Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 95-100 (1989) Kugler Publications, Amsterdam/Berkeley

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder, Pseudopleuronectes americanus Walbaum, associated with gonadal recrudescence Laurence W. Crim, Donna M. Evans and Kay Moreland Marine Sciences Research Laboratory, Memorial University of Newfoundland, St. John's, NFLD. AIC5S7, Canada Keywords: norepinephrine, dopamine, hypothalamus, teleost fish, reproduction, neurotransmitters

Abstract High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).

Introduction Many studies attest to the presence of monoamines in nervous tissue of vertebrate and invertebrate animals (Parent 1984). A large number of microscopical studies of fish aminergic neuronal systems have used formaldehyde-induced histochemical fluorescence to visualize neurotransmitter pathways, e.g. salmonids (Terlou et al. 1978) and a sculpin, Myoxocephalusscorpius (Watson 1980); more recently, a number of papers report the results of using immunohistochemical localization methods for determining the CNS distribution of catecholamines, indolamines and/or key enzymes for synthesis for these compounds, e.g. tyrosine hydroxylase, in the brains of goldfish (Yoshida et al. 1983; Kah and Chambolle 1983; Kah et al. 1984; Karasawa et al. 1984; Nagatsu et al. 1984 and Hornby et al. 1987), three-spined sticklebacks (Ekstrom and van

Veen 1984; Ekstrom et al. 1986), catfish (Corio et al. 1985), platyfish (Halpern-Sebold et al. 1985) and a cartilaginous fish, the sandy ray (Meredith and Smeets 1987). One particularly prominent feature of nonmammalian brain monoaminergic systems is the paraventricular organ (PVO), which consists of clusters of cell bodies (CSF-contracting cells) within the hypothalamus with axonal projections leading to the median eminence or directly to the pituitary in the case of the teleost fishes. The PVO-hypophysical tract, according to Parent (1984) is thought to be involved in regulation of various neuroendocrine events e.g. ovulation. Watson (1980) described green and yellow types of fluorescence located in PVO monoaminergic neurones in Myoxocephalus scorpius. It was suggested that the green fluorescence represents catechol substances while the indolealkylamines are responsible for the fast-

96 fading yellow type of fluorescent material. Specific quantitative analyses for the catecholamine (CA) content of teleost brain tissues has been accomplished by means of spectrofluorescent techniques in cod brain (Johansson and Henning 1981), eel brain (Le Bras 1979), and goldfish brain (Sauerbier and Meyer 1977); similar methodology was used to measure the serotonin content in the brain of Fundulus grandis (Fingerman 1976) and the hypothalamus of the rainbow trout (Olcese et al. 1981). Le Bras (1984) used radioenzymatic methodology to determine seasonal/circadian variations of catecholamine levels in the brain of the eel. Sloley et al. (1986) demonstrated the effectiveness of HPLC-EC for measurement of monoamine levels in the rainbow trout brain. The brain of the flagfish, Jordanellafloridae, contains HPLC-EC detectable levels of norepinephrine, dopamine and serotonin (Holdway et al. 1988) and these workers have also found elevations in brain serotonin in sexually active males compared with the resting male flagfish. The aims of our current studies included the following: (1) to examine the convenience, sensitivity and selectivity of HPLC-EC for estimation of biogenic amine substances in fish brain tissues (2) to investigate brain changes in monoamine levels in winter flounder during the period of seasonal reproductive development (3) to compare the biogenic amine profiles in the fish hypothalamus compared to a mammalian hypothalamus e.g. the laboratory rat.

Materials and methods Tissue preparationand chromatography The hypothalamus, a block of tissue without the pituitary containing the medial and lateral hypothalamic lobes (cutting began immediately behind the optic chiasma, tissue depth approximately 3 mm above the sacous vasculosus) was obtained from individual flounder. Since CA levels in the fish brain are known to vary with the time of day (Le Bras 1984), flounder brain sample collections (N= 513) were confined to 12:00 p.m. + 1 h. Both the hypothalamic tissues and whole brains minus the

