Quantitative Analysis by Reverse Phase High Performance Liquid Chromatography of 5-Hydroxytryptamine in the Central Nervous System of the Red Swamp Crayfish, Procambarus clarkii

7 Pages·1992·3.1 MB·English

by  G K Kulkarni

Reference: Biol Bull 182: 341-347. (June. 1992)
Quantitative Analysis by Reverse Phase High
Performance Liquid Chromatography of
5-Hydroxytryptamine in the Central Nervous System
of the Red Swamp Crayfish, Procambams clarkii
GUNDERAO K. KULKARNI AND MILTON FINGERMAN
Department ofEcology. Evolution, andOrganismal Biology,
Tulane University, New Orleans, Louisiana 70118
Abstract. The concentrations of 5-hydroxytryptamine Procambamsclarkii, contains, in the laminaganglionaris
(5-HT) in central nervous organs ofthe red swamp cray- ofits eyestalks, a set ofaxons with 5-HT-like immuno-
fish, Procambams clarkii, were determined by reverse reactivity. These investigatorsalso found that the respon-
phase high performance liquid Chromatography (RP- siveness ofthe retinal photoreceptors ofthis crayfish to
HPLC) with electrochemical detection. The quantity light is enhanced by exposure to 5-HT, both //; vivo and
ranged between 54 and 168 pg/mg wet weight oftissue. in vitro. With respect to othercrayfishes, 5-HT-containing
Theamount is highest in thebrain, followed in decreasing neurons have been reported in the optic lobes and
order by the thoracic ganglia, subesophageal ganglion, proto-, deuto-, and tritocerebral regions of the brain of
eyestalks, and abdominal nerve cord. Significant increases Pacifastacusleniusculiis(Myhrbergetal.. 1979; Elofsson,
in thelevelsof5-HTin theeyestalks, brain, subesophageal 1983), Orconectes virilis (Sandeman and Sandeman,
ganglion, and thoracic ganglia occurred in crayfish ex- 1987), and Cherax destructor(Sandeman et al.. 1988).
posed forthree days to continuous light on a white back- Kulkarni et al. (1991) have shown that 5-HTstimulates
ground, whereasthe 5-HT levels in thesetissuesdecreased oocyte maturation in Procambams clarkii. This obser-
incrayfish kept in darkness. Electrical stimulation ofcen- vation and the earlierstudy ofArechigaetal. (1990) have
tral nervousorgansin vitroproducedsignificantdecreases led us to determine, for the first time, the quantity of5-
in the levels of5-HT. Fenfluramine (5-HT releaser), 5,6- HT in the central nervous system ofthis crayfish. In ad-
DHT
(5-HT neurotoxin), and reserpine (5-HT depletor) dition, we have determined the effects, on the 5-HT con-
induced significant decreases in the 5-HT levels in the centration in components ofthe central nervous system
portions ofthe central nervous system tested. of (1) continuous exposure to light or darkness, (2) in
vitroelectrical stimulation, and(3)pharmacological agents
Introduction known to affect 5-HT levels in vertebrates.
