The postnatal development of ultrasonic vocalization-associated breathing is altered in glycine transporter 2-deficient mice.

KEY POINTS: Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2-knockout mice do not acquire adult ultrasonic vocalization-associated breathing patterns. Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read-out in translational research, these data are highly relevant for a broad range of research fields. ABSTRACT: Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post-inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2-knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2-KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild-type mice acquire different USV-associated breathing patterns that depend on post-inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2-KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.

Mixed miniature postsynaptic currents resulting from co-release of glycine and GABA recorded from glycinergic neurons in the neonatal respiratory network.

Inhibitory neurons are involved in the generation and patterning of the respiratory rhythm in the adult animal. However, the role of glycinergic neurons in the respiratory rhythm in the developing network is still not understood. Although the complete loss of glycinergic transmission in vivo is lethal, the blockade of glycinergic transmission in slices of the medulla has little effect on pre-Bötzinger complex network activity. As 50% of the respiratory rhythmic neurons in this slice preparation are glycinergic, they have to be considered as integrated parts of the network. We aimed to investigate whether glycinergic neurons receive mixed miniature inhibitory postsynaptic currents (mIPSCs) that result from co-release of GABA and glycine. Quantification of mixed mIPSCs by the use of different objective detection methods resulted in a wide range of results. Therefore, we generated traces of mIPSCs with a known distribution of mixed mIPSCs and mono-transmitter-induced mIPSCs, and tested the detection methods on the simulated data. We found that analysis paradigms, which are based on fitting the sum of two mIPSC templates, to be most acceptable. On the basis of these protocols, 20-40% of all mIPSCs recorded from respiratory glycinergic neurons are mixed mIPSCs that result from co-release of GABA and glycine. Furthermore, single-cell reverse transcriptase polymerase chain reaction revealed that 46% of glycinergic neurons co-express mRNA of glycine transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Bötzinger complex that might be involved in the shaping of synaptic inhibition of respiratory glycinergic neurons.

Development of synaptic inhibition in glycine transporter 2 deficient mice.

Mice deficient for the neuronal glycine transporter subtype 2 (GlyT2) die during the second postnatal week after developing neuromotor deficiencies, which resembles severe forms of human hyperekplexia. This phenotype has been attributed to a dramatic reduction in glycinergic neurotransmission. In the present study we analyzed the development of GABAergic and glycinergic synaptic transmission in GlyT2-knockout mice during early postnatal life. Anti-glycine immunohistochemistry in spinal cord and brainstem slices and whole-cell voltage-clamp recordings of glycinergic inhibitory postsynaptic currents (IPSCs) from hypoglossal motoneurons revealed strikingly reduced levels of synaptic glycine already at birth. Since GABA and glycine use the same vesicular inhibitory amino acid transporter (VIAAT or VGAT) we also analysed GABAergic neurotransmission. No increase of GABA immunoreactivity was observed in the spinal cord and brainstem of GlyT2(-/-) mice at any stage of postnatal development. Correspondingly no up-regulation of GABAergic IPSCs was detected in GlyT2(-/-) hypoglossal motoneurons. These data suggest that in the first postnatal week, loss of the glycine transporter 2 is neither compensated by glycine de-novo synthesis nor by up-regulation of the GABAergic transmission in GlyT2(-/-) mice.

Hyperekplexia phenotype of glycine receptor alpha1 subunit mutant mice identifies Zn(2+) 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.

Packing characteristics of a model system mimicking cytoplasmic bacterial membranes.

The phase diagram of fully hydrated mixtures of dipalmitoylphosphatidylethanolamine and -phosphatidylglycerol was constructed and the coexistence lines of the solidus and liquidus curve calculated based on regular solution theory using two nonideality parameters for each of the phase to account for nonideal and nonsymmetric mixing. Both lipids show nonideal miscibility in the liquid-crystalline phase, while a region of immiscibility exists in the lamellar-gel phase between the mole fraction x(DPPE)=0.05-0.4. Two lines of three-phase coexistence around 35 and 40 degrees C reflects the presence of lipid domains predominantly composed of phosphatidylglycerol as well as of the mixed lipid system. This is reflected in the positive nonideality parameters of the gel phase obtained from the simulation of the phase diagram. Moreover, segregation of pure phosphatidylethanolamine domains was detected in mixtures x(DPPE)>0.9, which formed multilamellar liposomes, while unilamellarity was observed for the mixed lipid systems owing to the presence of the negatively charged phosphatidylglycerol. The packing constraints of these phospholipids, major components of cytoplasmic bacterial membranes, may be of importance in the interaction with various solutes like antimicrobial peptides, and were explained based on the nature of the headgroups and the molecular geometry of the phospholipids.

Structural aspects of the interaction of peptidyl-glycylleucine-carboxyamide, a highly potent antimicrobial peptide from frog skin, with lipids.

The interaction of PGLa (peptidyl-glycylleucine-carboxyamide), a 21-amino-acid residue cationic peptide, isolated from the skin of the South African clawed frog, Xenopus laevis, with model membrane systems was investigated. Our studies focussed on the importance of the difference in the phospholipid composition of bacterial and erythrocyte membranes. This is of particular interest to gain information on the specificity of membranolysis exhibited by this peptide against bacteria but not against erythrocytes. In phosphate buffer at physiological pH, as well as in the presence of the zwitterionic phosphatidylcholine and sphingomyelin. the peptide had a random structure but it adopted an alpha-helical conformation in the presence of negatively charged lipids. Furthermore, calorimetric experiments showed that PGLa had no effects on the thermotropic phase behavior of liposomes composed of the choline phosphatides, while separation of a distinct peptide-rich domain was observed for phosphatidylglycerol liposomes. In addition to the main transition of pure 1,2-dipalmitoylglycerophosphoglycerol at 40 degrees C a second transition owing to the peptide-perturbed lipid domains was found at 41 degrees C. This conclusion is supported by X-ray diffraction experiments which indicated that PGLa penetrates into the hydrophobic core of the bilayer inducing an untilting of the hydrocarbon chains as observed in the gel phase of the pure lipid. These results demonstrate that this antibacterial peptide specifically interacts with negatively charged lipid membranes, which are characteristic of bacterial membranes. This can be explained based on the structural features of PGLa.

Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems.

alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.