Olivier Pierrefiche

Neurotransmitters and neuromodulators controlling the hypoxic respiratory response in anaesthetized cats

1 The contributions of neurotransmitters and neuromodulators to the responses of the respiratory network to acute hypoxia were analysed in anaesthetized cats. 2 Samples of extracellular fluid were collected at 1–1.5 min time intervals by microdialysis in the medullary region of ventral respiratory group neurones and analysed for their content of glutamate, γ‐aminobutyric acid (GABA), serotonin and adenosine by high performance liquid chromatography. Phrenic nerve activity was correlated with these measurements. 3 Levels of glutamate and GABA increased transiently during early periods of hypoxia, coinciding with augmented phrenic nerve activity and then fell below control during central apnoea. Serotonin and adenosine increased slowly and steadily with onset of hypoxic depression of phrenic nerve activity. 4 The possibility that serotonin contributes to hypoxic respiratory depression was tested by microinjecting the 5‐HT‐1A receptor agonist 8‐OH‐DPAT into the medullary region that is important for rhythmogenesis. Hypoxic activation of respiratory neurones and phrenic nerve activity were suppressed. Microinjections of NAN‐190, a 5‐HT‐1A receptor blocker, enhanced hypoxic augmentation resulting in apneustic prolongation of inspiratory bursts. 5 The results reveal a temporal sequence in the release of neurotransmitters and neuromodulators and suggest a specific role for each of them in the sequential development of hypoxic respiratory disturbances.

Blockade of synaptic inhibition within the pre‐Bötzinger complex in the cat suppresses respiratory rhythm generation in vivo

1 The role of synaptic inhibition in respiratory rhythm generation was analysed by microinjections of GABAA and glycine receptor antagonists into the bilateral pre‐Bötzinger complex (PBC) of anaesthetized cats. Central respiratory activity was monitored by phrenic nerve recordings. 2 Bilateral injections of bicuculline (50 or 100 μm) irreversibly slowed respiratory frequency and induced apneustic patterns. 3 Bilateral injections of strychnine (50 or 100 μm) greatly reduced phrenic burst amplitudes leading to increased burst frequency or irreversibly blocked rhythmic phrenic discharges. After unilateral tetrodotoxin (TTX) blockade in the PBC, strychnine injection into the contralateral PBC blocked rhythmic phrenic discharges. 4 Bilateral blockade of both GABAergic and glycinergic inhibition abolished rhythmic burst discharges and only tonic phrenic activity remained. Such tonic activity was blocked only by TTX (1 μm). 5 Potentiation of synaptic inhibition by the serotonin 1A receptor agonist 8‐hydroxydipropylaminotetralin (8‐OH‐DPAT; 50 μm) restored rhythmic activity only when given shortly after strychnine and bicuculline applications. It was, however, ineffective after blockade of synaptic inhibition was complete. 6 The study demonstrates the significance of synaptic inhibition in the process of respiratory generation in the adult cat in vivo .

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 (700u2003µm) of neonatal rats (P0–P4). Rhythmic activity was absent at 3u2003mm[K+]o but could be triggered by either the elevation of [K+]o to 5–7u2003mm or application of potassium current blockers (4‐AP, 50–200u2003µm, or TEA, 2 or 4u2003mm), or by blocking aerobic metabolism with NaCN (2u2003mm). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50u2003µm). 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.

Intracellular signal pathways controlling respiratory neurons

Medullary respiratory neurons are influenced by a variety of neuromodulators, but there is a lack of information about the specific intracellular signal pathways involved. In this report we describe the modulatory effects of the cyclic adenosine-triphosphate (cAMP)-dependent protein kinase and of protein kinase C pathways on voltage- and ligand-controlled ionic conductances and demonstrate their functional significance in regulating the excitability of medullary respiratory neurons of the vivo cat. Evidence is presented that PKA and PKC pathways are persistently activated. PKA regulates current flow through persistently activated and GABAB receptor-controlled potassium channels as well as GABAA receptor-controlled chloride channels. PKC also depresses persistent potassium currents but it potentiates excitatory and inhibitory synaptic currents. The clinical significance of these intracellular signal pathways is demonstrated in a case of a child suffering from apneustic breathing, who was successfully treated with a 5HT-1A receptor agonist.

