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Keywords:

  • adenylate cyclase;
  • Aplysia;
  • protein kinase C;
  • serotonin (5-hydroxytryptamine);
  • serotonin (5-hydroxytryptamine) receptor;
  • synaptic plasticity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

J. Neurochem. (2010) 115, 994–1006.

Abstract

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT2, 5HT4, and 5HT7 families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT1Apl(a), 5HT2Apl, and 5HT7Apl were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT2Apl could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT2 receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT1Apl(a) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

Abbreviations used:
8-OH-DPAT

8-Hydroxy-2-(di-n-propylamino) tetralin

5HT

serotonin (5-hydroxytryptamine)

aa

amino acids

AC

adenylate cyclase

AP

action potential

cAMP

cyclic AMP

DAG

diacylglycerol

eGFP

enhanced green fluorescent protein

ERK

extracellular signal-regulated kinase

GPCR

G protein-coupled receptor

MN

motor neuron

mRFP

monomeric red fluorescent protein

ORF

open reading frame

PA

phosphatidic acid

PKA

protein kinase A

PKC

protein kinase C

PLC

phospholipase C

PLD

phospholipase D

PSP

post-synaptic potential

SF9

Spodoptera frugiperda 9

SN

sensory neuron

TCB-2

(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide

Aplysia is a powerful model system to reveal molecular events that control synaptic plasticity underlying learning and memory. There are three simple forms of non-associative learning studied in Aplysia; sensitization, habituation, and dishabituation of the gill-, tail-, or siphon-withdrawal reflex. Experimentally, sensitization of the withdrawal reflex is achieved by giving an aversive shock to a part of the body, such as the tail, which produces a stronger reflex to a previously innocuous stimulation (Pinsker et al. 1973). Habituation, however, is achieved when the innocuous stimulus is repeatedly presented, which decreases the reflexive response. Dishabituation takes place following habituation when a noxious stimulation is applied after habituating the animal, which restores the withdrawal response. These changes in behavioral response are reflected in concurrent changes in synaptic efficacy at sensory neuron (SN) to motor neuron (MN) synapses as facilitation, homosynaptic depression, and reversal of depression, all of which can be replicated at synapses between cultured sensory and MNs (Zhao et al. 2009). The cellular analog for sensitization is synaptic facilitation induced by the application of serotonin 5-hydroxytryptamine (5HT) to a SN-MN synapse, and habituation can be replicated in culture as synaptic depression induced by low frequency stimulation (Castellucci et al. 1970; Pinsker et al. 1970). This synaptic depression can be reversed by 5HT application, called reversal of synaptic depression, and is thought to underlie dishabituation (Hochner et al. 1986). Interestingly, the mechanism for reversal of depression is distinct from that of facilitation at naïve synapses as the former is mediated by protein kinase C (PKC), whereas the latter depends on protein kinase A (PKA) (Braha et al. 1990; Goldsmith and Abrams 1991; Ghirardi et al. 1992). The role of PKC has been confirmed behaviorally, PKC inhibitors in the SN block dishabituation (Antonov et al. 2010).

Protein kinase C is a family of serine/threonine kinases that mediate a wide variety of cellular processes including synaptic plasticity. There are four families of PKC isoforms: classical PKCs (α, β, and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι) (Sossin 2007). The classical PKCs are activated by Ca2+ and diacylglycerol (DAG), whereas the novel PKCs are still activated by DAG, but are Ca2+-independent, and the atypical PKCs are activated by neither Ca2+ nor DAG (Sossin 2007). In Aplysia, three isoforms of PKCs have been identified; PKC Apl I (classical PKC), PKC Apl II (novel type I PKC), and PKC Apl III (atypical PKC) (Kruger et al. 1991; Bougie et al. 2009). Among these PKCs, Ca2+-independent PKC Apl II is translocated by 5HT in Aplysia SNs (Zhao et al. 2006) and is required for 5HT-induced reversal of synaptic depression (Manseau et al. 2001). PKC Apl II translocation is mediated by coincident production of phosphatidic acid (PA) produced by phospholipase D (PLD), and DAG produced by phospholipase C (PLC) (Farah et al. 2008). However, 5HT receptors that are coupled to the PLC or PLD pathways have not been identified in Aplysia. The requirement of both the PLC and PLD pathways brings into question whether multiple receptors are required to activate PKC Apl II.

Serotonin (5-hydroxytryptamine) is a major facilitatory neurotransmitter in Aplysia and a crucial mediator of synaptic facilitation. The 5HT responding G protein coupled receptors (GPCRs) are known to be involved in synaptic plasticity by regulating activation of PKA and PKC. In vertebrates, 5HT1 and 5HT5 receptors are negatively coupled to adenylyl cyclase (AC) leading to the inhibition of PKA, whereas 5HT4, 5HT6, and 5HT7 are positively coupled to AC leading to the activation of PKA through the increased production of cyclic AMP (cAMP; Raymond et al. 2001; Barbas et al. 2003). 5HT2 receptors activate PLC, resulting in the generation of DAG and inositol triphosphate (Conn and Sanders-Bush 1986). DAG is bound to the plasma membrane and activates PKC, and inositol triphosphate leads to the release of Ca2+ from intracellular stores (Nichols 2004). In Aplysia, there were only two 5HT GPCRs that had been identified at the time this study was started, called 5HTApl1 and 5HTApl2 both negatively coupled to AC (Angers et al. 1998; Barbas et al. 2002).

