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Biol. Bull. 209: 67-74. (August 2005)
© 2005 Marine Biological Laboratory

Pharmacological Manipulation of Serotonin Levels in the Nervous System of the Opisthobranch Mollusc Tritonia diomedea

David J. Fickbohm, Nadja Spitzer and Paul S. Katz{dagger}

Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, Georgia 30302-4010

* Present Address: Santa Monica College, Life Sciences, 1900 Pico Boulevard, Santa Monica, California 90405. E-mail: fickbohm_david{at}smc.edu

{dagger} To whom correspondence should be addressed. E-mail: pkatz{at}gsu.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Serotonin-related disorders can be treated by manipulating serotonin synthesis with the serotonin precursor 5-hydroxytryptophan (5-HTP) or other pharmacological agents. The mollusc Tritonia diomedea is a model for investigating the effects of altering serotonin content on the functions of identified neurons. We used high-performance liquid chromatography and immunohistochemistry to examine the amount and localization of 5-HTP, serotonin, and the serotonin breakdown product 5-hydroxyindolacetic acid (5-HIAA) in the Tritonia brain after various pharmacological treatments. Exposure to 5-HTP (2 mM for 30 min–1 h) caused an immediate and massive increase in total 5-HTP content, which lasted more than 20 h, and the widespread appearance of 5-HTP immunoreactivity in neurons. Serotonin levels rose gradually, but only a restricted number of additional neurons displayed serotonin immunoreactivity. 5-HTP treatment also caused an increase in the total amount of 5-HIAA and the appearance of 5-HIAA immunoreactivity throughout the brain. Treatment with the synthesis cofactor tetrahydrobiopterin, the initial precursor tryptophan, or serotonin itself had no persistent effect on total serotonin content. The amino acid decarboxylase inhibitor hydroxybenzylhydrazine (NSD-1015) also had no effect on the total serotonin content, although it caused an accumulation of 5-HTP. Thus, serotonin levels in the brain of T. diomedea appear to be maintained by a homeostatic mechanism that can be disrupted by 5-HTP.

Abbreviations: AADC, aromatic amino acid decarboxylase • BH4, tetrahydrobiopterin • DSI, dorsal swim interneuron • 5- HTP, 5-hydroxytryptophan • 5-HIAA, 5-hydroxyindolacetic acid • NSD-1015, hydroxybenzylhydrazine


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Some clinical therapies for serotonin-related disorders involve altering the levels of serotonin by affecting its biosynthesis or breakdown (van Praag, 1981; Byerley et al., 1987; Birdsall, 1998). Yet the effect of changing serotonin levels on the functions of serotonin neurons is not well understood. We are using a molluscan nervous system that contains identified neurons in well-characterized neuronal circuits as a model for understanding how altering serotonin levels affects the operation of neuronal circuits that use serotonin. Previously, we showed that in the opisthobranch mollusc Tritonia diomedea, exposure of the brain to the serotonin precursor 5-hydroxytryptophan (5-HTP) causes a prolonged (>20 h) enhancement of the actions of identified serotonergic neurons called the dorsal swim interneurons (DSIs) (Fickbohm and Katz, 2000). Since both the pattern of activity expressed by the DSIs and their synaptic connections are known, these serotonergic neurons in T. diomedea can serve as a model for examining the effects of 5-HTP on neurons in defined neuronal circuits. We sought to more fully characterize the effect of 5-HTP treatment on T. diomedea and to examine other means of altering serotonin levels in this model system.

To compare this molluscan system to mammals, our laboratory examined the effect of oral administration of 5-HTP to rats (Lynn-Bullock et al., 2004). It was found that 5-HTP causes only a transient increase in serotonin, as measured by excretion of serotonin metabolites in the urine and by changes in serotonin immunoreactivity in the brain (Lynn- Bullock et al., 2004). The maximum change seems to occur within 2 h of administration. By 24 h, serotonin returns to control levels. Yet, in T. diomedea, the outputs of the DSIs were enhanced for more than 20 h after treatment (Fickbohm and Katz, 2000), suggesting a difference in serotonin metabolism between mammals and molluscs. Therefore, in this study we examined the changes in serotonin levels in the nervous system of T. diomedea after exposure to 5-HTP. We also investigated whether other methods for altering serotonin levels are effective in this mollusc.

