Olga Alexandrova

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity

BackgroundApoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.ResultsWe have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected.ConclusionsOur results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.

Tuning of Ranvier node and internode properties in myelinated axons to adjust action potential timing

Action potential timing is fundamental to information processing; however, its determinants are not fully understood. Here we report unexpected structural specializations in the Ranvier nodes and internodes of auditory brainstem axons involved in sound localization. Myelination properties deviated significantly from the traditionally assumed structure. Axons responding best to low-frequency sounds had a larger diameter than high-frequency axons but, surprisingly, shorter internodes. Simulations predicted that this geometry helps to adjust the conduction velocity and timing of action potentials within the circuit. Electrophysiological recordings in vitro and in vivo confirmed higher conduction velocities in low-frequency axons. Moreover, internode length decreased and Ranvier node diameter increased progressively along the distal axon segments, which simulations show was essential to ensure precisely timed depolarization of the giant calyx of Held presynaptic terminal. Thus, individual anatomical parameters of myelinated axons can be tuned to optimize pathways involved in temporal processing.

Experience-Dependent Refinement of the Inhibitory Axons Projecting to the Medial Superior Olive

Neurons in the medial superior olive (MSO) analyze interaural time differences (ITDs) by comparing the arrival times of the two excitatory inputs from each ear using a coincidence detection mechanism. They also receive a prominent inhibitory, glycinergic projection from the ipsilateral medial nucleus of the trapezoid body (MNTB), which contributes to the fine‐tuning of ITD analysis. Here, we investigated developmental changes of the axonal arborisation pattern of single Microruby‐labeled MNTB neurons projecting to the MSO region. During the first 2 weeks after hearing onset, the axonal arborisation of MNTB neurons was significantly refined resulting in a narrowed projection area across the tonotopic axis of the MSO and a redistribution of the axonal endsegments to a mostly somatic location. Rearing the animals in omnidirectional noise prevented the structural changes of single MNTB projections. These results indicate that the functional elimination of inhibitory inputs on MSO neurons after hearing onset, as described previously, is paralleled by a structural, site‐specific refinement of the inputs and is dependent on the normal acoustic experience of the animal.

GFP expression in Hydra: lessons from the particle gun.

Abstract. The cnidarian Hydra is an important model organism to study pattern formation and stem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have not been accessible to gene transfection studies. We have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.

Hydra and the Evolution of Apoptosis

Abstract Programmed cell death occurs in most, if not all life forms. It is used to sculpt tissue during embryogenesis, to remove damaged cells, to protect against pathogen infection and to regulate cell numbers and tissue homeostasis. In animals cell death often occurs by a morphologically and biochemically conserved process called apoptosis. A novel group of cysteine proteases, referred to as caspases, constitute the central component of this process. Caspases are activated following the induction of apoptosis and cleave a variety of cellular substrates, thus giving rise to the characteristic morphological events of apoptosis. Apoptosis is rapid and cell corpses are removed by phagocytosis. Recent work has shown that apoptosis also occurs in Cnidaria and Porifera, thus extending the origin of this evolutionary innovation down to the first metazoan animal phyla. Here, we review several examples of the role of apoptosis in cnidarians and then summarize new results on the subcellular localization of caspases and the control of apoptosis in Hydra. We show by immuncytochemistry that caspases in Hydra are localized in mitochondria. Following induction of apoptosis caspases are released from mitochondria as proenzymes and then activated by proteolytic cleavage in the cytoplasm. We also present evidence that apoptosis in Hydra is dramatically stimulated by inhibitors of PI3-kinase. Since PI3-kinase is a central component of growth factor signaling cascades in higher metazoans, this result suggests that control of apoptosis by growth factors is also evolutionarily conserved. We speculate on the role of growth factors in the evolution of apoptosis.

Adaptation in sound localization: from GABA B receptor–mediated synaptic modulation to perception

Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABAB receptor–mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABAB receptor–mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

Action Potential Generation in an Anatomically Constrained Model of Medial Superior Olive Axons

Neurons in the medial superior olive (MSO) encode interaural time differences (ITDs) with sustained firing rates of >100 Hz. They are able to generate such high firing rates for several hundred milliseconds despite their extremely low-input resistances of only few megaohms and high synaptic conductances in vivo. The biophysical mechanisms by which these leaky neurons maintain their excitability are not understood. Since action potentials (APs) are usually assumed to be generated in the axon initial segment (AIS), we analyzed anatomical data of proximal MSO axons in Mongolian gerbils and found that the axon diameter is <1 μm and the internode length is ∼100 μm. Using a morphologically constrained computational model of the MSO axon, we show that these thin axons facilitate the excitability of the AIS. However, for ongoing high rates of synaptic inputs the model generates a substantial fraction of APs in its nodes of Ranvier. These distally initiated APs are mediated by a spatial gradient of sodium channel inactivation and a strong somatic current sink. The model also predicts that distal AP initiation increases the dynamic range of the rate code for ITDs.