The goal of this study was to identify 5HT GPCRs that are coupled to PKC Apl II activation. To this end, we cloned Aplysia orthologs for the mammalian AC-coupled receptors 5HT4 (5HT4Apl) and 5HT7 (5HT7Apl) and PLC-coupled 5HT2 (5HT2Apl). The receptor almost identical to 5HT7Apl was recently cloned from Aplysia Kurodai (Lee et al. 2009). Although both 5HT7Apl and 5HT2Apl could translocate PKC Apl II in a heterologous system, neither appears to be critical for translocation in SNs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Aplysia neuronal cultures and plasmid injection

Aplysia Californica (50–125 g for SNs; 50–250 g for LFS MNs) was obtained from the Mariculture Facility of the University of Miami (Miami, FL, USA). Aplysia dissociated SN cultures were prepared based on a previously described method (Farah et al. 2008) and plated on MatTek glass-bottom culture dishes with a glass surface of 14 mm (MatTek Corporation, Ashland, MA, USA) for live imaging. The DNA plasmids were microinjected at a concentration ranging from 100 to 400 ng/μL together with 0.4–0.5% of fast green. SNs were injected 1–4 days after they were plated on MatTek dishes, and imaged 1–2 days after injection. For electrophysiological experiments, LFS MNs were isolated from the abdominal ganglion and identified by morphology and plated such that the axon(s) was in contact with the axon of SNs. After 4 days in culture and prior to electrophysiological recordings, the culture media was replaced with artificial sea water (NaCl 460 mm, MgCl2 55 mm, CaCl2 10 mm, KCl 10 mm, HEPES 10 mm, pH 7.6).

PCR cloning of 5HT2Apl, 5HT4Apl, and 5HT7Apl

To clone 5HT GPCRs from Aplysia Californica, we initially used sequences of cloned 5HT genes from other molluscs, Lymnaea 5HT2 and Helisoma 5HT7 sequences. When the limpet Lottia sequence became available, we also used putative 5HT receptors from this assembly, including members of the 2, 4, and 7 families. We initially performed Aplysia trace searches on the NCBI website from the initial reads of the genome isolating fragments of Aplysia genomic DNA sequences that are homologous to these sequences and when an assembly was available repeated the searches on the assembly. For the regions that were neither available nor homologous, in particular, for the 5′-end, the genomic DNA sequences were used as a template to get the upstream sequences assuming no introns between the first transmembrane domain and the initiating methionine. Finally, we assembled those genomic DNA sequences using the software Lasergene SeqMan (DNASTAR, Madison, WI, USA) and obtained a putative Aplysia genomic DNA sequence for 5HT2Apl, 5HT4Apl, and 5HT7Apl. Primers based on the putative sequences were used for PCR amplification using a cDNA library prepared from RNAs extracted from the nervous system of Aplysia Californica (Table S1). For 5HT2Apl and 5HT4Apl, overlap PCR was performed from 5 and 3, respectively, overlapped regions of the separate PCR products. The products of these PCR amplifications were subcloned into pJET1.2 vectors and sequenced. After the sequences were verified, the entire sequence of each protein was subcloned into pNEX3 vectors.

Production of eGFP-tagged 5HT receptors

The signal sequence of sensorin was PCR amplified using the following sense and anti-sense primers: 5′-AGCAACATGCCTTCCAGAG-3′ and 5′-TGTCGGATATGAAGACCTGC-3′, and ligated into enhanced green fluorescent protein (eGFP)-pNEX3 vectors digested with BamHI and NcoI (sen-eGFP). To produce eGFP-5HT7Apl and eGFP-5HT1Apl(a), 5HT7Apl-pNEX3 and 5HT1Apl(a)-pNEX3 were cut with SacI and BsiWI and ligated with sen-eGFP cut with SacI and BsrGI. To produce eGFP-5HT2Apl, both sen-eGFP and 5HT2Apl-pNEX3 were cut with SacI and BsrGI and ligated, which produced eGFP-5HT2Apl with 3′-end missing (Δ3′eGFP-5HT2Apl). To produce eGFP-5HT2Apl with an intact 3′-end, both Δ3′eGFP-5HT2Apl and 5HT2Apl-pNEX3 were cut with FspI and ligated and correct orientation was confirmed.

RT-PCR

Pleural SNs were isolated from ganglia as described (Farah et al. 2008) and then used for RNA extractions using RNAqueous-4PCR (Ambion, Austin, TX, USA) and reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexamers. The resulting cDNA was used as a template for subsequent PCR amplification using primers specific to each mRNA (Table S2). The PCR products were visualized on agarose gel.

Live imaging of PKC Apl II translocation in Spodoptera frugiperda 9 cells and neurons

Live cell imaging was done as previously described (Farah et al. 2008) and is detailed in the Appendix S1.

Measurement and analysis of PKC Apl II translocation

Translocation was measured as previously described (Farah et al. 2008) with slight modification (see Appendix S1).

Electrophysiology

Electrophysiology was done as previously described (Weatherill et al. 2010) and is detailed in the Appendix S1.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Molecular cloning and structural analysis of 5HT2Apl, 5HT4Apl, and 5HT7Apl

To identify all 5HT receptors other than previously cloned 5HTApl1 and 5HTApl2, we performed comprehensive bioinformatics screening using both vertebrate and invertebrate 5HT receptors. As a result, we found Aplysia genomic DNA sequences homologous to Lymnaea 5HT2, putative Lottia 5HT4, and Helisoma 5HT7 receptors. We first obtained putative Aplysia sequences for 5HT2Apl, 5HT4Apl, and 5HT7Apl based on the homology to Lymnaea 5HT2, Lottia 5HT4, and Helisoma 5HT7 receptor sequences, respectively (see Materials and methods for details). For isolating all these receptors, PCR reactions were performed from Aplysia nervous system cDNA using oligonucleotide primers derived from the putative sequences.

For isolating 5HT2Apl, five overlapping clones (5HT2Apl.1, 5HT2Apl.2, 5HT2Apl.3, 5HT2Apl.4, and 5HT2Apl.5) covering 2976 bp of the 5HT2Apl sequence were amplified and cloned by overlap PCR (Fig. S1a). Alternative splicing of 660 bp was found in the clone 5HT2Apl.2, which corresponds to the third intracellular loop. Thus, spliced and non-spliced (full-length) forms of 5HT2Apl exist in the Aplysia nervous system. However, there was difficulty in reconstituting the longer receptor, and we were only successful in cloning the shorter form that we refer to as 5HT2Apl throughout the article. The spliced region of the receptor shares some homology with Lymnaea 5HT2 and contains several repetitive amino acid sequences (Fig. S1b). 5HT2Apl(s) contains an open reading frame (ORF) that encodes a protein of 992 amino acids (aa) with a predicted molecular weight of 108 kDa (Fig. S1c). 5HT2Apl has a very long N-terminal of about 440 aa, which is not well conserved (33% homology to Lymnaea 5HT2). It also has a long third intracellular loop of 468 aa with 62% and 38% homology to Lymnaea and Lottia 5HT2, respectively. Phylogenetic analysis with other invertebrate and mammalian 5HT receptors revealed that the cloned 5HT2Apl is a member of the 5HT2 family of receptors (Fig. 1).