Some of these results were presented in abstract form (Fickbohm et al., 2000).


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Specimens of Tritonia diomedea Bergh (1894) were obtained from Living Elements (Delta, British Columbia, Canada). Techniques for dissection, immunohistochemistry, and high-performance liquid chromatography (HPLC) were previously described (Fickbohm and Katz, 2000; Fickbohm et al., 2001). In all experiments, matched control and experimental ganglia were processed side-by-side. During the initial, gross stage of dissection, the brains, comprising the bilaterally symmetric cerebropleural and pedal ganglia, were quickly removed from each animal in a test group and placed together in saline (consisting of [in mM] 420 NaCl, 10 KCl, 10 CaCl2, 50 MgCl2, 10 D-glucose and 10 HEPES, pH 7.6), chilled to 2 to 4 °C. The connective tissue was removed from each ganglion, and nerves were trimmed to minimize variability in HPLC. During the treatment period, saline temperature was raised to 10 °C, the temperature at which the animals were kept in the laboratory. Drugs were applied for 30 min to 1 h, and the preparation was then superfused with normal saline for the times indicated in each experiment. Matched control preparations were subjected to identical conditions with the exception of drug application.

For immunohistochemistry, the ganglia were treated with 0.5% type XIV protease (Sigma, MO) for 5–10 min, washed in normal saline, and fixed overnight at 4 °C in paraformaldehyde-lysine-periodate fixative (McLean and Nakane, 1974). The brains were washed by rinsing two times (30 min) with cacodylate buffer (0.2 M cacodylic acid [Na salt] in 0.3 M NaCl, pH 7.5), followed by two rinses (30 min) with 4% Triton X-100 in phosphate-buffered saline (PBS, consisting of 50 mM Na2HPO4, 140 mM NaCl, pH 7.2). The ganglia were then incubated in antiserum diluent (consisting of 0.5% Triton X-100, 1% normal goat serum, 1% bovine serum, in PBS) for 1 h. The preparations were incubated for 72 h in primary antiserum (rabbit polyclonal anti-5HT, anti-5HTP, or 5-HIAA: DiaSorin, Inc. (Immunostar), Stillwater, MN) diluted 1:200 to 1:10,000 in antiserum diluent as indicated. After rinsing in 0.5% Triton X-100 in PBS (6 h), the ganglia were exposed overnight to goat anti-rabbit antiserum conjugated to Cy2, Cy3, Alexa 488, or Alexa 546 fluorochromes (Molecular Probes, Eugene, OR) diluted 1:50 or 1:100 in antiserum diluent. After this step, the ganglia were washed with 0.5% Triton X-100 (in PBS) for 6 h, dehydrated in a graded series of ethanol washes, cleared in methyl salicylate, and mounted and cover-slipped on microscope slides by using Cytoseal 60 (Stephens Scientific, Kalamazoo, MI).

Fluorescence images were visualized by using a laser scanning confocal microscope (LSM 510, Carl Zeiss, Inc., Thornwood, NY) with a 10x or 20x objective. For experiments involving comparisons of staining intensity in different animals, ganglia from an experimental group were processed for immunohistochemistry in parallel, using aliquots of the same solutions, and visualized using identical confocal microscope settings. Photomicrographs were acquired using LSM 510 software and manipulated with Adobe Photoshop or Corel Photopaint to adjust the overall brightness and contrast. All images within each treatment group were manipulated identically. Staining intensity was measured by pixel-intensity averaging as previously described (Fickbohm and Katz, 2000).

For HPLC, excess moisture was blotted from the brains, and they were weighed before placement in mobile-phase; see below. NV-methyl-5-hydroxytryptamine (NMS [oxalate salt]; Sigma, MO) was added to samples as an internal standard, and perchloric acid (J.T. Baker, NJ) was added (0.1 N final concentration). The tissue was homogenized with a Teflon pestle (Kontes, NJ) and centrifuged at 5585 x g for 5 min, at 4 °C (VWR Model V benchtop microfuge). The supernatant was filtered through a 0.22-µm centrifuge filtration device (Ultrafree-MC; Millipore, MA). Samples were adjusted to a known volume with mobile phase.

