Pii: s0165-0270(00)00259-4

Journal of Neuroscience Methods 101 (2000) 141 – 148 The determination of histamine in the Drosophila head J. Borycz a,*, M. Vohra c, G. Tokarczyk a, I.A. Meinertzhagen a,b a Life Sciences Centre, Dalhousie Uni6ersity, Halifax, NS, Canada B3H 4J1 b Neuroscience Institute, Dalhousie Uni6ersity, Halifax, NS, Canada c Department of Pharmacology, Sir Charles Tupper Building, Dalhousie Uni6ersity Medical School, 5859 Uni6ersity A6enue, Halifax, NS, Canada B3H 4H7 Received 28 March 2000; received in revised form 31 May 2000; accepted 2 June 2000 Abstract
Histamine is a neurotransmitter at arthropod photoreceptors. Even though the fruit fly, Drosophila melanogaster, is a widely used model in neuroscience research, the histamine content of its nervous system has not so far been reported. We have developeda high performance liquid chromatography (HPLC) method with pre-column o-phtaldialdehyde-mercaptoethanol (OPA-ME)derivatization and electrochemical detection, to determine this amine in Drosophila. The histamine content of the fly’s headaverages about 2.0 ng per head. In heads of the mutant hdc JK910, a presumed null for the gene encoding the enzyme thatsynthesizes histamine, histamine was not detected in measurable amounts. In heads of the mutant sine oculis, which lackscompound eyes, only 28% of this amine was found compared with wild type flies, so histamine is mainly present in the compoundeye photoreceptors. Also observed in histamine-deficient mutants was a decrease in the peak which contains a substance havingthe same retention time as carcinine (b-alanyl-histamine). Our method was not able to detect compounds previously reported ashistamine metabolites in insects. In spite of this, the method we have developed enables the fast and accurate measurement ofhistamine in the heads of Drosophila, suitable for screening mutants. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Drosophila melanogaster; Mutant, sine oculis; Mutant, hdcJK910; Compound eye; Visual system; High performance liquid chromatogra-phy (HPLC) 1. Introduction
partly blocked by histamine H -receptor antagonists (promethazine, mepyramine) and also decreased by his- There is significant evidence that histamine acts as a tamine H -receptor antagonists (cimetidine, metiamide) transmitter at the synaptic terminals of photoreceptors (Hardie, 1987, 1988). At a different site, histamine H -specific receptor antagonists (cimetidine, ranitidine) Callaway and Stuart, 1989; Stuart, 1999). In flies, for block the inhibitory action of histamine on a descend- example, not only does the action of ionophoresed ing interneuron in a locust extraocular pathway (Lundquist et al., 1996). However, the characteristics of (Hardie, 1987) but light-evoked release of radiolabelled the ion-channel coupled histamine receptors in the in- histamine has been observed from eye-cup preparations sect’s lamina seem to be quite different from those in (Sarthy, 1991). Histamine H binding sites have been vertebrates, which are coupled to G-proteins. As in found in the optic lobe of the locust (Elias et al., 1984; vertebrates, histamine is synthesized by the single-step Roeder, 1990). Histamine acts at a ligand-gated ion decarboxylation of histidine, under the influence of channel to cause a selective increase in chloride conduc- histidine decarboxylase (Burg et al., 1993). The tance at the first-order interneurons (Hardie, 1989) in metabolism of histamine in invertebrates is, by contrast, the first optic neuropile, or lamina, an action that is not yet well established. There are only two previousstudies, and these show that the main histaminemetabolites in insects are: imidazole-4-acetic acid and * Corresponding author. Tel.: + 1-902-4942131; fax: acetylhistamine (Elias and Evans, 1983; Sarthy, 1991).
E-mail address: [email protected] (J. Borycz).
On the other hand, in crab tissue b-alanyl histamine 0165-0270/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 5 - 0 2 7 0 ( 0 0 ) 0 0 2 5 9 - 4 J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 (carcinine), the b-alanine conjugate of histamine, was found to be the main metabolite (Arnould, 1985, 1987).