image

Figure 1.  Phylogenetic analysis of selected serotonin 5-hydroxytryptamine (5HT) and dopamine receptors from invertebrates and vertebrates. The core regions (surrounding TM1-TM7 without non-consensus N- and C-terminals) were analyzed and aligned with the Phylip programs, and bootstrap numbers are given in each node and represent the percentage of total trees that give the tree shown (see Appendix S1 for more details and Table S3 for the nomenclature and original source). All of the receptors cloned in this study fall into well-defined classes of 5HT receptors. It should be noted that there appears to be an invertebrate specific class of dopamine receptors, separate from the orthologs of the D1 receptor. Aplysia has a member of this class that has not yet been characterized. The abbreviations are as followed: Lot (Lottia), Apl (Aplysia), Lym (Lymnaea), Dro (Drosophila), Hel (Helisoma), Cap (Capitella), Dinv (invertebrate dopamine receptor). Rat M3 (muscarinic acetylcoline receptor) was included as an outgroup.

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For isolating 5HT4Apl, three overlapping clones (5HT4Apl.1, 5HT4Apl.2, and 5HT4Apl.3) covering 1152 bp of the 5HT4Apl sequence were amplified and cloned by overlap PCR (Fig. S2a). The putative ORF of 5HT4Apl encodes a protein of 384 aa with a predicted molecular weight of 43 kDa (Fig. S2b). Phylogenetic analysis with other invertebrate and mammalian 5HT receptors revealed that the cloned 5HT4Apl belongs within the 5HT4 family of receptors (Fig. 1). 5HT7Apl was obtained by single PCR reaction, and the putative 1455 bp ORF encodes a protein of 485 aa with a predicted molecular weight of 54 kDa (Fig. S2c and d). Phylogenetic analysis with other invertebrate and mammalian 5HT receptors revealed that the cloned 5HT7Apl belongs within the 5HT7 family of receptors (Fig. 1).

All three receptors have seven putative transmembrane domains characteristic of GPCRs and display several key features common to mammalian and invertebrate 5HT receptors. In particular, the tripeptide aspartatic acid, arginine, tyrosine (DRY) at the interface of the third transmembrane domain and the second intracellular loop required for G protein coupling are conserved for 5HT4Apl and 5HT7Apl. Interestingly, instead of aspartatic acid, arginine, tyrosine (DRY), glutamatic acid, arginine, tyrosine (ERY) appears at the end of the third transmembrane domain, which is conserved in Lymnaea 5HT2. For all the receptors, the NPXXY motif required for receptor desensitization and internalization in the seventh transmembrane domain is conserved.

Using phylogenetic analysis, all the receptors identified are members of the previously described 5HT receptor families defined for vertebrate 5HT receptors. Thus, to name the receptor, we use the common convention of the subtype followed by the species as a subscript; i.e. 5HT2Apl receptor. For receptors that are recently duplicated, a subtype after the species name is used (e.g. 5HT1Apl(a)). For consistency, we use these names for all the receptors. In Table S3, the new names and previously used names are shown.

Expression of endogenous receptors and eGFP-tagged 5HT receptors

To determine which Aplysia 5HT receptors were candidates for mediating translocation of PKC Apl II during facilitation, single cell RT-PCR analysis was performed on pleural SNs, the same neurons used for our previous studies on activation of PKC Apl II and PKC Apl II mediated reversal of depression (Manseau et al. 2001; Zhao et al. 2006; Farah et al. 2008). This included the previously cloned 5HTap1 and 5HTap2, which are negatively coupled to AC, and we call 5HT1Apl(a) and 5HT1Apl(b), respectively, for the purpose of consistency with other receptors (Table S3). The results showed that 5HT1Apl(a), 5HT2Apl, and 5HT7Apl were expressed in SNs (Fig. 2a). For 5HT2Apl, both the spliced and non-spliced forms of receptors were expressed in SNs (data not shown). However, 5HT1Aplb and 5HT4Apl were not expressed. Thus, we decided to further examine the receptors that are expressed in SNs; 5HT2Apl, 5HT7Apl, and 5HT1Apl(a).

image

Figure 2.  Expression of serotonin 5-hydroxytryptamine (5HT) receptors in Aplysia sensory neurons (SNs) and construction of enhanced green fluorescent protein (eGFP)-tagged receptors. (a) Single cell RT-PCR analysis for 5HT receptors from Aplysia SNs. Sensorin was used as a positive control for SNs. 5HT1Apl(a), 5HT2Apl, and 5HT7Apl were expressed in SNs, whereas 5HT1Apl(b) and 5HT4Apl were not. All the receptors were expressed in Aplysia central CNS. (b) Schematic representation of an eGFP-tagged receptor construct. N-terminal of a receptor was truncated and replaced by eGFP following the signal sequence (SS) of sensorin. (c) Confocal images of over-expressed eGFP-tagged receptors, eGFP-5HT1Apl(a), eGFP-5HT2Apl, and eGFP5HT7Apl, in Spodoptera frugiperda 9 (SF9) cells and in Aplysia SNs. (d) Ratio of the plasma membrane to cytoplasmic localization of the over-expressed receptors in Aplysia SNs. eGFP-5HT1Apl(a) (n = 4) and eGFP-5HT7Apl (n = 13) were more than three times enriched at the plasma membrane, whereas eGFP-5HT2Apl (n = 27) showed similar levels of plasma membrane and cytoplasmic expression.

