The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of rats

Journal of Chemical Neuroanatomy 27 (2004) 129–138

The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of rats Christina P. Lynn-Bullock, Kristy Welshhans, Sarah L. Pallas, Paul S. Katz∗ Department of Biology, MSC 8L0389, Georgia State University, 33 Gilmer St SE Unit 8, Atlanta, GA 30303-3088, USA Received 23 May 2003; received in revised form 24 November 2003; accepted 2 February 2004

Abstract 5-Hydroxytryptophan (5-HTP), which is the rate-limiting precursor in serotonin (5-hydroxytryptamine (5-HT)) biosynthesis, is used as an oral supplement to enhance serotonin levels in humans. To evaluate its effects on serotonin levels and localization, 5-hydroxytryptophan was administered to Sprague–Dawley rats either orally or via intraperitoneal injection. 5-Hydroxytryptophan-immunoreactivity was co-localized with serotonin-immunoreactivity in the serotonergic dorsal raphe nucleus of control animals and this was not changed in animals given 5-hydroxytryptophan. Oral 5-HTP administration increased the intensity of both 5-HTP and serotonin immunoreactivity in raphe neurons. However, 5-HTP treatment also caused ectopic 5-hydroxytryptophan-immunoreactivity and serotonin-immunoreactivity in normally dopaminergic neurons of the substantia nigra par compacta. Serotonin-immunoreactivity was confined to neurons that also displayed amino acid decarboxylase immunoreactivity, but in a small percentage of substantia nigra neurons, serotonin immunoreactivity was not co-localized with tyrosine hydroxylase-immunoreactivity. The intensity of the immunoreactivity to serotonin and 5-hydroxytryptophan in the substantia nigra was maximal within 2 h of 5-hydroxytryptophan administration and returned to control levels by 24 h. This time course mirrored changes in HPLC measurements of 5-hydroxytryptophan, serotonin, and the metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the urine. 5-Hydroxytryptophan administration did not cause ectopic appearance of either serotonin or 5-hydroxytryptophan in the noradrenergic locus coeruleus. These results suggest that a single oral dose of 5-HTP increases the 5-HTP and serotonin content of serotonergic neurons and causes the transient ectopic appearance of serotonin in some normally non-serotonergic neurons. © 2004 Elsevier B.V. All rights reserved. Keywords: Double label immunohistochemistry; Confocal fluorescence microscopy; Co-localization; Serotonin; 5-Hydroxytryptophan; HPLC

1. Introduction Decreased levels of serotonin (5-hydroxytryptamine (5-HT)) in the central nervous system (CNS) are correlated with many behavioral and mental conditions such as depression (Byerley and Risch, 1985; Jesberger, 1985), regulation of sleep cycles (Wyatt et al., 1971; Imeri et al., 2000), and stress (Hashimoto et al., 1999). Serotonin itself cannot cross the blood–brain barrier (Bouchaud, 1972), therefore, the biosynthetic precursor of serotonin, 5-hydroxytryptophan (5-HTP), has been used as a dietary supplement to treat these serotonin-linked disorders (Sahelian, 1998; Murray, 1998). Animal studies have shown that 5-HTP treatment ∗ Corresponding author. Tel.: +1-404-651-0922; fax: +1-404-651-2509. E-mail address: [email protected] (P.S. Katz).

0891-0618/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2004.02.003

can raise 5-HT levels in the CNS (Denoyer et al., 1989; Bogdanski et al., 1958; Arai et al., 1995; Kitahama et al., 2002). 5-HTP is converted into 5-HT in serotonergic neurons by the enzyme aromatic amino acid decarboxylase (AADC) (Boadle-Biber, 1982; Zhu and Juorio, 1995). However, AADC is also present in catecholaminergic neurons, where it normally converts l-DOPA into dopamine. Thus, oral consumption of 5-HTP may lead to the ectopic appearance of 5-HT in catecholaminergic neurons. Intraperitoneal (IP) injection of 5-HTP causes 5-HT to appear in the dopaminergic substantia nigra pars compacta (SNC) (Arai et al., 1994, 1995) but not the noradrenergic locus coeruleus (LC) unless monoamine oxidase (MAO) inhibitors are administered concurrently (Arai et al., 1995). The present study sought to determine whether oral administration of 5-HTP, the common method of administration used by humans, alters the distribution of 5-HT and 5-HTP im-

130

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

munoreactivity (-ir) in a manner similar to that caused by IP injection. This study also sought to further characterize the extent to which 5-HTP is co-localized with 5-HT. 5-HTP, which is synthesized in serotonergic neurons from tryptophan by the enzyme tryptophan hydroxylase, is normally present at low levels in the nervous system because it is rapidly converted into 5-HT (Boadle-Biber, 1982; Sloley and Juorio, 1995). 5-HTP immunoreactivity (5-HTP-ir) can be observed in the serotonergic raphe nuclei (RN) (Touret et al., 1987; Geffard et al., 1987; Brownfield et al., 1998), but the extent of co-localization of 5-HTP with serotonin had not been determined previously. Furthermore, the distribution of 5-HTP-ir after 5-HTP administration had not been examined previously. 5-HTP administration might result in widespread uptake of 5-HTP into neurons and cause the appearance of 5-HTP in neurons that lack AADC and are, thus, incapable of converting it into 5-HT, as was seen in a molluscan nervous system (Fickbohm and Katz, 1999). Therefore, we examined the co-localization of 5-HTP-ir and 5-HT-ir to determine if it was affected by oral 5-HTP treatment. We found that oral 5-HTP administration increased the intensity of 5-HTP and 5-HT immunoreactivity in the normally serotonergic dorsal raphe nucleus and caused transient ectopic expression of 5-HTP-ir and 5-HT-ir in the normally dopaminergic SNC but not in the noradrenergic LC. We did not observe widespread uptake of 5-HTP into neurons that do not convert it to 5-HT. Some of these results have been previously presented in abstract form (Lynn-Bullock et al., 2001).

