Three functional isoforms of GAR-2, a Caenorhabditis elegans G-protein-linked acetylcholine receptor, are produced by alternative splicing.

We have previously isolated a cDNA clone from Caenorhabditis elegans that encodes a novel form of G-protein-linked acetylcholine receptor, termed GAR-2. GAR-2 is similar to but pharmacologically distinct from muscarinic acetylcholine receptors. Here we report the identification of two gar-2 cDNA clones that are different from the previous one. These newly identified cDNAs encode polypeptides of 664 and 627 amino acids, whereas the previous one encodes a polypeptide of 614 amino acids. The three GAR-2 isoforms, which differ only in the third intracellular loop, arise from alternative splicing. Electrophysiological analyses using the Xenopus oocyte system showed that all three GAR-2 isoforms couple to the activation of G-protein-gated inwardly rectifying K+ (GIRK1) channel with similar drug specificity. Our results indicate that alternative splicing plays an important role in promoting molecular diversity of G-protein-linked acetylcholine receptors in C. elegans.

Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses.

In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.

Characterization of GAR-2, a novel G protein-linked acetylcholine receptor from Caenorhabditis elegans.

We have previously identified two G protein-linked acetylcholine receptors (GARs), GAR-1 and GAR-3, in the nematode Caenorhabditis elegans. Whereas GAR-3 is a homologue of muscarinic acetylcholine receptors (mAChRs), GAR-1 is similar to but pharmacologically distinct from mAChRs. In the current work we isolated a new type of GAR using C. elegans genome sequence information. This receptor, named GAR-2, consists of 614 amino acid residues and has seven putative transmembrane domains. Database searches indicate that GAR-2 is most similar to GAR-1 and closely related to GAR-3/mAChRs. The overall amino acid sequence identities to GAR-1 and GAR-3 are approximately 32 and approximately 23%, respectively. When GAR-2 was coexpressed with the G protein-activated inwardly rectifying K(+) (GIRK1) channel in Xenopus oocytes, acetylcholine was able to evoke the GIRK current in a dose-dependent fashion. Oxotremorine, a classical muscarinic agonist, had little effect on the receptor, indicating that GAR-2 is pharmacologically different from mAChRs but rather similar to GAR-1. GAR-2 differs from GAR-1, however, in that it showed virtually no response to muscarinic antagonists such as atropine, scopolamine, and pirenzepine. Expression studies using green fluorescent protein reporter gene fusion revealed that GAR-2 is expressed in a subset of C. elegans neurons, distinct from those expressing GAR-1. Together with our previous reports, this study demonstrates that diverse types of GARs are present in C. elegans.

Genomic analysis and functional expression of canine dopamine D2 receptor.

Dopamine D2 receptor (DRD2) is one of the five dopamine receptors with seven transmembrane domains that are coupled to the G protein. We have cloned and characterized the genomic and cDNA sequences of the canine DRD2 gene, which are 12.7 and 2.7 kb in size, respectively. The genomic DNA is composed of seven exons and six introns, encoding a 443 amino acid protein with 95% amino acid identity to other mammalian D2 receptors. A length polymorphism was detected in intron 3 of the receptor gene. We also characterized alternatively spliced forms of DRD2 cDNAs, DRD2L and DRD2S. They showed a higher level of expression in midbrain and thalamus. The ratio between the long and short form is similar in RT-PCR reaction. In human and rodent, the same two spliced forms are known to be coupled to G(i)-type heterotrimeric GTP binding protein, thereby opening an inwardly rectifying potassium channel, GIRK1. When the canine DRD2L and DRD2S were heterologously expressed in Xenopus oocytes, both forms activated GIRK1 potassium channels through coupling with G(i) protein. This activation was dose-dependent, demonstrating its ligand specificity.

The cAMP-dependent kinase pathway does not sensitize the cloned vanilloid receptor type 1 expressed in xenopus oocytes or Aplysia neurons.

