Glutamate transporters in the central nervous system of a pond snail

Abstract

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm.

Glial solute carrier transporters in drosophila and mice

“…The conclusion that particular human astrocytes sequester glutamate to facilitate gluta-matergic signaling is based entirely on cross-species inferences and the fact that certain cells express specific transporter proteins. The same inferences have been applied to zebrafish (Rico et al, 2010), fruit flies (Besson et al, 1999;Soustelle et al, 2002), mosquito (Umesh et al, 2003), honeybee (Kucharski et al, 2000), leech (Hirth and Deitmer, 2006), snails (Hatakeyama et al, 2010), and other species. So the approach advocated here is nothing new.…”

Molecular and functional characterization of an evolutionarily conserved CREB‐binding protein in the LymnaeaCNS

“…After insertion of this PCR product into pGEM®‐T Easy Vector, the plasmid was linearized with Eco R V and Bam H I to generate digoxigenin‐labeled antisense and sense RNA probes, respectively. The procedures for generating the probes, hybridization, and coloring were the same as those that were previously reported 32,33 . Our main target in these experiments was the paired cerebral giant cells that can be easily and accurately identified visually even without staining them with a dye or a specific probe.…”

“…The procedures for generating the probes, hybridization, and coloring were the same as those that were previously reported. 32 , 33 Our main target in these experiments was the paired cerebral giant cells that can be easily and accurately identified visually even without staining them with a dye or a specific probe. This is because they are the largest neurons on the ventral side of the anterior lobes of the cerebral ganglia and occupy a very characteristic position in it, as shown in the diagram in Figure 3F .…”

“…The procedures for this experiment were modified from previous reports. 30,31,33 Non-functional region (from 278th to 1146th of nucleotide sequence) of LymCBP was isolated by RT-PCR [see Table S1 (No. 21,22) for nucleotide sequences of primers].…”

Electrical stimulation of cultured neurons using a simply patterned indium-tin-oxide (ITO) glass electrode

“…Intracellular recordings and patch clamp methods have been widely used to measure neuronal activity and to stimulate individual neurons, (Cohen et al, 2008;Hatakeyama et al, 2010;Wallach and Marom, 2012). Expanding on this approach, multielectrode arrays (MEAs) enable the stimulation of cells whilst simultaneously measuring their activity, allowing the network properties of multiple neurons dissociated in culture or tissue to be investigated (Wagenaar et al, 2005;Furukawa et al, 2013).…”