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To identify over-expressed receptors, eGFP-tagged constructs were generated by replacing most of the N-terminal of the receptors with eGFP (Fig. 2b). The long N-terminal seems to interfere with high expression of the receptor in 5HT2Lym, and the N-terminal truncated mutant, ΔN-5HT2Lym, expresses high levels of the receptor protein (Gerhardt et al. 1996). However, N-terminus secondary structure of a G protein-coupled receptor (GPCR) is suggested to be required for targeting into the endoplasmic reticulum, and therefore replacing its N-terminal with eGFP might not lead to successful surface expression of the receptor. This problem has been solved by adding an artificial signal sequence at the N-terminus, which led to efficient surface expression of the cannabinoid CB1 receptor (McDonald et al. 2007). Therefore, a signal sequence was placed in front of eGFP at the 5′-end to induce insertion of the receptor into endoplasmic reticulum (Fig. 2b). The resulting constructs, eGFP-5H1Apl(a), -5HT2Apl, and -5HT7Apl, were expressed in Spodoptera frugiperda 9 (SF9) cells and Aplysia SNs. The tagged eGFP-5HT2Apl receptor expressed at much higher levels than the non-tagged receptor including the N-terminus (Fig. S3), which could not be detected, necessitating the use of the tagged receptor. Strong plasma membrane localization of the receptor was observed in eGFP-5HT1Apl(a) and -5HT7Apl expressed cells, while more internal cytoplasmic localization was observed in eGFP-5HT2Apl expressed cells (Fig. 2c). In Aplysia SNs, the plasma membrane localization compared with the cytoplasm was more than threefold for eGFP-5HT1Apla and -5HT7Apl, whereas that for eGFP-5HT2Apl was close to one (Fig. 2d). It is not clear why some GPCRs are more basally internalized than others but in most cases, this is because of protein–protein interactions with either chaperones, anchoring proteins, or adaptors that enhance endocytosis (Bockaert et al. 2010). The large third intracellular loop and extended carboxy-terminus of eGFP-5HT2Apl provides a large number of sites for these interactions.

Translocation of PKC Apl II by 5HT2Apl and 5HT7Apl in Spodoptera frugiperda 9 cells

To determine the ability of 5HT receptors to translocate PKC Apl II to the plasma membrane, we used a SF9 cell system where there are no known endogenous 5HT receptors. In fact, PKC Apl II cannot be translocated by 5HT in SF9 cells in the absence of a co-transfected receptor (Fig. 3a and b). We have previously used this system to examine translocation of PKC Apl II to the plasma membrane by lipids and results from this system closely parallel results from SNs, suggesting PKC Apl II is functional in this system (Zhao et al. 2006; Farah et al. 2008). SF9 cells were cotransfected with one of eGFP-5HT1Apl(a), eGFP-5HT2Apl, or eGFP-5HT7Apl and monomeric red fluorescent protein (mRFP)-PKC Apl II, and the effects of addition of 5HT (10 μm) on PKC Apl II translocation was examined. Over-expression of 5HT2Apl and 5HT7Apl led to a significant translocation of PKC Apl II after 5HT treatment compared with pre-treatment (p < 0.05; Fig. 3a and b). Over-expression of 5HT1Apl(a), however, was not sufficient for 5HT-mediated translocation of PKC Apl II (Fig. 3a and b). There was no effect of any of the receptors on the initial distribution of PKC Apl II.

image

Figure 3.  Translocation of protein kinase C (PKC) Apl II in Spodoptera frugiperda 9 (SF9) cells by serotonin 5-hydroxytryptamine (5HT)2Apl and 5HT7Apl. SF9 cells were transfected with monomeric red fluorescent protein (mRFP)-PKC Apl II alone (no receptor), or cotransfected with either enhanced green fluorescent protein (eGFP)-5HT1Apl(a), eGFP-5HT2Apl, or eGFP-5HT7Apl and mRFP-PKC Apl II. (a) Confocal images of SF9 cells showing eGFP-tagged receptors and mRFP-PKC Apl II pre- and post-5HT (10 μm) treatment. (b) Quantification of PKC Apl II translocation ratio (Post-/Pre-5HT). Post-5HT is an average of 60, 90, 120, and 150 s after 5HT treatment (No receptor, n = 9; 5HT1Apl(a), n = 6; 5HT2Apl, n = 11; 5HT7Apl, n = 7). An asterisk (*) denotes p < 0.05 (Student’s paired t-test). (c) Confocal images of SF9 cells showing the effect of U-73122 or 1-Butanol on 5HT-induced translocation of mRFP-PKC Apl II in cells expressing eGFP-5HT2Apl (left) or eGFP-5HT7Apl (right). U-73122 (10 μm) or 1-Butanol (1%) was applied pre- and during 5HT (10 μm) treatment. (d) Quantification of PKC Apl II translocation ratio (Post-/Pre-5HT) with the inhibitors in SF9 cells expressing 5HT2Apl (Left: Control, n = 10; U-73122, n = 11; 1-butanol, n = 14) or 5HT7Apl (Right: Control, n = 11; U-73122, n = 7; 1-butanol, n = 12). U-73122 treatment significantly blocked 5HT-induced translocation in SF9 cells expressing 5HT2Apl; One-way anova, F(2,32) = 4.45, p = 0.0197; Tukey’s post hoc test, *p < 0.05 between control and U-73122, #p = 0.06 between control and 1-butanol. Both U-73122 and 1-butanol treatments blocked the translocation in cells expressing 5HT7Apl; One-way anova, F(2,27) = 7.31, p = 0.003; Tukey’s post hoc test, *p < 0.05 between control and U-73122, *p < 0.01 between control and 1-butanol. (e) Confocal images of SF9 cells showing the effect of phosphatidic acid (PA) in 5HT-induced translocation of mRFP-PKC Apl II in cells expressing eGFP-5HT2Apl (left) or eGFP-5HT7Apl (right). 1,2-Dioctanoyl-sn-glycero-3-phosphate (DiC8-PA) (PA, 2.5-5 μg/mL) was applied together with 5HT (10 μm). (f) Quantification of PKC Apl II translocation (Post-/Pre-5HT) with PA in SF9 cells expressing 5HT2Apl (Left: control, n = 17; PA, n = 23) or 5HT7Apl (Right: control, n = 15; PA, n = 13). Addition of PA increased 5HT-induced translocation of PKC Apl II in SF9 cells expressing 5HT2Apl; Student’s unpaired t-test, *p < 0.05.