Capsaicin-activated channels present in sensory neurons are ligand-gated cation channels that largely account for mediating some types of pain. The cAMP-dependent protein kinase (PKA) signal pathway was suggested to mediate the prostaglandin-induced enhancement of capsaicin-evoked inward current (I(CAP)) in rat sensory neurons. It is not clear, however, whether PKA acts directly on the capsaicin-sensitive channel that is responsible for I(CAP). To address this issue, we overexpressed the cloned capsaicin receptor, VR1, in heterologous expression systems such as Xenopus oocytes or Aplysia R2 neuron and stimulated PKA pathways. As a result, activation of PKA by applying either 8-bromo-cAMP or forskolin with 3-isobutyl-1-methylxanthine or through activation of beta(2) adrenergic receptors failed to enhance I(CAP) in oocytes or R2 neurons expressing VR1. Our results raise two possibilities. (1) Direct phosphorylation of VR1 by PKA may not be responsible for the sensitization; instead, phosphorylation of regulatory proteins associated with VR1 would account for the sensitization of I(CAP) evoked by prostaglandin E(2) in dorsal root ganglion (DRG) neurons. (2) DRG neurons may have a different PKA signaling mechanism that is not replicable in Xenopus oocytes or Aplysia R2 neurons.

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in aplysia sensory neurons.

Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa(1), that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

Alternative splicing of gar-1, a Caenorhabditis elegans G-protein-linked acetylcholine receptor gene.

We have recently identified a gene, designated gar-1, coding for a novel form of G-protein-linked acetylcholine (ACh) receptor in Caenorhabditis elegans. Although this receptor is most closely related to muscarinic ACh receptors (mAChRs), electrophysiological analyses have shown that ligand binding specificity of the receptor is distinct from that of mAChRs. Here we report that three receptor isoforms are generated by alternative splicing of the gar-1 transcript. These receptor isoforms differ only in the third intracellular loop that is considered to be important for G protein coupling. The three splice variants, when expressed in Xenopus oocyte, displayed similar pharmacological profiles and signaling activities. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the three gar-1 mRNAs are present at all developmental stages examined. The results in this study provide evidence that alternative splicing is involved in promoting molecular diversity of G-protein-linked ACh receptors.

Expression of a non-inactivating K+ channel driven by a rat heat shock promoter increased the resting potential in Aplysia silent neurons.

We assessed the role of a non-inactivating K+ channel (aKv5.1) in the resting potential by overexpressing this channel by heat shock in the neurons. A reporter gene lacZ linked to a promoter region spanning from the -285 to the +88 base of the rat HSP70ib gene was induced 62.5-fold when this DNA construct was microinjected into the neurons of the marine mollusk Aplysia and treated with heat shock at 30 degrees C for 3 h. Using this efficient induction system, we induced the expression of aKv5.1 by heat shock in cultured, electrically silent neurons of Aplysia and examined its effect on the resting potential. The channel expression increased the resting potential by approximately 10 mV. This increase was specific to heat shock induction and abolished by treatment with TEA, a specific K+ channel blocker. These results provide the direct evidence that a low voltage-activated, non-inactivating K+ channel can contribute to the resting potential.

Cloning and expression of a G protein-linked acetylcholine receptor from Caenorhabditis elegans.

We have isolated a cDNA clone from the nematode Caenorhabditis elegans that encodes a protein of greatest sequence similarity to muscarinic acetylcholine receptors. This gene codes for a polypeptide of 682 amino acids containing seven putative transmembrane domains. The amino acid identities, excluding a highly variable middle portion of the third intracellular loop, to the human m1-m5 receptors are 28-34%. When this cloned receptor was coexpressed with a G protein-gated inwardly rectifying K+ channel (GIRK1) in Xenopus oocyte, acetylcholine was able to elicit the GIRK current. This acetylcholine-induced current was substantially inhibited by the muscarinic antagonist atropine in a reversible manner. However, another muscarinic agonist oxotremorine and antagonists scopolamine and pirenzepine had little or negligible effects on this receptor. Taken together, these results suggest that the cloned gene encodes a G protein-linked acetylcholine receptor that is most similar to but pharmacologically distinct from muscarinic acetylcholine receptors.