2.2. Urine collection and analysis Animals were housed in metabolic cages three to four days prior to administration of treatment so that baseline urine samples could be collected. Urine was collected every 24 h for three days before treatment to establish baseline values of 5-HTP, 5-HT, and hydroxyindoleacetic acid (5-HIAA). After treatment, available urine was collected every hour. To avoid overestimates of concentration, urine was discarded from analysis if the time between collections exceeded 1 h. Five microliters of N-methyl-serotonin (NMS, oxalate salt; Sigma, St. Louis, MO), an internal standard, and 1.7 ␮l of perchloric acid, which prevents the oxidation of amines present in the samples, were added to 100 ␮l aliquots of urine samples. The mixtures were centrifuged at 5585×g for 5 min at 4 ◦ C. The supernatant was passed through a 0.22 ␮m centrifuge filtration device (Ultrafree-MC; Millipore, Bedford, MA), brought up to a known volume (100 ␮l), and diluted as necessary with mobile phase (MP) consisting of 75 mM sodium dihydrogen phosphate, monohydrate, 1.7 mM sodium octyl sulfate (SOS), 0.01% (v/v) triethylamine (Fisher, New Jersey), 25 ␮M EDTA, and 15% acetonitrile (pH 3.0). The HPLC system consisted of an ESA Model 528 pump and Coulochem II detector with a flow-through Model 5011 Analytical Cell (ESA Inc., Chelmsford, MA) and a guard cell set at 350 mV with the screen and analytical electrodes set at 50 and 325 mV, respectively. The column was a 150 mm × 3.2 mm, 3 ␮m RP-C18 (MD-150; ESA Inc., Chelmsford, MA). The amounts of 5-HTP, 5-HT, and 5-HIAA were compared to standard curves run concurrently.

2. Methods

2.3. Tissue preparation

2.1. 5-HTP administration

Animals that received an oral gavage of 5-HTP or water were euthanized (sodium pentobarbitol, 50 mg/kg) at 1, 2, 4, or 24 h following 5-HTP administration. Additional animals received IP injections of 5-HTP or water and were then euthanized at 1 or 24 h following treatment. Table 1 lists the number of animals used for each treatment and survival time. The animals were perfused first with 0.1 M phosphate buffer (PB, pH = 7.4) and then with cold 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH = 7.4). The brains were post-fixed overnight in the same solution and then immersed in 30% sucrose for approximately 24 h for cryoprotection. Frozen 50 ␮m coronal sections were cut using a sliding microtome and collected in phosphate buffer. Every fourth section was processed for Nissl staining with cresyl violet to determine the location of the brain regions to be tested.

Thirty-four Sprague–Dawley rats (200–230 g; Charles River Laboratories, Wilmington, MA) received an oral gavage or an IP injection of either 5-HTP (10 mg/kg of body weight, in 2 ml water) or water alone (sham-treatment; Table 1). Prior to 5-HTP treatment, the animals were maintained under a 12 h light: 12 h dark cycle and were provided food and water ad libtum. All procedures used met or exceeded the standards of accepted care developed by the Institutional Animal Care and Use Committee, the National Institutes of Health, and the Society for Neuroscience. Table 1 Numbers of animals for each treatment and survival time Treatment

Oral 5-HTP Oral sham IP 5-HTP IP sham

Survival time (h) 1

2

4

24

4 3 2 1

3 3 – –

4 3 – –

3 3 3 2

2.4. Fluorescence immunohistochemistry Free-floating sections were rinsed three times (15 min each) in phosphate buffered saline with 0.02% Na Azide