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It was somewhat surprising that not only 5HT2Apl, but also 5HT7Apl was able to translocate PKC Apl II. We hypothesized that while 5HT2Apl translocates PKC Apl II by the PLC pathway through the production of DAG, 5HT7Apl may be able to translocate PKC Apl II by the PLD pathway through the production of PA. To test this hypothesis, we used the PLC inhibitor U-73122 and the PLD inhibitor 1-butanol as done previously to examine PKC Apl II translocation in neurons (Farah et al. 2008). 5HT-induced translocation of PKC Apl II in 5HT2Apl over-expressing cells was blocked by the application of U-73122 (10 μm), and strongly reduced by the application of 1-butanol (1%) (p < 0.01 between control and U-73122, p = 0.06 between control and 1-butanol; Tukey’s post hoc test; Fig. 3c and d). 5HT-induced translocation of PKC Apl II in 5HT7Apl over-expressing cells was completely blocked by the addition of either U-73122 or 1-butanol (p < 0.05 between control and U-73122, p < 0.01 between control and 1-butanol, Tukey’s post hoc test; Fig. 3c and d). Although suggestive of activation of both PLC and PLD by both receptors, it is possible that basal activation of PLC and PLD are also involved (Farah et al. 2008). We have previously shown that low concentrations of DAG and PA, which do not normally cause the translocation of PKC Apl II on their own, can synergistically translocate PKC Apl II when combined (Farah et al. 2008). If 5HT2Apl mainly activates PLC, addition of low concentrations of PA would synergize with DAG produced by PLC and increase the translocation of PKC Apl II. Indeed, addition of 2.5–5 μg/mL of 1,2-dioctanoyl-sn-glycero-3-phosphate (a cell-permeable form of PA), which does not cause translocation on its own (Farah et al. 2008) and data not shown, significantly increased the level of 5HT-induced PKC Apl II translocation in 5HT2Apl over-expressed cells (p < 0.05; Fig. 3e and f). The same treatment did not further increase the translocation in 5HT7Apl over-expressed cells (Fig. 3e and f), suggesting that PA levels are already saturating under these conditions. Taken together, these results suggest that the translocation of PKC Apl II by 5HT2Apl and by 5HT7Apl requires both PLC and PLD activation, but that activation by 5HT2Apl does not saturate PLD activation, whereas activation by 5HT7Apl does.

Identification of antagonists for 5HT2Apl and testing a role for 5HT2Apl in translocation of PKC Apl II in SNs

Selective antagonists are useful tools for determining the role of specific subtypes of GPCRs in physiological processes. The translocation of PKC Apl II in SF9 cells that is dependent on the co-expression of 5HT2Apl allowed us to test antagonists for this receptor. Previously, it was reported that reversal of synaptic depression was blocked by spiperone (Dumitriu et al. 2006), which is known to be an antagonist for 5HT2 receptors, in particular 5HT2A subtype, in mammals (Baxter et al. 1995), although it was a poor antagonist for the Lymnaea 5HT2 receptor (Gerhardt et al. 1996). Spiperone (even at the maximum concentration of 200 μm) failed to block the translocation of PKC Apl II (Fig. S4) in SF9 cells co-expressing 5HT2Apl, demonstrating that spiperone is not an effective antagonist for 5HT2Apl. Surprisingly, in our experiments, spiperone did not block 5HT-induced translocation of PKC Apl II in SNs nor 5HT-induced reversal of depression of SN-MN synapses (Fig. S4). Possible explanations for this difference from previous results can be found in the discussion.

Pirenperone is a specific 5HT2 receptor antagonist in vertebrates, and it strongly inhibited the translocation of PKC Apl II mediated by 5HT2Apl in SF9 cells (p < 0.05; Fig. 6). We hypothesized that 5HT2Apl would be the receptor coupled to the PKC Apl II translocation in SNs, which was tested using pirenperone. Contradictory to our hypothesis, pirenperone failed to inhibit 5HT-induced translocation of PKC Apl II in SNs (Fig. 4). It is possible that receptors are different in isolated SNs and sensory cells making synapses or that the receptors at synapses are distinct from those in the cell body (Sun et al. 1996). Thus, to determine if the 5HT2Apl receptor was important for synaptic activation of PKC Apl II, we determined if the 5HT2Apl antagonist, pirenperone, could block reversal of depression, which requires synaptic activation of PKC Apl II in SNs paired with MNs. However, pirenperone did not block reversal of depression, suggesting that 5HT2Apl is not the receptor required for the PKC Apl II translocation in SN cell soma or synapses (Fig. 4). Removal of the 5HT7Apl receptor using siRNA also did not block reversal of depression (Lee et al. 2009), suggesting that this is also not the receptor coupled to PKC activation in SNs.

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Figure 6.  Involvement of tyrosine kinase signaling in protein kinase C (PKC) Apl II translocation and reversal of synaptic depression. (a) Confocal images of sensory neurons (SNs) showing enhanced green fluorescent protein (eGFP)-PKC Apl II pre- and post-serotonin 5-hydroxytryptamine (5HT) (10 μm, 5 min) in the presence or absence of genistein (100 μm). (b) Quantification of PKC Apl II translocation (Post-/Pre-5HT). Genistein significantly inhibited 5HT-induced translocation of PKC Apl II (control, n = 16; genistein, n = 18); Student’s unpaired t-test, *, p < 0.05. (c) Effect of genistein in 5HT-induced reversal of synaptic depression. (d) Quantification of facilitation after 5HT treatment. Genistein significantly inhibited 5HT-induced reversal of synaptic depression (control, n = 6; genistein, n = 6); Student’s unpaired t-test, *, p < 0.05.