In the opisthobranch Aplysia californica another D. melanogaster, Oregon-R wild type and mutants metabolic pathway has been described in which his- (sine oculis, hdcJK910), were used from stocks held at tamine is inactivated by conversion to g-glutamylhis- 24°C in a 12 h light/dark cycle. Flies were raised on a standard cornmeal/molasses medium (Lewis, 1960).
Compatible with evidence for histamine as a trans- mitter, photoreceptors of the compound eye also ex- hibit histamine-like immunoreactivity, which is welldemonstrated in flies (Na¨ssel et al., 1988; Pollack and For histamine determinations the flies were taken 2 h Hofbauer, 1991), amongst many other arthropods. His- after lights on, and quickly killed by freezing on dry ice.
tamine-like immunoreactivity is also found among pho- This procedure was standardized as far as possible, and toreceptors of the ocellus (Simmons and Hardie, 1988; the heads were then separated from the bodies by gentle Schlemermeyer et al., 1989). In the Drosophila head, sifting through S.I.K. stainless steel sieves (IIDA test histamine-like immunoreactivity also occurs in extraoc- sieves: IIDA Seisakusho Co. Ltd., Osaka, Japan) with a ular photoreceptors (Hofbauer and Buchner, 1989; Ya- mesh size of 710 mm to retain the bodies and 425 mm to collect the heads, after which the heads were stored at integumentary mechanoreceptors (Buchner et al., 1993).
− 80°C until the time of assay. Heads from the par- The function of these systems, photoreceptor (Burg et tially penetrant sine oculis stock were inspected to re- al., 1993) and mechanoreceptor (Melzig et al., 1998), is move those with partially developed compound eyes, moreover elimininated in a presumed genetic null for the heads finally assayed lacking all externally visible the histidine decarboxylase gene (hdc), hdcJK910.
compound eye tissue. For a sample preparation, 50 For neuron systems of flies that either contain, or are heads were mixed with 75 ml of 0.1 M perchloric acid thought to contain, histamine, there is no information containing 0.1% EDTA and 1.125 mg 3-methylhis- about the quantity of histamine involved, or whether tamine (as an internal standard), and homogenized.
the contents are indeed chemically histamine. Such The homogenate was centrifuged (16 000 × g) and the evidence is necessary before physiologically more im- supernatant was filtered through a 0.2 mm cellulose portant parameters, such as data on histamine release, can be evaluated. Two previously described procedures For each assay 40 ml of filtrate was added to the condensation mixture containing: 480 ml of mobile (HPLC) to determine the endogenous histamine con- phase plus 30 ml orthophthaldialdehyde (0.25% v/v in tents, in the heads of the cockroach (Pirvola et al., methanol) and 30 ml 2-mercaptoethanol (0.25% v/v in 1988) or the eyes of the horseshoe crab Limulus (Bat- methanol). After condensation, the sample was adjusted telle et al., 1991), require complicated HPLC equipment to pH 11 with approximately 20 ml of 2M KOH. Each or preparative procedures. We have therefore modified sample thus finally contained 600 ml. From this sample, a previously published method for the detection of aliquots of 20 ml were injected into the HPLC system.
histamine in vertebrate systems (Han and Vohra, 1991) The histamine recovery of the method, evaluated from using HPLC, for use with insect brains. With this the loss of the internal standard, 3-methylhistamine, method we are able to confirm the presence of his- added to the homogenization mixture along with the tamine in the fly’s brain, and quantify the total his- internal standard solution, was greater than 90%. Each tamine contents. For the present study, we have chosen aliquot corresponded to the original number of heads, the fruit fly Drosophila melanogaster, because of the 50, divided by two ratios: that of the perchloric acid to availability of the hdcJK910 mutant, which because it is filtrate volumes (75/40 ml=1.875 : 1) and that of the unable to synthesize histamine (Burg et al., 1993), pro- sample to aliquot volumes (600/20 ml=30 : 1). Thus vides a genetic control for the chemical identification of each aliquot corresponded to an extract from 50/ 1.875 × 30, or approximately 0.89 head.
2. Materials and methods
The buffer was prepared from high purity Millipore water (resistivity: 18.2 Megohm-cm) containing the fol- Histamine determinations were performed according lowing (mmoles/l): sodium acetate: 100; citric acid to the method described by Han and Vohra (1991), but with several modifications adapting this method to the sodium salt (Sigma): 0.32; Na EDTA (BDH): 0.15.
differences that exist in insect species and eye tissues we Other components of the mobile phase were: acetoni- have used. All reagents were HPLC grade.
trile (Fisher) 18% and methanol 7% (v/v). Buffer was J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 Fig. 1. Detector response to increasing concentrations of injected standard histamine (HA), after condensation with OPA/ME. As an internalstandard 7.5 ng 3-methylhistamine (3-MeHA) was added to each sample. HPLC condition: 5 mm C18, 100 A, Nucleosil column (150×4.6 mm)coupled with a 5 mm C18, 100 A, Nucleosil guard column (10×4.6 mm); mobile phase: sodium acetate 100 mM; citric acid monohydrate 20 mM;1-octane-sulfonic acid, sodium salt 0.32 nM; Na EDTA 0.15 mM, acetonitrile 18% v/v and methanol 7% v/v; flow rate 1 ml/min.
filtered through a 0.2 mm cellulose acetate membrane injection. The internal standard 3-methylhistamine was filter (BAS) and was degassed prior to use in the HPLC added to each sample in an amount of 7.5 ng, and induced almost identical detector responses for eachsample. For injected histamine standards (1.25; 2.5; 5.0; 7.5 ng) an approximately linear dose-dependent detec-tor response was observed (Fig. 2).
A BAS 480 liquid chromatograph equipped with an Much lower concentrations of injected histamine isocratic pump (BAS PM-80) and amperometric elec- standards were examined to establish the detection limit trochemical detector (BAS LC-4C) was used. Histamine of the method (Fig. 3). With increasing dilutions, the was separated with a 5 mm C18, 100 A, Nucleosil lowest detection limit was established to be better than column (150 × 4.6 mm) coupled with a 5 mm C 18, 100 25 pg per sample (Fig. 3A). At this concentration, the A, Nucleosil guard column (10×4.6 mm) (Alltech). The height of the much reduced peak was still more than column was maintained at ambient room temperature twice the height of the baseline noise, providing a (19 – 21°C) and had a flow rate of 1 ml/min. The criterion signal: noise ratio of at least 2 : 1. The internal working electrode potential was maintained at + 0.7 V standard 3-methylhistamine (500 pg) was added to the histamine containing samples, and in all cases evoked Determinations were tabulated as means 9SE for each group of flies (i.e. each genotype). Tests of statisti-cal significance were used to assess differences betweenthe histamine contents in different Drosophila mutantsusing an unpaired t-test.
3. Results
3.1. Sensiti6ity of the method Examples of chromatograms of injected standards are shown in Fig. 1. Under our HPLC conditions, the Fig. 2. Detector response as a function of the amount of injected retention time for histamine was about 18 min after histamine. HPLC conditions as Fig. 1.
J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 Fig. 3. Lower limit of detection for histamine (HA). A,B,C: three chromatographs for increasing amounts of histamine (25, 50, 100 pg: arrows)which, as here, sometimes occur against a drifting baseline. As an internal standard, 3-methylhistamine (3-MeHA) 500 pg is added to each sample.
U: unknown peak, also visible in an injection of 0.1 M perchloric acid (in which standards were dissolved) after condensation with OPA-ME (D).
