Peter M. Lalley

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.

Reflex firing in the lumbar sympathetic outflow to activation of vesical afferent fibres

1. Activation of vesical afferent fibres in the Aγδ range by electrical stimulation of the pelvic nerve or by bladder distension elicited reflex firing in hypogastric nerves and in preganglionic nerves to the inferior mesenteric ganglion.

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.

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.

Serotonin targets inhibitory synapses to induce modulation of network functions

The cellular effects of serotonin (5-HT), a neuromodulator with widespread influences in the central nervous system, have been investigated. Despite detailed knowledge about the molecular biology of cellular signalling, it is not possible to anticipate the responses of neuronal networks to a global action of 5-HT. Heterogeneous expression of various subtypes of serotonin receptors (5-HTR) in a variety of neurons differently equipped with cell-specific transmitter receptors and ion channel assemblies can provoke diverse cellular reactions resulting in various forms of network adjustment and, hence, motor behaviour. Using the respiratory network as a model for reciprocal synaptic inhibition, we demonstrate that 5-HT1AR modulation primarily affects inhibition through glycinergic synapses. Potentiation of glycinergic inhibition of both excitatory and inhibitory neurons induces a functional reorganization of the network leading to a characteristic change of motor output. The changes in network operation are robust and help to overcome opiate-induced respiratory depression. Hence, 5-HT1AR activation stabilizes the rhythmicity of breathing during opiate medication of pain.

Spatial profiles of store‐dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca2+ buffering capacity and an exceptional vulnerability to Ca2+‐mediated excitotoxic events. In this report, we investigated local Ca2+ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca2+ release events, which displayed an initial peak followed by a slow ‘shoulder’ lasting several seconds. Peak amplitudes were paralleled by Ca2+‐activated, apamin‐sensitive K+ currents (IKCa), demonstrating a functional link between Ca2+ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently IKCa, indicating that functionally intact mitochondria were critical for store‐dependent modulation of HMN excitability. Taken together, our results indicate localized Ca2+ release profiles in HMNs, where low buffering capacities enhance the role of Ca2+‐regulating organelles as local determinants of [Ca2+]i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS‐related, cellular disturbances.

Serotonergic modulation of intracellular calcium dynamics in neonatal hypoglossal motoneurons from mouse

(1) Serotonin (5HT)-mediated calcium signaling was investigated in hypoglossal motoneurons (HGMs) in brain stem slices of neonatal mice. Electrical activity and associated calcium signaling were studied by simultaneous patch clamp recordings and high resolution calcium imaging. (2) Bath application of 5HT (5-50 microM) depolarized membrane potential of HGMs and generated action potential discharges that were accompanied by elevations in intracellular calcium concentrations ([Ca2+]i) in the soma and dendrites. Current-evoked bursts of action potentials were more intense in the presence of 5HT; however, the corresponding calcium signals were reduced. (3) The 5HT2 receptor agonist alpha-Methyl-5HT (25, 50 microM) had effects on membrane potential, discharge properties and [Ca]i that were identical to those observed for 5HT, whereas the 5HT3 receptor agonist 1-(m-chlorophenyl) biguanide (50 microM) had no effect on membrane properties or intracellular calcium levels. (4) 8-OHDPAT (25, 50 microM), a 5HT1A receptor agonist, was without effect on steady-state membrane potential or basal [Ca]i. Similar to 5HT and alpha-Methyl-5HT, 8-OHDPAT depressed stimulus-evoked calcium transients in current and voltage clamp mode. (5) Our results suggest that calcium profiles in hypoglossal motoneurons are differentially regulated by 5HT1A and 5HT2 receptors. Activation of 5HT1A receptors primarily reduced voltage-activated Ca2+ signals without a significant impact on basal [Ca]i. In contrast, activation of 5HT2 receptors initiated a net inward current followed by membrane depolarization, where the resulting pattern of action potential discharges represents the essential determinant of global elevations in [Ca2+]i. Taken together, our results therefore identify 5HT-dependent signal pathways as a versatile tool to modulate hypoglossal motoneuron excitability under various physiological and pathophysiological conditions.

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.

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).

Activating convergent signal pathways in respiratory neurons of the ventral medullary group

Opiates are known to disrupt the respiratory rhythm by binding to μ- and δ-type opioid receptors of respiratory neurons within the ventral medullary group and by activating signal pathways that induce depression of neuronal excitability and synaptic interaction. In previous experiments we demonstrated that opioid depression of respiration can be treated by a variety of drugs that increase intracellular cAMP levels. In the present study, we investigated how activation of μ-type opioid receptors can be counteracted and respiratory depression be treated by activation of convergent signal pathways targeting the same second messenger systems of the neurons. Our starting point was the observation that a compensatory effect can be achieved with Buspirone, a drug purported to activate 5-HT1A receptors and, consequently, to reduce intracellular cAMP levels [1]. If these effects were confirmed, this observation would indicate a profound non-specificity of the 5-HT-1A directed drug. The task then would be to identify the serotonin receptor isoforms responsible. In various preparations, including the anaesthetized in vivo cat, the perfused mouse or rat brain stem and the brain stem slice of the mouse or rat, we performed current and voltage clamp measurements with fine tipped or patch electrodes to measure electrophysiological parameters. RT-PCR methods were applied to identify the mRNA encoding for serotonin receptor isoforms, while immunocytochemical techniques were used to verify receptor expression. We verified the stabilizing effect of cAMP [2,3] and confirmed the protective effect of 5-HT1A-receptor agonists 8-OHDPAT and Buspirone against opioid depression of neural respiratory activity [4]. A detailed inspection of the results obtained with the commonly used 5-HT1A-agonists, 8-OH-DPAT and Buspirone, indicated ambiguous effects of these drugs. Our conclusion was that these drugs probably act on other serotonin receptor isoforms which are expressed in addition to the known 5-HT1A and 5-HT2A subtypes. Therefore, we developed two novel antibodies and demonstrated the expression of 5-HT4 and 5-HT7 receptors in neurons of the VRG and the hypoglossal nuclei. These immunocytochemical findings were verified by quantitative RT-PCR analysis of the VRG region and confirmed in single cell RT-PCR analysis on identified respiratory neurons. Finally, we demonstrated that these findings provide a basis for novel strategies for the treatment of respiratory depression induced by opioids. The same strategy seems to be efficient in the treatment of respiratory disturbances induced by barbiturates.