hypothalamus were quickly removed from spinal sectioned fish and immediately frozen on dry ice prior to storage at -80°C until HPLC-EC analysis according to the methods of Mefford (1981). In brief, frozen tissue samples were extracted in the presence of 200 ng DHBA standard by sonication in 0.2M perchloric acid. Purification of the neuroamines was accomplished by selective adsorption of the CAs on 10 mg acid-washed alumina. Following Tris buffer washing of alumina, CAs were recovered with 0.2M perchloric acid and injected onto the HPLC-EC system for detection. The isocratic HPLC-EC system was composed of a Waters M-45 solvent delivery pump, a C-8 reverse phase precolumn, a 15 cm C-8 reverse phase column and a Bioanalytical Systems LC-4A amperometric detector equipped with glassy carbon electrode set at 0.6V vs. Ag/AgCl. The solvent system mobile phase for CA analysis was made of 150 mM monochloroacetic acid, 117 mM sodium hydroxide, 2 mM EDTA and 0.13 mM sodium -pentane sulfonate, pH 3.0 with a flowrate of 1.0 ml/min. Flounder brain neurotransmitter substances were identified by coelution of sample peaks with standard catecholamines and by coelution of sample peaks following the overloading of flounder brain extracts with standards. Monoamine substances can also be analyzed in perchloric acid brain extracts without the alumina purification step, by directly injecting the perchloric acid extracts of the vertebrate brain onto the HPLC-EC (conditions II). We used a Waters HPLC-EC system consisting of a M-510 solvent delivery pump, the Wisp 710A automatic injector system and the 5-um RCM 100 radial-pak column protected by a Guard-Pak precolumn and the LC-4A amperometric detector mentioned above. The solvent system mobile phase, pH 4.7, for direct injections was composed of 0.1 M sodium acetate, 0.02 M citric acid, 0.15 mM EDTA, 0.43 mM octyl sodium sulfate and 0.3-7.0% acetonitrile and the flowrate was maintained at 1.0 ml/min.

Results A study of brain catecholamine fluctuations asso-

97 Fn w z

K

I;12

I

to10

E

C 4 0

.8

2

z

,I I /A

,

C

6

"NE

z L

4 a -1.0

…. 4

AUG

SEP

OCT

NOV

DEC

-0.5 2 DA

10

AUG E 8

-

~

-10

I

4

4

I/`

1I - -,

U

L--- -- .. i

0.5 O

2 2

I'T

-05 2

IA

I\A 't,

o 0

I

AUG 0

DEC

NE

4 C

4 0

NOV

C -I.

~,.I' ~ ~ ~~~~~~ I

OCT

LIIU

, '

6

SEP

i AUG

~ SEP

I OCT

I NOV

I

SEP

I

OCT

i

NOV

i

DEC

I DEC

Fig. 1. Female brain and hypothalamic CA concentrations during the months of August-December, the normal period of gonadal recrudescence for the winter flounder. Values are X ± SEM. %0GSI= Gonadosomatic index (gonad weight/body weight x 100).

ciated with the period of seasonal reproductive development in male and female winter flounder revealed that, in general, the concentrations of NE and DA are higher in the flounder hypothalamus compared with extracts of the whole brain (Figs. 1 and

Fig. 2. Male brain and hypothalamic CA concentrations during the months of August-December, the normal period of gonadal recrudescence for the winter flounder. Values are X + SEM. %GSI = Gonadosomatic index (gonad weight/body weight x 100).

2). By collecting female brain samples throughout the period of gonadal recrudescence (AugustDecember), it was noted that brain and hypothalamic NE concentrations reached their highest levels (p < 0.05) in October (samples 4 and 5) relative to the lower monoamine values before and after

98 (p < 0.05) in late October compared with lower NE values e.g. August and September (brain); September (hypothalamus). Although relatively small changes in brain DA concentrations were observed, there was a significant seasonal increase in brain dopamine (late October value significantly higher than other times). Figure 3 shows representative diagrams of typical HPLC-EC chromatograms for the pure monamine standards, norepinephrine (NE), dopamine (DA) and serotonin (5-HT), compared with monoamine profiles for hypothalamic extracts of three species of fish and the monoamine profile obtained with and extract of the hypothalamus of the rat. Under our chromatographic conditions (II) stated above for direct injections of perchloric acid brain extracts, four hypothalamic electroactive compounds were readily separated and detectable in all fish species tested and the rat hypothalamus as well including peak (1), (norepinephrine), peak (2), (an unidentified compound), peak (3), (dopamine) and peak (4), (serotonin).