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

and 0.03% Triton X (PBS∗ ) and then incubated for 1 h in blocking buffer (PBS∗ with 3% normal goat or donkey serum). Sections were incubated for 72 h at 4 ◦ C in primary antiserum diluted 1:1000 with blocking buffer (PBS∗ with 3% normal goat or donkey serum). Rabbit anti-AADC was purchased from Affinity Research Products (Exeter, United Kingdom). All other primary antisera were purchased from Diasorin Inc. (Stillwater, MN). Following incubation in primary antisera, sections were rinsed three times (15 min each) in PBS∗ and incubated for 4 h in secondary antiserum diluted 1:250 with blocking buffer at room temperature. All secondary antisera were purchased from Molecular Probes (Eugene, OR). Sections were rinsed three times in PBS∗ , mounted, dried overnight, cleared with xylene, and coverslipped using CytosealTM (Stephens Scientific, Kalamazoo, MI). Immunofluorescence was visualized using a laser scanning confocal microscope (LSM 510, Carl Zeiss Inc., Thornwood, NY). Images were viewed as a maximum projection of three optical sections. As a control for the specificity of the fluorescence from the secondary antibody, the primary antisera or the secondary antisera were omitted in some sections. No cell-specific or background labeling was observed in these preparations. It was previously shown that this 5-HTP antiserum does not cross-react with 5-HT; staining for 5-HT was abolished by preabsorption with conjugated 5-HT, whereas 5-HTP staining was unaffected (Fickbohm et al., 2001). Immunofluorescence is inherently difficult to quantify, therefore, comparisons were made only between sections that were treated identically. Sections to be compared were processed for immunohistochemistry in parallel and imaged with identical confocal microscope settings on the same day. All sections used in the analysis were double-labeled for 5-HTP and 5-HT. The analysis was performed using Zeiss LSM 510 Software (Carl Zeiss). At least two different brain sections, imaged at 40×, were quantified for fluorescence intensity in each animal. A minimum of 15 cells per section and 45 cells per animal, chosen randomly were sampled. The background level of fluorescence was quantified and subtracted from the fluorescence value for each cell prior to comparison. Statistical analysis of differences in both 5-HTP and 5-HT immunofluorescence was performed using a t-test (between cells in control and treated sections) in SigmaStat software (SPSS, Chicago, IL). For images in which we did not compare the relative intensity, confocal settings were chosen to optimize the signal to noise ratio in that section.

131

2.5. Double label immunohistochemistry To examine co-localization of antigens, sections were treated as described above except that two different primary antisera were used (DiaSorin Inc.), each diluted 1:1000 with blocking buffer and followed by the secondary antiserum (Molecular Probes) diluted 1:250 with blocking buffer. All primary and secondary antisera used for double label immunohistochemistry are listed in Table 2. Primary antisera were tested individually before being used in the double label protocol.

3. Results 3.1. Oral 5-HTP treatment increased the intensity of 5-HTP and 5-HT immunoreactivity in the dorsal raphe nucleus We found that the intensity of 5-HTP immunoreactivity was increased in rats euthanized 1 h after receiving a single oral dose (10 mg/kg) of 5-HTP, a dosage equivalent to the upper range of suggested doses for humans (Sahelian, 1998), compared to animals given a water gavage. Comparable regions of the dorsal raphe nucleus were chosen in each animal using the Aqueduct of Sylvius as a guide. In control animals, the average intensity above background in arbitrary units derived from the eight-bit intensity scale for 5-HTP immunofluorescence was 18.2 ± 1.3 (mean ± S.E.M., N = 135 from three animals). This more than doubled in rats that received the oral 5-HTP treatment to 49.3 ± 2.0 (N = 221 from four animals), a highly significant change (P < 0.001, unpaired t-test). Examples of 5-HTP immunofluorescence from control and treated animals are presented in Fig. 1A and C. The insets show the raw images from which the intensities were measured. A cumulative histogram of fluorescence intensities from individual cells shows that the increase in fluorescence was due to a broadening of the distribution, rather than a shift in intensity (Fig. 1E), which might indicate an offset in the baseline fluorescence. Serotonin immunofluorescence also increased, but to a lesser extent than 5-HTP (Fig. 1B, D and F). In control rats, the average intensity of serotonin immunofluorescence over background was 69.1 ± 1.8 (N = 135). This increased to 80.3 ± 2.0 (N = 221) in rats euthanized 1 h after 5-HTP treatment, a highly significant difference (P < 0.001, unpaired t-test). As with 5-HTP immunofluorescence, 5-HTP

Table 2 Double label antibody combinations Combinations

5-HTP and 5-HT AADC and 5-HT 5-HT and TH AADC and TH

First set

Second set

Primary Ab

Secondary Ab

Primary Ab

Secondary Ab

Goat anti-5-HT Goat anti-5-HT Goat anti-5-HT Rabbit anti-AADC

Donkey anti-goat Alexa 546 Donkey anti-goat Alexa 546 Donkey anti-goat Alexa 546 Goat anti-rabbit Alexa 488

Rabbit Rabbit Mouse Mouse

Goat-anti rabbit Alexa 488 Goat-anti rabbit Alexa 488 Rabbit anti-mouse Alexa 488 Goat anti-mouse CY3 or Alexa 350

anti-5-HTP anti-AADC anti-TH anti-TH

132

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

Fig. 1. Oral 5-HTP treatment increased 5-HTP and serotonin-immunoreactivity in the dorsal raphe nucleus (RN), but did not disrupt the co-localization of 5-HTP and serotonin. 5-HTP-ir (A and C) was co-localized with 5-HT-ir (B and D) in the RN of control (A and B) and oral 5-HTP treated (C and D) animals. 5-HTP treatment did not alter the co-localization of 5-HTP-ir and 5-HT-ir in the RN (arrows indicate some double-labeled neurons). Thus, 5-HTP was not taken up by non-serotonergic neurons. Scale bars = 50 ␮m. The inset in each panel shows the raw images used to compare intensities at one third scale. (E and F) The intensity of 5-HTP and 5-HT immunoreactivity increased in rats treated with 5-HTP and euthanized after 1 h. The cumulative histograms show the distribution of 5-HTP (E) and 5-HT (F) immunofluorescence intensities above background for cells from animals euthanized 1 h after control treatment (open circles) or oral 5-HTP treatment (filled squares). The vertical dashed lines show the median values for the two groups.