Cloning and functional characterization of a Caenorhabditis elegans muscarinic acetylcholine receptor.

A cDNA clone encoding a muscarinic acetylcholine receptor (mAChR) has been isolated from the nematode Caenorhabditis elegans. The nematode mAChR, consisted of 585 amino acids, displays a high degree of amino acid sequence homology to other invertebrate and vertebrate mAChRs. Excluding a highly variable middle portion of the third intracellular loop, the C. elegans mAChR shares about 51% amino acid sequence identity with a Drosophila mAChR and 42-44% identity with human m1-m5 mAChR subtypes. Comparison of the cDNA sequence with the corresponding genomic sequence reveals that the C. elegans mAChR gene contains ten introns, eight of them in the coding region. Pharmacological profiles of the C. elegans mAChR expressed in Chinese hamster ovary (CHO) cells were shown to be similar to those of mammalian counterparts, indicating that ligand binding domains of the receptor have been conserved during evolution. When this cloned receptor was expressed in Xenopus oocytes, acetylcholine evoked a transient Cl- current. Furthermore, activation of the receptor with oxotremorine, acetylcholine or carbachol resulted in the stimulation of phosphatidylinositol metabolism in CHO cells, suggesting that the receptor is coupled to phospholipase C activation.

Truncated green fluorescent protein mutants and their expression in Aplysia neurons.

We determined in detail the primary structure requirements for green fluorescence of the jellyfish green fluorescent protein (GFP) and of its improved mutants (S65T and 1167T). We performed a deletion mapping in combination with fluorescence-activated cell sorting (FACS) and spectrofluorometry. This showed that deletion of up to nine amino acids at the C-terminal end of GFP had no deleterious effect on the fluorescent activity of GFP; in fact the deletion increased intensity of fluorescence. Further truncation of up to 11 amino acids resulted in partial impairment in maximal fluorescence. The GFP fluorescence was completely disrupted when more than 12 amino acids were deleted out of the C-terminal. Truncated mutants or their fusion genes with lacZ emitted fluorescence when plasmids encoding them were introduced by microinjection into Aplysia neurons.

Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons.

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.

Partial anatomical and physiological characterization and dissociated cell culture of the nervous system of the marine mollusc Aplysia kurodai.

Snail nervous systems are powerful tools for neurobiological studies as the biophysical properties of the giant neurons and their neural circuits can be examined in relation to specific behaviors of animals. The marine mollusc Aplysia californica is particularly useful for analyzing the components of learning and memory at the molecular and cellular levels. Here we partially examined the nervous systems of two species (A. kurodai and A. juliana) commonly found along the Korean coast in comparison with that of A. californica, one of the American marine snails. A. kurodai appeared to be identical to A. californica in both anatomical and physiological properties of the nervous system. A. juliana could be distinguished from A. californica in certain morphological aspects of the nervous system. The hemolymph either from A. kurodai or from A. juliana was required for effectively elongating neurite outgrowth of A. kurodai neurons in dissociated cell culture. The cultured cells retained neuronal properties such as neurite outgrowth, synapse formation, and generation of action potentials. The sensory cells of A. kurodai in dissociated cultures showed a response to serotonin (5-HT) of spike broadening and enhanced membrane excitability as in intact ganglia. Therefore, the nervous system and dissociated neuronal culture of A. kurodai may be useful for studying learning and memory in the context-of well-defined neural circuits of A. californica.

Parameters influencing ectopic gene expression in Aplysia neurons.

DNA microinjection for expressing exogenous genes in Aplysia neurons has been very useful for analyzing not only functions of encoded proteins, but also regulations of gene expression in the nervous system. Some of the factors that affect expression of foreign genes microinjected into Aplysia neurons are described. The effect of the DNA form (supercoiled or linear) and promoter modification in the expression vectors are analyzed as well as buffer composition and DNA concentration in the microinjection solution. The time course of reporter gene expression was also monitored. Reporter gene expression was first detected as early as 1 h and maintained at a high level even until 7 days after microinjection. The presence of AP-1 enhancer in the promoter region of the expression vectors was essential in driving a high-level constitutive expression of reporter genes. Particularly, a pNEX derivative containing eight copies of AP-1 enhancer drove constitutive overexpression more effectively than ones harboring either four or 12 copies of AP-1 enhancer. We also found that a prokaryotic promoter/operator from E. coli lacZ gene placed upstream from an eukaryotic enhancer/promoter repressed the expression of the downstream reporter gene in Aplysia neurons.

