Diethelm W. Richter

Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition.

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.

Deletion of the Mouse Glycine Transporter 2 Results in a Hyperekplexia Phenotype and Postnatal Lethality

The glycine transporter subtype 2 (GlyT2) is localized in the axon terminals of glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.

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.

Selective lesioning of the cat pre‐Bötzinger complex in vivo eliminates breathing but not gasping

1 To examine the functional importance of the pre‐Bötzinger complex for breathing we micro‐injected, under in vivo conditions, the calcium channel blocker ω‐conotoxin GVIA and the sodium channel blocker tetrodotoxin (TTX) into the ventrolateral medulla of adult cats, while monitoring respiratory rhythmic motor output in the phrenic nerve. 2 ω‐Conotoxin GVIA caused a highly localized synaptic ablation by blocking presynaptic N‐type calcium channels. When injecting 5–60 fmol ω‐conotoxin GVIA unilaterally, the amplitude of phrenic nerve activity decreased bilaterally and sometimes disappeared, indicating central apnoea. These effects were reversible and could only be induced in a very localized area of the pre‐Bötzinger complex. By injecting ω‐conotoxin GVIA several times during an experiment and analysing the areas where injections affected respiratory activity, it was possible to map exactly the anatomical extent of the area critical for respiratory rhythm generation. 3 Following the precise localization of the pre‐Bötzinger complex with ω‐conotoxin GVIA, we injected TTX to induce an irreversible inactivation of this region. TTX injected unilaterally into the pre‐Bötzinger complex irreversibly reduced the amplitude of phrenic nerve activity. Bilateral TTX injections eliminated respiratory rhythmic activity, causing a persistent central apnoea. 4 After bilateral lesioning of the pre‐Bötzinger complex, it was still possible to induce gasping during hypoxia or asphyxia, indicating that respiration and gasping are generated by two different neuronal networks. 5 We propose that ω‐conotoxin GVIA as employed in this study to investigate the functional role of the pre‐Bötzinger complex can also be used as a general pharmacological approach to map other neuronal networks. We call this the ‘ω‐conotoxin GVIA tracing’ method.

The neuronal mechanisms of respiratory rhythm generation

New, improved in vivo and in vitro approaches have led to a better understanding of the mechanisms that generate respiratory rhythm, which depends on a complex interaction between network and intrinsic membrane properties. The pre-Bötzinger complex in the ventrolateral medulla is particularly important for respiratory rhythm generation. This complex can be studied in isolation, and it contains all the known types of respiratory neurons that are now amenable to detailed cellular and molecular analyses.

Mechanisms of respiratory rhythm generation

Abstract In mammals, a three-phasic respiratory rhythm is generated by a network of various types of neurons in the lower brainstem. The cellular mechanisms of rhythmogenesis involve cooperative interactions between synaptic processes and specific membrane properties. The network seems to be driven by extrinsic sources in mature animals, whereas in the immature network pacemaker neurons might be involved.

Response of the medullary respiratory network of the cat to hypoxia.

1. The effect of systemic hypoxia was tested in anaesthetized, immobilized, thoracotomized and artificially ventilated cats with peripheral chemoreceptor afferents either intact or cut. Extracellular recordings from different types of medullary respiratory neurones and intracellular recordings from stage 2 expiratory neurones were made to determine the hypoxia‐induced changes in neuronal discharge patterns and postsynaptic activity as an index for the disturbances of synaptic interaction within the network. 2. The general effect of systemic hypoxia was an initial augmentation of respiratory activity followed by a secondary depression. In chemoreceptor‐denervated animals, secondary depression led to central apnoea. 3. The effects of systemic hypoxia were comparable with those of cerebral ischaemia following occlusion of carotid and vertebral arteries. 4. In chemoreceptor‐denervated animals, all types of medullary respiratory neurones ceased spontaneous action potential discharge during hypoxia. 5. Reversal of inhibitory postsynaptic potentials (IPSPs) and/or blockade of IPSPs was seen after 2‐3 min of hypoxia. 6. During hypoxia, the membrane potential of stage 2 expiratory neurones showed a slight depolarization to ‐45 to ‐55 mV and then remained stable. 7. The neurone input resistance increased initially and then decreased significantly during central apnoea. 8. Rhythmogenesis of respiration was greatly disturbed. This was due to blockade of IPSPs and, in some animals, to more complex disturbances of phase switching from inspiration to expiration. 9. Central apnoea occurred while respiratory neurones were still excitable as shown by stimulus‐evoked orthodromic and antidromic action potentials. 10. The results indicate that the medullary respiratory network is directly affected by energy depletion. There is indication for a neurohumoral mechanism which blocks synaptic interaction between respiratory neurones in chemoreceptor‐intact animals.

Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats.

1. O2‐, K(+)‐ and pH‐sensitive microelectrodes were used to measure extracellular oxygen pressure (PO2), K+ activity (aKo) and pH (pHo) in ventral regions of the medulla oblongata containing respiratory neurons in the in vitro brainstem‐spinal cord preparation from 0 to 4‐day‐old rats. 2. The location of respiratory neurons was mapped by extracellular recordings with conventional microelectrodes, or with the reference barrel of ion‐sensitive microelectrodes. The major populations of respiratory neurons were distributed in the ventrolateral reticular formation near the nucleus ambiguus at depths of 300‐600 microns. In this area, aKo baseline increased from 3.2 to 3.8 mM whereas steady‐state values of PO2 and pHo fell from 120 to 7 mmHg and from 6.9 to 6.7, respectively. 3. During rhythmic inspiratory discharges recorded with suction electrodes from ventral roots of spinal (C3‐C5) and cranial (IX, X, XII) nerves, aKo transiently increased by up to 100 microM, and PO2 fell maximally by 0.4 mmHg. During episodes of non‐rhythmic neuronal discharge, aKo increased by as much as 0.4 mM and PO2 decreased by about 10 mmHg. In contrast, no variations in pHo could be detected during such activities. 4. Activation of medullary neurons by tetanic electrical stimulation of axonal tracts in the ventrolateral column of the spinal cord at the level of the phrenic motoneuron pool produced aKo elevations of up to 5 mM, decreases of PO2 by up to 50 mmHg, and pHo increases by a maximum of 0.07 pH units. These aKo and PO2 transients were reduced by more than 80% during blockade of synaptic transmission with 5 mM manganese (Mn2+) and completely blocked by 1 microM tetrodotoxin (TTX). 5. The tissue PO2 gradient as well as activity‐related decreases of PO2 were completely abolished after block of oxidative cellular metabolism by addition of 2‐10 mM cyanide (CN‐) to the bathing solution. 6. Inhibition of the Na(+)‐K+ pump by addition of 3‐50 microM ouabain (3‐10 min) caused a reversible increase of aKo by 0.8‐3 mM, a delayed recovery of stimulus‐induced aKo elevations, and produced a disturbance of the respiratory rhythm. 7. The sensitivity of the respiratory network to oxygen depletion was tested by superfusing the neuraxis with hypoxic solutions gassed with N2 instead of O2 (5‐20 min).(ABSTRACT TRUNCATED AT 400 WORDS)

5-HT7 Receptor Is Coupled to Gα Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

Breathing dysfunctions associated with impaired control of postinspiratory activity in Mecp2−/y knockout mice

Rett syndrome (RTT) is an inborn neurodevelopmental disorder caused by mutations in the X‐linked methyl‐CpG binding protein 2 gene (MECP2). Besides mental retardation, most patients suffer from potentially life‐threatening breathing arrhythmia. To study its pathophysiology, we performed comparative analyses of the breathing phenotype of Mecp2−/y knockout (KO) and C57BL/6J wild‐type mice using the perfused working heart–brainstem preparation (WHBP). We simultaneously recorded phrenic and efferent vagal nerve activities to analyse the motor pattern of respiration, discriminating between inspiration, postinspiration and late expiration. Our results revealed respiratory disturbances in KO preparations that were similar to those reported from in vivo measurements in KO mice and also to those seen in RTT patients. The main finding was a highly variable postinspiratory activity in KO mice that correlated closely with breathing arrhythmias leading to repetitive apnoeas even under undisturbed control conditions. Analysis of the pontine and peripheral sensory regulation of postinspiratory activity in KO preparations revealed: (i) prolonged apnoeas associated with enhanced postinspiratory activity after glutamate‐induced activation of the pontine Kölliker‐Fuse nucleus; and (ii) prolonged apnoeas and lack of reflex desensitization in response to repetitive vagal stimulations. We conclude that impaired network and sensory mediated synaptic control of postinspiration induces severe breathing dysfunctions in Mecp2−/y KO preparations. As postinspiration is particularly important for the control of laryngeal adductors, the finding might explain the upper airway‐related clinical problems of patients with RTT such as apnoeas, loss of speech and weak coordination of breathing and swallowing.