Changing dendritic field size of mouse retinal ganglion cells in early postnatal development

During early postnatal development, dendrites of retinal ganglion cells (RGCs) extend and branch in the inner plexiform layer to establish the adult level of stratification, pattern of branching, and coverage. Many studies have described the branching patterns, transient features, and regulatory factors of stratification of the RGCs. The rate of RGC dendritic field (DF) expansion relative to the growing retina has not been systematically investigated. In this study, we used two methods to examine the relative expansion of RGC DFs. First, we measured the size of RGC DFs and the diameters of the eyeballs at several postnatal stages. We compared the measurements with the RGC DF sizes calculated from difference of the eyeball sizes based on a linear expansion assumption. Second, we used the number of cholinergic amacrine cells (SACs) circumscribed by the DFs of RGCs at corresponding time points as an internal ruler to assess the size of DFs. We found most RGCs exhibit a phase of faster expansion relative to the retina between postnatal day 8 (P8) and P13, followed by a phase of retraction between P13 and adulthood. The morphological α cells showed the faster growing phase but not the retraction phase, whereas the morphological ON–OFF direction selective ganglion cells expanded in the same pace as the growing retina. These findings indicate different RGCs show different modes of growth, whereas most subtypes exhibit a fast expansion followed by a retraction phase to reach the adult size. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 397–407, 2010     

Mice with selective elimination of striatal acetylcholine release are lean,show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)^{D2‐Cre‐flox/flox} mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT^{D2‐Cre‐flox/flox} mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT^{D2‐Cre‐flox/flox} mice showed increased physical activity. Interestingly, VAChT^{D2‐Cre‐flox/flox} mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.     

Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration

Alpha-motoneurons appear to be exceedingly affected in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Morphological and physiological degeneration of this neuronal phenotype is typically characterized by a marked decrease of neuronal markers and by alterations of cholinergic metabolism such as reduced choline acetyltransferase (ChAT) expression. The motoneuron-like cell line NSC-34 is a hybrid cell line produced by fusion of neuroblastoma with mouse motoneuron-enriched primary spinal cord cells. In order to further establish this cell line as a valid model system to investigate cholinergic neurodegeneration, NSC-34 cells were differentiated by serum deprivation and additional treatment with all-trans retinoic acid (atRA). Cell maturation was characterized by neurite outgrowth and increased expression of neuronal and cholinergic markers, including MAP2, GAP-43 and ChAT. Subsequently, we used differentiated NSC-34 cells to study early degenerative responses following exposure to various neurotoxins (H₂O₂, TNF-α, and glutamate). Susceptibility to toxin-induced cell death was determined by means of morphological changes, expression of neuronal marker proteins, and the ratio of pro-(Bax) to anti-(Bcl-2) apoptotic proteins. NSC-34 cells respond to low doses of neurotoxins with increased cell death of remaining undifferentiated cells with no obvious adverse effects on differentiated cells. Thus, the different vulnerability of differentiated and undifferentiated NSC-34 cells to neurotoxins is a key characteristic of NSC-34 cells and has to be considered in neurotoxic studies. Nonetheless, application of atRA induced differentiation of NSC-34 cells and provides a suitable model to investigate molecular events linked to neurodegeneration of differentiated neurons.     

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.     

From the cholinergic gene locus to the cholinergic neuron

The cholinergic gene locus (CGL) was first identified in 1994 as the site (human chromosome 10q11.2) at which choline acetyltransferase and a functional vesicular acetylcholine transporter are co-localized. Here, we present recent neuroanatomical, developmental, and evolutionary insights into the chemical coding of cholinergic neurotransmission that have been gleaned from the study of the CGL, and its protein products VAChT and ChAT, which comprise a synthesis-sequestration pathway that functionally defines the cholinergic phenotype.

Résumé

Le locus génétique cholinergique (cholinergic gene locus, CGL) a été identifié en 1994 et regroupe les gènes de la choline acétyltransférase et d'un récepteur vésiculaire d'acétylcholine (chromosome humain 10q11.2). Nous présentons ici des données sur la neuroanatomie, le développement et l'évolution de la neurotransmission cholinergique obtenus à partir de l'étude du CGL et de ses produits protéiques, VAChT et ChAT: ce système de synthèse-séquestration définit fonctionnellement le phénotype cholinergique.     

Unusual Regulation of Splicing of the Cholinergic Locus in Caenorhabditis elegans

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.     

Glutamatergic neurotransmission in the synapsin I and II double knock-out mouse

The synaptic vesicle-associated synapsin proteins may participate in synaptic transmission, but their exact functional role(s) here remain(s) uncertain. We here briefly describe the important characteristics of the synapsin proteins, and review recent studies on transgenic mice devoid of the gene products encoded by the synapsin I and II genes, where both neurochemical, cell biological and electrophysiological methods have been employed. We present evidence for synapsin effects on both neurotransmitter synthesis and homeostasis, as well as on synaptic vesicle development and functions. Moreover, we describe physiological analyses of excitatory glutamatergic hippocampal synapses where a novel synapsin-dependent delayed response enhancement (DRE) phase occurs, and demonstrate the postnatal developmental patterns of both frequency facilitations and DRE responses. Finally, we report synapsin I and II effects in distinct excitatory glutamatergic synapses in the hippocampus, and indicate that synapsin-dependent modulations of synaptic function may use distinct presynaptic response patterns in order to induce different classes of presynaptic plasticity.     

Exploring the Regulation of the Expression of ChAT and VAChT Genes in NG108-15 Cells: Implication of PKA and PI3K Signaling Pathways

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single cholinergic locus, can be differentially or synergistically regulated, depending on the activated signaling pathways.     

Regulation of vesicular acetylcholine transporter by the activation of excitatory amino acid receptors in the avian retina

1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior.2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions.3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor.4. Phorbol myristate acetate (PMA), but not -PMA, reduced in more than 85% the number of L-[³H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT.5. The data show that activation of glutamatergic receptors reduces [³H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier.