5-羟基色氨酸3

Journal of Chemical Neuroanatomy 27(2004)129–138The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of ratsChristina P.Lynn-Bullock,Kristy Welshhans,Sarah L.Pallas,Paul S.Katz ∗Department of Biology,MSC 8L0389,Georgia State University,33Gilmer St SE Unit 8,Atlanta,GA 30303-3088,USAReceived 23May 2003;received in revised form 24November 2003;accepted 2February 2004Abstract5-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 dopamin-ergic 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 substan-tia nigra was maximal within 2h of 5-hydroxytryptophan administration and returned to control levels by 24h.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.©2004Elsevier B.V .All rights reserved.Keywords:Double label immunohistochemistry;Confocal fluorescence microscopy;Co-localization;Serotonin;5-Hydroxytryptophan;HPLC1.IntroductionDecreased levels of serotonin (5-hydroxytryptamine (5-HT))in the central nervous system (CNS)are correlated with many behavioral and mental conditions such as depres-sion (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:pkatz@ (P.S.Katz).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 neu-rons by the enzyme aromatic amino acid decarboxylase (AADC)(Boadle-Biber,1982;Zhu and Juorio,1995).How-ever,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 appear-ance 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 ad-ministered 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-0891-0618/$–see front matter ©2004Elsevier B.V .All rights reserved.doi:10.1016/j.jchemneu.2004.02.003130 C.P.Lynn-Bullock et al./Journal of Chemical Neuroanatomy27(2004)129–138munoreactivity(-ir)in a manner similar to that caused by IP injection.This study also sought to further characterize the extent to which5-HTP is co-localized with5-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 into5-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 of5-HTP with serotonin had not been determined previously.Furthermore,the distribution of 5-HTP-ir after5-HTP administration had not been examined previously.5-HTP administration might result in widespread uptake of5-HTP into neurons and cause the appearance of 5-HTP in neurons that lack AADC and are,thus,incapable of converting it into5-HT,as was seen in a molluscan nervous system(Fickbohm and Katz,1999).Therefore,we examined the co-localization of5-HTP-ir and5-HT-ir to determine if it was affected by oral5-HTP treatment.We found that oral5-HTP administration increased the intensity of5-HTP and5-HT immunoreactivity in the nor-mally serotonergic dorsal raphe nucleus and caused transient ectopic expression of5-HTP-ir and5-HT-ir in the normally dopaminergic SNC but not in the noradrenergic LC.We did not observe widespread uptake of5-HTP into neurons that do not convert it to5-HT.Some of these results have been previously presented in abstract form(Lynn-Bullock et al., 2001).2.Methods2.1.5-HTP administrationThirty-four Sprague–Dawley rats(200–230g;Charles River Laboratories,Wilmington,MA)received an oral gav-age or an IP injection of either5-HTP(10mg/kg of body weight,in2ml water)or water alone(sham-treatment; Table1).Prior to5-HTP treatment,the animals were main-tained under a12h light:12h dark cycle and were provided food and water ad libtum.All procedures used met or ex-ceeded the standards of accepted care developed by the Institutional Animal Care and Use Committee,the National Institutes of Health,and the Society for Neuroscience. Table1Numbers of animals for each treatment and survival timeTreatment Survival time(h)12424Oral5-HTP4343Oral sham3333IP5-HTP2––3IP sham1––22.2.Urine collection and analysisAnimals 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 ev-ery24h for three days before treatment to establish base-line values of5-HTP,5-HT,and hydroxyindoleacetic acid (5-HIAA).After treatment,available urine was collected ev-ery hour.To avoid overestimates of concentration,urine was discarded from analysis if the time between collections ex-ceeded1h.Five microliters of N-methyl-serotonin(NMS, oxalate salt;Sigma,St.Louis,MO),an internal standard, and1.7␮l of perchloric acid,which prevents the oxidation of amines present in the samples,were added to100␮l aliquots of urine samples.The mixtures were centrifuged at5585×g for5min at4◦C.The supernatant was passed through a 0.22␮m centrifugefiltration device(Ultrafree-MC;Milli-pore,Bedford,MA),brought up to a known volume(100␮l), and diluted as necessary with mobile phase(MP)consist-ing of75mM sodium dihydrogen phosphate,monohydrate, 1.7mM sodium octyl sulfate(SOS),0.01%(v/v)triethy-lamine(Fisher,New Jersey),25␮M EDTA,and15%ace-tonitrile(pH3.0).The HPLC system consisted of an ESA Model528pump and Coulochem II detector with aflow-through Model5011 Analytical Cell(ESA Inc.,Chelmsford,MA)and a guard cell set at350mV with the screen and analytical electrodes set at 50and325mV,respectively.The column