Calcium-dependent conductances control neurones involved in termination of inspiration in cats

Intracellular injection of the calcium chelator BAPTA into postinspiratory (PI) and late inspiratory neurones (late-I) of the ventral respiratory group of anaesthetised cat was performed to study the role of intracellular free calcium in patterning the activity of neurones controlling termination of inspiration. BAPTA injection into neurones resulted in an increase of input resistance and prolongation of action potential discharge with reduced adaptation. In addition, late-I neurones developed a secondary burst of action potentials during the postinspiratory phase of the cycle. We conclude that intracellular free calcium controls (1) the duration of activation and the degree of adaptation of PI neurones and (2) repolarisation of late-I neurones during postinspiration.

Voltage-clamp analysis of neurons within deep layers of the brain.

Single electrode whole cell current- and voltage-clamp techniques in conjunction with intra- and extracellular phoresis and extracellular application of pharmacological agents were applied to study neurons in deep layers of the brainstem of anesthetized, paralyzed and artificially ventilated cats. We compared slow rhythmic changes and stimulus-evoked postsynaptic current and voltage responses of neurons as they were recorded with fine-tipped microelectrodes filled with 2-3 M microelectrode solutions or with 0.3 M patch solutions, or with patch electrodes. The experimental data were then compared with the effects of somatic and dendritic conductance changes simulated in a cell model. A new method was introduced for alternating current and voltage-clamp measurements performed at 300 Hz, which provided quasi-simultaneous measurements of slow changes of spontaneous synaptic currents and potentials. During current or voltage clamp, chemicals which affect voltage- and receptor-controlled conductances were ionophoresed intracellularly through single or theta-type glass electrodes. We show examples of activation of low-voltage activated Ca2+ responses after blockade of Na+ currents by intracellular QX 314 and K+ currents by intracellular Cs+ injections in addition to Sp-cAMPs to activate protein kinase A. TEA, NMDA and GABA were used to demonstrate the effectiveness of extracellular application of drugs through multibarrel electrodes or local application through a bath. The various tests demonstrated that single electrode whole cell current- and voltage-clamp methods, in combination with various techniques for drug application, can be well applied to study the biophysical properties and pharmacological sensitivities of neurons embedded in in vivo networks within deep layers of the brain.

Hypoxic response of hypoglossal motoneurones in the in vivo cat.

1 In current and voltage clamp, the effects of hypoxia were studied on resting and synaptic properties of hypoglossal motoneurones in barbiturate‐anaesthetized adult cats. 2 Twenty‐nine hypoglossal motoneurones with a mean membrane potential of −55 mV responded rapidly to acute hypoxia with a persistent membrane depolarization of about +17 mV. This depolarization correlated with the development of a persistent inward current of 0.3 nA at holding potentials close to resting membrane potential. 3 Superior laryngeal nerve (SLN) stimulation‐evoked EPSUPs were reduced in amplitude by, on average, 46%, while IPSUP amplitude was reduced by 31 %. SLN stimulation‐evoked EPSCs were reduced by 50–70%. 4 Extracellular application of adenosine (10 mm) hyperpolarized hypoglossal motoneurones by, on average, 5.6 mV, from a control value of –62 mV. SLN stimulation‐evoked EPSUPs decreased by 18% and IPSUPs decreased by 46% during adenosine application. 5 Extracellular application of the KATP channel blocker glibenclamide led to a blockade of a persistent outward current and a significant increase of SLN stimulation‐evoked EPSCs. 6 We conclude that hypoglossal motoneurones have a very low tolerance to hypoxia. They appear to be under metabolic stress even in normoxia and their capacity to activate protective potassium currents is limited when compared with other brainstem neurones. This may help to explain the rapid disturbance of hypoglossal function during energy depletion.

ATP-sensitive K+ channels are functional in expiratory neurones of normoxic cats.