HPLC conditions for column, mobile phase, and flow rate, as in Fig. 1.
an identical detector response (Fig. 3 A, B, C). How- response, almost twice as high as carcinine and three ever, the shape of this peak suffered some interference times as high as 3-methylhistamine injected in the same from the nearby peak of an unknown substance, which concentration. Injections of 0.1 M perchloric acid- was also observed in a standard-free, perchloric acid- OPA-ME reaction product (Fig. 5B) indicated that OPA-ME derivatization sample (Fig. 3D). This un- none of the other peaks detected (Fig. 5A) was acetyl- known peak was however small compared with the histamine or 4-imidazoleacetic acid.
amount of 3-methylhistamine standard normally in-jected, and moreover it did not exactly coincide with 3.4. Validation of histamine peak in HPLC the peak for the latter, so its contribution to the peak amplitude for 3-methylhistamine was consequentlysmall. As a result, however, the shoulder for the 3- In our conditions the retention time of the last major methylhistamine peak was somewhat asymmetrical.
compound within Drosophila head extracts was approx-imately 35 min. Even though the chromatograms exhib- 3.2. Stability of the product of OPA-ME-histamine ited considerably more peaks than for corresponding injections of histamine standards, the peak having the When protected from direct light, the reaction product of OPA-ME-histamine was stable for at least60 min as measured by the detector response (Fig. 4).
Exposure to light induced a rapid decrease in thedetector response to the same reaction product, andthis was ten times smaller 60 min after the reactionproduct was first formed (Fig. 4).
3.3. Standards of histamine and its possible metabolites Fig. 5 shows the response to injecting five related compounds: histamine, acetylhistamine, carcinine, 4-imidazolacetic acid and 3-methylhistamine, each in anamount of 2.5 ng. Only carcinine, histamine and 3-methylhistamine were detected. Acetylhistamine and 4- Fig. 4. Stability of histamine standards (2.5 ng) measured as detectorresponse to injections of OPA-ME derivatization product at 15 min imidazoleacetic acid were non-detectable over a wide intervals after reaction. Solid line samples protected from direct light.
range of doses between 1 and 200 ng. Among the Dashed line indicates the same substance exposed to fluorescent substances detected, histamine gave the highest detector J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 hdcJK910, head extracts of which lacked a clear peak atthe same retention time as the histamine peak in wild-type extracts (Fig. 6). Although some of the hdcJK910samples did in fact show a visible histamine peak, thiswas extremely reduced and never greater than the peakgiven by a 100 pg histamine standard.
3.5. Histamine content of the fly’s head method averaged in wild type flies 1.98 90.15 ng (17.8pmol) per head. In sine oculis, the peak height wasreduced to 0.56 90.07 ng per head, giving a histaminedetermination for this amine that was 28% of the wildtype, corresponding to the loss of the compound eyesand their contributions to the innervation of the opticlobes. This difference was significant (P B0.01, t-test).
The lack of a measurable histamine peak in hdcJK910, which lacks detectable expression of the gene for his- Fig. 5. (A). Chromatograph of injection containing 2.5 ng of each of tamine synthesis, indicated the lack of significant five standards: histamine (HA), carcinine (CA), n-acetylhistamine, amounts of histamine ( B100 pg/sample), so that this 4-imidazoleacetic acid and 3-methylhistamine (3-MeHA). Only three mutant contained less than 4% of the wild-type his- compounds: carcinine, histamine and 3-methylhistamine are de-tectable. (B). Chromatograph of injection of the same mixture deriva- tamine content. A previous report (Melzig et al., 1998) tized with OPA-ME with histamine and related agents replaced with has indicated that the photoreceptors of hdcJK910 can 0.1 M perchloric acid. Mobile phase and other conditions as in Fig.
take up exogenous histamine from the fly’s food 1. The small final peak at a retention time \20 min is the unknown medium. We therefore considered the possibility that the greatly diminished peak seen in hdcJK910 was at-tributable to that cause, and also measured histamine in same retention time as the standard was nevertheless freshly prepared medium on which the flies fed. The sharp and clearly separated from nearby peaks (Fig. 6).
medium contained only negligible amounts of histamine We could thus be sure that this peak corresponded to (0.29 mg/g wet weight, less than 0.29 ppm).