(I) (2) (e)

() (4)

i~~~ Ui,(3

, Ax

_

(d)

_

w C) z

t

0

0. o

w i.I

(c)j

U 0 mw b. a:

J

(b)

irllivinn

The present winter flounder study was designed to estimate seasonal variations in CAs during the period of gonadal recrudescence. Significant variations

(a)

in CA cntpnt nf hnth thp hrain andl the hvnnthala...

0

8

16

24

32

40

TIME (min) Fig. 3. Resketched chromatograms conducted at 0.5 nanoamperes of (a) standard catecholamine (2.0, 1.64 aand 1.97 ng of NE, DA and 5-HT, respectively, and perchloric a icid extracts of the hypothalamus of (b) rainbow trout, 1.24 mg (c) winter flounder, 1.63 (d) cod, 2.54 mg and (e) the labor atory rat, 2.23 mg fresh tissue.

this particular time of the year; althoug]hin August, hypothalamic dopamine levels were ssignificantly higher (p < 0.05) compared with subsequent seasonal values for dopamine, in geineral, compared with NE, DA levels remained stallble throughout the study period. In males the brain and hypothalamic levels of NE were signific antly higher

mlc 111U

·rrn Wle;

-1

.

... '

-.

-_

...

n tm ··n mFalf~ ahe>^ro/or UU;SCI VtJU 111 111iai[; dllUIililili

th;^ llsrc xl WILLI LII1

highest CA levels observed during the period of rapid gonadal development in October. In principal, these data agree with the flagfish studies reported by Holdway et al. (1988) showing that

brain dopamine and serotonin concentrations are higher in sexually mature male flagfish compared with lower brain neurotransmitter levels found in resting males. Clearly, since monoaminergic substances in flounder brain tissues both rise and fall during the period of gonadal recrudescence, we find no simple direct correlation between progressive development of the gonads (rising GSI) and increases or decreases taking place in whole brain/ hypothalamic CA neurotransmitter content. Our HPLC-EC studies of the brain monoaminergic substances indicate that this technology is con-

99 venient for yielding rapid and sensitive determinations of the brain levels of neurotransmitter compounds by direct injections of perchloric acid extracts of teleost brain tissues. By comparing hypothalamic neurotransmitter profiles in three different species of fish with the hypothalamic profile of the rat, one may predict the presence of similar CNS monoamine profiles in teleosts and mammals. Sloley et al. (1986) described the presence of NE, DA and 5-HT among the monoamines they found in the brain of the rainbow trout and we have confirmed that these substances are represented in chromatograms of the hypothalamus from three species of fish (two marine teleost species) and the rat. In addition to the above mentioned compounds, we also consistently observed a second peak (peak 2), eluting between NE and DA, which coelutes with both homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), metabolites of dopamine and serotonin, respectively. Epinephrine (E), which has a similar retention time compared with peak (2), consistently runs just ahead of peak 2; thus, the unidentified peak (2) compound may not be a catechol but, more likely, is one of the metabolites (HVA or 5-HIAA) mentioned above. The disadvantages of alumina CA purification of perchloric acid extracts of the teleost brain, e.g. inconvenience and a lack of retention of indoleamine neurotransmitter compounds necessitating double injections to acquire complete brain neurotransmitter profiles, leads us to conclude the direct HPLCEC analysis of perchloric acid brain extracts of the teleost brain is the preferred analytical method. Our future studies of the role of brain neurotransmitters in teleost seasonal reproductive cycles will be intended to examine biogenic amine concentrations in specific brain nuclei during the period of gonadal recrudescence in winter flounder.

Acknowledgements This work supported by NSERC Grant A9729 to L.W. Crim. Ocean Sciences Centre Contribution Number 28.

