Histological Analysis of SLC38A6 (SNAT6) Expression in Mouse Brain Shows Selective Expression in Excitatory Neurons with High Expression in the Synapses

SLC38A6 is one of the newly found members of the solute carrier 38 family consisting of total 11 members, of which only 6 have been characterized so far. Being the only glutamine transporter family expressed in the brain, this family of proteins are most probably involved in the regulation of the glutamate-glutamine cycle, responsible for preventing excitotoxicity. We used immunohistochemistry to show that SLC38A6 is primarily expressed in excitatory neurons and is not expressed in the astrocytes. Using proximity ligation assay, we have quantified the interactions of this SLC38 family protein with other proteins with known localization in the cells, showing that this transporter is expressed at the synapses. Moreover, this study has enabled us to come up with a model suggesting sub-cellular localization of SLC38A6 at the synaptic membrane of the excitatory neurons.     

Expression of Synaptophysin During Postnatal Development of the Mouse Brain

The expression of the synaptic vesicle membrane protein, synaptophysin, was analyzed during postnatal development of the mouse cerebrum using a quantitative immunoblotting procedure. From birth to adulthood, the relative contents of synaptophysin increased 80-fold, reaching a final level of 3.5 micrograms/mg of total protein. The time course of accumulation suggests that synaptophysin expression is correlated with synaptogenesis. Thus synaptophysin may be used as a reliable marker of nerve terminal differentiation.     

Modulation of LPA Receptor Expression in the Human Brain Following Neurotrauma

Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR₂ and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR₁ colocalized with astrocytes and that LPAR₂ is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.     

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

Analysis of Pax6 Contiguous Gene Deletions in the Mouse,Mus musculus,Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca⁺²-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.CONTIGUOUS gene deletions account for a significant portion of human genetic syndromes. The application of fluorescence in situ hybridization (FISH) cytogenetics and array comparative genome hybridization (array-CGH) technologies have enabled more accurate localization of deletion breakpoints. This deletion information combined with the annotation of the human genome structure provides critical information to identify genes responsible for particular phenotypes associated with a syndrome. For example, deletions of the 11p11p12 and 11p13 regions on the short arm of human chromosome (Chr) 11 have been identified in the Potocki–Shaffer syndrome (Shaffer et al. 1993; Bartsch et al. 1996; Potocki and Shaffer 1996) and the Wilm''s tumor- aniridia- genitourinary abnormalities- mental retardation (WAGR) syndrome (Riccardi et al. 1978; Francke et al. 1979; Hittner et al. 1979; Fryns et al. 1981), respectively. Deletion analyses were important in identifying genes associated with clinical features of the syndromes: EXT2 for multiple exostoses and ALX4 for parietal foramina in Potocki–Shaffer syndrome (Ligon et al. 1998; Wu et al. 2000; Wakui et al. 2005), WT1 for Wilm''s tumor, and PAX6 for aniridia in WAGR syndrome (van Heyningen et al. 1985; Glaser et al. 1986, 1992; Fantes et al. 1992). Deletion analyses have also defined the extent of the deleted region in patients with combined Potocki–Shaffer and WAGR syndromes (McGaughran et al. 1995; Brémond-Gignac et al. 2005) as well as microdeletions 3′ to PAX6, which prevent expression of PAX6 and cause aniridia (Lauderdale et al. 2000; D''elia et al. 2007; Davis et al. 2008).The mouse Chr 2 region homologous to the human WAGR region contains the genes Wt1, Rcn1, Pax6, and Elp4. An extensive allelic series at Pax6 has been identified (Bult et al. 2008). Heterozygote Pax6 intragenic null mutants express microphthalmia, iris anomalies, corneal opacities, lens opacities, and lens-corneal adhesions. Homozygotes are anophthalmic and die shortly after birth (Roberts 1967; Hogan et al. 1986). Five deletions in the region have been identified: Pax6^Sey-Dey, Pax6^Sey-H, Pax6^Sey-2H, Pax6^Sey-3H, Pax6^Sey-4H of which two, Pax6^Sey-H (Hogan et al. 1986; Kent et al. 1997; Kleinjan et al. 2002; Webb et al. 2008) and Pax6^Sey-Dey (Theiler et al. 1978; Hogan et al. 1987; Glaser et al. 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations. Homozygotes for both deletions are lethal at an early embryonic stage.We were particularly interested in the extreme eye phenotype associated with the Pax6 deletions and considered two alternative hypotheses. Either Pax6 deletions are functionally different from Pax6 intragenic null mutations or deletion of a region linked to but distinct from the Pax6 structural gene affects the eye phenotype.In the present study we identify three new deletions encompassing the Pax6 region of the mouse. They have been assigned the mutant allele symbols Del(2)Pax6^11Neu/1Neu, Del(2)Pax6^12Neu/2Neu, and Del(2)Pax6^13Neu/3Neu and will be referred to throughout this publication as Pax6^11Neu, Pax6^12Neu, and Pax6^13Neu, respectively. All three deletions are homozygous lethal at an early embryonic stage. The deletions differentiate for the extent of the eye abnormality expressed: Pax6^11Neu heterozygotes express extreme microphthalmia similar to that observed in the Pax6^Sey-Dey and Pax6^Sey-H deletions. Pax6^12Neu and Pax6^13Neu heterozygotes express the milder eye abnormality seen in heterozygous intragenic null mutants. For all three deletions, heterozygotes do not express belly spotting. Genetic, phenotypic, and molecular characterization of the deletions allowed us to identify regions associated with the array of phenotypes in these contiguous gene deletions.     