The HPLC with electrochemical detection system consisted of a model 528 pump and Coulochem II detector with a flow-through model 5011 analytical cell (ESA, Inc., MA). The guard cell was set at +350 mV, whereas the screen and analytical electrodes were set at +50 and +325 mV, respectively. The column was 150 x 3.2 mm, 3 µm RP-C18 (MD-150; ESA, Inc.), and the mobile phase consisted of 75 mM NaH2PO4 · H2O (Sigma, St. Louis, MO), 1.7 mM SOS, 0.01% (v/v) triethylamine (TEA; Fisher, NJ), 25 mM EDTA, 15% acetonitrile, pH 3.2. The amounts of the monoamines and 5-HT-related compounds were determined using software provided with the system and standard curves run on the same day. The amounts were adjusted for loss during preparation and processing by using the NMS internal standard.

All values are given as mean ± SEM. Unpaired student’s t tests were used for comparisons between control and experimental groups. Differences were considered significant if P < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Effects of 5-HTP: HPLC results
In the brain of Tritonia diomedea, 5-hydroxytryptophan (5-HTP) normally is found at very low levels, close to the detection level of our HPLC system (Fickbohm et al., 2001). Treatment with 2 mM 5-HTP caused a massive increase in 5-HTP concentration that was observable immediately after the 30-min exposure period (Fig. 1A). This dose of 5-HTP is consistent with that used to elicit physiological effects in mollusc brains and behavioral effects in freely moving Aplysia (Fickbohm and Katz, 2000; Marinesco et al., 2004). In addition, 5-HTP has no nonspecific effects at serotonin receptors at this concentration in T. diomedea (Fickbohm and Katz, 2000). 5-HTP concentration remained elevated after 1 h of continuous washing in normal saline, falling to 20% of its maximum after 20 h of washing (Fig. 1A). The amount of 5-HTP remaining after 20 h (270 ± 29 pmol/mg tissue, n = 3) was still tremendously elevated over the amount measured in untreated brains ({approx}0.1 pmol/mg tissue, n = 6). Thus, 5-HTP was apparently taken up by the brain and was still present after 20 h.



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  		Figure 1. Treatment with 5-hydroxytryptophan (5-HTP) elevates levels of 5-HTP, serotonin, and 5-hydroxyindolacetic acid (5-HIAA) and alters immunolabeling in Tritonia diomedea brains. (A–C) HPLC measurements of 5-HTP (A), serotonin (B), and 5-HIAA (C) in untreated brains (n = 6), after 30-min exposure of the brain to 2 mM 5-HTP (n = 3), 1 h of wash (n = 3), and more than 20 h of wash (n = 3). (D–K) Immunohistochemical comparison of the effect of 5-HTP treatment (2 mM for 1 h, with 20 h wash; right column) on 5-HTP immunoreactivity (D, E), serotonin-immunoreactivity (F–I), and 5-HIAA immunoreactivity (J, K). The immunohistochemical procedure was as previously described (Fickbohm and Katz, 2000; Fickbohm et al., 2001). Secondary antiserum (Molecular Probes: Goat Anti Rabbit Alexa 488) was diluted 1:50 in each case. Scale bars are 200 µm. Pairs were imaged together under identical settings, allowing comparison of relative fluorescence. Lighter colors indicate greater fluorescence, as indicated on the scale bar at the bottom of the figure. (D, E) 5-HTP treatment caused widespread appearance of 5-HTP immunoreactivity throughout the brain, which consists of the bilaterally symmetric cerebral (Ce), pleural (Pl), and pedal (Pd) ganglia. The giant serotonergic neuron C1 (arrow) and the medial serotonergic group containing the dorsal swim interneurons (DSIs) (circle) are indicated on the left side. 5-HTP antiserum (DiaSorin, Stillwater, MN) dilution 1:600. (F–I) 5-HTP treatment caused an increase in the intensity of serotonin immunofluorescence. The dorsal view (F, G) shows C1 (arrow) and the DSIs (circle). In the 5-HTP-treated brain, three additional neurons exhibit serotonin immunoreactivity (small arrowhead). The ventral view (H, I) shows additional neurons that become serotonin-immunoreactive after 5-HTP treatment (circled). One group is lateral to C1; the other in the pedal ganglion is found between two large serotonergic neurons. Serotonin antiserum (DiaSorin, Stillwater, MN) dilution 1:8000. (J, K) 5-HTP treatment caused the appearance of immunoreactivity to 5-HIAA. 5-HIAA antiserum (DiaSorin, Stillwater, MN) dilution 1:200. Similar results were obtained with 1:1000 dilution.