References cited Corio, M., Peute, J., Steinbusch, H.W.M. and Doerr-Schott, J. 1985. Immunoreactive dopaminergic neurons in the hypothalamus of a fish, Clariaslazera and an amphibian, Xenopus laevis. Neurosci. Lett. (suppl.) 22: S543. Ekstrom, P. and van Veen, Th. 1984. Distribution of 5-hydroxytryptamine (serotonin) in the brain of the teleost Gasterosteus aculeatus L. J. Comp. Neurology 226: 307-320. Ekstrom, P., Reschke, M., Steinbusch, H. and van Veen, Th. 1986. Distribution of noradrenaline in the brain of the teleost Gasterosteus aculeatus L.: An immunohistochemical analysis. J. Comp. Neurology 254: 297-313. Fingerman, S.W. 1976. Circadian rhythms of brain 5-hydroxytryptamine and swimming activity in the teleost, Fundulus grandis. Comp. Biochem. Physiol. 54C: 49-53. Halpern-Sebold, L.R., Margolis-Kazan, H., Schreibman, M.P. and Joh, T.H. 1985. Immunoreactive tyrosine hydroxylase in the brain and pituitary gland of the platyfish. Proc. Soc. Exp. Biol. Med. 178: 486-489. Holdway, D.A., Sloley, B.D., Munkittrick, K.R., Downer, R.G.H. and Dixon, D.G. 1988. Effect of gender and reproductive status on brain catecholamine and indoleamine levels of flagfish (Jordanella floridae). Fish Physiol. Biochem. 5: 153-158. Hornby, P.J., Piekut, D.T. and Demski, L.S. 1987. Localization of immunoreactive tyrosine hydroxylase in the goldfish brain. J. Comp. Neurology 261: 1-14. Johansson, P. and Henning, M. 1981. The effect of L-DOPA on blood pressure and catecholamine content in the brain and peripheral organs in the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. 70C: 249-253. Le Bras, Y.M. 1979. Circadian rhythm in brain catecholamine concentrations in the teleost, Anguilla anguilla L. Comp. Biochem. Physiol. 62C: 115-117. Kah, 0. and Chambolle, P. 1983. Serotonin in the brain of the goldfish, Carassiusauratus: An immunocytochemical study. Cell Tiss. Res. 234: 319-333. Kah, O., Chambolle, P., Thibault, J. and Geffard, M. 1984. Existence of dopaminergic neurons in the preoptic region of the goldfish. Neurosci. Lett. 48: 293-298. Karasawa, N., Yoshida, M., Kawakami-Kondo, Y., Okumara, A., Sato, T. and Nagatsu, I. 1984. Immunohistochemical demonstration of "big monoaminergic neurons" of the goldfish hypothalamus. Biogenic Amines 1: 133-141. Le Bras, Y.M. 1984. Circadian variations of catecholamine levels in brain, heart and plasma in the eel, Anguilla anguilla L., at three different times of year. Gen. Comp. Endocrinol. 55: 472-479. Mefford, I.N. 1981. Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: Measurement of catecholamines, serotonin and metabolites in rat brain. J. Neurosci. Methods 3: 207-224. Meredith, G.E. and Smeets, W.J.A.J. 1987. Immunocytochemical analysis of the dopamine system in the forebrain and mid-

100 brain of Raja radiata:Evidence for a substantia nigra and ventral tegmental area in cartilaginous fish. J. Comp. Neurology 265: 530-548. Nagatsu, I., Karasawa, N., Kawakami, Y. and Yoshida, M. 1984. Studies on monamine-containing neurons by immunoenzyme-histocytochemistry and immunohistocytochemistry with special reference to goldfish brain. Acta Histochem. Cytochem. 17: 151-160. Parent, A. 1984. Functional anatomy and evolution of monoaminergic systems. Am. Zool. 24: 783-790. Olcese, J., Figueroa, H., Hall, T.R., Yurgens, P., Kiebzak, G., Meyer, R. and de Vlaming, V. 1981. Effects of para-chlorophenylalanine, a brain serotonin depletor, on pituitary cyclic AMP levels in the rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 43: 462-466. Sauerbier, I. and Meyer, W. 1977. Circadian rhythms in catecholamine concentrations in organs of the common goldfish (Carassiusauratus L.) Comp. Biochem. Physiol. 57C: 117120.

Sloley, B.D., Hickie, B.E., Dixon, D.G., Downer, R.G.H. and Martin, R.J. 1986. The effects of sodium pentachlorophenate, diet and sampling procedure on amine and tryptophan concentrations in the brain of rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 28: 267-277. Terlou, M., Ekengren, B. and Hiemstra, K. 1978. Localization of monoamines in the forebrain of two salmonid species, with special reference to the hypothalamo-hypophysial system. Cell Tiss. Res. 190: 417-434. Watson, A.H.D. 1980. The distribution of aminergic neurones and their projections in the brain of the teleost, Myoxocephalus scorpius. Cell Tiss. Res. 208: 299-312. Yoshida, M., Nagatsu, I., Kawakami-Kondo, Y., Karasawa, N., Spatz, M. and Nagatsu, T. 1983. Monoaminergic neurons in the brain of goldfish as observed by immunohistochemical techniques. Experientia 39: 1171-1174.