Limulus Central Nervous System Research Articles

Cloning and partial characterization of four plasmalemmal-associated syntaxin isoforms in Limulus.

We describe herein the cloning of a group of syntaxins in Limulus that are associated with the plasma membrane. Initially, multiple degenerate oligonucleotide primers (DOP) and probes were designed from sequences of known plasma membrane associated syntaxins. Combined experiments using reverse transcriptase-polymerase chain reaction (RT-PCR), colony hybridization and reverse dot blot yielded three distinct probes. Subsequently, two cDNA libraries derived from the Limulus central nervous system (CNS) were screened and four distinct isoforms, designated Limulus syntaxin (Lim-syn) 1A, 1B, 1C and 1D, were obtained from forty cloned full-length sequences. The predicted amino acid (aa) sequences 1-265 were identical for Lim-syn 1A, 1C and for Lim-syn 1B, 1D, respectively. A comparison of the 265 aa cytoplasmic segments for the two subgroups Lim-syn 1A/1C and Lim-syn 1B/1D differed at 13 aa residues within this sequence. Lim-syn 1A and 1B contained 290 aa residues, and both contained a transmembrane domain (TMD, 267-288) and a myristylation-like site (286-290) at the C-termini. Lim-syn 1C (291 residues) contained only the TMD whereas Lim-syn 1D was truncated (277 residues) and had neither a TMD nor a myristylation-like site. All Lim-syn isoforms showed great identity with syntaxin 1-homologs (syntaxin 1A/1B) from various other species. Ribonuclease protection assay (RPA) analyses revealed distinctive expression patterns for individual Lim-syn transcripts but all were detectable in the CNS. Moreover, the antibody (anti-Lim-syn-1) produced against aa 133-145 epitope of Lim-syn identified a protein of approximately 35 kDa found only in CNS tissues.

Molecular cloning and identification of a putative PKCε cDNA from Limulus polyphemus brain

The protein kinase C (PKC) family of enzymes is broadly distributed and has been implicated in a diverse array of cellular functions. Recent evidence supporting PKC involvement in the regulation of the Limulus choline cotransporter prompted us to clone PKC from a Limulus central nervous system (CNS) cDNA library. An Aplysia californica calcium independent PKC (Apl II) cDNA probe was used to screen the library and 5′ RACE SMART™ PCR was used to obtain the full-length sequence. The resulting cDNA, which included 5′ and 3′ nontranslation regions, was 4675 bp. Analysis of the encoded peptide sequence using the Swiss–prot database revealed at least 58% identity to PKCε. A commercial polyclonal antibody against PKCε was used in Western blots to positively label a 30 kDa protein from Limulus CNS and the expressed fusion protein of the encoded sequence. These data support the presence of a newly identified PKC-like homolog in Limulus which likely represents a PKCε equivalent.

Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS

It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci. 3, 120–125) recently reported the successful cloning of the choline co-transporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herein the cloning of the choline co-transporter in the horseshoe crab, Limulus polyphemus. Through the use of a series of degenerate primers selected from consensus sequences of CHO-1 and CHT1, we generated two probes that were used to search a Limulus cDNA library produced from central nervous system (CNS) tissue. The full length nucleotide sequence of the Limulus homolog consists of 3368 bp which includes an open reading frame (ORF) that predicts a protein of 579 amino acids and two non-translation regions (NTR), one at the 3′ end and the other at the 5′ end. The amino acid sequence has 46% identity with rat CHT1 and 50% identity with both CHO-1 in C. elegans and the recently cloned human co-transporter (hCHT; Apparsundaram et al., 2000. Biochem. Biophys. Res. Commun. 276, 862–867; Okuda and Haga, 2000. FEBS Lett. 484, 92–97). Hydropathy plot analysis predicts the Limulus choline co-transporter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analyses using cDNA probes designed from LChCoT cDNA sequences revealed its distribution specifically in central nervous system structures. On the other hand it was not found in non-nervous tissues. The successful cloning of LChCoT, which was shown to be a member of the sodium-dependent glucose transporter family (SLGT), should prove useful in the determination of its physiological regulation, including its intracellular trafficking.

The quantitative distribution of a putative PKCε mRNA in Limulus central nervous system by modified competitive RT-PCR

Recently, a full length cDNA for the epsilon (ε) isoform of protein kinase C (PKC) was cloned and sequenced from a cDNA library for the horseshoe crab, Limulus polyphemus. This multifunctional enzyme has been implicated in the modulation of the choline cotransporter in Limulus and the epsilon isoform has been identified in homogenates from its central nervous system (CNS). RT-PCR has proven to be a very useful method for quantifying even a few molecules of mRNA in tissue samples. A modified competitive RT-PCR was used here to quantify a putative PKCε mRNA in Limulus CNS preparations. First, we replaced normally used oligo dT and random primers generated from mRNA with a PKCε specific (3′ end) primer P4. Then we used modified nucleotides to extend sample life in storage and finally, we used only annealing and denaturing temperatures during PCR to reduce background. The modified method was used for the first time to quantify PKCε mRNA from three distinct areas of the CNS in Limulus. Results revealed high levels of PKCε mRNA in the corpora pedunculata, in the abdominal ganglia and in the brain ring. These results indicate that PKCε mRNA is broadly distributed throughout the Limulus CNS. Importantly, this modified competitive RT-PCR technique was successfully applied to the quantitation of specific mRNA from Limulus nervous tissue for which no internal standard is available commercially.

The effect of neuropeptides from Limulus on its circadian rhythm in retinal sensitivity.

