GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

Genetic ablation of VIAAT in glycinergic neurons causes a severe respiratory phenotype and perinatal death.

Both glycinergic and GABAergic neurons require the vesicular inhibitory amino acid transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic glycine content depends on the plasma membrane glycine transporter 2 (GlyT2). Although severely impaired, glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of glycine uptake or de novo synthesis of glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.

Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.

Deletion of the cell adhesion adaptor protein vinculin disturbs the localization of GFAP in Bergmann glial cells.

Astrocytes operate in close spatial relationship to other cells including neurons. Structural interaction is controlled by a dynamic interplay between actin-based cell motility and contact formation via cell-cell and cell-extracellular matrix adhesions. A central player in the control of cell adhesion is the cytoskeletal adaptor protein Vinculin. Incorporation of Vinculin affects mechanical properties and turnover of cell adhesion sites. To study the in vivo function of Vinculin in astrocytes, a mouse line with astrocyte specific and inducible deletion of vinculin was generated. Deletion of vinculin decreased the expression of the glial acidic fibrillary protein (GFAP) in Bergmann glial cells in the cerebellum. In addition, localization of GFAP to Bergmann glial endfeet was disturbed, indicating a role for vinculin in controlling its expression and localization. In contrast, vimentin expression, morphology, activation state and polarity of the targeted cells as well as the localization of the extracellular matrix protein laminin was not compromised. Furthermore, stab wound lesions were performed in the cerebellar cortex. In both wildtype and vinculin knockout mice GFAP expression was upregulated in Bergmann glial cells of the lesioned area with no differences observed between genotypes in expression and localization of GFAP. These results propose a selective requirement for vinculin in cellular events related to cell adhesion in vivo. As in vitro data suggested a major role for vinculin in the control of the cytoskeletal connection affecting mechanical stability and cell motility, our data add a note of caution to the extrapolation of in vitro data to in vivo function.

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.

Ca²âº signals of astrocytes are modulated by the NAD⁺/NADH redox state.

Astrocytes are important glial cells in the brain providing metabolic support to neurons as well as contributing to brain signaling. These different functional levels have to be highly coordinated to allow for proper cell and brain function. In this study, we show that in astrocytes the NAD(+) /NADH redox state modulates dopamine-induced Ca(2+) signals thereby connecting metabolism and Ca(2+) signaling. Application of dopamine induced a dose-dependent increase in Ca(2+) signal frequency in these cells, which was dependent on D(1) -receptor signaling, glycolytic activity, an increase in cytosolic NADH and inositol 1,4,5-triphosphate receptor operated intracellular Ca(2+) stores. Application of dopamine at a low concentration (1 µM) did not induce an increase in Ca(2+) signal frequency by itself. However, simultaneously increasing cytosolic NADH content either by direct application of NADH or by application of lactate resulted in a pronounced increase in Ca(2+) signal frequency. This increase could be blocked by co-application of pyruvate, suggesting that indeed the NAD(+) /NADH redox state is regulating Ca(2+) signals. We conclude that at the NAD(+) /NADH redox state metabolic and signaling information is integrated in astrocytes, thereby most likely contributing to precisely coordinate these different tasks of astrocytes.

Olfactory receptors and signalling elements in the Grueneberg ganglion.

The Grueneberg ganglion (GG) is a cluster of neurones present in the vestibule of the anterior nasal cavity. Although its function is still elusive, recent studies have shown that cells of the GG transcribe the gene encoding the olfactory marker protein (OMP) and project their axons to glomeruli of the olfactory bulb, suggesting that they may have a chemosensory function. Chemosensory responsiveness of olfactory neurones in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) is based on the expression of either odorant receptors or vomeronasal putative pheromone receptors. To scrutinize its presumptive olfactory nature, the GG was assessed for receptor expression by extensive RT-PCR analyses, leading to the identification of a distinct vomeronasal receptor which was expressed in the majority of OMP-positive GG neurones. Along with this receptor, these cells expressed the G proteins Go and Gi, both of which are also present in sensory neurones of the vomeronasal organ. Odorant receptors were expressed by very few cells during prenatal and perinatal stages; a similar number of cells expressed adenylyl cyclase type III and G(olf/s), characteristic signalling elements of the main olfactory system. The findings of the study support the notion that the GG is in fact a subunit of the complex olfactory system, comprising cells with either a VNO-like or a MOE-like phenotype. Moreover, expression of a vomeronasal receptor indicates that the GG might serve to detect pheromones.

A novel population of neuronal cells expressing the olfactory marker protein (OMP) in the anterior/dorsal region of the nasal cavity.

The olfactory marker protein (OMP) is expressed in mature chemosensory neurons in the nasal neuroepithelium. Here, we report the identification of a novel population of OMP-expressing neurons located bilaterally in the anterior/dorsal region of each nasal cavity at the septum. These cells are clearly separated from the regio olfactoria, harboring the olfactory sensory neurons. During mouse development, the arrangement of the anterior OMP-cells undergoes considerable change. They appear at about stage E13 and are localized in the nasal epithelium during early stages; by epithelial budding, ganglion-shaped clusters are formed in the mesenchyme during the perinatal phase, and a filiform layer directly underneath the nasal epithelium is established in adults. The anterior OMP-cells extend long axonal processes which form bundles and project towards the brain. The data suggest that the newly discovered group of OMP-cells in the anterior region of the nasal cavity may serve a distinct sensory function.