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

treatment broadened the distribution of 5-HT immunofluorescence intensities. In sham-treated and untreated animals, 5-HTP was co-localized with 5-HT (Fig. 1A and B). There continued to be co-localization of 5-HTP-ir and 5-HT-ir following oral 5-HTP treatment (Fig. 1C and D) at all of the survival times tested (n = 14, see Table 1, data not shown). Similar results were obtained using IP injection of 5-HTP (n = 5). Thus, immunofluorescence measurements suggest that 5-HTP treatment appeared to increase 5-HTP and 5-HT levels in serotonergic cells of the RN and did not result in the appearance of 5-HTP in non-serotonergic neurons. 3.2. Oral 5-HTP administration resulted in the appearance of 5-HT-ir in the SNC The presence of AADC in dopaminergic neurons should cause these neurons to display 5-HT-ir when presented with 5-HTP. In all animals euthanized 1 h after oral 5-HTP administration (n = 4), 5-HT-ir was observed in neurons of the SNC (Fig. 2A, C and E). This is similar to previously reported results using IP injection (Arai et al., 1994, 1995), which we repeated (n = 2). 5-HT immunolabeling was not observed in the SNC of any of the animals that received oral sham treatments (n = 12), IP sham-treatments (n = 3), or no treatment (n = 1). Not all of the neurons in the SNC are dopaminergic. Therefore, to determine whether 5-HT-ir was restricted to dopaminergic neurons, we double-labeled brain sections with an antiserum against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. In all three animals that we examined, which received an oral 5-HTP and were euthanized after 1 h, neurons positive for TH-ir also displayed 5-HT-ir (Fig. 2A and B, open arrowheads) indicating that dopaminergic neurons were able to synthesize serotonin as a result of 5-HTP treatment. In two of the three animals examined, 1–2% of the neurons exhibiting 5-HT-ir did not show TH-ir (Fig. 2A and B, solid arrow), indicating that non-catecholaminergic neurons became 5-HT immunoreactive. These neurons correspond to a population of neurons in the SNC, the so-called “D-type” neurons, that were previously shown to be AADC positive, but TH negative (Kitahama et al., 1998). Consistent with this identification, all SCN neurons observed to be 5-HT-immunoreactive also displayed AADC-ir in animals euthanized 1 h after oral 5-HTP administration (n = 4) (Fig. 2C and D; open arrowheads) or IP injection (n = 2). This result is also consistent with the hypothesis that 5-HT is not taken up into dopaminergic terminals, but is synthesized from 5-HTP through the action of AADC. 3.3. Co-localization of 5-HTP and 5-HT in the SNC If 5-HTP is taken up, then a further question arises as to whether its uptake is confined to neurons that convert it to 5-HT or whether there are neurons that take up 5-HTP but

133

do not convert it into 5-HT. To distinguish between these alternatives, we performed double-label immunohistochemistry and looked for co-localization of 5-HTP-ir and 5-HT-ir. 5-HTP labeling was not observed in the SNC of animals receiving no treatment (n = 1) or those euthanized 1 h after either oral sham treatment (n = 3) or IP sham treatment (n = 1). However, 1 h after oral or IP 5-HTP administration there was clear cell-specific 5-HTP-ir in SNC neurons (Fig. 2F). Almost all 5-HTP-positive neurons also displayed 5-HT-ir (Fig. 2E and F; open arrowheads), which suggests that these neurons took up 5-HTP and converted it into 5-HT. There was variability in the extent of co-localization of 5-HTP and 5-HT. A small number of neurons that were 5-HTP-immunoreactive were not 5-HT-immunoreactive (Fig. 2E and F; filled arrowheads). This could indicate that these neurons were not capable of converting 5-HTP into 5-HT or that synthesis was slower in these neurons. Conversely, a small percentage of neurons that expressed 5-HT-ir were not 5-HTP immunoreactive (Fig. 2E and F; solid arrows). These neurons exhibited the highest intensity of 5-HT labeling. Thus, the lack of 5-HTP-ir could indicate that these neurons converted 5-HTP into 5-HT more rapidly and completely than neighboring cells. 3.4. 5-HTP-ir and 5-HT-ir in the SNC was transient We examined the time course over which ectopic 5-HT-ir and 5-HTP-ir were observable in the SNC. As noted previously, there were no 5-HT or 5-HTP labeled neurons in the SNC of untreated or sham-treated control animals (Fig. 3A), but in animals euthanized 1 h after oral 5-HTP administration, neurons were labeled strongly for both 5-HTP and 5-HT (Fig. 3B; solid arrows). The intensity of 5-HTP and 5-HT staining in individual neurons was not correlated either positively or negatively; that is, some neurons that stained strongly for 5-HTP stained weakly for 5-HT, whereas others stained strongly for both. Two hours after oral treatment, the intensity of specific 5-HTP labeling had substantially decreased (Fig. 3Ci) compared to 1 h post-treatment (Fig. 3Bi). In contrast, the intensity of 5-HT-ir remained the same or increased (Fig. 3Cii) compared to staining from animals euthanized 1 h after 5-HTP administration (Fig. 3Bii). Some strongly labeled 5-HT-immunoreactive neurons in the 2 h preparations showed weak or no 5-HTP-ir (Fig. 3C, open arrowheads). As was observed in animals euthanized 1 h after treatment, there were neurons that were 5-HTP-immunoreactive but not 5-HT immunoreactive at 2 h post-treatment (Fig. 3C, filled arrowheads). Four hours after 5-HTP administration, the intensity of the 5-HTP-ir had decreased to control levels (Fig. 3Di), however, the relative intensity of 5-HT-ir remained elevated above control (Fig. 3Dii). Twenty-four hours after 5-HTP administration, few if any immunopositive neurons could be discerned and the relative intensity of both 5-HTP and 5-HT labeling was visually indistinguishable from control animals