 
In contrast to 5-HTP, serotonin levels were easily measured in untreated preparations, averaging 60 ± 11 pmol/mg tissue (n = 6). Exposure to 5-HTP caused a gradual increase in serotonin content in the brain, reaching 214 ± 4 pmol/mg tissue (n = 3) by 20 h (Fig. 1B), consistent with previous studies in the opisthobranch Aplysia californica (McCaman et al., 1984). Thus serotonin synthesis was enhanced by exposure to 5-HTP, and serotonin continued to accumulate more than 20 h after 5-HTP treatment.

Presumably at least some of the decline in 5-HTP during the washout was due to its conversion to 5-HT; however, 5-HTP levels decreased by about 700 pmol/mg tissue, whereas 5-HT increased by only about 150 pmol/mg tissue. Therefore, either the serotonin that was produced was broken down or not all of the 5-HTP was converted to serotonin. In opisthobranch molluscs, serotonin is catabolized through several different pathways (Sloley and Juorio, 1995; Stuart et al., 2003, Stuart et al.,2004). Monoamine oxidase, which plays just a minor role in serotonin catabolism, produces the breakdown product 5-hydroxyindolacetic acid (5-HIAA), which we were able to measure simultaneously with serotonin and 5-HTP. We found that 5-HIAA was barely detectable in untreated brains, consistent with previous studies in other opisthobranchs (Takeda, 1992; Sloley and Juorio, 1995; Fuller et al., 1998; Stuart et al., 2003, Stuart et al.,2004) and our own previous study in T. diomedea (Fickbohm et al., 2001). However, immediately after 5-HTP exposure, 5-HIAA concentration increased to 21 ± 2 pmol/mg tissue (n = 3) and continued to rise after 20 h in wash to 38 ± 3 pmol/mg tissue (n = 3, Fig. 1C). The rise in 5-HIAA paralleled that of serotonin. Thus, oxidation of serotonin appears to occur in response to a large buildup of serotonin.

Effects of 5-HTP: immunohistochemistry
To further examine the effect of 5-HTP on T. diomedea brain neurochemistry, we compared 5-HTP, serotonin, and 5-HIAA immunoreactivity in untreated (Fig. 1D, F, H, J) and 5-HTP treated brains (Fig. 1E, G, I, K). For each treatment, the brains were dissected and divided into two groups: one group received 2 mM 5-HTP for 1 h; the other did not. Both groups were then fixed 20 h after dissection. The brains were processed together for immunohistochemistry. They were then imaged on the confocal microscope using identical settings so that the relative fluorescence could be compared.

The 5-HTP treatment caused changes in immunoreactivity that were consistent with the HPLC results. Following 5-HTP treatment, there was a widespread increase in the number of neurons displaying 5-HTP immunoreactivity (compare Fig. 1D and E). Three separate trials were performed, involving a total of 17 animals. For the example shown, two groups consisting of four untreated and three 5-HTP treated brains each were compared. The other two trials contained a total of 10 additional brains equally divided into untreated and 5-HTP treated groups. In untreated brains, 5-HTP immunoreactivity is primarily localized to known serotonergic neurons (compare Fig. 1D and F) such as the giant cerebral neuron, C1 (Fig. 1D, F; white arrows), the dorsal swim interneurons (DSIs) (Fig. 1D, F; circle), and unidentified lateral pedal neurons, as shown previously (Fickbohm et al., 2001). After 5-HTP treatment, 5-HTP immunoreactivity was observed in most neurons, including neurons that are not ordinarily serotonin-immunoreactive.

The relative intensity of 5-HTP immunoreactivity in some serotonergic neurons, such as C1 and the DSIs, sometimes decreased after 5-HTP treatment (compare Fig. 1D and E). This may indicate that the conversion of 5-HTP to serotonin is affected by the overall availability of 5-HTP, perhaps through an up-regulation of the enzyme that converts 5-HTP to serotonin, aromatic amino acid decarboxylase (AADC).

