Deficiency in endothelin receptor B reduces proliferation of neuronal progenitors and increases apoptosis in postnatal rat cerebellum.

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.

Activation of alpha 2A adrenoceptors alters dendritic spine development and the expression of spinophilin in cultured cortical neurones.

alpha2 adrenoceptors have been shown to regulate the development of dendrites in mammalian cortical neurones. In this study we have investigated how agonists of alpha2 adrenoceptors affect length and density of dendritic spines in cultured cortical neurones from C57/B6 mice. A twenty-four hour incubation of 14 day old cultured neurones with UK 14304, an alpha2-adrenoceptor agonist, resulted in a significant increase in the average length and density of dendritic spines. Furthermore, incubation of neurones with the selective alpha 2A agonist guanfacine resulted in 1.2-fold increase in spine length and 1.8-fold increase in spine density. These effects were blocked by RX 821002 and BRL 44408, alpha2- and alpha 2A-adrenoceptor antagonists, respectively. The observed changes in the density and length of dendritic spines were correlated with increased expression of spinophilin, a key cytoskeletal protein in the formation and maintenance of dendritic spines, and a decrease in the phosphorylation of spinophilin on serine residues. The latter finding points to a possible mechanism by which adrenoceptors may regulate spinophilin function in dendritic spine development and structure in cortical neurones in vitro.

Expression of mRNA and functional alpha(1)-adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons.

1. Locus coeruleus neurons in adult rats express binding sites and mRNA for alpha(1)-adrenoceptors even though the depolarizing effect of alpha(1)-adrenoceptor agonists on neonatal neurons disappears during development. 2. In this study intracellular microelectrodes were used to record from locus coeruleus neurons in brain slices of adult rats and reverse transcription-polymerase chain reaction (RT - PCR) was used to investigate the mRNA expression of alpha(1)- and alpha(2)-adrenoceptors in juvenile and adult rats. 3. The alpha(1)-adrenoceptor agonist phenylephrine had no effect on the membrane conductance of locus coeruleus neurons (V(hold) -60 mV) but decreased the G protein coupled, inward rectifier potassium (GIRK) conductance induced by alpha(2)-adrenoceptor or mu-opioid agonists. The GIRK conductance induced by noradrenaline was increased in amplitude when alpha(1)-adrenoceptors were blocked with prazosin. 4. RT - PCR of total cellular RNA isolated from microdissected locus coeruleus tissue demonstrated strong mRNA expression of alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptors in both juvenile and adult rats. However, only mRNA transcripts for the alpha(1b)-adrenoceptors were consistently detected in cytoplasmic samples taken from single locus coeruleus neurons of juvenile rats, suggesting that this subtype may be responsible for the physiological effects seen in juvenile rats. 5. Juvenile and adult locus coeruleus tissue expressed mRNA for the alpha(2a)- and alpha(2c)-adrenoceptors while the alpha(2b)-adrenoceptor was only weakly expressed in juveniles and was not detected in adults. 6. The results of this study show that alpha(1)-adrenoceptors expressed in adult locus coeruleus neurons function to suppress the GIRK conductance that is activated by mu-opioid and alpha(2)-adrenoceptors.

Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera.

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

Eph receptor and ephrin signaling in developing and adult brain of the honeybee (Apis mellifera).

Roles for Eph receptor tyrosine kinase and ephrin signaling in vertebrate brain development are well established. Their involvement in the modulation of mammalian synaptic structure and physiology is also emerging. However, less is known of their effects on brain development and their function in adult invertebrate nervous systems. Here, we report on the characterization of Eph receptor and ephrin orthologs in the honeybee, Apis mellifera (Am), and their role in learning and memory. In situ hybridization for mRNA expression showed a uniform distribution of expression of both genes across the developing pupal and adult brain. However, in situ labeling with Fc fusion proteins indicated that the AmEphR and Amephrin proteins were differentially localized to cell body regions in the mushroom bodies and the developing neuropiles of the antennal and optic lobes. In adults, AmEphR protein was localized to regions of synaptic contacts in optic lobes, in the glomeruli of antennal lobes, and in the medial lobe of the mushroom body. The latter two regions are involved in olfactory learning and memory in the honeybee. Injections of EphR-Fc and ephrin-Fc proteins into the brains of adult bees, 1 h before olfactory conditioning of the proboscis extension reflex, significantly reduced memory 24 h later. Experimental amnesia in the group injected with ephrin-Fc was apparent 1 h post-training. Experimental amnesia was also induced by post-training injections with ephrin-Fc suggesting a role in recall. This is the first demonstration that Eph molecules function to regulate the formation of memory in insects.

Analysis of EphB receptors and their ligands in the developing retinocollicular system of the wallaby reveals dynamic patterns of expression in the retina.

The expression of EphB1 and B2 receptors and ephrins-B1, -B2 and -B3 in the retina and superior colliculus of the wallaby (Macropus eugenii) was examined during the development of the retinocollicular projection, using reverse transcription-polymerase chain reaction and immunohistochemistry. There was an early transient differential expression of EphB2 that was higher in ventral retina and restricted to the outer neuroblast layer, whereas a high ventral to low dorsal gradient of ephrin-B2 expression occurred there throughout the study period. However, there was no dorsoventral gradient of receptors or ligands in retinal ganglion cells or a mediolateral gradient of ephrins in the colliculus. These findings suggest a limited role for these molecules in topographic mapping across the mediolateral colliculus in the wallaby. Early in retinal development there is a complementary pattern of expression of ephrin-B1 and -B2 in the outer neuroblast layer that overlaps with expression of EphB2. Ganglion and amacrine cells also express EphB2. As development proceeds subpopulations of putative horizontal and bipolar cells, also expressing EphB2, come to reside in the inner nuclear layer and ephrin-B1 is expressed throughout the outer nuclear layer. At the same time cells expressing ephrin-B2, and subpopulations of horizontal and bipolar cells come to reside in the inner nuclear layer and there is a corresponding decrease in ephrin-B2 expression in the outer nuclear layer. This pattern of coexpression of receptors and ligands suggests a role for them in cell migration and maintenance of laminar boundaries.