The biogenic amines, norepinephrine. dopamine, his-
tamine. octopamine, 5-hydroxytryptamine (5-HT), and Materials and Methods
gamma aminobutyricacid, function asneurotransmitters
Animals
in various animals (Werman, 1966; Gerschenfeld, 1973;
Krnjevic, 1974; Fingerman, 1985), and have been found Red swamp crayfish, Procambams clarkii, were pur-
in crustacean central nervous organs (Beltz and Kravitz, chased from a local seafood dealerand maintained in the
1983; Elofsson, 1983: Laxmyr, 1984; Fingerman, 1985; 24C
Sandeman el al. 1988). Arechiga el al. (1990) showed Tlahbeoyratwoerryeatacclimatiinzeadretcoirctuhleatilnagbofrraetsohrwyatceorndsiysttieomn.s
thatthe species used in thisstudy, the red swampcrayfish (12:12 L:D) for at least two days before being used in an
experiment. Medium sized (carapace length 40-50 mm),
Received 21 October 1991:accepted 26 March 1992. intermolt (Stage C4, Reddy et al.. 1990) crayfish ofboth
341
342 G. K. KULKARNI AND M. FINGERMAN
sexeswereused. Thecrayfishwerefedcommercial crayfish 10%. Theelution reagent wasmethanesulphonicacid (40
food. Fiddler crabs, Uca piigilator. were obtained from mA/) and phosphoric acid (30 mM} in 17% methanol,
the GulfSpecimen Co., Panacea, Florida, and acclima- pH 2.5, and wasthoroughly degassed before use (Hansson
tized for three days to the laboratory conditions under and Rosengren. 1978; Elofsson el ai, 1982; Nassel and
12:12 L:D in a recirculating artificial seawater system. Laxmyr, 1983). The pressure applied was 1500 psi with
a flow rate of 1 ml/min. The detectorwasaglassy carbon
Tissuepreparation andhomogenizationfor 5-HT electrode, and the working potential was set at +0.75 V
del'emunations against the reference electrode. The 5-HT concentrations
were determined by comparing the peak height in the
In this section and the following two, we describe the
procedure for determining the 5-HT concentrations in ealnudtiaornepprroefsielentoefdtahsepsga/mmpgleofwwietth ttihsastueo.fOtnhee satdadnidtairodnsa,l
tmhaeinctoamipnoendeinntsthoefstthoeckcetnatnrkasl.nTehrevoeuysesstyaslktse,mborfaicnr,aysfuibs-h ctrhiete5r-ioHnT, poethaekrinthtahne selaumtpiloens.tiBmeef,orweaasnaulsyseids,toweidaedntdiefdy
esophageal ganglion, thoracic ganglia, and abdominal a small amount (10 /ug/ml) ofsynthetic 5-HT tothe sam-
anserpvoesscioblredionfco1l0d0VcraanyfHiasrhrweveerled'dsiscsreacytfeidshopuhtysaisolroagpiicdally gpleense.itIyndnuoectaosethdeiadddthietisoanmopfle5-pHeTaktosthhoewbiaonlyogiicnahlommao--
saline(van Harreveld, 1936).Thesecomponentswerethen terialwhen compared with thepeak ofthestandard. With
Mdistpreirbcuhtleodr,ictenacpiedratnudbe,soinnictautbeeds cionntfaouirnicnygcl1esmlofof300.4s this analytical system we could detect as little as 25 pg
of5-HT.
each with a sonicator (Biosonik-II. Bronwill Scientific)
equipped with narrow probe for small volumes. The ho-
Calibration curve
mogenates were centrifuged (10,000 X g) for 15 min at
4C. The pH oftheMsupernatant, afterdecanting, wasad- The calibration curvewas prepared as follMows. To nine
justedto 6.0with 2 potassium carbonate, and the mix- clean glasstubes, each containing2 ml of0.4 perchloric
ture was again centrifuged for 10 min; the supernatants acid, wasadded a known amount of5-HTcreatinine suMl-
were used forfurtherpurification. Thewet weight ofeach fate monohydrate (0-51.2 ng/ml free base) and 10~5
tissue was recorded. The averages for the ten determi- 3,4-dihydroxybenzylamine hydrobromide (DHBA) (in-
nations of the 5-HT contents of each nervous system ternal standard). The samples were loaded onto the ion
component were then calculated. exchange column (Amberlite IRP-64) and treated as
above. Samples ( 100 /^l) were collected and 25 /ul ofthe
Purification and analysis of the sample eluatewasinjected ontothe RP-HPLCcolumn. Thepeak
The supernatantswere filtered and purified on a weakly choeingchetnstrwaetrieonroefco5r-dHeTd farneedbfaistet.edThinearegternatpihonagtaiimnes,twitthhe
ascoindacnadtiRoonseemxncgmhreann.ge1r9c78o)l.uTmnh.eAcmobleurmlnitweasIRgPla-s6s4,H3V(0Hamnsm- 51-mHlT/mainndf3l.o8wmriantefaonrdDaHtB1A500(Fipgs.i.1w).as 8.8-8.9 min for