the endogenous histamine of the fly’s head. The identity An unknown peak with a similar retention time to of the peak was further confirmed in the mutant that of carcinine was observed (Fig. 6), which was Fig. 6. Chromatograms for samples of Drosophila heads for wild type (Oregon R), and for the mutants hdcJK910 and sine oculis. The retentiontimes for histamine (arrows, HA) reveal peaks for wild type and sine oculis samples, whereas the hdcJK910 sample lacks a detectable peak at thecorresponding retention time (arrow). 3-MeHA: peak of internal standard; U: unknown peak close to carcinine retention time; Un: secondunknown peak, missing from sine oculis samples. HPLC conditions for column, mobile phase, flow rate and other conditions, as in Fig. 1.
J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 significantly reduced in both hdcJK910 and sine oculis methods are suitable for samples of larger volume they mutants, suggesting that it was related to histamine are not applicable to the small volume of the fly’s head.
(Fig. 6). On the other hand, the determinations it Moreover, using cation-exchange resins to separate his- provided were different in each sample analyzed from tamine from other amines means that relatively low the same fly mutant, possibly because in the pH range rates of recovery of this amine from the sample are adopted here for histamine determinations carcinine is typical, between 70 and 80%, unlike the recovery rates more pH-sensitive than histamine (data not shown).
reported here, in excess of 90%. There are, however, Because the same peak was also seen in hdcJK910, how- previously described methods which do not require ever, a compound other than carcinine probably had prior extraction of histamine (Scofitsch et al., 1981; the same retention time. The sine oculis peak had a Yamatodani et al., 1985; Saito et al., 1992; Jensen and paradoxically smaller height than hdcJK910, which lacks Marley, 1995), but none of these is specifically applica- all histamine and should therefore lack carcinine as ble to insect tissues and moreover all either require well, but its peak was less asymmetrical than in hdcJK910 more expensive apparatus or utilize a more complex procedure. The conditions for OPA/ME derivatization Finally, the peak of an unknown substance which of histamine and the HPLC detection parameters of the was present in both wild-type and hdcJK910 samples was current method are both the same as in the method missing from each sample of sine oculis (Fig. 6). Thus, described by Han and Vohra (1991) and these features the condition of eyelessness removes an eye-specific of our method therefore require no further discussion.
peak as well as diminishing the peak for histamine.
The lower limit of detection established with our mod-ification of the method, 25 pg/20 ml of sample, issomewhat less sensitive than the two most sensitive 4. Discussion
methods described thus far (Yamatodani et al., 1985;Jensen and Marley, 1995). One of these HPLC methods We report the modification of a method for the (Yamatodani et al., 1985) has previously been used to determination of histamine by HPLC which is applica- determine histamine in an insect brain (Pirvola et al., ble to insects, and we use it to confirm that histamine is 1988) and has a claimed sensitivity of 5 pg, roughly five present in the fly’s head, and to derive the head his- times more sensitive than our method. The criterion tamine content of D. melanogaster. The utility of this peak supporting that claim is about the same as our method to determine the histamine content of the entire criterion (a signal/noise ratio of at least 2). Although fly’s head will, we think, become especially obvious the method of Yamatodani et al. (1985) is more sensi- when the recently released Drosophila genome database tive than the method described here, it uses fluorescence (Adams et al., 2000) becomes used to create new mu- detection and is more complex, and thus less convenient tants of the histamine cycle through P-element mutage- for routine operation. A previous HPLC method using nesis in this species. In common with previous studies electrochemical detection to determine histamine con- (e.g. Elias and Evans, 1983), we report total histamine tent in the lateral and ventral eyes of Limulus (Battelle content per head, rather than as a specific concentra- et al., 1991) is similar to ours, but the sample prepara- tion, given both the inaccuracies that would be intro- tion differs, requiring more steps that include vacuum duced when weighing individual heads in Drosophila, drying. The method has a detection limit of 0.5 pmol and the limited extent of the indeterminacy introduced (55.5 pg) per sample, two-fold less sensitive than our by small variations in eye size, as given by facet number method, and lacks complete separation between the (Krafka, 1920). Compared with existing HPLC meth- histamine peak and neighboring peaks.