134

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

Fig. 2. Co-localization of serotonin immunoreactivity (5-HT-ir) (A, C and E) with that of tyrosine hydroxylase (TH) (B), amino acid decarboxylase (AADC) (D), and 5-hydroxytryptophan (5-HTP) (F) in the substantia nigra pars compacta (SNC) of animals euthanized 1 h after oral 5-HTP treatment. (A and B) Serotonin immunoreactivity was present in all neurons that displayed TH-ir (open arrowheads). However, a small number of serotonin-immunoreactive neurons did not display TH-ir (closed arrows). The patterns of labeling for TH and 5-HT were similar in distribution throughout the SNC. (C and D) AADC-ir and 5-HT-ir were co-localized in neurons of the SNC (open arrowheads). (E and F) Serotonin-ir was co-localized with 5-HTP-ir in most 5-HT-immunoreactive neurons (open arrowheads), however, some neurons displayed 5-HT-ir without 5-HTP-ir (closed arrows). Additionally, a few neurons were 5-HTP-immunoreactive but not 5-HT-immunoreactive (filled arrowheads). Scale bars = 50 ␮m.

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

135

Fig. 3. The ectopic expression of 5-HTP and 5-HT in the SNC of 5-HTP treated animals was transient. Rats were euthanized at varying time points following a single oral gavage of 5-HTP. SNC sections were obtained from animals euthanized at each time point. The sections were processed in parallel for immunohistochemistry. They were then imaged using identical settings on the confocal microscope. The images are displayed at equal intensity and contrast settings, using a color scale to indicate relative intensity. (A) In control animals, no cell-specific labeling or background labeling was observed. (B) One hour after 5-HTP administration, there was strong 5-HTP and 5-HT cell-specific labeling and co-localization of the staining (solid arrows). (C) Two hours post-treatment, 5-HTP-ir had begun to decrease (i) while somatic 5-HT-ir continued to be elevated (ii). Filled arrowheads indicate neurons that labeled for 5-HTP but not 5-HT. Open arrowheads indicate neurons that labeled for 5-HT but not 5-HTP. (D) At 4 h post-treatment cell-specific staining had decreased. (E) By 24 h the immunoreactivity had returned to control-like intensities. Scale bar = 50 ␮m. The color scale bar on the right indicates the range of colors corresponding to relative immunofluorecence intensity.

(Fig. 3Ei and Eii). Thus, these data indicate a turnover rate of under 24 h for 5-HT in these non-serotonergic neurons of the SNC. 3.5. 5-HTP administration caused a transient elevation of 5-HTP, 5-HT, and 5-HIAA in the urine To determine the rate at which 5-HTP is cleared from the body, we examined the effects of oral 5-HTP administration on the concentration of 5-HTP, 5-HT, and the 5-HT metabolite, 5-hydroxyindolacetic acid, in the urine of 5-HTP treated rats. Prior to treatment and in sham-treated animals, low levels of 5-HIAA could be measured in the urine (mean value 27 ± 3 pmol/␮l; n = 9), but no 5-HTP or 5-HT could be detected with our method. Urine was sampled at 1 h intervals following a single oral dose of 5-HTP (10 mg/kg; see Section 2). 5-HTP, 5-HT, and 5-HIAA levels increased within the first hour after 5-HTP administration (Fig. 4). The concentrations of 5-HT and 5-HIAA in the urine were of similar magnitude to each other, but 5-HTP concentration was 100 fold lower. Some of the 5-HT in the urine is likely due to decarboxylase activity within the kidney converting 5-HTP to 5-HT (Stier and Itskovitz, 1985). Thus, the actual amount of 5-HTP in the blood is likely to be higher than indicated by the excreted concentration. The time course of the appearance of 5-HT in the urine roughly mirrored the relative intensity of 5-HT-ir in the SNC following 5-HTP treatment. The urine concentrations of 5-HTP, 5-HT, and 5-HIAA reached their maximum levels between 2 and 4 h post-treatment and declined thereafter. By 24 h post-treatment, the levels of 5-HTP, 5-HT, and 5-HIAA had returned to pre-treatment levels. However, in

one animal, the 5-HIAA concentration remained elevated 24 h post-treatment (Fig. 4Aiii). The effect of IP injection on the levels of 5-HT and 5-HIAA in the urine was similar to that of oral 5-HTP administration (Fig. 4B). However, IP injection caused more 5-HTP to appear in the urine than did oral gavage (Fig. 4Ai inset); the amount of 5-HTP 1–2 h after IP injection of 5-HTP was approximately equivalent to the amount of 5-HT excreted in both oral and IP treated animals (Fig. 4Bi). No other differences between IP and oral administration of 5-HTP were observed. 3.6. 5-HTP and 5-HT-ir in the LC A previous study reported that IP injection of 5-HTP did not cause ectopic 5-HT-ir in the noradrenergic LC unless the animal was also treated with an MAO inhibitor (Arai et al., 1995). We examined the effect of oral 5-HTP treatment on 5-HTP-ir and 5-HT-ir in the LC in the absence of MAO inhibitors. The LC was positively identified by TH-ir (Fig. 5A). No 5-HTP-ir or 5-HT-ir was observed in the LC of control animals (n = 3). Oral 5-HTP treatment did not cause neurons in the LC to display 5-HTP-ir or 5-HT-ir (n = 4; Fig. 5B and C). 4. Discussion We found that oral administration to rats of the dietary supplement, 5-hydroxytryptophan, increased the intensity of serotonin and 5-HTP immunoreactivity in neurons of the dorsal raphe nucleus. Furthermore, it caused changes in immunohistochemical staining similar to those previ-