long with 5 i.d.. equipped with a Millipore 0.45
nm filter (Nihon Millipore Kogyokk) at the bottom and
filled with 300 /ul ofthe resin. The supernatant from each Experimentalprotocols
1 ml extract was divided into two 500 ^1 aliquots, and The 5-HT content in the central nervous organs ofthe
each aliquot wasloaded separately onto the resin ata rate crayfish were initially determined using specimens that
of 50 ^1/min. The 5-HT adsorbed onto the resin from had been exposed for 2 days to 12:12: L:D. In addition,
each aliquot was eluted with 500 n\ of 1.2 A' HC1. The 20 equal-sized crayfish were held continuously foran ad-
two eluted fractions were combined for fasteranalysis of ditional three days either under a fluorescent light (450-
only one sample rather than two, and we obtained high 500 lux) or in darkness. After these three days, all ofthe
recovery which was always in the range of80-82%. The central nervous organs from ten crayfish were dissected
data presented havebeen corrected to reflect the recovery out for5-HTdeterminations. In addition, thecentral ner-
percentage. Theremainingsampleswere storedat 70C vous organs were removed from the rest ofthese crayfish
(Hansson and Rosengren. 1978; Elofsson. ct ai. 1982). and homogenized in 2 ml of crab physiological saline
A Waters RP-HPLC unit. Model 501, fittedwitha U6K (Cooke ct ai. 1977). pH 7.4. These extracts were then
universal LC injectorand a 3.9 X 150 mm 5 ^m silica C- centrifuged ( 10,000 X g) at 4C. and the supernates were
18 steel column with a small guard column, coupled to bioassayed for red pigment-dispersing activity in eye-
mm
a Waters electrochemical detector (Model 460) was used stalkless fiddler crabs, L'cu piigilator. of 10-15 car-
forthequantitative analysis of5-HT in the samples. Five apace width. The pigment in the erythrophores ofthese
aliquots (25 /ul) ofeach sample were run. and the results eyestalkless crabs was initially maximally concentrated.
wereaveraged. The variation amongsampleswas lessthan The erythrophores were staged according to the method
5-HYDROXYTRYPTAMINE IN P. CLARKII 343
and varying voltage (10. 15. 20. and 25 V). Stimulation
wasdelivered by a stimulator(Model S44)with a stimulus
isolation unit (Model SIU 5A; both from the Grass In-
strument Co.) Aftera stimulation bout of2 min, the ner-
M
vous tissues were homogenized in 0.4 perchloric acid
and processed for5-HTdetermination asdescribed above.
A total of75 crayfish were used, divided equally among
one group of unstimulated controls and four groups of
voltage stimulated preparations.
Experiments on the effects ofpharmacological agents,
such as fenfluramine (5-HT releaser, Consolo et a/..
02468 02468 1979), fluoxetine (5-HT potentiator, Wong et al.. 1975),
10 10 5,6-dihydroxytryptamine (5,6-DHT) (5-HT neurotoxin,
MINUTES Baumgarten et al., 1982), and reserpine (5-HT depletor,
Figure 1. Chromatogramsof(A)brainsupernatantofPracambarus Myhrbergeta/., 1979: Elofsson et al., 1982), on the levels
clarkiiand(B)aqueousstandardcontaining5-HTandDHBA.Column: of 5-HT in the central nervous tissues of Procambarus
5^m silicaC-18steel. Mobilephase: methanesulphonicacidandphos- clarkii, were performed according to the procedure of
phoric acid in methanol. Detector: glassy carbon electrode at +0.75 V Myhrbergetal. (1979).Crayfishweredividedinto5groups
potential.Retentiontimefor5-HT8.8-8.9minandforDHBA 3.8min. of 15 each. The first group received physiological saline
aloneand served asthecontrol. Thecrayfish in thesecond
through fifth groups were administered various concen-
of Hogben and Slome (1931) wherein stage 1 indicates trations ( 10-25 /ug/g body weight) ofeither fenfluramine.
maximal pigment concentration, stage 5 maximal pig- fluoxetine. 5.6-DHT, or reserpine. All injections were
ment dispersion, and stages 2, 3, and 4 the intermediate given once in a dose of50 ^1, and the crayfish were sac-
conditions. The Hogben and Slome stages for the exper- rificed after 2 h and their nervous tissues removed and
imental and control animals were then used to calculate processed for 5-HT analysis as described earlier.
