Figure 1.
Sequence of the apUII precursor and its comparison to known homologous sequences from vertebrates.
(A) Consensus cDNA (plain font), translated protein sequence (bold font). Right: open reading frame map (full vertical line, stop codon; half line, met, beginning of protein) shows three reading schemes: 1. as read shown at left, 2. shift right by 1 nucleic acid, 3. shift right by 2 nucleic acids. The map shows Scheme 1 is the most appropriate reading scheme. (B) A comparison between known UII precursor sequences from different species; for human, mouse and rat, urotensin-2B prohormone sequences (Q76510, Q76511, Q76512, respectively) were included in the alignment in addition to UII sequences For carp, two sequences found in UniProtKB and differing by a few single amino acid substitutions were also included. Protein accession numbers are shown at left. The BLOSUM62 identity scoring is shaded; the lighter shade represents an identity of >50%, darker shade is 100%. Symbols: “*” indicates fully conserved amino acid positions, “:” and “.” indicate strong (>0.5) and weak (≤0.5) positively scoring groups that occur in the Gonnet Pam250 matrix. (C) Sequence alignment of apUII and contulakins from the lettered cone snail shows similarity highlighted by the BLOSUM62 method. (D) the urotensin II precursor phylogenetic tree calculated by the neighbor-joining method from the multiple sequence alignment.
Figure 2.
Distribution of apUII positive neurons in the buccal and cerebral ganglia.
(A) Diagram of the buccal ganglion neurons involved in generating feeding behavior, highlighting the sensory neuron cluster. The larger motoneurons are present as a cluster on the dorsal surface of the ganglion (the larger orange cells above), while the smaller sensory neurons are present in a cluster on the dorsal-medial aspect of the ganglion. The population of buccal sensory neurons is heterogeneous. They are grouped into at least three clusters with distinct sensory properties and transmitter contents. The radula mechanoafferent neurons (green) innervate the subradular tissue and contain SCP and enterin. The S1 sensory neuron cluster (purple) is present only on the caudal surface and is distinguished by a slightly larger size than the S2 sensory neuron cluster (brown). The S1 cluster contains primarily FMRFamide, while S2 contains primarily FRFamide. Here, we show that apUII is expressed in both the S1 and S2 clusters. Note that the commissure is twisted, so that on the left, the rostral surface of the buccal ganglion is shown, while the right side shows the caudal surface. Nerve abbreviations, Com: buccal commissure; CBC: cerebral-buccal connective; EN, esophageal nerve; N, buccal nerve; RN: radular nerve. (B) In-situ hybridization staining of the buccal and cerebral ganglion shows the distribution of apUII mRNA expressing neuron somata. The buccal sensory neurons express the mRNA for apUII. Abbreviations, rBG: rostral side of the buccal ganglion; cBG: caudal side of the buccal ganglion; dCG: dorsal side of the cerebral ganglion; vCG: ventral side of the cerebral ganglion. S1, S2, A-, F- and C-neuronal clusters are labeled.
Figure 3.
Mass spectrometric analysis of apUII prohormone processing and structure verification.
(A) Representative MALDI MS spectrum of individual cultured buccal sensory neurons expressing FMRFa, FRFa and apUII prohormones. apUII-derived peptides (in bold), FMRFa, FRFa peptides (a–e) and an apUII linker peptide are labeled. The inset table specifies the accuracy of the apUII peptides measurement; (M+H) is the monoisotopic mass of the protonated molecular ions, (*) denotes the theoretical mass corrected for the difference from the disulfide bond. (B) Fragmentation spectrum of apUII peptide S [93]-S [112] detected in a +2 charge state with the two red Cys residues highlighted (lower case c). (C) Fragmentation spectrum of apUII peptide S [45]-Q [82] detected in a +2 charge state.
Figure 4.
Immunostaining of apUII-positive neurons and fibers in the Aplysia CNS.
(A) The distribution of immunostained neurons in the buccal ganglion is similar to the in-situ hybridization (ISH) staining distribution shown in Figure 2B. Abbreviations, rBG: rostral surface of the buccal ganglion; cBG: caudal surface of the buccal ganglion. Arrowheads point to individual stained neurons. (B) The distribution of immunostained neurons in the cerebral ganglion is similar to the ISH staining distribution shown in Figure 2B. Abbreviations, dCG: dorsal surface of the cerebral ganglion; vCG: ventral surface of the cerebral ganglion; CPC cerebral-pedal connective; A, C, F, neuronal clusters. (C) The distribution of immunostained neurons in the pedal, pleural and abdominal ganglia. Abbreviations, rdPPG: right dorsal pleural-pedal ganglia; ldPPG: left dorsal pleural-pedal ganglia (for these two images, the pleural ganglia are on the left); rvPPG: right ventral pleural-pedal ganglia; lvPPG: left ventral pleural-pedal ganglia (for these two images, the pleural ganglia are on the right); dAG: dorsal abdominal ganglion; vAG: ventral abdominal ganglion. Arrowheads point to stained neurons. Calibration bar for all panels : 200 µm.
Figure 5.
apUII actions on motor programs elicited by a command-like interneuron CBI-2.
(A) Representative examples. CBI-2 was stimulated throughout the protraction phase (open bar) to elicit single cycle motor programs; the inter-trial interval is 1 min. In control condition (A1), B8 is predominantly active during the retraction phase (filled bar), while B61 is only active during protraction. Perfusion of 10−6 M (A2) and 10−5 M (A3) apUII’ reduced the firing frequency of both B8 and B61, but had a lesser effect on the firing frequency of B4/5. Protraction is monitored via activity of I2, retraction via activity of BN2. (B) Group data showing the suppressive effects of 10−6 M and 10−5 M apUII’ on B8 firing frequency during protraction (B1), or during retraction (B2), and B61/62 firing frequency during protraction (B3). (C) Group data showing the lengthening of the latency to protraction initiation, measured as the time that elapsed from the onset of CBI-2 stimulation to the onset of I2 nerve activity (C1). apUII’ also reduced protraction duration (C2) and retraction duration (C3). *, p<0.05; **, p<0.01; ***, p<0.001; error bars represent SEM.
Figure 6.
apUII inhibits the generation of spontaneously occurring programs.
(A) In control condition, motor programs occurred spontaneously. (B) Perfusion of 10−5 M apUII reduced the frequency of motor programs. (C) Upon washout of the peptide, motor programs occurred more frequently. Each cycle of motor programs consists of a protraction (open bar) and retraction (filled bar) sequence.
Figure 7.
Summary data showing the effects of 10−6 M and 10−5 M apUII on the frequency of spontaneously-occurring motor programs.
The frequency of motor programs is the number of cycles per minute measured over a period of 10 minutes or more in each experimental condition. There is a dose-dependent reduction in the frequency of spontaneously-occurring motor programs. *, p<0.05; error bars represent SEM.