The effect of 5-HTP treatment on serotonin immunoreactivity was examined by comparing two groups of four brains: one group was untreated; the other group was exposed to 5-HTP. Although 5-HTP treatment caused 5-HTP to be taken up nonspecifically, it did not cause a widespread change in serotonin immunoreactivity (Fig. 1F–I). This indicates that although 5-HTP is taken up by most neurons, those neurons must lack AADC, the enzyme that converts 5-HTP into serotonin. 5-HTP treatment increased the intensity of serotonin immunoreactivity in the DSIs and unidentified pedal neurons, consistent with previous observations (Fickbohm and Katz, 2000). It also caused the appearance of ectopic serotonin immunoreactivity in two to three neurons located anterolateral to the DSIs (Fig. 1F, G; white arrowhead) as previously reported (Fickbohm and Katz, 2000). In this study, we also examined the ventral surface of the brain and found additional clusters of neurons that displayed serotonin immunoreactivity only after 5-HTP treatment (Fig. 1H, I; circled).

The 5-HTP treatment also caused the appearance of 5-HIAA immunoreactivity (Fig. 1J, K), which is consistent with the HPLC results showing an increase in its concentration. In untreated preparations, 5-HIAA immunoreactivity was uniformly weak throughout the tissue (n = 2). By 20 h after 5-HTP treatment, the intensity of 5-HIAA labeling was increased (n = 2). The labeling appeared to be primarily outside of the cytoplasm of the neuronal somata, possibly in glial cells and connective tissue.

Serotonin levels were not increased by other agents
We examined the effect of a variety of other pharmacological agents on the total serotonin content in the brain. Exposure of the brain to tryptophan (1 mM for 30 min, n = 3) increased the 5-HTP content of the brain but had no effect on the total serotonin content (Fig. 2A). 5-HTP consistently increased 10-fold, from 0.13 ± 0.04 to 1.34 ± 0.42 pmol/mg tissue (n = 3, P = 0.04; t test). In contrast, the amount of serotonin under control conditions was 57.5 ± 5.2 and 53.3 ± 3.0 pmol/mg tissue after tryptophan treatment (n = 3, P = 0.52; t test). Tryptophan treatment increased 5-HIAA levels from 0.11 ± 0.03 to 0.53 ± 0.12 pmol/mg tissue (n = 3, P = 0.03; t test), suggesting an increase in serotonin catabolism. In other trials, 2 mM or 4 mM tryptophan for 2 h also did not increase the total serotonin content (n = 2 for each condition). Thus, serotonin levels were maintained even though 5-HTP levels increased in the presence of tryptophan.



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  		Figure 2. Other means of enhancing serotonin synthesis did not affect serotonin levels in Tritonia diomedea brains, as determined by HPLC. (A) L-tryptophan (1 mM for 30 min) increased levels of 5-HTP and 5-HIAA (asterisks), but did not affect serotonin. Serotonin treatment (5-HT, 0.2 mM for 1 h) also had no lasting effect on serotonin levels. However, 5-HT did cause an increase in 5-HIAA levels (asterisk). (B) The serotonin synthesis cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) applied for 1 h either alone (0.1 mM) or together with the monoamine oxidase inhibitor pargyline (0.1 mM) did not significantly affect levels of 5-HTP, serotonin, or 5-HIAA.

 
Exposure to serotonin itself (0.2 mM for 1 h) also did not affect the total serotonin content in the brain. Two trials were conducted with a total of 5 experimental treatments and 5 controls. The results of the two trials were similar, but are reported separately to maintain the within-trial comparisons. One of the trials was run simultaneously with the tryptophan group (Fig. 2A, right) and produced a serotonin level of 70.5 ± 3.1 pmol/mg tissue, which was not significantly different from control (n = 3, P = 0.10; t test). In that trial, 5-HTP was unaffected, but 5-HIAA increased to 2.4 ± 0.2 pmol/mg protein (n = 3, P < 0.001; t test), indicating that some of the excess serotonin was oxidized. In the other trial, the value for serotonin was 56.0 ± 11.2 pmol/mg tissue for untreated and 47.4 ± 1.88 pmol/mg tissue for serotonin-treated brains (n = 2). These results are consistent with our immunohistochemical staining, where we found that exposure of the brain to serotonin (0.1 mM for 30 min, n = 2 or 0.2 mM for 1 h, n = 2) did not increase the intensity of serotonin immunoreactivity over matched untreated controls (data not shown). This suggests that uptake of serotonin does not contribute greatly to the total serotonin content of the brain. Furthermore, we did not observe the appearance of serotonin immunoreactivity in normally non-serotonergic neurons after serotonin treatment.