Standard Integrated Responses (SIR) ofthe erythrophores Fenfluramine hydrochloride, 5.6-DHT. reserpine. and
ofUcapugilatortothe nervoustissue extractsofProcam- Amberlite IRP-64 werepurchased from Sigma. Fluoxetine
barus clarkii according to the method of Fingerman el hydrochloride wasagift from Lilly Research Laboratories,
al. (1967). Briefly, when pigment dispersion occurs, the whereas the 5-HT creatinine sulfate monohydrate and
sum of the Hogben and Slome stages recorded for the DHBA were purchased from Aldrich. All drugs were dis-
duration ofthe experiment for the control group is sub- solved in isosmotic crayfish physiological saline.
stracted from the corresponding sum forthe experimental The data obtained from these experiments were ana-
group. The difference is the SIR. The SIR integrates the lyzedstatistically bycalculatingthestandard errorforeach
amplitude, which is based on the observed Hogben and ofthe means (SEM).
Slome stages, and duration of the response of the ery-
throphores. A dose of50 n\ containing the tissue extract Results
equivalent to either one eyestalk. brain, subesophageal,
or thoracic ganglion was injected into each crab. The measurements quantifying 5-HT in the central
For experiments involving in vitro electrical stimula- nervousorgans, eyestalks, brain, subesophageal ganglion.
tion, the entire optic tract, including the major ganglia thoracic ganglia, and abdominal nerve cord of crayfish
and sinus gland, was removed from the eyestalk and maintained under laboratory conditions (12:12 L:D;
maintained in 50 ^1 physiological saline. The remaining 24nC) in recirculating freshwater for two days are sum-
nervoustissue from thebrain totheend ofthoracic nerve marized in Figure 2. The 5-HTconcentration washighest
cord was also dissected out intact and carefully placed in in the brain (168 pg/mg) and lowest in the abdominal
100 /jl of physiological saline. Electrical stimulation of nerve cord (54 pg/mg). with the eyestalk, subesophageal
the isolated tissue was performed as described in detail ganglion, and thoracic ganglia having intermediate con-
by Quackenbush and Fingerman ( 1984). Briefly, the eye- centrations. Because the concentration of 5-HT in the
stalk tissue was held in place with a suction electrode at- abdominal nerve cord is small relative to the rest ofthe
tachedtothestump ofthe optic nerve, whereasthecentral central nervous organs, only eyestalks. brains, subesoph-
nervetract, from the brain to theend ofthethoracic nerve ageal ganglia, and thoracic ganglia were used in the rest
cord, was held in place by a suction electrode attached at ofthe experiments.
thebrain end. The stimulation given via the suction elec- The pigment in the erythrophores ofthe crayfish (P.
trodewas5 pps. 4msdelay, with pulsesof40 msduration clarkii)that were illuminated whileonawhitebackground
344 G. K. KULKARNI AND M. F1NGERMAN
fish. Interestingly, thepercentagedecreasein 5-HTcontent
decreased progressively in the tissues along the central
170 nervous chain from the brain to the thoracic ganglia.
Of the concentrations tested, the smallest concentra-
tions that produced significant effects on the 5-HT level
1 50 after 2 h were 15 ^g/g body weight of fenfluramine, 10
c? AJg/g body weight of5,6-DHT. and 15 yug/g body weight
ofreserpine. Fenfluramine induced a significant decrease
130 of5-HT from all ofthe nervous tissues (Fig. 5). The de-
crease was maximum in the eyestalks and least in the
110 thoracic ganglia. None ofthe concentrations (10-25 ^g/
gbodyweight)offluoxetineproducedanysignificanteffect
on the 5-HTconcentration inanyofthetissues. 5,6-DHT
90 and reserpine produced significant decreases in the 5-HT
concentration ofall the nervous tissues tested. The max-
imum decrease was produced in the eyestalks, whereas
70 the minimum decrease occurred in the thoracic ganglia.
Discussion
50 Histochemical studies by means of fluorescence mi-
ES BR SG ThG ANC
croscopy have revealedthe presenceofyellow-fluorescing
TISSUE
Figure 2. 5-HTconcentration in pg/mgoftissue,assayed by HPLC
andelectrochemical detection, in eyestalks(ES), brain (BR), subesoph-
agealganglion (SG). thoracicganglia(ThG). andabdominal nervecord
(ANC)ofProcambarusclarkii. ErrorbarsareSEM forten separateex-
tractsoftissue pooled from ten animalseach.
wasconcentrated, whereasthe red pigment ofthecrayfish
kept in darknesswasdispersed. The 5-HT concentrations
in the eyestalks, brain, subesophageal ganglion, and tho-
racic ganglia ofcrayfish held under continuous light for
three days on a white background increased significantly
(Fig. 3). The corresponding red pigment-dispersing SIR
values evoked in the fiddler crabs by these extracts also
increased. In contrast, both the 5-HT levels and the SIR
values in the eyestalks, brains, subesophageal ganglia,
and thoracic ganglia ofcrayfish held in darkness showed
significant decreases when compared to the controls
(Fig. 3).