Evidence for synaptotagmin as an inhibitory clamp on synaptic vesicle release in Aplysia neurons.

While previous studies have demonstrated that synaptotagmin plays an essential role in evoked neurotransmitter release, it has been difficult to determine whether it acts to facilitate or inhibit release. To address this question, we used acute genetic manipulations to alter the expression of synaptotagmin in Aplysia neurons. Transient overexpression of synaptotagmin in acutely dissected cholinergic neurons and in cultured glutaminergic neurons decreased the amplitude of the excitatory postsynaptic potential (EPSP) by 32% and 26%, respectively. In contrast, treatment of cultured presynaptic neurons with synaptotagmin antisense oligonucleotides increased the amplitude of the EPSP by 50-75%. These results are consistent with a role of synaptotagmin as an inhibitor of release.

Proteins functioning in synaptic transmission at the sensory to motor synapse of Aplysia.

Over expression of Aplysia synaptotagmin in acutely dissected cholinergic neurons from the buccal ganglia, or in primary co-cultures of glutaminergic sensory neurons and motor neurons, causes a reduction synaptic transmission. Anti-sense oligonucleotide treatment of similar cultures produced an enhancement of synaptic transmission. The interaction between Aplysia VAMP/synaptobrevin and syntaxin is reconstructed using the yeast two hybrid system, and used to identify amino acid residues of VAMP/synaptobrevin that are required for this interaction. Point mutations around residue 50, close to the site of cleavage by botulinum toxins specifically disrupt the interaction with syntaxin. An additional VAMP/synaptobrevin binding protein, VAP33, is identified using the yeast two hybrid system. Intracellular injection of VAP33 specific antisera inhibits synaptic transmission in sensory-motor neuron co-cultures.

A new class of noninactivating K+ channels from aplysia capable of contributing to the resting potential and firing patterns of neurons.

From the nervous system of Aplysia, we have cloned a new class of noninactivating K+ channels (aKv5.1) that are activated at low voltage and are capable of contributing to the resting potential and firing patterns of neurons. Expression of aKv5.1 in Aplysia neuron R15 revealed that aKv5.1 exerts an unusual control over cell excitability; it increased the resting potential by more than 20 mV and abolished the spontaneous bursting activity of the cell. In its ability to suppress the endogenous rhythm of R15, aKv5.1 differs in its actions from transient, inactivating K+ channels such as aKv1.1a, an Aplysia homolog of Shaker. aKv1.1a shortens the duration of the spike and increases the afterpotential, but does not suppress bursting. Thus, by expressing different classes of K+ channels, it is possible to redesign, in specific ways, the signaling capabilities of specific, identified neurons.

Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.

Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons.

One of the hallmarks of long-term memory in both vertebrates and invertebrates is the requirement for new protein synthesis. In sensitization of the gill-withdrawal reflex in Aplysia, this requirement can be studied on the cellular level. Here, long-term but not short-term facilitation of the monosynaptic connections between the sensory and motor neurons requires new protein synthesis and is reflected in an altered level of expression of specific proteins regulated through the cAMP second-messenger pathway. Using gene transfer into individual sensory neurons of Aplysia, we find that serotonin (5-HT) induces transcriptional activation of a lacZ reporter gene driven by the cAMP response element (CRE) and that this induction requires CRE-binding proteins (CREBs). The induction by 5-HT does not occur following a single pulse, but becomes progressively more effective following two or more pulses. Moreover, expression of GAL4-CREB fusion genes shows that 5-HT induction requires phosphorylation of CREB on Ser119 by protein kinase A. These data provide direct evidence for CREB-modulated transcriptional activation with long-term facilitation.