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Figure 4.  Effect of pirenperone on protein kinase C (PKC) Apl II translocation and reversal of synaptic depression. (a) Confocal images showing the effect of pirenperone in serotonin 5-hydroxytryptamine (5HT)-induced translocation of monomeric red fluorescent protein (mRFP)-PKC Apl II in Spodoptera frugiperda 9 (SF9) cells expressing enhanced green fluorescent protein (eGFP)-5HT2Apl (left) or 5HT-induced translocation of eGFP-PKC Apl II in Aplysia sensory neurons (SNs; right). Pirenperone (100 μm) was applied pre- and during 5HT (10 μm) treatment to SF9 cells expressing 5HT2Apl or pre- and during 5HT (20 μm) treatment to SNs. (b) Quantification of PKC Apl II translocation (Post-/Pre-5HT) with pirenperone in SF9 cells expressing 5HT2Apl (Left: control, n = 12; pirenperone, n = 10) or SNs (Right: control, n = 11; pirenperone, n = 11). Post-5HT is an average of 60, 90, 120, and 150 s after 5HT treatment in SF9 cells and 5 min after 5HT treatment in SNs. Pirenperone blocked 5HT-induced translocation of PKC Apl II in SF9 cells expressing 5HT2Apl; Student’s unpaired t-test, *p < 0.05 Pirenperone against control. However, it did not block 5HT-induced translocation of PKC Apl II in SNs. (c) Effect of pirenperone in 5HT-induced reversal of synaptic depression. 5HT was added after 40th post-synaptic potential (PSP). Pirenperone (100 μm) was applied pre- and during 5HT (10 μm) treatment. PSP amplitude was normalized to the first PSP. (d) Quantification of facilitation after 5HT treatment. % Facilitation = 100 × (Ave. PSP#41-43/Ave. PSP#38-40). Pirenperone did not have an effect in 5HT-induced reversal of synaptic depression (control, n = 2; pirenperone, n = 2); Student’s unpaired t-test, p < 0.05.

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Inhibition of PKC Apl II translocation by over-expression of 5HT1Apl(a) in SNs

Next, we examined if over-expression of 5HT receptors would increase the translocation of mRFP-tagged PKC Apl II in SNs as it did in SF9 cells. SNs over-expressing eGFP-tagged 5HT2Apl and 5HT7Apl translocated mRFP-PKC Apl II to the similar extent as the control group without receptor over-expression (Fig. 5). There was no correlation with the level of receptor (eGFP) and the amount of translocation (data not shown). Translocation is not saturated as the amount of the PKC Apl II translocation varies markedly between preparations of SNs and in these experiments translocation was not saturating (Farah et al. 2008). In contrast to over-expression of eGFP-tagged 5HT2Apl and 5HT7Apl, over-expression of 5HT1Apl(a) completely blocked PKC Apl II translocation (p < 0.01 compared with the control; Fig. 5b). Over-expression of eGFP-tagged 5HT1Apl(a) also completely blocked the reversal of synaptic depression (Fig. 5c). This was not because of a dominant negative effect on endogenous 5HT1Apl(a), as an agonist of this receptor, 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (Angers et al. 1998), did not cause translocation of eGFP-tagged PKC Apl II (Fig. S5).

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Figure 5.  Protein kinase C (PKC) Apl II translocation and reversal of synaptic depression blocked by over-expression of serotonin 5-hydroxytryptamine (5HT)1Apl(a) in sensory neurons (SNs). SNs were injected and expressed with monomeric red fluorescent protein (mRFP)-PKC Apl II alone (No receptor), or coexpressed with either enhanced green fluorescent protein (eGFP)-5HT1Apl(a), eGFP-5HT2Apl, or eGFP-5HT7Apl and mRFP-PKC Apl II. (a) Confocal images of SNs showing eGFP-tagged receptors and mRFP-PKC Apl II pre- and post-5HT (10 μm, 5 min) treatment. (b) Quantification of PKC Apl II translocation ratio (Post-/Pre-5HT). 5HT2Apl, and 5HT7Apl over-expression translocated PKC Apl II to the same extent as the control, whereas 5HT1Apl(a) over-expression blocked the translocation (control, n = 9; 5HT1Apl(a), n = 15; 5HT2Apl, n = 21; 5HT7Apl, n = 13). An asterisk (*) denotes p < 0.01 (Student’s paired t-test). (c) Effect of eGFP-5HT1Apl(a) over-expression in 5HT-induced reversal of synaptic depression compared to over-expression of eGFP only. (d) Quantification of facilitation after 5HT treatment. Over-expression of eGFP-5HT1Apl(a) significantly decreased 5HT-induced reversal of synaptic depression (eGFP, n = 5; eGFP-5HT1Apl(a), n = 5); Student’s unpaired t-test, p < 0.05.

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Over-expression of eGFP-tagged 5HT1Apl(a), which is known to couple to Gi and inhibit AC (Angers et al. 1998) would also be expected to inhibit the 5HT responses dependent on PKA, such as the increase in excitability (Lee et al. 2009). Indeed 5HT increased excitability in control injected neurons, observed as a significant reduction in the amount of current needed to generate an action potential (AP) in pairs with the SN expressing eGFP (71.4 ± 7.7% of the current required before 5HT to reach AP threshold, p < 0.05 paired t-test). In contrast, at synaptic pairs with the SN over-expressing eGFP-tagged 5HT1Apla, 5HT resulted in a significant reduction in SN excitability requiring almost twice as much current to fire a single AP (192.2 ± 28.4% of current required before 5HT, p < 0.05 paired t-test). This negative effect suggests an important role for basal AC activity for regulating levels of excitability.

Initial synaptic strength is highly variable at cultured synapses between Aplysia SN and MN, but there was the tendency for weaker synapses when the SN over-expressed 5HT1Apl(a)-GFP (14.2 ± 7.0 mV vs. 28.0 ± 7.0 mV in SN over-expressing eGFP). Although initial synaptic strength was weaker at synapses over-expressing eGFP-5HT1Apl(a), following 30 post-synaptic potentials (PSPs) at 0.05 Hz, average PSP amplitude at these synapses was stronger than synapses over-expressing eGFP (Fig. 5c, 3.6 ± 0.9 mV with eGFP and 4.7 ± 2.1 mV with eGFP-tagged 5HT1Apla, p < 0.05), suggesting less synaptic depression in this group. This decrease in the degree of synaptic depression was associated with a difference in the variance in PSP amplitude during depression. The average variance in PSP amplitude between PSP#20 and PSP#40, was over twofold greater at synapses over-expressing eGFP-5HT1Apl(a) in the SN (1.89 ± 0.55 with eGFP and 4.27 ± 2.39 with 5HT1Apla-GFP). Thus, eGFP-5HT1Apl(a) over-expression blocked the 5HT-mediated facilitation at depressed synapses, but also affected the kinetics and degree of depression.