In the experiments in which eyestalk, neural ganglia,
and the central nerve tract (from brain to the end ofthe
thoracic nerve cord)wereelectrically stimulated with var-
ious voltages (10. 15, 20, and 25 V). 25 V was found to
be most effective. The data in Figure 4 are for tissues
stimulated with 25 V. The stimulation produced signifi-
cant decreases in the concentration of 5-HT in all the
tissues, with the maximum decrease occurring in the
eyestalks( 37.4^ )andthe minimum in thethoracicgan-
glia (22.8%). Furthermore, when extracts ofthe electri-
cally stimulated tissues were bioassayed for red pigment-
dispersingactivity in crabs, it was found that the SIR val-
ues evoked by these extracts were significantly decreased
incomparison tothetissueextractsfrom thecontrolcray-
5-HYDROXYTRYPTAMINE IN P. CLARK/I 345
170
346 G. K. KULKARNI AND M. FINGERMAN
awhite background notonlycontained higher 5-HTlevels persion than did the tissues ofthe unstimulated controls.
than the tissues ofthe control crayfish, but also evoked In theirstudies, both Quackenbush and Fingerman (1984)
higher red pigment-dispersing SIR values, observations and Kulkarni and Fingerman (1991) bioassayed only the
that are consistent with a pigment-dispersing hormone bathing fluid and not the actual stimulated tissue. In the
releasing role of5-HT. Presumably, because the red pig- present study, the stimulated tissues were extracted and
ment was concentrated, neither 5-HT nor pigment-dis- bioassayed forred pigment-dispersing activity by injecting
persing hormone was being used, thereby accounting for the extracts into eyestalkless crabs, Vcapugilator.
the increased levelsofboth substances. Earlier, Fingerman Fenfluramine (5-HT releaser), 5,6-DHT (5-HT neu-
et al. (1964) reported the presence oferythrophorotropic rotoxin), and reserpine (5-HT depletor) decreased the
hormones in the eyestalks and brain of juveniles and amount of5-HT in the central nervous system, although
adultsofPwcambarmc/arkii. They observed thatthe in- their modes ofaction are different (Fig. 5). However, the
jection of an extract containing one-third of an organ 5-HT potentiator fluoxetine had no appreciable effect on
complement per dose significantly evoked pigment mi- the 5-HTconcentration. These findingsareconsistentwith
gration ineyestalklessanimals. Morerecently, McCallum earlier 5-HTdepletion studies in which reserpine wasused
et al. (1988, 1989)confirmedthepreviousfindingsofFin- with crustaceans. Myhrberg el ul. (1979), using the his-
german el al. (1964) by isolating and sequencing the pig- tochemical fluorescence method ofFalckand Hillarp, and
ment-dispersing hormone (PDH). which is an octadeca- Elofsson et al. (1982). using HPLC, both found that re-
peptide, from the eyestalks ofProcambams clarkii. serpine decreases the 5-HT content ofnervous tissues in
Rao and Fingerman (1975) later reported that 5-HT, thecrayfish, Pacifastacusleniiisenhis. Likewise, the 5-HT
when injected into the dwarfcrayfish Camharellus s/ni- content of the brain and eyestalks of Vca pugilator de-
feldti, dispersed the red pigment in the erythrophores, as creased after injection of reserpine and 5,6-DHT (Fin-
in the fiddlercrab, butwas ineffective when tested in vitro german et al.. 1974).
on isolated chromatophore-bearing pieces ofthe crayfish
carapace. In crayfish on a white background with their Acknowledgments
redpigmentconcentrated, 5-HTturnoverwouldpresum-
ably have decreased because, with the red pigment con- This research was supported by Grant No. 1-1435-88
centrated, 5-HT would not be used to stimulate release from BARD. The United States-Israel Binational Agri-
ofred pigment-dispersing hormone, soa rise in the intra- cultural Research & Development Fund. We thank Mr.
neuronal concentration of this neurotransmitter would Chayan Chakraborti for his technical assistance.
occur. On the other hand, because darkness fosters red
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