136

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

Fig. 4. The concentrations of 5-HTP, 5-HT, and the 5-HT metabolite 5-hydroxyindolacetic acid (5-HIAA) transiently increase in the urine following oral gavage (A) (n = 11) and IP injections (B) (n = 3) of 5-HTP. All three substances increased in concentration, peaking within 2–4 h after 5-HTP administration and returning to control values within 24 h. Inset in Ai represents an enlargement of the y-axis for the 5-HTP data from animals receiving oral administration; although the increase in 5-HTP followed the same time course as 5-HT (Aii) and 5-HIAA (Aiii), the concentration was about 100 fold lower. The animals that received IP injections had 5-HTP concentrations (Bi) that were similar to the concentrations of 5-HT and 5-HIAA (Bii and Biii). Arrow indicates the day of 5-HTP treatment. Dashed line indicates baseline levels of 5-HTP, 5-HT, and 5-HIAA. Each symbol represents a different animal.

Fig. 5. No 5-HTP-ir or 5-HT-ir was observed in the locus coeruleus (LC) 1 h following oral treatment. (A) Tyrosine hyroxylase (TH) immunoreactivity delineates the extent of the LC (arrows). An adjacent serial section was double-labeled for 5-HTP (B) and 5-HT (C). There was no cell-specific labeling throughout the LC. Large neurons in the neighboring region, mescenphalic region 5 (Me5), exhibited background fluorescence, which persisted in the absence of the primary antisera (not shown). Subsequent serial sections displayed TH-ir, indicating that this section fell within the LC. All images were taken from the same animal. Scale bar = 100 ␮m.

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

ously described to be produced by IP injection (Arai et al., 1994, 1995). Specifically, there was an ectopic appearance of 5-HTP-ir and 5-HT-ir in the SNC. In contrast, 5-HTP did not induce the appearance of 5-HT-ir in another catecholaminergic area, the locus coeruleus. 4.1. 5-HTP administration enhanced 5-HTP and 5-HT immunoreactivity in the dorsal raphe nucleus The clinical rationale for ingesting 5-HTP supplements is to boost the serotonin content of the serotonergic neurons in the raphe nuclei. We found that oral 5-HTP treatment caused an increase in the intensity of 5-HTP and 5-HT immunoreactivity in neurons of the dorsal raphe nucleus. The fact that the increase in 5-HTP was greater than the increase for 5-HT is probably related to the fact that 5-HTP is normally present at such low levels (Boadle-Biber, 1982; Sloley and Juorio, 1995), making it easier to detect a small change with immunohistochemistry. 5-HTP-ir and 5-HT-ir were co-localized to an equivalent extent both in control and in 5-HTP treated animals, thus, there did not appear to be an increase in the number of 5-HTP-immunoreactive neurons. Therefore, 5-HTP treatment appears to have the effect of increasing serotonin levels in serotonergic neurons of the RN. We did not examine the time course of these effects. 4.2. 5-HT in the substantia nigra In addition to increasing serotonin and 5-HTP in serotonergic neurons, 5-HTP treatment also caused serotonin and 5-HTP to appear in normally non-serotonergic neurons in the substantia nigra. The effects of 5-HTP treatment on the SNC were transient. Levels of 5-HT and 5-HTP staining observed in the SNC were reduced within 4 h and they were similar to control levels by 24 h. The intensity of 5-HTP-ir decreased more rapidly than the intensity of 5-HT-ir and was noticeably reduced within 2 h after administration. 5-HT is normally present in at least two classes of neurons in the SNC: catecholaminergic neurons and AADC-positive, but TH-negative neurons. Based on double-label immunohistochemistry with antisera against 5-HT and tyrosine hydroxylase, all of the dopaminergic neurons in the SNC displayed 5-HT-ir after 5-HTP treatment. However, a few 5-HTimmunoreactive neurons did not contain TH-ir. After 5-HTP treatment, all 5-HT-ir was co-localized with AADC-ir. These AADC-positive but TH-negative neurons correspond to the so-called “D-type” neurons (Kitahama et al., 1998). Ectopic serotonin has been shown to appear in dopaminergic neurons in the SNC and ventral tegmental area (VTA) as a result of uptake via dopamine transporters (Zhou et al., 2002). Our data suggest that ectopic 5-HT in the SNC after 5-HTP treatment may result from the synthesis of 5-HT from 5-HTP rather than uptake of excess 5-HT. First, as mentioned above, all neurons that stained for 5-HT also stained for AADC, and thus, were capable of converting 5-HTP into