ods for histamine determination, our method is rapid, The lack of a detectable histamine peak in the null easy, of sufficient sensitivity, and adapted specifically to mutant hdcJK910 confirms two critical pieces of evi- work with these flies. Given the current interest in the dence. First, it provides good evidence that the detected visual system of Drosophila (Heisenberg and Wolf, peak in Drosophila tissue lacking the synthetic enzyme 1984; Zipursky and Rubin, 1994; Ranganathan et al., for histamine is, indeed, that of histamine itself. Sec- 1995), and in transmitter systems (Na¨ssel, 1991; Restifo ond, the lack of a detectable histamine peak means that and White, 1990) in this genetically manipulable organ- such a peak in wild-type tissue does not obscure peaks ism, we developed an HPLC method specifically for with similar retention times corresponding to other histamine, because this is the reported transmitter for substances in the sample. Melzig et al. (1998) have fly photoreceptors (Hardie, 1987; Sarthy, 1991).
shown by immunocytochemical means that hdcJK910 can Most older methods for histamine assay require the take up exogenous histamine into the photoreceptors.
prior purification of this amine from biological samples The fact that we failed to find a measurable histamine before injecting the sample into the HPLC system peak in hdcJK910 therefore implies that our flies were (Davis et al., 1979; Yamatodani et al., 1982; Harsing et unable to concentrate exogenous histamine to a signifi- al., 1986; Han and Vohra, 1991). Thus, although such cant extent from their diet. This in turn suggests that J. Borycz et al. / Journal of Neuroscience Methods 101 (2000) 141 – 148 there is a low concentration of histamine in our the fluorescent or electrochemical detection of such medium, as indeed was shown empirically. On the other compounds (Simons and Johnson, 1978; Allison et al., hand, the presence of a small residual histamine peak in 1984). On the other hand, L-histidine, which does react some chromatograms, close to the detection limit, could with OPA-ME, is also undetectable in the range of indicate either the uptake of exogenous histamine at 1 – 80 ng per sample (data not shown). Most probably low concentrations from the medium, or possible resid- L-histidine has a short retention time and disappears ual expression of the hdc gene.
into the initial noise of the chromatograph. The same Previous determinations of total histamine, using bio- may also be true for other histamine-related com- chemical methods (Elias and Evans, 1983) or HPLC pounds which are not detectable with our method.
(Pirvola et al., 1988), report values for larger species of Another proposed metabolite, carcinine, which is de- insect, the locust and sphinx moth (Elias and Evans, tected clearly in standard solutions, fails to give a 1983) and the cockroach (Periplaneta: Elias and Evans, consistent peak in samples. Even so, the determination 1983; Blaberus: Pirvola et al., 1988). Most histamine of histamine using a simple HPLC system equipped occurs in the compound eyes (Elias and Evans, 1983), with electrochemical detection is both easy and rapid in about 150 times more than the amount occurring in the the method we report that is especially adapted to central brain (Pirvola et al., 1988). The determinations reported here now show for the first time the accuratecontent of histamine in the Drosophila head. Our resultssupport in quantitative terms previous immunocyto- Acknowledgements
chemical studies (Pollack and Hofbauer, 1991; Sarthy,1991; Na¨ssel and Elekes, 1992) which show that most This work was supported by grants from MRC histamine-like immunoreactivity occurs in the fly’s vi- (MOP-36453), NIH (EY-03592) and NSERC (A- sual system, corresponding to the strong reduction in 0000065). I.A.M. is also supported by the Killam Trust the content of this amine in eyeless sine oculis. Thus most histamine is contained in the compound eye’sphotoreceptors and their synaptic terminals. There isalso an action of eyelessness on the interneurons of the References
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