In a further, though unsuccessful, attempt to increase serotonin levels, we treated the brain with the essential cofactor necessary for serotonin synthesis, tetrahydrobiopterin (BH4) (Boadle-Biber, 1982). Exposure of the brain to 0.1 mM BH4 for 3 h did not significantly affect levels of 5-HTP, serotonin, or 5-HIAA (n = 3, Fig. 2B). Addition of the monoamine oxidase inhibitor pargyline together with BH4 also did not increase serotonin levels (n = 3, Fig. 2B).

Serotonin was not decreased by inhibiting synthesis
Since serotonin levels could not be enhanced by any of the manipulations except 5-HTP, we attempted to decrease serotonin by blocking synthesis. We tested blocking the conversion of 5-HTP to serotonin with the commonly used AADC inhibitor 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015). NSD-1015 treatment did not significantly alter serotonin or 5-HIAA (Fig. 3). However, it did increase 5-HTP more than 50-fold, going from 0.8 ± 0.3 pmol/mg tissue in untreated brains to 47.5 ± 3.4 pmol/mg tissue in brains exposed to 10 mM NSD-1015 for 1 h and allowed to wash for 14 h (n = 3). Similar results were obtained with 3.3 mM NSD-1015 (n = 2), where 5-HTP was below our detection level in controls and increased to 41.7 ± 4.9 pmol/mg tissue after treatment. The increase in 5-HTP suggests that NSD-1015 effectively blocked AADC, causing an accumulation of 5-HTP. Combining NSD-1015 and the tryptophan hydroxylase inhibitor p-chlorophenylalanine also did not decrease serotonin levels (n = 2). Thus, attempts to block synthesis of serotonin did not decrease serotonin levels.



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  		Figure 3. Inhibiting serotonin synthesis did not affect serotonin levels in Tritonia diomedea brains, as determined by HPLC. Blocking aromatic amino acid decarboxylase with 10 mM 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015) (1-h application, 14-h wash) had no significant effect on the amount of serotonin or 5-HIAA, but caused a large increase in 5-hydroxytryptophan (5-HTP).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
In the mollusc Tritonia diomedea, serotonin levels were resistant to change except by application of the immediate precursor, 5-hydroxytryptophan (5-HTP). A brief exposure of the brain to 5-HTP caused a gradual increase in 5-HT content that lasted over 20 h. This contrasts with results in rats (Lynn-Bullock et al., 2004), where 5-HTP enhancement of serotonin lasted only about 2 h. Thus, it is possible that the turnover rate for serotonin in molluscs is significantly slower than in mammals. This may be partially due to the difference in physiological temperatures of the two animals. Alternatively, the experimental procedures may contribute to the difference. Rats were given a single injection or were fed 5-HTP, whereas in T. diomedea, 5-HTP was applied directly to the brain at a high concentration. Thus, either phyletic or experimental differences might account for the different results. Although the 5-HTP applications in these studies were relatively acute compared to the chronic dietary ingestion seen clinically, they underline the importance of serotonin homeostasis in nervous systems and show that attempts to change that homeostasis may have unexpected effects on other parts of the brain.

Normally, 5-HTP is localized only to serotonergic neurons (Fickbohm et al., 2001), but after the brain was bathed in 5-HTP, it appeared to be taken up widely by neurons, causing 5-HTP immunoreactivity to be seen in practically every neuron in the brain. This is consistent with HPLC measurements of 5-HTP that show its concentration to be greatly elevated over the same time course. The widespread appearance of 5-HTP immunoreactivity is fundamentally different than the effect of 5-HTP treatment in rats, where 5-HTP immunoreactivity was confined to groups of neurons that synthesize 5-HT (Lynn-Bullock et al., 2004). This suggests that mechanisms of 5-HTP uptake are different in the two phyla: in molluscs, 5-HTP is taken up by a transporter that is common to all neurons; in rats, it is taken up by a transporter found only in catecholaminergic or serotonergic neurons.