Involvement of tyrosine kinase signaling in PKC Apl II translocation and reversal of synaptic depression

The vertebrate ortholog of PKC Apl II, PKC-epsilon, is translocated in vertebrate SNs through Ras-mediated activation of PLC-epsilon (Oestreich et al. 2009). Indeed, translocation of PKC Apl I mediating intermediate-term facilitation and memory was shown to be blocked by genistein, although this is presumably mediated by tyrosine kinase activation of extracellular signal-regulated kinase (ERK) ERK since ERK inhibitors also blocked translocation of PKC Apl I (Shobe et al. 2009). Inhibition of ERK did not affect translocation of PKC Apl II (Shobe et al. 2009). To determine if the translocation of PKC Apl II in SNs requires tyrosine kinase activity, we tested genistein, the kinase inhibitor used in the aforementioned study (Shobe et al. 2009). Interestingly, genistein significantly decreased both the 5HT-induced translocation of PKC Apl II and reversal of synaptic depression (p < 0.05 both compared with the control; Fig. 6). These results thus suggest involvement of tyrosine kinase activity in the PKC Apl II translocation and the reversal of synaptic depression.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Phylogeny of 5HT2Apl, 5HT4Apl, and 5HT7Apl

In this study, we isolated three novel 5HT receptors from Aplysia Californica that belong to the GPCR family. Evolutionary analysis revealed that the cloned receptors, 5HT2Apl, 5HT4Apl, and 5HT7Apl are members of the PLC-coupled 5HT2, AC-coupled 5HT4 and 5HT7 receptors, respectively. During our study, cloning of AC-coupled 5HTAplAC1 was reported from Aplysia Kurodai (Lee et al. 2009). 5HTAplAC1 is almost identical to 5HT7Apl except for a few amino acid differences, as would be expected from different Aplysia species. Phylogeny does not necessarily determine G protein activation. However, 5HTAplAC1 has been shown to couple to AC (Lee et al. 2009). The Lymnaea ortholog of the 5HT2Apl is coupled to PLC (Gerhardt et al. 1996), and our data with expression of the receptor in SF9 cells is also consistent with PLC coupling. None of the invertebrate 5HT4 receptors have been characterized and it will be interesting to determine if 5HT4Apl is coupled to AC.

Are we missing other 5HT receptors?

We have tried to be exhaustive in searching the Aplysia genome assembly for all biogenic amine receptors using mammalian and invertebrate 5HT receptors as probes. For each sequence, we used reverse blast approaches to determine the family member. We found fragments of receptors homologous to other biogenic amine receptors (octopamine and dopamine) and even some non-biogenic amine receptors such as muscarinic receptors using this strategy (data not shown). However, the genome assembly is not complete and we cannot rule out additional Aplysia receptors. Indeed, we did not find an Aplysia D2-like receptor, although there are D2 orthologs in Drosophila and Lottia (Fig. 3). Moreover, some Aplysia receptors have recently duplicated, for example, the 5HT1Apl(a) receptor (previously 5HT1Apla), while a member of the 5HT1 family appears to have originated recently as the 5HT1Apl(b) receptor (previously 5HTApl2) is closer to the lone 5HT1 receptor identified in Lottia, Capitella, and Lymnaea (Fig. 3).

The Aplysia B receptors (Li et al. 1995) also appear to be recently duplicated receptors as they also have no ortholog in the assembly. They have clearly duplicated from the Aplysia dopamine receptor Apl D1 or Apdop1 (Barbas et al. 2006). This is seen in Fig. 1, but can also be clearly seen from a simple Blast search where the Aplysia dopamine receptor is the first protein identified by a large margin. However, these receptors are evolving quickly and the Aplysia D1 receptor is actually more similar to other invertebrate D1 receptors than to the B receptors. In general, the biogenic amine families do not segregate by ligand (see Fig. 1). A good example is the 5HT4 receptors that are no more similar to any of the other 5HT receptor families than they are to any other biogenic amine receptors (data not shown). Thus, the B receptors, although they were recently duplicated from dopamine receptors, could be 5HT receptors. Indeed, there are actually at least seven members of this family, mostly found in tandem on a single genomic assembly (data not shown). While the data supporting the initial identification of these receptors as 5HT receptors could not be replicated (Li et al. 2003) whether these are 5HT or dopamine receptors or receptors for an unknown ligand is still not clear. It should be noted that dopamine does not induce PKC Apl II translocation in SNs (data not shown).

5HT receptors coupled to PKC Apl II translocation

In SF9 cells, both 5HT2Apl and 5HT7Apl could couple to PKC Apl II translocation. Removal of 5HT7Apl does not block the reversal of depression (Lee et al. 2009), but Apl II translocation at synapses is completely blocked by the inhibitor of PLD, 1-butanol (Farah et al. 2008), so either 5HT7Apl is not coupled to PLD in sensory cells, or there are additional mechanisms involved in the activation of PLD in SNs. For 5HT2Apl, an antagonist that completely blocked the ability of 5HT to translocate PKC Apl II in SF9 cells had no effect in SNs. Over-expression of 5HT2Apl did not increase translocation, despite the clear presence of the receptor. This could be owing to the saturation of the PLC pathway be endogenous receptors, even if the translocation was not saturated. PA may be rate limiting under normal conditions, and activation of 5HT2Apl does not seem to be very effective at activating PLD. It is possible that the removal of the N-terminal of the receptor altered its transduction or pharmacology. This seems unlikely since binding of agonists and antagonists to biogenic amine GPCRs occurs in the transmembrane domains. Moreover, the truncated receptor was activated by 5HT in SF9 cells. However, since the N-terminal containing receptor did not express, we cannot rule out differences between this receptor and the truncated receptor. As one additional test, we tried a 5HT2 agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2). However, TCB-2 did not cause translocation of PKC Apl II in SNs, but in the same neurons 5HT caused a robust translocation (1.05 ± 0.07 for 10 μm TCB-2, n = 5 and 1.91 ± 0.57 for 10 μm 5HT, n = 5). Overall, although our data cannot completely rule out 5HT2Apl as a receptor involved in PKC Apl II translocation, it is likely that other pathways are more important, at least in isolated SNs.