Calcium Modulation of Activation and Desensitization of Nicotinic Receptors from Mouse Brain

Abstract: The effects of extracellular calcium on functional properties of nicotinic receptors from mouse thalamus were investigated. Previous studies have reported that calcium modulates the function of several neuronal nicotinic receptors. A ⁸⁶Rb⁺ ion efflux assay was developed to measure nicotinic receptor function from brain tissue, and data indicate that α₄β₂ receptors may mediate this response. Using the ⁸⁶Rb⁺ efflux assay, calcium effects on receptor activation, desensitization induced by high, activating and low, subactivating concentrations of agonist, and recovery from desensitization were examined. Effects of calcium on the kinetics of ligand binding were also investigated. Calcium modulated receptor activation by increasing the maximal response to nicotine in a concentration-dependent manner, without affecting the EC₅₀ of nicotine. Barium, but not magnesium, mimicked the effects of calcium on receptor activation. The increase in receptor activation could not be explained by changes in the ratio of activatable to desensitized receptors as assessed by the kinetics of ligand binding. Desensitization following activation was unaffected by calcium. Calcium, barium, and magnesium, however, increased the potency of nicotine for desensitization induced by exposure to low, subactivating concentrations of nicotine. Recovery from desensitization was not modulated by calcium. These data suggest that calcium modulates various functional aspects of nicotinic receptors from mouse brain and may do so via different mechanisms.     

Plectin Rodless Isoform Expression and Its Detectiona in Mouse Brain

Summary The widely expressed cytolinker protein plectin shows extensive isoform diversity both at the N-terminus and in the central part of the molecule. Judged on mRNA data, plectin variants lacking the central rod domain are expressed at a ∼20-fold lower level than full-length proteins and their detection on the protein level can be difficult. Here we present data on the expression of plectin rodless isoforms in mouse brain and in rat glioma C6 cells on RNA and protein levels. Our data indicate that among the rodless variants expressed in neuronal tissues, those starting with exon 1c (plectin 1c) seem to be the most prominent ones. In addition, we show that similar to other monoclonal antibodies reported in the literature, the widely used mAb 7A8 recognizes an epitope within plectin's rod domain and therefore is unsuited to detect rodless variants of plectin.     

Developmental Regulation of Microtubule-Associated Protein 2 Expression in Regions of Mouse Brain

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.