Retinal responses of the Limulus lateral eyes to light are greater at night than during the day. A circadian clock in the brain of the horseshoe crab controls these rhythmic changes of light sensitivity. The increase in sensitivity (as measured by the amplitude of the electroretinogram) is mediated at least in part by octopamine that is released from efferent axons terminating in the visual cells. Earlier studies indicate that certain factors in Limulus hemolymph can act in conjunction with octopamine. More recently, five neuropeptides (LP1-LP4 and Lip-HP) had been isolated from acetone extracts of the Limulus central nervous system using HPLC fractionation and radioimmunoassay with antisera against FMRFamide-like peptides for detection. Presently, we have injected into the Limulus lateral eye these five peptides and observed changes in retinal sensitivity. Injection during daytime had no immediate effect on that daytime electroretinogram but decreased the electroretinogram amplitude for the entire subsequent night (12 h). However, upon injection at night, we observed an immediate but only transitory decrease in electroretinogram amplitude for about 1 h without effect on the subsequent daytime electroretinogram. We suggest that the peptides act antagonistically to octopamine and are highly dependent upon the activity state of the efferent nerve terminals.

Excitatory actions of FMRFamide-related peptides (FaRPs) on the neurogenic Limulus heart.

The actions of FMRFamide-related peptides (FaRPs) on the neurogenic heart of the horseshoe crab, Limulus polyphemus, were investigated. Excitatory chronotropic effects were produced by application of TNRNFLRFamide, SDRNFLRFamide, GYNRS-FLRFamide, or pQDPFLRFamide to the intact heart preparation. Effects were dose-dependent with a threshold of 10(-9) M or less. TNRNFLRFamide and SDRNFLRFamide increased the burst rate of the isolated Limulus cardiac ganglion. Synthetic FaRPs produced inotropic excitation of the heartbeat as well. GYNRSFLRFamide, TNRNFLRFamide, SDRNFLRFamide, and pQDPFLRFamide increased heart contraction strength at a threshold dose of approximately 10(-8) M. TNRNFLRFamide and SDRNFLRFamide enhanced electrically evoked contractions of the Limulus myocardium, elicited contracture in some preparations, and increased the excitability of cardiac muscle fibers. The presence of cardioactive FaRPs in the Limulus central nervous system was suggested by reverse phase HPLC of acidified methanol extracts of Limulus nervous tissue. Four peaks of FaRP-like bioactivity were detected with the Busycon radula protractor muscle bioassay. These peaks also contained FaRP-like immunoreactivity. Two of these partially purified peaks produced excitatory chronotropic effects on the intact Limulus heart preparation similar to those produced by synthetic FaRPs.

Selective protection of high affinity [ 3H] choline uptake sites by hemicholinium-3 against N-ethylmaleimide binding in Limulus synaptosomes

1. Synaptosomes from Limulus central nervous system (CNS) were preincubated with the sulfhydryl reagent N-ethylmaleimide (NEM) both in the presence and in the absence of hemicholinium-3 (HC-3), a competitive inhibitor of sodium-dependent high affinity choline transport. 2. Synaptosomes preincubated with HC-3 and NEM together retained a significantly higher level of high affinity [ H]choline uptake (P < 0.05) than did those that were incubated with only NEM. 3. Additionally, the synaptosomes incubated simultaneously with HC-3 and NEM also retained a significantly larger population of available binding sites for [ H]NEM binding upon subsequent exposure to [ H]NEM than did synaptosomes incubated with only NEM. 4. The results indicate the possible use of HC-3 as a protective binding agent for the specific labeling of high affinity choline transport protein.

The activity of cis and trans -2- (aminomethyl) cyclopropane carboxylic acids on gaba — related mechanisms in the central nervous system of limulus and periplanata and of mammals

Some pharmacological actions of (±) — cis — and (±) — trans -2-(aminomethyl) cyclopropane carboxylic acids, conformationally restricted analogues of GABA, are described. The trans isomer (TAMP) was 17 and 4.5 times less active than GABA in hyperpolarising central neurones of Limulus and Periplanata and 10.8 times less active in displacing [ 3H]-muscimol from rat brain membranes. The cis isomer (CAMP) was ineffective on Limulusneurones at 35 μmoles, 650 times less active on Periplanata neurones and only weakly displaced [ 3H]-muscimol at 100 μm. Neither isomer inhibited synaptosomal uptake of [ 3H]-GABA at 100 μM. These data suggest that the invertebrate receptors studied respond to GABA in an extended conformation.

The effects of curare in Limulus central nervous system.

Exposure of Limulus abdominal ganglia to (+)-tubocurarine before or after cotton-mouth moccasin venom treatment resulted in a decrease of spontaneous activity in individual fibers in dorsal nerve roots and an inhibition of heart rate, but did not decrease responsiveness of the dorsal nerve to tactile stimulation. No effects of curare were obtained in nonsynaptic regions of nerve bundles either with or without venom pretreatment. Combining acetylcholine perfusion with (+)-tubocurarine resulted in a suppression of the stimulating effect of acetylcholine on dorsal nerve activity.

Cardioregulatory properties of the abdominal ganglia in Limulus.

The abdominal ganglia of the Limulus central nervous system exert a net inhibitory effect on heart rate. This influence is mediated mainly by the dorsal nerves in the first three ganglia. When the dorsal nerves are sectioned, cardioacceleration results; when these nerves are stimulated, a reduction in rate is obtained. However, cardioaccelerators can be unmasked by splitting a ganglion. This selectively removes the inhibitory output, leaving only a cardioaccelerator influence. Inhibition of bioelectrical activity in the intact abdominal ganglia with GABA also resulted in an increased heart rate, confirming their net inhibitory influence on heart function. Possible models of abdominal ganglia organization are discussed.