Dacks AM et al. (2013), A characterization of the Manduca sexta seroton...

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PLoS One 2013 Jan 01;87:e69422. doi: 10.1371/journal.pone.0069422.

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A characterization of the Manduca sexta serotonin receptors in the context of olfactory neuromodulation.

Dacks AM , Reale V , Pi Y , Zhang W , Dacks JB , Nighorn AJ , Evans PD .

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Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of Manduca sexta. To this end we cloned all four known insect serotonin receptor types from Manduca (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype.

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Species referenced: Xenopus laevis
Genes referenced: camp cftr htr1a htr7 sod1

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	Figure 2. Phylogenetic relationships between the Ms5HTRs and other amine receptors.Ms5HTRs are indicated with bold text (A) Phylogenetic tree of all available serotonin, tyramine, octopamine and dopamine receptors from 7 species of insects. This figure is arbitrarily rooted on the 5-HT2 clade. Strongly supported clades for 5-HT1, 2, 7 Dopamine 1, 2, Octopamine A, B, and Tyramine are generated as shown. The newly characterized Ms5HT2R sequence is present in the 5-HT2 clade and Ms5HT7R sequence in the 5-HT7 clade. In this and all subsequent phylogenetic trees, the best Bayesian topology is shown with node support values listed in the order of Posterior probability values, Maximum Likelihood bootstrap support values for PhyML and Maximum Likelihood bootstrap support values for RAxML. Other values are replaced with symbols as shown in the figure. Vertical bars with labels denote the reconstructed clades. The scale bar represents the number of changes per site. (B) Phylogenetic analysis of serotonin receptors from insects (I) and vertebrates (V), with the 5-HT2 receptors excluded. The figure is arbitrarily rooted on the vertebrate 5-HT3 clade, but should be treated as unrooted. Note the reconstruction of the various named insect and vertebrate receptor sub-types.
	Figure 3. Responses of the Ms5HTRs for 5-HT and the dose response curves for selective agonists.(A) Currents (normalized to maximal responses) evoked by increasing concentrations of 5-HT (from 0.1nM to 10 µM) in Xenopus laevis oocytes expressing the Ms5HT1A (black circle), Ms5HT1B (grey triangle), Ms5HT2 (open triangle), Ms5HT7 (black diamond). (B) Xenopus oocytes expressing the Ms5HT2 receptor (open triangle) respond to 5-nonyloxytryptamine at 0.1 µM and above, while oocytes expressing the Ms5HT1A (black circle), Ms5HT1B (grey triangle) or Ms5HT7 (black diamond) receptors do not respond at any of the concentrations tested. The symbols for the Ms5HT1A, 1B and 7 receptors have been staggered for clarity. In (A) and (B) the 5HT1A responses were measured in oocytes co-injected with receptor cRNA and cRNA for the CFTR channel whilst the 5HT1B, 5HT2 and 5HT7 responses were measured in oocytes injected with the receptor cRNA alone. Oocytes were exposed to 2 min pulses of the agonists at various concentrations. (C) HEK293 cells expressing the Ms5HT2 receptor respond (measured as nM concentration of IP-1/well of a 96 well plate) to 5-HT and DOI (4-Iodo-2,5-dimethoxy-α-methylbenzene ethanamine). (D) HEK293 cells expressing the Ms5HT7 receptor respond (measured as nM concentration of cAMP/well of a 96 well plate) to 5-HT, but not DOI. Shades of grey in (C) and D) indicate concentration of pharmacological agent applied. All error bars represent SEM.
	Figure 4. All four Ms5HTRs are expressed in both the antennal lobes and the antennae.RT-PCR for the four Ms5HTRs using cDNA from (A) antennal lobes and (B) the antennae. For each receptor, the left-hand lane depicts the RT-PCR with the reverse transcriptase included during cDNA production and right-hand lane depicts the RT-control to ensure no genomic DNA contamination.
	Figure 5. The Ms5HT1A receptor is expressed in a subset of GABA-like immunoreactive local interneurons.(A) Western blots of the AM3-A antibody against Manduca protein and in which the AM3-A antibody was omitted (no 1o) produced no labeling. (B) Frontal section through an AL labeled using an antibody against a 5-HT1A receptor from prawn (AM3-A) generously provided by Dr. Maria Sosa. Note cell bodies (arrowheads; 24±2) and fine processes (arrows). (C) Manduca ALs labeled with AM3-A antibody pre-adsorbed with the Ms5HT1A receptor sequence (KDPDYLARITQQQKCLVSQD) homologous to the antigenic sequence used to generate the AM3-A antibody (KDPDFLVRVNEHKKCLVSQD) resulted in no labeling. (D) Double labeling against the Ms5HT1A receptor (green) and 5-HT (magenta) show no overlap. (E) Backfills of projection neurons (PNs) (magenta) reveal no co-localization of the Ms5HT1A receptor (green) in the lateral cell cluster of the AL. Magenta arrows indicate PNs and green arrows indicate Ms5HT1A-ir neurons. (F) Double labeling against the Ms5HT1A receptor (green) and GABA (magenta) revealed a subset of Ms5HT1A-ir cell bodies co-localizing GABA (arrows). All scale bars=100µm.
	Figure 1. ClustalW sequence alignments for the Manduca 5-HT2 and 5-HT7 receptor homologues.Grey rectangles enclosing amino acid sequences indicate conserved sequence. For both receptors the putative 7 transmembrane domains are indicated by the black bars. Potential PKA/PKC phosphorylation sites are indicated by a “†”, tyrosine kinase phosphorylation sites by a “‡” and N-glycosylation sites by a “*”. (A) Alignment of the Ms5NT2R with 5-HT2 type receptors from Apis mellifera (Am5HT2), Drosophila melanogaster (DM5HT2) and Anopheles gambiae (Ag5HT2). (B) Alignment of the Ms5HT7 receptor with 5-HT7 receptors Drosophila melanogaster (Dm5HT7), Aedes aegypti (Ae5HT1) and A. mellifera (Am5HT7).

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