Swen Hülsmann

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.

Astroglial processes show spontaneous motility at active synaptic terminals in situ.

Within the tripartite structure of vertebrate synapses, enwrapping astroglial processes regulate synaptic transmission by transmitter uptake and by direct transmitter release. We applied confocal and two‐photon laser scanning microscopy to acutely isolated slices prepared from the brainstem of transgenic TgN(GFAP‐EGFP) mice. In transversal sections fluorescently labelled astrocytes are evenly distributed throughout the tissue. Astroglial processes contacted neuronal somata and enwrapped active synaptic terminals as visualized using FM1‐43 staining in situ. Here, at these synaptic regions astroglial process endings displayed a high degree of dynamic morphological changes. Two defined modes of spontaneous motility could be distinguished: (i) gliding of thin lamellipodia‐like membrane protrusions along neuronal surfaces and (ii) transient extensions of filopodia‐like processes into the neuronal environment. Our observations highlight the active role of astrocytes in direct modulation of synaptic transmission.

Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice

Collybistin (Cb) is a brain‐specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and GABAergic synapses. Here we show that Cb‐deficient mice display a region‐specific loss of postsynaptic gephyrin and GABAA receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long‐term potentiation is enhanced, and long‐term depression reduced, in Cb‐deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin‐dependent clustering of a specific set of GABAA receptors, but not required for glycine receptor postsynaptic localization.

Hyperekplexia Phenotype of Glycine Receptor α1 Subunit Mutant Mice Identifies Zn2+ as an Essential Endogenous Modulator of Glycinergic Neurotransmission

Zn(2+) is thought to modulate neurotransmission by affecting currents mediated by ligand-gated ion channels and transmitter reuptake by Na(+)-dependent transporter systems. Here, we examined the in vivo relevance of Zn(2+) neuromodulation by producing knockin mice carrying the mutation D80A in the glycine receptor (GlyR) alpha1 subunit gene (Glra1). This substitution selectively eliminates the potentiating effect of Zn(2+) on GlyR currents. Mice homozygous for Glra1(D80A) develop a severe neuromotor phenotype postnatally that resembles forms of human hyperekplexia (startle disease) caused by mutations in GlyR genes. In spinal neurons and brainstem slices from Glra1(D80A) mice, GlyR expression, synaptic localization, and basal glycinergic transmission were normal; however, potentiation of spontaneous glycinergic currents by Zn(2+) was significantly impaired. Thus, the hyperekplexia phenotype of Glra1(D80A) mice is due to the loss of Zn(2+) potentiation of alpha1 subunit containing GlyRs, indicating that synaptic Zn(2+) is essential for proper in vivo functioning of glycinergic neurotransmission.

Diversity of Functional Astroglial Properties in the Respiratory Network

A population of neurons in the caudal medulla generates the rhythmic activity underlying breathing movements. Although this neuronal network has attracted great attention for studying neuronal aspects of synaptic transmission, functions of glial cells supporting this neuronal activity remain unclear. To investigate the role of astrocytes in the respiratory network, we applied electrophysiological and immunohistochemical techniques to characterize astrocytes in regions involved in the generation and transmission of rhythmic activity. In the ventral respiratory group and the hypoglossal nucleus (XII) of acutely isolated brainstem slices, we analyzed fluorescently labeled astrocytes obtained from TgN(GFAP-EGFP) transgenic mice with the whole-cell voltage-clamp technique. Three subpopulations of astrocytes could be discerned by their distinct membrane current profiles. A first group of astrocytes was characterized by nonrectifying, symmetrical and voltage-independent potassium currents and a robust glutamate transporter response to d-aspartate. A second group of astrocytes showed additional A-type potassium currents, whereas a third group, identified by immunolabeling for the glial progenitor marker NG2, expressed outwardly rectifying potassium currents, smaller potassium inward currents, and only minimal d-aspartate-induced transporter currents. Astrocytes of all groups showed kainate-induced inward currents. We conclude that most of the astrocytes serve as a buffer system of excess extracellular glutamate and potassium; however, a distinct cell population (NG2-positive, A-type potassium currents) may play an important role for network plasticity.

Metabolic coupling between glia and neurons is necessary for maintaining respiratory activity in transverse medullary slices of neonatal mouse

The respiratory rhythm is generated and regulated by a neuronal network within the lower brainstem. While the neuronal mechanisms of rhythm generation have been extensively investigated, the contribution of glial cells remains to be determined. Here we report the effect of specific blockade of the glial Krebs cycle and glutamine synthetase on the neuronal activity of the respiratory network. Application of 5 mm fluoroacetate, which selectively blocks the glial Krebs cycle, suppressed rhythmic respiratory burst activity. Substitution of either the Krebs cycle substrate isocitrate (3 mm) or glutamine (3 mm) restored rhythmic network activity. Blockade of glutamine synthetase by methionine sulfoximine (0.5 mm) suppressed rhythmic burst activity as well. Resubstitution of glutamine (3 mm) was able to restore rhythmic activity in the presence of methionine sulfoximine. This data demonstrates that the glutamate–glutamine cycle in astrocytes and their supply of glutamine to neuronal glutamatergic terminals is essential for the rhythm generation in the respiratory centre.

Insulin-Like Growth Factor-1 Exerts Ca2+-Dependent Positive Inotropic Effects in Failing Human Myocardium

Abstract— Myocardial generation of insulin-like growth factor-1 (IGF-1) is altered in hypertrophy and heart failure, but there are no reports on acute functional effects of IGF-1 in human cardiac muscle. We examined inotropic responses and signal transduction mechanisms of IGF-1 in human myocardium. Experiments were performed in isolated trabeculae or cardiomyocytes from 46 end-stage failing hearts. The effect of IGF-1 (0.001 to 0.2 &mgr;mol/L) on isometric twitch force (37°C, 1 Hz), intracellular Ca2+ transients (aequorin method), sarcoplasmic reticulum (SR) Ca2+ content (rapid cooling contractures), L-type Ca2+ current (whole-cell voltage clamp), and cAMP concentrations was assessed. In addition, the effects of blocking IGF-1 receptors, phosphoinositide 3-kinase (PI3-kinase), protein kinase C (PKC), or transsarcolemmal Ca2+ entry were tested. IGF-1 exerted concentration-dependent positive inotropic effects (twitch force increased to maximally 133±4% of baseline values at 0.1 &mgr;mol/L;P <0.05). The IGF-1 receptor antibody &agr;IR3 or the PI3-kinase inhibitor wortmannin prevented the functional effects. The inotropic response was paralleled by increases in Ca2+ transients and SR Ca2+ content. IGF-1 (0.1 &mgr;mol/L) increased L-type Ca2+ current amplitude by 24±7% (P <0.05). Blockade of SR function did not affect the inotropic response to IGF-1. In contrast, L-type Ca2+ channel blockade with diltiazem partially prevented (≈50%) the inotropic response to IGF-1. Inhibition of PKC (GF109203X), Na+-H+ exchange (HOE642), or reverse-mode Na+-Ca2+ exchange (KB-R7943) reduced the response to IGF-1 by ≈60% to 70%. IGF-1 exerts Ca2+-dependent positive inotropic effects through activation of IGF-1 receptors and a PI3-kinase-dependent pathway in failing human myocardium. The increased [Ca2+]i with IGF-1 originates from both enhanced L-type Ca2+ currents and enhanced Na+-H+ exchange-dependent reverse-mode Na+-Ca2+ exchange. These nongenomic functional effects of IGF-1 may be of clinical relevance.

Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls breathing in mice

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.

Progressive loss of a glial potassium channel (KCNJ10) in the spinal cord of the SOD1 (G93A) transgenic mouse model of amyotrophic lateral sclerosis

Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1G93A) were used to investigate the role of glial inwardly rectifying K+ (Kir)4.1 channels, which buffer extracellular K+ increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1G93A mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K+. To understand the functional implications of extracellular K+ increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K+ concentrations. Exposure to high extracellular K+ induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K+ homeostasis and may contribute to motor neurone degeneration in SOD1G93A mutants by K+ excitotoxic mechanisms.