was a150mm×3.2mm,3␮m RP-C18(MD-150;ESA Inc.,Chelmsford, MA).The amounts of5-HTP,5-HT,and5-HIAA were com-pared to standard curves run concurrently.2.3.Tissue preparationAnimals that received an oral gavage of5-HTP or water were euthanized(sodium pentobarbitol,50mg/kg)at1,2,4, or24h following5-HTP administration.Additional animals received IP injections of5-HTP or water and were then euthanized at1or24h following treatment.Table1lists the number of animals used for each treatment and survival time.The animals were perfusedfirst with0.1M phosphate buffer(PB,pH=7.4)and then with cold4%paraformalde-hyde in0.1M phosphate buffered saline(PBS,pH=7.4). The brains were post-fixed overnight in the same solution and then immersed in30%sucrose for approximately24h for cryoprotection.Frozen50␮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.2.4.Fluorescence immunohistochemistryFree-floating sections were rinsed three times(15min each)in phosphate buffered saline with0.02%Na AzideC.P .Lynn-Bullock et al./Journal of Chemical Neuroanatomy 27(2004)129–138131and 0.03%Triton X (PBS ∗)and then incubated for 1h in blocking buffer (PBS ∗with 3%normal goat or donkey serum).Sections were incubated for 72h at 4◦C in primary antiserum diluted 1:1000with 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 pri-mary antisera,sections were rinsed three times (15min each)in PBS ∗and incubated for 4h in secondary antiserum di-luted 1:250with 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 cover-slipped using Cytoseal TM (Stephens Scientific,Kalamazoo,MI).Immunofluorescence was visualized using a laser scan-ning confocal microscope (LSM 510,Carl Zeiss Inc.,Thorn-wood,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 sec-tions.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 510Software (Carl Zeiss).At least two different brain sections,imaged at 40×,were quantified for fluorescence intensity in each animal.A minimum of 15cells per section and 45cells 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 SigmaS-tat 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.Table 2Double label antibody combinations CombinationsFirst set Second set Primary AbSecondary AbPrimary Ab Secondary Ab5-HTP and 5-HT Goat anti-5-HT Donkey anti-goat Alexa 546Rabbit anti-5-HTP Goat-anti rabbit Alexa 488AADC and 5-HT Goat anti-5-HT Donkey anti-goat Alexa 546Rabbit anti-AADC Goat-anti rabbit Alexa 4885-HT and TH Goat anti-5-HT Donkey anti-goat Alexa 546Mouse anti-TH Rabbit anti-mouse Alexa 488AADC and THRabbit anti-AADCGoat anti-rabbit Alexa 488Mouseanti-THGoat anti-mouse CY3or Alexa 3502.5.Double label immunohistochemistryTo examine co-localization of antigens,sections were treated as described above except that two different primary antisera were used (DiaSorin Inc.),each diluted 1:1000with blocking buffer and followed by the secondary antiserum (Molecular Probes)diluted 1:250with blocking buffer.All primary and secondary antisera used for double label im-munohistochemistry are listed in Table 2.Primary antisera were tested individually before being used in the double la-bel protocol.3.Results3.1.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 1h after receiving a sin-gle oral dose (10mg/kg)of 5-HTP,a dosage equivalent to the upper range of suggested doses for humans (Sahelian,1998),compared to animals given a water para-ble 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 =135from three animals).This more than doubled in rats that received the oral 5-HTP treatment to 49.3±2.0(N =221from 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 flu-orescence 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 1h after 5-HTP treatment,a highly significant difference (P <0.001,un-paired t -test).As with 5-HTP immunofluorescence,5-HTP132 C.P.Lynn-Bullock et al./Journal of Chemical Neuroanatomy27(2004)129–138Fig.1.Oral5-HTP treatment increased5-HTP and serotonin-immunoreactivity in the dorsal raphe nucleus(RN),but did not disrupt the co-localization of5-HTP and serotonin.5-HTP-ir(A and C)was co-localized with5-HT-ir(B and D)in the RN of control(A and B)and oral5-HTP treated(C and D) animals.5-HTP treatment did not alter the co-localization of5-HTP-ir and5-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 of5-HTP and5-HT immunoreactivity increased in rats treated with5-HTP and euthanized after1h.The cumulative histograms show the distribution of5-HTP(E)and5-HT(F)immunofluorescence intensities above background for cells from animals euthanized1h after control treatment(open circles)or oral5-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 Neuroanatomy27(2004)129–138133treatment broadened the distribution of5-HT immunofluo-rescence intensities.In sham-treated and untreated animals,5-HTP was co-localized with5-HT(Fig.1A and B).There continued to be co-localization of5-HTP-ir and5-HT-ir following oral5-HTP treatment(Fig.1C and D)at all of the