137

5-HT. Second, 5-HT-ir was found primarily in neurons that also exhibited 5-HTP-ir, indicating that the neurons that had 5-HT also had access to 5-HTP. However, it is possible that the ectopic appearance of 5-HT in the SNC is not due to uptake of 5-HTP and synthesis into 5-HT, but rather is caused by uptake, via the dopamine transporter, of excess 5-HT released from serotonergic terminals, as occurs in response to blockade of 5-HT transporters (Zhou et al., 2002). 4.3. Effect of 5-HTP on urinary metabolites Urinalysis was performed as an independent confirmation that oral 5-HTP treatment was effective at delivering 5-HTP into the bloodstream and to estimate approximately how long it remained elevated. We found that the time course of changes in 5-HTP, 5-HT, and 5-HIAA in the urine mirrored the time course of immunoreactivity in the SNC. These metabolites increased rapidly and returned to baseline values within 24 h. Following oral 5-HTP administration, the concentration of 5-HTP found in the urine was about two orders of magnitude lower than the concentration of either 5-HT or 5-HIAA, suggesting that most of the 5-HTP is decarboxylated prior to excretion. In contrast to oral administration, IP injection of 5-HTP produced concentrations of 5-HTP in the urine that were of the same order of magnitude as 5-HT and 5-HIAA, suggesting that IP injection overwhelmed the decarboxylation process, allowing an overflow of 5-HTP to the urine. The presence of 5-HT in the urine may be due to the decarboxylation of 5-HTP in the kidneys (Stier and Itskovitz, 1985) or it may represent excretion of 5-HT synthesized elsewhere. The appearance of 5-HT in the urine following 5-HTP administration has clinical implications for the use of 5-HTP because it potentially could cause problems with kidney function; 5-HTP administration in rats can result in renal damage, possibly due to 5-HT toxicity (Hirai and Nakajima, 1979). 4.4. 5-HTP administration did not cause ectopic 5-HT staining in the locus coeruleus No cell-specific 5-HT or 5-HTP staining was seen in the locus coeruleus of animals given oral 5-HTP treatment. This is consistent with a previous study, which showed that intraperitoneal injections of 5-HTP caused the appearance of 5-HT-ir in this area only when combined with an MAO inhibitor (Arai et al., 1995). The differences between staining in the LC and that in the SNC may be due to high levels of MAO in the LC (Arai et al., 1998). 4.5. Clinical implications Serotonin can act as a false transmitter in dopaminergic neurons, where it can be released by Ca2+ -mediated exocytosis (Stamford et al., 1990; Vanhatalo and Soinila, 1995). However, it is not known whether the decarboxylation of 5-HTP and the subsequent release of 5-HT by dopaminer-

138

C.P. Lynn-Bullock et al. / Journal of Chemical Neuroanatomy 27 (2004) 129–138

gic neurons leads to a decreased production of dopamine. If dopamine synthesis is impaired, symptoms that are similar to Parkinson’s disease could be induced. This idea is supported by results from case studies in which Parkinson’s patients were treated with 5-HTP alone or in combination with peripheral decarboxylase inhibitors. There was a worsening in patient akinesia and rigidity with 5-HTP treatment that returned to the former state once 5-HTP treatment was stopped (Klein et al., 1986; Chase, 1970). In summary, we showed that a single oral dose of 5-HTP, without the administration of MAO inhibitors, was sufficient to increase 5-HTP and 5-HT immunoreactivity in the RN and also cause the ectopic appearance of serotonin in normally non-serotonergic brain regions. Acknowledgements We would like to thank Laura O’Farrell for her help with the maintenance of the animals and with the oral gavage technique. We would also like to thank Nadja Spitzer for help with the HPLC instrumentation and Birgit Neuhaus, Ann C. Reedy, and Steve Belinga for help with confocal imaging. In addition, we would like to thank Stefan Clemens for helpful comments and suggestions on this project. Supporting grant: NIH NS35371 to P.S.K., NIH EY12696, NSF IBN0078110 to S.L.P. and a GSU Research Program Enhancement Grant are acknowledged. References Arai, R., Horiike, K., Hasegawa, Y., 1998. Dopamine-degrading activity of monoamine oxidase is not detected by histochemistry in neurons of the substantia nigra pars compacta of the rat. Brain Res. 812, 275–278. Arai, R., Karasawa, N., Geffard, M., Nagatsu, T., Nagatsu, I., 1994. Immunohistochemical evidence that central serotonin neurons produce dopamine from exogenous l-DOPA in the rat, with reference to the involvement of aromatic l-amino acid decarboxylase. Brain Res. 667, 295– 299. Arai, R., Karasawa, N., Nagatsu, T., Nagatsu, I., 1995. Exogenous l-5hydroxytryptophan is decarboxylated in neurons of the substantia nigra par compacta and locus coeruleus of the rat. Brain Res. 669, 145–149. Boadle-Biber, M.C., 1982. Biosynthesis of serotonin. In: Osborne, N.N. (Ed.), Biology of Serotonergic Transmission. Wiley, Chichester, pp. 63–94. Bogdanski, D.F., Wissbach, H., Udenfriend, S., 1958. Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharmacol. Exp. Ther. 122, 182–194. Bouchaud, C., 1972. Demonstration par radioautographie de l’existence d’une barriere hematoencephalique pour la 5-hydroxytryptamine. C.R. Acad. Sci. Hebd. Seances Acad. Sci. D 275, 975–978. Brownfield, M.S., Yracheta, J., Chu, F., Lorenz, D., Diaz, A., 1998. Functional chemical neuroanatomy of serotonergic neurons and their targets: antibody production and immunohistochemistry (IHC) for 5-HT, its precursor (5-HTP) and metabolite (5-HIAA), biosynthetic enzyme (TPH), transporter (SERT), and three receptors (5-HT2A, 5-HT5a, 5-HT7). Proc. Natl. Acad. Sci. 861, 232–233. Byerley, W.F., Risch, S.C., 1985. Depression and serotonin metabolism: rationale for neurotransmitter precursor treatment. J. Clin. Psychopharm. 5, 191–206. Chase, T.N., 1970. 5-Hydroxytryptophan in Parkinsonism. Lancet 2, 1029– 1030.