5HT1Apl(a) did not lead to translocation in SF9 cells, and an agonist of 5HT1Apl(a) did not cause translocation in SNs, suggesting that this receptor is not positively coupled to PKC Apl II translocation. However, this receptor is negatively coupled to translocation as over-expression of 5HT1Apl(a) blocked the translocation in SNs and blocked the reversal of synaptic depression. The dramatic reduction in SN excitability indicates antagonism of AC as expected (Ghirardi et al. 1992; Angers et al. 1998), and that some basal AC activity increases SN excitability in the absence of 5HT stimulation. Whether the effect of 5HT1Apla over-expression on PKC Apl II translocation and reversal of depression is related to the inhibition of AC or an alternate pathway will need to be determined. It is not clear if this receptor is present in all SNs; using in situ hybridization 5HT1Apl(a) was only expressed in a subset of pleural SNs (Barbas et al. 2005).

Thus, none of the 5HT receptors we identified as being present in SNs appears to be the important receptor coupled to translocation of PKC. As discussed before, there may be additional 5HT receptors expressed in SNs that we have not yet tested. The B receptors need to be re-examined, and as more sequences become available, there may indeed be new receptors to be seen.

The inhibition of both translocation and reversal of synaptic depression by genistein points to an important role for activation of a tyrosine kinase. Although genistein has a number of known targets other than tyrosine kinases, none of them can easily explain the acute effect of blocking PKC Apl II translocation. Activation of tyrosine kinases could be through transactivation of a GPCR, as occurs for ApTrkl (Nagakura et al. 2008), or by GPCR activation of an intracellular tyrosine kinase, such as Src (Yan et al. 2010). We did not observe activation of ERK (a downstream target of most tyrosine kinases) after 5HT treatment of SF9 cells expressing the receptors (data not shown).

Tyrosine kinase activation leads to activation of ras and thus suggests a ras-activated PLC, such as PLC-epsilon is involved. PLC-epsilon has been reported to activate PLD as well, perhaps through its rap-guanine exchange factor activity and thus is an attractive mechanism for PKC Apl II activation (Hucho et al. 2005). Aplysia has a PLC-epsilon ortholog (Sossin and Abrams 2009). If PLC-epsilon is involved, it could also suggest that PKC activation could be downstream of Epac, a cAMP-activated guanine exchange factor. Such activation mechanism by Epac has been reported for PKC-epsilon, the vertebrate ortholog of PKC Apl II (Hucho et al. 2005; Oestreich et al. 2009). A pathway with cAMP dependence would provide a mechanism for the observed effects of 5HT1Apla over-expression, a receptor known to be negatively coupled to AC. However, as discussed before, in cultured sensory-MNs, the AC-coupled 5HT7 receptor does not appear to play a role in the PKC Apl II-mediated recovery from synaptic depression (Lee et al. 2009).

Discrepancies from previous results

The major discrepancy from previous results is the inability of spiperone to block the reversal of synaptic depression in our experiments compared to the block seen in Dumitriu et al. (2006). There are a number of differences between the two experiments, including the type of SN (pleural SNs vs. abdominal LE SNs) and cultured neurons versus neurons in isolated ganglia. This could suggest that different SNs, or neurons in distinct environments could use different receptors to activate PKC Apl II. Interestingly, one difference between pleural SNs and abdominal LE neurons is the expression of 5HTApl1(a) that is reportedly not expressed in LE neurons, but is expressed in a subset of pleural SNs (Barbas et al. 2005).

The spiperone-sensitive receptor is unlikely to be 5HT2Apl as this receptor is insensitive to spiperone, as is the closely related Lymnaea ortholog. Spiperone blocks dopamine receptors, and although dopamine does not cause PKC Apl II translocation in cultured SNs (data not shown), it has not been examined for the reversal of synaptic depression in ganglia. This would be particularly important if the B receptors are responsible. Since spiperone does not block PKA-mediated effects in the ganglia experiments (Dumitriu et al. 2006), it is unlikely to act through 5HTApl7 receptors.

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

We have cloned several novel 5HT receptors from Aplysia. mRNA encoding the 5HT2Apl and 5HT7Apl receptors are expressed in SNs, but they do not appear to contribute to 5HT-mediated translocation of PKC Apl II or the reversal of synaptic depression. We have confirmed that mRNA encoding the 5HT1Apl(a) receptor is present in isolated pleural SNs and this receptor appears to play an inhibitory role in PKC Apl II translocation and reversal of synaptic depression. Finally, tyrosine kinases were shown to play a role in both translocation of PKC Apl II and reversal of synaptic depression. Surprisingly, 5HT-mediated translocation of PKC Apl II appears to require a complex signal transduction pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the Canadian Institutes of Health Research (CIHR) operating grant MOP 12046 to WSS. IN is a recipient of the Jeanne Timmins-Costelllo Fellowship. CAF is a postdoctoral fellow of the Fonds de la Recherche en Santé du Québec (FRSQ) and a Conrad Harrington fellow. TWD is supported by a fellowship from CIHR. WSS is a James Mcgill Scholar and a Chercheur national of the FRSQ.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Appendix   S1.     Supplementary Materials and methods.

Figure   S1.     Molecular cloning of 5HT2Apl.

Figure   S2.     Molecular cloning of 5HT4Apl and 5HT7Apl.

Figure   S3.     Comparison of xxpression of tagged eGFP-5HT2Apl and unmodified 5HT2Apl receptors.

Figure   S4.     Effect of spiperone on PKC Apl II translocation and reversal of synaptic depression.

Figure   S5.     Effect of 8-OH-DPAT on PKC Apl II translocation.

Table   S1.     List of PCR primers for cloning of 5HT receptors.

Table   S2.     List of primers for single cell RT-PCR for 5HT Receptors.

Table   S3.     Nomenclature of receptors.

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