sur-vival times tested(n=14,see Table1,data not shown). Similar results were obtained using IP injection of5-HTP (n=5).Thus,immunofluorescence measurements suggest that5-HTP treatment appeared to increase5-HTP and5-HT levels in serotonergic cells of the RN and did not result in the appearance of5-HTP in non-serotonergic neurons.3.2.Oral5-HTP administration resulted in the appearance of5-HT-ir in the SNCThe presence of AADC in dopaminergic neurons should cause these neurons to display5-HT-ir when presented with 5-HTP.In all animals euthanized1h after oral5-HTP ad-ministration(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 whether5-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 after1h,neurons positive for TH-ir also displayed5-HT-ir(Fig.2A and B,open arrow-heads)indicating that dopaminergic neurons were able to synthesize serotonin as a result of5-HTP treatment.In two of the three animals examined,1–2%of the neurons exhibiting5-HT-ir did not show TH-ir(Fig.2A and B,solid arrow),indicating that non-catecholaminergic neurons became5-HT immunoreactive.These neurons cor-respond 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).Con-sistent with this identification,all SCN neurons observed to be5-HT-immunoreactive also displayed AADC-ir in animals euthanized1h after oral5-HTP administration (n=4)(Fig.2C and D;open arrowheads)or IP injection (n=2).This result is also consistent with the hypothesis that5-HT is not taken up into dopaminergic terminals,but is synthesized from5-HTP through the action of AADC.3.3.Co-localization of5-HTP and5-HT in the SNCIf5-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 up5-HTP but do not convert it into5-HT.To distinguish between these alternatives,we performed double-label immunohistochem-istry and looked for co-localization of5-HTP-ir and5-HT-ir. 5-HTP labeling was not observed in the SNC of animals re-ceiving no treatment(n=1)or those euthanized1h after either oral sham treatment(n=3)or IP sham treatment (n=1).However,1h after oral or IP5-HTP administra-tion there was clear cell-specific5-HTP-ir in SNC neurons (Fig.2F).Almost all5-HTP-positive neurons also displayed 5-HT-ir(Fig.2E and F;open arrowheads),which suggests that these neurons took up5-HTP and converted it into5-HT. There was variability in the extent of co-localization of 5-HTP and5-HT.A small number of neurons that were 5-HTP-immunoreactive were not5-HT-immunoreactive (Fig.2E and F;filled arrowheads).This could indicate that these neurons were not capable of converting5-HTP into5-HT or that synthesis was slower in these neurons. Conversely,a small percentage of neurons that expressed 5-HT-ir were not5-HTP immunoreactive(Fig.2E and F; solid arrows).These neurons exhibited the highest intensity of5-HT labeling.Thus,the lack of5-HTP-ir could indicate that these neurons converted5-HTP into5-HT more rapidly and completely than neighboring cells.3.4.5-HTP-ir and5-HT-ir in the SNC was transientWe examined the time course over which ectopic5-HT-ir and5-HTP-ir were observable in the SNC.As noted previ-ously,there were no5-HT or5-HTP labeled neurons in the SNC of untreated or sham-treated control animals(Fig.3A), but in animals euthanized1h after oral5-HTP adminis-tration,neurons were labeled strongly for both5-HTP and 5-HT(Fig.3B;solid arrows).The intensity of5-HTP and 5-HT staining in individual neurons was not correlated either positively or negatively;that is,some neurons that stained strongly for5-HTP stained weakly for5-HT,whereas others stained strongly for both.Two hours after oral treatment,the intensity of specific 5-HTP labeling had substantially decreased(Fig.3Ci)com-pared to1h post-treatment(Fig.3Bi).In contrast,the inten-sity of5-HT-ir remained the same or increased(Fig.3Cii) compared to staining from animals euthanized1h after 5-HTP administration(Fig.3Bii).Some strongly labeled 5-HT-immunoreactive neurons in the2h preparations showed weak or no5-HTP-ir(Fig.3C,open arrowheads). As was observed in animals euthanized1h after treatment, there were neurons that were5-HTP-immunoreactive but not5-HT immunoreactive at2h post-treatment(Fig.3C,filled arrowheads).Four hours after5-HTP administration,the intensity of the5-HTP-ir had decreased to control levels(Fig.3Di), however,the relative intensity of5-HT-ir remained elevated above control(Fig.3Dii).Twenty-four hours after5-HTP administration,few if any immunopositive neurons could be discerned and the relative intensity of both5-HTP and5-HT labeling was visually indistinguishable from control animals134 C.P.Lynn-Bullock et al./Journal of Chemical Neuroanatomy27(2004)129–138Fig.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),and5-hydroxytryptophan(5-HTP)(F)in the substantia nigra pars compacta(SNC)of animals euthanized1h after oral5-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 and5-HT were similar in distribution throughout the SNC.