1. We analysed spontaneously active expiratory neurones (n = 48) of anaesthetized cats for the presence of ATP‐sensitive K+ (KATP) channels. 2. Intracellular injection of ATP reversibly depolarized neurones during all phases of the respiratory cycle. During expiration, membrane potential depolarized by an average of 1.5 +/‐ 0.1 mV leading to a 25% increase of discharge frequency. During inspiration, ATP induced a 1.8 +/‐ 0.2 mV depolarization, which was accompanied by a maximum of 20% increase of input resistance (Rn). 3. Extracellular application of diazoxide, an agonist of KATP channels, resulted in reversible membrane hyperpolarization in 68% of neurones (n = 19). This hyperpolarization (2.5 mV during expiration and 3.1 mV during inspiration) was accompanied by a 22% decrease in Rn. 4. Extracellular application of tolbutamide and glibenclamide, two antagonists of KATP channels, evoked reversible depolarizations in 76% of neurones (n = 21). The depolarization was relatively constant throughout the respiratory cycle (1.4 mV during expiration and 2.3 mV during inspiration). Rn increased by 22%. 5. The same sulphonylureas also changed the steepness of membrane depolarization when neurones escaped spontaneous synaptic inhibition during postinspiration. Extracellularly applied tolbutamide and glibenclamide increased the steepness of depolarization by 21%, while diazoxide reduced it by 20%. 6. Antagonism of drugs was verified by simultaneous extra‐ and intracellular application of diazoxide and glibenclamide, respectively. 7. During voltage clamp at holding potential at ‐60 to ‐67 mV, intracellular or extracellular application of tolbutamide and glibenclamide blocked a persistent outward current. 8. We conclude that KATP channels are functional in expiratory neurones of adult cats and contribute to the control of excitability even during normoxia.

Protein kinase C pathways modulate respiratory pattern generation in the cat.

1. The significance of protein kinase C (PKC) in respiratory pattern generation was investigated in forty‐three expiratory neurones of anaesthetized cats. 2. Intracellular injection of R‐2,6‐diamino‐N‐([1‐(oxotridecyl)‐2‐piperidinyl]‐methyl)‐hexana mide dihydrochloride reversibly hyperpolarized twenty‐six neurones. Respiratory drive potentials decreased to 92% of control, and action potential discharges were reduced. Neuronal input resistance (Rin) decreased during inspiration and increased during expiration. 3. Voltage clamp revealed that blockade of PKC induced an increase of inhibitory drive currents and a decrease of excitatory drive currents in sixteen neurones. The amplitude of respiratory drive currents was decreased to 91% of control. The slope of synaptic inward currents during postinspiration was reduced. 4. After blockade of K+ conductances by TEA, additional blockade of PKC caused a hyperpolarization during postinspiration and expiration, but depolarization during inspiration in fourteen neurones. The respiratory drive currents were reduced to 61% of control. Respiratory drive potentials decreased to 72% of control, leading to reduced spontaneous discharge. Rin was increased throughout the respiratory cycle. 5. Stimulus‐evoked postsynaptic currents and potentials decreased after blockade of PKC with and without TEA. 6. The results indicate that PKC is endogenously active in expiratory neurones, modulating their excitability in three different ways: (a) it downregulates persistent K+ currents, (b) it upregulates Cl(‐)‐mediated inhibitory postsynaptic currents (IPSCs), and (c) it upregulates excitatory postsynaptic currents (EPSCs).

Modelling respiratory rhythmogenesis: focus on phase switching mechanisms.

It has been established that the normal respiratory pattern (“eupnoea”) in mammals is generated in the lower brainstem1,2 and may involve several medullary and pontine regions. Although some researchers suggest that a smaller region within the medulla (e.g., the pre-Botzinger Complex (pre-BotC) may be sufficient for the respiratory rhythm generation3, 4, 5, the eupnoeic respiratory rhythm (as well as apneustic breathing) has never been reproduced in reduced medullary preparations without the pons. At the same time, the specific ponto-medullary interactions related to genesis, shaping and control of the respiratory pattern have not been well characterized so far. Here we present a preliminary computational model of the ponto-medullary respiratory network that is considered a basis for the future interactive modeling-experimental studies. The model has been developed using a series of assumptions. Specifically, we have suggested that, under normal conditions in vivo, the eupnoeic respiratory rhythm is generated by a ponto-medullary network. Hence, although the pre-BotC is a necessary part of this network, the intrinsic oscillations in this region are suppressed during eupnoea by ponto-medullary interactions. These endogenous oscillations, however, may be released under some specific conditions, e.g., in vitro, because of the lack of the pons, or during hypoxia in vivo 6. We have also assumed that the medullary part of the respiratory network contains special neural circuits performing the respiratory phase switching. Moreover, these circuits are also targets for pulmonary feedback and inputs from the pons and major afferent nerves, which use the same medullary switching circuits to regulate the timing of phase transitions and modulate the respiratory motor pattern7.