Synthesis of pre-rRNA and mRNA is directed to a chromatin-poor compartment in the macronucleus of the spirotrichous ciliate Stylonychia lemnae

In contrast to the chromosomal genome organization common to most eukaryotes, DNA in the macronucleus of spirotrichous ciliates like Stylonychia lemnae is organized into small gene-sized nanochromosomes. We intended to elucidate whether a spatial organization of nucleoli similar to other eukaryotes can be found in absence of typical chromosomes. Whereas micronuclei of Stylonychia exhibit homogenously stained heterochromatin and possess no nucleoli, macronuclear chromatin is compartmentalized and contains numerous putative nucleoli. Since the identity of these spherical structures has never been unequivocally demonstrated to date, we applied immunofluorescence techniques together with confocal laser scanning microscopy to identify nucleolar bodies in the macronucleus of Stylonychia and to analyse their spatial organization. We found that multiple spherical bodies, which fulfil nucleolar function, occupy a peripheral localization in mature macronuclei. Using fibrillarin/Nop1p as a nucleolar marker, we monitored the assembly of such nucleolar bodies during macronuclear differentiation. 3D-FISH experiments revealed that rRNA genes are mostly concentrated adjacent to but not inside of fibrillarin/Nop1p-containing bodies. We further showed that transcription sites for rRNA synthesis but also for mRNA synthesis occur predominantly at surfaces of nucleolar bodies and chromatin-poor spaces bordering condensed chromatin. Our data suggest that transcription of rRNA genes in the macronucleus of Stylonychia does not rely on a classical nucleolus-type organization. We assume that vectorial synthesis and processing of rRNA and mRNA is directed to a functional interchromatin compartment.

Urocortin-expressing olivocochlear neurons exhibit tonotopic and developmental changes in the auditory brainstem and in the innervation of the cochlea†

The mammalian cochlea is under direct control of two groups of cholinergic auditory brainstem neurons, the medial and the lateral olivocochlear neurons. The former modulate the electromechanical amplification in outer hair cells and the latter the transduction of inner hair cells to auditory nerve fibers. The lateral olivocochlear neurons express not only acetylcholine but a variety of co‐transmitters including urocortin, which is known to regulate homeostatic responses related to stress; it may also be related to the ontogeny of hearing as well as the generation of hearing disorders. In the present study, we investigated the distribution of urocortin‐expressing lateral olivocochlear neurons and their connectivity and distribution of synaptic terminals in the cochlea of juvenile and adult gerbils. In contrast to most other rodents, the gerbils audiogram covers low frequencies similar to humans, although their communication calls are exclusively in the high‐frequency domain. We confirm that in the auditory brainstem urocortin is expressed exclusively in neurons within the lateral superior olive and their synaptic terminals in the cochlea. Moreover, we show that in adult gerbils urocortin expression is restricted to the medial, high‐frequency processing, limb of the lateral superior olive and to the mid and basal parts of the cochlea. The same pattern is present in juvenile gerbils shortly before hearing onset (P 9) but transiently disappears after hearing onset, when urocortin is also expressed in low‐frequency processing regions. These results suggest a possible role of urocortin in late cochlear development and in the processing of social calls in adult animals. J. Comp. Neurol. 519:2758–2778, 2011.

Input timing for spatial processing is precisely tuned via constant synaptic delays and myelination patterns in the auditory brainstem

Significance Neural computation depends on precisely timed synaptic inputs, but the way that the timing of inputs is tuned to match postsynaptic processing requirements is not well understood. Here, we studied the same brainstem sound localization pathway in two species with dissimilar temporal processing requirements. Two factors that limit precise timing are synaptic delay and axonal conduction time. In gerbils, which depend on precise timing for sound localization, synaptic delays in fast conducting axons are stable across activity level, and axon myelination is adapted to minimize conduction delays. In mice, which do not depend on precise timing, these specializations are absent. Our results suggest that both axonal and synaptic properties are optimized to the specific functional requirements of neural computation, advancing our understanding of the mechanisms that optimize neural circuits. Precise timing of synaptic inputs is a fundamental principle of neural circuit processing. The temporal precision of postsynaptic input integration is known to vary with the computational requirements of a circuit, yet how the timing of action potentials is tuned presynaptically to match these processing demands is not well understood. In particular, action potential timing is shaped by the axonal conduction velocity and the duration of synaptic transmission delays within a pathway. However, it is not known to what extent these factors are adapted to the functional constraints of the respective circuit. Here, we report the finding of activity-invariant synaptic transmission delays as a functional adaptation for input timing adjustment in a brainstem sound localization circuit. We compared axonal and synaptic properties of the same pathway between two species with dissimilar timing requirements (gerbil and mouse): In gerbils (like humans), neuronal processing of sound source location requires exceptionally high input precision in the range of microseconds, but not in mice. Activity-invariant synaptic transmission and conduction delays were present exclusively in fast conducting axons of gerbils that also exhibited unusual structural adaptations in axon myelination for increased conduction velocity. In contrast, synaptic transmission delays in mice varied depending on activity levels, and axonal myelination and conduction velocity exhibited no adaptations. Thus, the specializations in gerbils and their absence in mice suggest an optimization of axonal and synaptic properties to the specific demands of sound localization. These findings significantly advance our understanding of structural and functional adaptations for circuit processing.