(C and D) AADC-ir and5-HT-ir were co-localized in neurons of the SNC(open arrowheads).(E and F)Serotonin-ir was co-localized with5-HTP-ir in most 5-HT-immunoreactive neurons(open arrowheads),however,some neurons displayed5-HT-ir without5-HTP-ir(closed arrows).Additionally,a few neurons were5-HTP-immunoreactive but not5-HT-immunoreactive(filled arrowheads).Scale bars=50␮m.C.P.Lynn-Bullock et al./Journal of Chemical Neuroanatomy27(2004)129–138135Fig.3.The ectopic expression of5-HTP and5-HT in the SNC of5-HTP treated animals was transient.Rats were euthanized at varying time points following a single oral gavage of5-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 after5-HTP administration,there was strong5-HTP and5-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 somatic5-HT-ir continued to be elevated(ii).Filled arrowheads indicate neurons that labeled for5-HTP but not5-HT.Open arrowheads indicate neurons that labeled for5-HT but not5-HTP.(D)At4h post-treatment cell-specific staining had decreased.(E)By24h 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 under24h for5-HT in these non-serotonergic neurons of the SNC.3.5.5-HTP administration caused a transient elevation of 5-HTP,5-HT,and5-HIAA in the urineTo determine the rate at which5-HTP is cleared from the body,we examined the effects of oral5-HTP admin-istration on the concentration of5-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 of5-HIAA could be measured in the urine(mean value27±3pmol/␮l;n=9),but no5-HTP or 5-HT could be detected with our method.Urine was sam-pled at1h intervals following a single oral dose of5-HTP (10mg/kg;see Section2).5-HTP,5-HT,and5-HIAA levels increased within thefirst hour after5-HTP administration (Fig.4).The concentrations of5-HT and5-HIAA in the urine were of similar magnitude to each other,but5-HTP concentration was100fold lower.Some of the5-HT in the urine is likely due to decarboxylase activity within the kid-ney converting5-HTP to5-HT(Stier and Itskovitz,1985). Thus,the actual amount of5-HTP in the blood is likely to be higher than indicated by the excreted concentration. The time course of the appearance of5-HT in the urine roughly mirrored the relative intensity of5-HT-ir in the SNC following5-HTP treatment.The urine concentrations of5-HTP,5-HT,and5-HIAA reached their maximum lev-els between2and4h post-treatment and declined there-after.By24h post-treatment,the levels of5-HTP,5-HT,and 5-HIAA had returned to pre-treatment levels.However,in one animal,the5-HIAA concentration remained elevated 24h post-treatment(Fig.4Aiii).The effect of IP injection on the levels of5-HT and 5-HIAA in the urine was similar to that of oral5-HTP ad-ministration(Fig.4B).However,IP injection caused more 5-HTP to appear in the urine than did oral gavage(Fig.4Ai inset);the amount of5-HTP1–2h after IP injection of 5-HTP was approximately equivalent to the amount of5-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 and5-HT-ir in the LCA previous study reported that IP injection of5-HTP did not cause ectopic5-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 oral5-HTP treat-ment on5-HTP-ir and5-HT-ir in the LC in the absence of MAO inhibitors.The LC was positively identified by TH-ir (Fig.5A).No5-HTP-ir or5-HT-ir was observed in the LC of control animals(n=3).Oral5-HTP treatment did not cause neurons in the LC to display5-HTP-ir or5-HT-ir (n=4;Fig.5B and C).4.DiscussionWe found that oral administration to rats of the dietary supplement,5-hydroxytryptophan,increased the intensity of serotonin and5-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 Neuroanatomy27(2004)129–138Fig.4.The concentrations of5-HTP,5-HT,and the5-HT metabolite5-hydroxyindolacetic acid(5-HIAA)transiently increase in the urine following oral gavage(A)(n=11)and IP injections(B)(n=3)of5-HTP.All three substances increased in concentration,peaking within2–4h after5-HTP administration and returning to control values within24h.Inset in Ai represents an enlargement of the y-axis for the5-HTP data from animals receiving oral administration;although the increase in5-HTP followed the same time course as5-HT(Aii)and5-HIAA(Aiii),the concentration was about100fold lower.The animals that received IP injections had5-HTP concentrations(Bi)that were similar to the concentrations of5-HT and5-HIAA(Bii and Biii). Arrow indicates the day of5-HTP treatment.Dashed line indicates baseline levels of5-HTP,5-HT,and5-HIAA.Each symbol represents a different animal.Fig.5.No5-HTP-ir or5-HT-ir was observed in the locus coeruleus(LC)1h following oral treatment.(A)Tyrosine hyroxylase(TH)immunoreactivity delineates the extent of the LC(arrows).An adjacent serial section was double-labeled for5-HTP(B)and5-HT(C).There was no cell-specific labeling throughout the rge neurons in the neighboring region,mescenphalic region5(Me5),exhibited backgroundfluorescence,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.。