Denoyer, M., Kitahama, K., Sallanon, M., Touret, M., Jouvet, M., 1989. 5-Hydroxytryptophan uptake and decarboxylating neurons in the cat hypothalamus. Neuroscience 31, 203–211. Fickbohm, D.J., Katz, P.S., 1999. 5-HT content alters the modulatory actions of serotonergic neurons in the Tritonia swim CPG. Soc. Neurosci. Abstr. 25, 168. Fickbohm, D.J., Lynn-Bullock, C.P., Spitzer, N., Katz, P.S., 2001. Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia. J. Comp. Neurol. 437, 91–105. Geffard, M., Touret, M., Kitahama, K., 1987. First characterization of 5−hydroxytryptophan in rat brain by using specific antibodies. Brain Res. 426, 191–196. Hashimoto, S., Inoue, T., Koyama, T., 1999. Effects of conditioned fear stress on serotonin neurotransmission and freezing behavior in rats. Eur. J. Pharm. 378, 23–30. Hirai, M., Nakajima, T., 1979. Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-l-tryptophan between Sprague Dawley and Wistar rats. J. Biochem. (Tokyo) 86, 907–913. Imeri, L., Mancia, M., Bianchi, S., Opp, M.R., 2000. 5-Hydroxytryptophan, but not l-tryptophan, alters sleep and brain temperature in rats. Neuroscience 95, 445–452. Jesberger, J.S., 1985. Neurochemical aspects of depression: the past and the future. Int. J. Neurosci. 27, 19–47. Kitahama, K., Ikemoto, K., Jouvet, A., Nagatsu, I., Sakamoto, N., Pearson, J., 1998. Aromatic l-amino acid decarboxylase- and tyrosine hydroxylase-immunohistochemistry in the adult human hypothalamus. J. Chem. Neuroanat. 16, 43–55. Kitahama, K., Jouvet, A., Fujimiya, M., Nagatsu, I., Arai, R., 2002. 5-Hydroxytryptophan (5-HTP) uptake and decarboxylation in the kitten brain. J. Neural. Transm. 109, 683–689. Klein, P., Lees, A., Stern, G., 1986. Consequences of chronic 5-hydroxytryptophan in Parkinsonian instability of gait and balance and in other neurological disorders. Adv. Neurol. 45, 603–604. Lynn-Bullock, C.P., Welshhans, K., Reedy, A., Belinga, S.F., Pallas, S.L., Katz, P.S., 2001. 5-HTP- and 5-HT-immunoreactivity in the raphe nucleus, substantia nigra, and locus coeruleus after oral 5-HTP administration. Soc. Neurosci. Abstr. 27, 414. Murray, M., 1998. 5-HTP: the Natural Way to Overcome Depression, Obesity and Insomnia. Bantam Books, New York. Sahelian, R., 1998. 5-HTP: Nature’s Serotonin Solution. Avery Publishing Group, New York. Sloley, B.D., Juorio, A.V., 1995. Monoamine neurotransmitters in invertebrates and vertebrates: an examination of the diverse enzymatic pathways utilized to synthesize and inactivate biogenic amines. Int. Rev. Neurobiol. 38, 253–303. Stamford, J.A., Kruk, Z.L., Millar, J., 1990. Striatal dopamine terminals release serotonin after 5-HTP pretreatment: in vivo voltammetric data. Brain Res. 515, 173–180. Stier, C.T., Itskovitz, H.D., 1985. Formation of serotonin by rat kidneys in vivo. Proc. Soc. Exp. Biol. Med. 180, 550–557. Touret, M., Kitahama, K., Geffard, M., Jouvet, M., 1987. 5-Hydroxytryptophan (5-HTP)-immunoreactive neurons in the rat brain tissue. Neurosci. Lett. 80, 263–267. Vanhatalo, S., Soinila, S., 1995. Release of false transmitter serotonin from the dopaminergic nerve terminals of the rat pituitary intermediate lobe. Neurosci. Res. 22, 367–374. Wyatt, R.J., Zarcone, V., Engelman, K., Dement, W.C., Snyder, F., Sjoerdsma, A., 1971. Effects of 5-hydroxytrytophan on the sleep of normal human subjects. Electroencephalogr. Clin. Neurophysiol. 30, 505–509. Zhou, F.C., Lesch, K.-P., Murphy, D.L., 2002. Serotonin uptake into dopamine neurons via dopamine transporters: a compensatory alternative. Brain Res. 942, 109–119. Zhu, M.Y., Juorio, A.V., 1995. Aromatic l-amino acid decarboxylase: biological characterization and functional role. Gen. Pharmacol. 26, 681–696.