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Exp Neurobiol 2015; 24(3): 246-251
Published online September 30, 2015
© The Korean Society for Brain and Neural Sciences
Deok-Jin Jang1,#, Hyoung F. Kim2,#, Jae-Hoon Sim2, Chae-Seok Lim2 and Bong-Kiun Kaang2,*
1Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sangju 37224, Korea 2Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-880-7525, FAX: 82-2-884-9577
#These authors contributed equally to this work.
Phosphodiesterases (PDEs) play a key role in the regulation of cyclic adenosine monophosphate (cAMP), which in turn mediates various cellular functions including learning and memory. We previously cloned and characterized three PDE4 isoforms (ApPDE4) from
Keywords: Aplysia, phosphodiesterase 4, bag cell, in situ hybridization
Phosphodiesterases (PDEs) are key regulators of cyclic adenosine monophosphate (cAMP), an important signaling molecule implicated in various physiological functions including addiction and memory [1,2,3]. We previously cloned long-, short- and supershort-isoform of
Previously, using reverse transcription polymerase chain reaction (RT-PCR) with isoform-specific primer sets, mRNAs of ApPDE4 isoforms were found to be primarily expressed in various tissues including the central nervous system . However, the detailed distribution of ApPDE4s mRNAs in
A digoxigenin-labeled ApPDE4 RNA anti-sense or sense probe from the PDE catalytic domain corresponding to 510~842 amino acids of ApPDE4 long-form was synthesized using T7 or T3 RNA polymerase with the ApPDE4 cDNA clone as the template, respectively. The probe was dissolved in hybridization buffer (1 µg/ml), and the ganglia were hybridized overnight at 50℃ and then washed at 60℃ in washing solution A (50% formamide, 5× SSC, 1% SDS), washing solution B (50% formamide, 2× SSC, 1% SDS), and washing solution C (0.2× SSC).
The ganglia were washed with PBS and then blocked for 2 h with 10% normal goat serum in PBS at RT. After blocking, the ganglia were incubated overnight with alkaline-phosphatase-conjugated anti-digoxigenin antibody diluted 1:1500 in PBS containing 1% goat serum at 4℃. These ganglia were rinsed with detection buffer (100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, 1 mM lavamisol, 10 mM Tris-HCl, pH 9.5) and then developed with 4.5 µl of NBT and 3.5 µl of BCIP in 1 ml of detection buffer. The staining reaction was monitored and subsequently stopped with 4% formaldehyde in methanol when the signal became readily apparent. The ganglia were observed and photographed using a microscope (Nikon) attached with a digital camera (Nikon CoolPix 995). We used three animals for each experimental groups and picked the typical one for the presentation.
In this study, we examined distributions of all PDE4 mRNA isoforms in
ISH-positive cells were also observed in cerebral, pleural and pedal ganglion. First, the dorsal and ventral surfaces of the cerebral ganglion contained clusters positive for ApPDE4s, but the signals were weaker than the bag cells' (Fig. 2). On the dorsal surface of the cerebral ganglion (Fig. 2A), A, B and G cluster cells were stained strongly and symmetrically. Interestingly, metacerebral (MCC) neurons were stained in the G cluster. C, D and F cluster cells were stained less than the other clusters, but the ISH-positive neurons were detected symmetrically on both sides. On the ventral surface of the cerebral ganglion, ISH-positive neurons were observed in the outer layers of the A, B, and G clusters. The serotonergic MCC neurons are involved in food-induced arousal by direct modulation of ingestion-related B21 neurons in the buccal ganglion . The MCC neurons receive long-lasting excitatory synaptic input by nitric oxide (NO) and histamine which induce cAMP-mediated signaling pathways . Therefore, ApPDE4 might play a role in food-induced arousal via indirect pathways such as cAMP-dependent signaling.
Second, we found ISH-positive neurons in each pleural ganglion (Fig. 3A-D). The giant cell, LP1 which was a homologous giant neuron of R2, was stained on the dorsal surface of the left pleural ganglion as similar intensity level with R2 (Fig. 3A and 2A). The LP1 and R2 are cholinergic neurons involved in mucus release from the body wall . This suggests that ApPDE4 has a role in mucus release. Some ISH-positive cells were observed near the pleura-abdominal, cerebro-pleural and pleura-pedal connections of the right pleural ganglion. On the ventral surface of both the pleural ganglia, several mechanosensory neurons from the sensory cluster were stained (Fig. 3C and D). Last, ISH-positive cells were detected in the pedal ganglion and these also showed the symmetrical staining pattern (Fig. 3E-H). On the dorsal surface of both pedal ganglia, ISH-positive neurons were located near the base of P3 and the middle pedal nerve (P9) (Fig. 3E and F). Additional ISH-positive neurons were found on the ventral surface near the pedal-pleural connective, cerebral connective, and P1 nerve in a symmetrical pattern (Fig. 3G and H). Previous study using RT-PCR showed that the short- and long-form of ApPDE4 were strongly expressed in the pleural ganglion, but the supershort-form was weakly expressed . In consistent with our previous results, we found the mRNA expressions of ApPDE isoforms in mechanosensory neurons of pleural ganglion (Fig. 3-C and D). These data suggest that short- and long-form of ApPDE4 in the pleural ganglion have main functions in 5-HT-induced synaptic plasticity in