_1AR activation can produce a disinhibition of inspiratory neurons, leading to the recovery of respiratory rhythm from opioid‐induced apnoeas.     

Stimulation of the midbrain periaqueductal gray modulates preinspiratory neurons in the ventrolateral medulla in the rat in vivo

The midbrain periaqueductal gray (PAG) is involved in many basic survival behaviors that affect respiration. We hypothesized that the PAG promotes these behaviors by changing the firing of preinspiratory (pre‐I) neurons in the pre‐Bötzinger complex, a cell group thought to be important in generating respiratory rhythm. We tested this hypothesis by recording single unit activity of pre‐Bötzinger pre‐I neurons during stimulation in different parts of the PAG. Stimulation in the dorsal PAG increased the firing of pre‐I neurons, resulting in tachypnea. Stimulation in the medial part of the lateral PAG converted the pre‐I neurons into inspiratory phase‐spanning cells, resulting in inspiratory apneusis. Stimulation in the lateral part of the lateral PAG generated an early onset of the pre‐I neuronal discharge, which continued throughout the inspiratory phase, while at the same time attenuating diaphragm contraction. Stimulation in the ventral part of the lateral PAG induced tachypnea but inhibited pre‐I cell firing, whereas stimulation in the ventrolateral PAG inhibited not only pre‐I cells but also the diaphragm, leading to apnea. These findings show that PAG stimulation changes the activity of the pre‐Bötzinger pre‐I neurons. These changes are in line with the different behaviors generated by the PAG, such as the dorsal PAG generating avoidance behavior, the lateral PAG generating fight and flight, and the ventrolateral PAG generating freezing and immobility. J. Comp. Neurol. 521: 3083–3098, 2013. © 2013 Wiley Periodicals, Inc.     

El complejo pre-Bötzinger: generación y modulación del ritmo respiratorio

IntroductionIn mammals, the preBötzinger complex (preBötC) is a bilateral and symmetrical neural network located in the brainstem which is essential for the generation and modulation of respiratory rhythm. There are few human studies about the preBötC and, its relationship with neurological diseases has not been described. However, the importance of the preBötC in neural control of breathing and its potential participation in neurological diseases in humans, has been suggested based on pharmacological manipulation and lesion of the preBötC in animal models, both in vivo and in vitro.MethodIn this review, we describe the effects of some drugs on the inspiratory activity in vitro in a transverse slice that contains the preBötC, as well as some in vivo experiments. Drugs were classified according to their effects on the main neurotransmitter systems and their importance as stimulators or inhibitors of preBötC activity and therefore for the generation of the respiratory rhythm.ConclusionClinical neurologists will find this information relevant to understanding how the central nervous system generates the respiratory rhythm and may also relate this information to the findings made in daily practice.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.