Stefan Trifonov

GPR155: Gene organization, multiple mRNA splice variants and expression in mouse central nervous system

Emerging evidence suggests that GPR155, an integral membrane protein related to G-protein coupled receptors, has specific roles in Huntington disease and autism spectrum disorders. This study reports the structural organization of mouse GPR155 gene and the generation of five variants (Variants 1-5) of GPR155 mRNA, including so far unknown four variants. Further, it presents the level of expression of GPR155 mRNA in different mouse tissues. The mRNAs for GPR155 are widely expressed in adult mouse tissues and during development. In situ hybridization was used to determine the distribution of GPR155 in mouse brain. The GPR155 mRNAs are widely distributed in forebrain regions and have more restricted distribution in the midbrain and hindbrain regions. The highest level of expression was in the lateral part of striatum and hippocampus. The expression pattern of GPR155 mRNAs in mouse striatum was very similar to that of cannabinoid receptor type 1. The predicted protein secondary structure indicated that GPR155 is a 17-TM protein, and Variant 1 and Variant 5 proteins have an intracellular, conserved DEP domain near the C-terminal.

Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

BackgroundGABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete.ResultsIn the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1.ConclusionsGeneration of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.

Overview and assessment of the histochemical methods and reagents for the detection of β-galactosidase activity in transgenic animals

Bacterial β-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect β-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing β-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional β-galactosidase histochemistry. Here, we compare the traditional β-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for β-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of β-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection neurons.

The GABA‐synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium‐sized projection neurons with high‐level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5′‐noncoding, 3′‐noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia‐thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium‐sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.

Lineage-Specific Purification of Neural Stem/Progenitor Cells From Differentiated Mouse Induced Pluripotent Stem Cells

Since induced pluripotent stem (iPS) cells have differentiation potential into all three germ layer‐derived tissues, efficient purification of target cells is required in many fields of iPS research. One useful strategy is isolation of desired cells from differentiated iPS cells by lineage‐specific expression of a drug‐resistance gene, followed by drug selection. With this strategy, we purified neural stem/progenitor cells (NSCs), a good candidate source for regenerative therapy, from differentiated mouse iPS cells. We constructed a bicistronic expression vector simultaneously expressing blasticidin S resistance gene and DsRed under the control of tandem enhancer of a 257‐base pair region of nestin second intron, an NSC‐specific enhancer. This construct was efficiently inserted into the iPS genome by piggyBac transposon‐mediated gene transfer, and the established subclone was differentiated into NSCs in the presence or absence of blasticidin S. Consequently, incubation with blasticidin S led to purification of NSCs from differentiated iPS cells. Our results suggest that a lineage‐specific drug selection strategy is useful for purification of NSCs from differentiated iPS cells and that this strategy can be applied for the purification of other cell types.

Histological Determination of the Areas Enriched in Cholinergic Terminals and m2 and m3 Muscarinic Receptors in the Mouse Central Auditory System

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor‐expressing cell bodies. Anat Rec 293:1393–1399, 2010.

Reconstruction of calbindin-poor lateral striatum in the mouse

tic rules expand the diversity of sequence patterns. To enable such syntactic vocal communication, neural systems must extract the sequence patterns from auditory information and establish syntactic rules to generate motor commands for vocal organs. However, the neural processing of syntax for learned vocal signals remains largely unknown. We found that the basal ganglia projecting premotor neurons (HVCX neurons) in Bengalese finches represent syntactic rules. These songbirds assemble a fixed number of vocal elements called syllables based upon a finite-state type of syntax, which defines “alternative transitions” to or from syllables. When vocalizing such an alternative transition segment between different syllables, activities of HVCX neurons represent a specific syllable type and a specific transition direction among the alternative trajectories. When vocalizing a variable repetition sequence of the same syllable, HVCX neurons signal the initiation and termination of the repetition sequence and the progress and stateof-completeness of the repetition, in addition to the identity of repeated syllables. These different types of syntactic information are frequently integrated within the activity of single HVCX neurons, suggesting that syntactic attributes of the individual HVCX neurons are not determined as a basic cellular subtype in advance but acquired in the course of vocal learning and maturation. Furthermore, some auditory–vocal mirroring type HVCX neurons display transition selectivity in the auditory phase just as they do in the vocal phase, suggesting that these songbirds may extract syntactic rules from auditory experience and apply them to form their own vocal behaviors. Research fund: the Takeda Science foundation, the Uehara Memorial Foundation, the Strategic Research Program for Brain Science (the MEXT of Japan).

Purification of neural stem cells derived from mouse induced pluripotent stem cells by drug selection

ation propensity. Therefore, we have to evaluate each hiPS cell line to obtain safe NS/PCs with normal properties to avoid tumorigenesis and inappropriate pathophysiological analysis due to abnormal differentiation. In this study, we derived NS/PCs from hES cells and hiPS cells as neurospheres, and examined their differentiation potentials, functional properties, and gene expression profiles in vitro. We also examined the proportion of residual undifferentiated cells in derived NS/PCs, and the expression of retroviral transgenes which were used for the establishment of hiPS cells, during neural differentiation, to estimate their tumorigenic potentials. Finally, we injected hES, hiPS cell-derived NS/PCs into brains and testes of NOD/SCID mice to evaluate their differentiation properties and tumorigenicities in vivo. As a result of these analyses, we found that one of the hiPS cell lines we used could not differentiate to form neurospheres efficiently, and that NS/PCs derived from some of the hiPS cell lines formed tumors after transplantation, but without teratoma formation. Focusing on these distinct properties of hiPS cell lines, we further analyzed the underlying differences among hiPS cell lines, and evaluated the quality of hiPS cell lines. Research fund: JST-CIRM collaborative program, Keio Kanrinmaru Project.

Increase in GAD2 expression in the periolivary region of the mouse following cochlear ablation

O2P-BØ3 Strength of GABAergic transmission influences climbing fiber synapse elimination during cerebellar development Hisako Nakayama1, Kouichi Hashimoto1,2, Taisuke Miyazaki3, Yuchio Yanagawa4, Kunihiko Obata5, Masahiko Watanabe3, Masanobu Kano1 1 Graduate School of Medicine, Osaka University, Osaka, Japan; 2 CREST-JST, Kawaguchi, Japan; 3 Graduate School of Medicine, Hokkaido University, Sapporo, Japan; 4 Graduate School of Medicine, Gunma University, Maebashi, Japan; 5 RIKEN, BSI, Wako, Japan