Anne M. Bischoff

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 .

5-HT-1A receptor-mediated modulation of medullary expiratory neurones in the cat.

The involvement of the 5‐HT‐1A receptor in serotoninergic responses of stage 2 expiratory (E‐2) neurones was investigated in pentobarbitone‐anaesthetized, mechanically ventilated cats. The specific agonist of the 5‐HT‐1A receptor, 8‐hydroxy‐diproplaminotetralin (8‐OH‐DPAT), administered systemically or by ionophoresis directly on to the neurones, had a clear depressant effect. Administration of 8‐OH‐DPAT at doses of 10‐50 micrograms kg‐1 (I.V.) increased the membrane hyperpolarizations of E‐2 neurones during the inspiratory and postinspiratory phases, and shortened their duration of activity in association with shortening of phrenic nerve activity. Discharges of E‐2 neurones were also less intense. At doses of 50‐90 micrograms kg‐1, 8‐OH‐DPAT reduced or abolished inspiratory hyperpolarizations, and reduced expiratory depolarizations of membrane potential and discharge in parallel with inhibition of phrenic nerve discharges. The effects of the larger doses were reversed by I.V. injection of NAN‐190, an antagonist at the 5‐HT‐1A receptor. Dose‐dependent effects on the membrane potential and discharge of E‐2 neurones, but not on phrenic nerve activity, were also seen by ionophoretic administration of 8‐OH‐DPAT on to E‐2 neurones. At low currents, ejection of 8‐OH‐DPAT hyperpolarized the neurones without affecting the duration of inspiratory hyperpolarization and expiratory depolarization. This hyperpolarization depressed the intensity and the duration of expiratory discharges. Ejection with larger currents hyperpolarized the E‐2 neurones further, and depressed expiratory depolarization leading to blockade of expiratory discharges. The effects on membrane potential were accompanied by decreased neuronal input resistance. This depressed the excitability of E‐2 neurones as tested by discharge evoked by intracellular current injection. The amplitudes of action potentials decreased in parallel with the changes in input resistance. The effects were attributed to a postsynaptic effect of 8‐OH‐DPAT leading to a gradually developing inhibition by activation of 5‐HT‐1A receptors. Hyperventilatory apnoea depressed on‐going synaptic activity and unmasked the effect of ionophoretically applied 8‐OH‐DPAT. The responses of the E‐2 neurone were enhanced, as evidenced by increased membrane hyperpolarization and greater reduction of input resistance. Both responses faded appreciably, indicating receptor desensitization. The degree and rate of apparent desensitization depended on the dose/ejecting current. The greater sensitivity and faster desensitization to 8‐OH‐DPAT were attributed to the hyperventilatory alkalinization of the extracellular fluid, which might influence agonist binding to 5HT‐1A receptors and/or receptor properties.

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.

Nucleus raphe obscurus evokes 5-HT-1A receptor-mediated modulation of respiratory neurons

We analysed in vivo the synaptic mechanisms underlying serotonin-mediated depression of expiratory neuronal discharges and phrenic nerve activity. We report that nucleus raphe obscurus stimulation not only abolishes phrenic nerve activity, but also hyperpolarizes the membrane potential, depresses periodic synaptic drive potentials and thus action potential discharges in caudal medullary expiratory neurons. These effects originate from pre- and post-synaptic inhibitory processes that involve 5-HT-1A receptor activation.

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.

The respiratory rhythm in mutant oscillator mice.

Since glycinergic inhibition is important for respiratory rhythm generation in mature mammals, we tested the hypothesis that the loss of glycine receptors during postnatal development (P17-P23) of homozygous mutant oscillator mice (spd(ot)/spd(ot)) may result in serious impairment of respiratory rhythm. We measured breathing in a plethysmographic recording chamber on conscious oscillator mice and used an in situ perfused brainstem preparation to record phrenic nerve activity, as well as membrane properties of respiratory neurones. The deletion of glycinergic inhibition did not result in failure of respiratory rhythm: homozygous mutant oscillator mice continue to generate a disturbed respiratory rhythm until death. Postsynaptic activity and membrane potential trajectories of respiratory neurones revealed a persistence of GABAergic inhibition and changes in respiratory rhythm and pattern generation.

Respiratory Rhythm Generation: Plasticity of a Neuronal Network:

The exchange of gases between the external environment and the organism is controlled by a neural network of medullary neurons that produces rhythmic activity that ultimately leads to periodic contractions of thoracic, abdominal, and diaphragm muscles. This occurs in three neural phases: inspiration, postinspiration, and expiration. The present article deals with the mechanisms underlying respiratory rhythm generation and the processes of dynamic adjustment of respiratory activity by neuromodulation as it occurs during normoxia and hypoxia. The respiratory rhythm originates from the “pre-Bötzinger complex,” which is a morphologically defined region within the lower brainstem. There is a primary oscillating network consisting of reciprocally connected early-inspiratory and postinspiratory neurons, whereas various other subgroups of respiratory neurons shape the activity pattern. Rhythm generation and pattern formation result from neuronal interactions within the network, that is, from cooperative adjustments of intrinsic membrane properties and synaptic processes in the respiratory neurons. There is evidence that in neonatal mammals, as well as under certain pathological situations in adult mammals, the respiratory rhythm derives from early-inspiratory burster neurons that drive inspiratory output neurons. The respiratory network is influenced by a variety of neuromodulators. Stimulation of appropriate receptors mostly activates signal pathways that converge on cAMP-dependent protein kinase and protein kinase C. Both pathways exert modulatory effects on voltage- and ligand-controlled ion channels. Many neuromodulators are continuously released within the respiratory region or accumulated under pathological conditions such as hypoxia. The functional significance of such ongoing neuromodulation is seen in variations of network excitability. In this review, the authors concentrate on the modulators serotonin, adenosine, and opioids.

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‐HT1A receptor (5‐HT1AR) 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‐HT1AR‐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‐HT1AR activation can produce a disinhibition of inspiratory neurons, leading to the recovery of respiratory rhythm from opioid‐induced apnoeas.