In contrast to the widespread distribution of 5-HTP, 5-HTP treatment did not cause serotonin to be equally widely distributed. Instead, 5-HTP was converted to serotonin in only a small subset of neurons, presumably those with aromatic amino acid decarboxylase (AADC). This not only includes neurons that normally are serotonergic, but also additional particular clusters of neurons that may be catecholaminergic, as previously reported (Fickbohm and Katz, 2000). Catecholaminergic neurons have been observed in the homologous regions of other nudibranchs (Croll et al., 2001). It is difficult to confirm whether the newly serotonergic neurons are catecholaminergic, because the available antibodies for tyrosine hydroxylase require a different fixative and thus are incompatible with double-label immunohistochemistry with serotonin. In the snail Lymnaea stagnalis, it has been similarly observed that 5-HTP treatment produced serotonin-like glyoxylic acid fluorescence in non-serotonergic neurons (Audesirk, 1985). This is also similar to results in rats, which showed the appearance of ectopic serotonin in dopaminergic neurons of the substantia nigra after 5-HTP treatment (Lynn-Bullock et al., 2004). These results suggest that AADC, the enzyme necessary for converting 5-HTP to 5-HT, is found only in those neurons that converted 5-HTP to serotonin.

Other attempts to alter levels of serotonin and related molecules were less successful. Although treating ganglia with tryptophan did increase 5-HTP levels, the increase was less than with 5-HTP treatment. Additionally, tryptophan treatment did not affect serotonin levels. This is consistent with results from Aplysia (McCaman et al., 1984), but differs from results in mammals that suggest that tryptophan hydroxylase is not normally saturated and that increasing tryptophan availability increases serotonin synthesis (Hamon et al., 1981).

Application of serotonin itself also failed to increase the accumulation of serotonin in T. diomedea brains. There was no persistent increase in serotonin levels, as measured with HPLC, and no apparent increase in the intensity of serotonin immunoreactivity. Moreover, additional serotonergic neurons did not appear in response to serotonin treatment. Thus, the phenomenon of serotonin acting as a false or borrowed transmitter apparently does not occur in T. diomedea as it does in mammals (Feuerstein et al., 1986; Vanhatalo and Soinila, 1995, Vanhatalo and Soinila, 1998; Lebrand et al., 1996) and in other invertebrates (Richards et al., 2003).

Despite reports that monoamine oxidase levels are low in molluscs and that 5-HIAA is not normally a breakdown product (Sloley and Juorio, 1995; Stuart et al., 2003, Stuart et al.,2004), we found that 5-HIAA levels increased in T. diomedea when brains were treated with 5-HTP or 5-HT. Furthermore, 5-HIAA immunoreactivity appeared widespread after 5-HTP treatment (Fig. 1). These results suggest that serotonin is oxidized when it is in excess.

Together, these results suggest that serotonin levels in the T. diomedea brain are homeostatically regulated and that 5-HTP treatment disrupts that regulation. It is possible that serotonin levels in individual cells vary more than the total serotonin content of the brain or that challenging the neurons with changes in activity could also alter this homeostasis. In Aplysia, the activity of individual neurons can alter their somatic concentration of 5-HT (Meulemans et al., 1987). Homeostatic levels of serotonin could also vary with the animal’s circadian rhythm; serotonin levels in the hemolymph of Aplysia fluctuate diurnally (Levenson et al., 1999).

In summary, our results show that total serotonin levels in T. diomedea can be increased by 5-HTP, but that other methods for altering these levels are ineffective in this system. The time course over which 5-HTP affects serotonin levels is much longer than in mammals (Lynn-Bullock et al., 2004). However, the long time course matches the physiological effects of 5-HTP treatment that we observed in T. diomedea. We previously found that 20 h after 5-HTP treatment, the synaptic actions of serotonergic neurons were greatly enhanced, the release of serotonin was enhanced, and the participation of serotonergic neurons in the generation of particular behaviors was altered (Fickbohm and Katz, 2000). Thus, 5-HTP causes a prolonged increase in the serotonin content of neurons in T. diomedea, but in general, total serotonin levels are not easily altered.


   Acknowledgments
 
This work was supported by an NIH grant to PSK and by a Research Program Enhancement Grant from Georgia State University.


   Footnotes
 
Received 10 January 2005; accepted 25 May 2005.


   Literature Cited
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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E. V. Megalou, C. J. Brandon, and W. N. Frost
Evidence That the Swim Afferent Neurons of Tritonia diomedea Are Glutamatergic
Biol. Bull., April 1, 2009; 216(2): 103 - 112.
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