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397

Effect of Salinity on Melanin-Concentrating Hormone Gene Expression in the Rainbow Trout

KAREN FRANCIS, MASAKAZU SUZUKI, ANDREW LEVY,a ANDBRIDGET I. BAKER

School of Biology and Biochemistry, University of Bath, Bath, United KingdomaDepartment of Medicine, Bristol Royal Infirmary, Bristol, United Kingdom

Melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced byhypothalamic neurons. This highly conserved peptide serves as a neurotransmitteror neuromodulator within the brain, but additionally in teleost fish it is a neurohypo-physial hormone, with effects on skin pigmentation. Observations in mammals indi-cate that MCH may modulate salt and water balance because in rats preproMCHgene expression is depressed by salt loading and dehydration,1,2 whereas in sheepicv injections of MCH, or its co-peptide NEI, enhance salt and water diuresis.3 Introut, MCH has previously been mapped to two locations in the hypothalamus.4 Theprincipal group of magnocellular neurons is located in the nucleus lateralis tuberis(NLT), with axons projecting to the neural lobe of the pituitary gland and into thebrain. A second, smaller group of parvocellular neurons lies above the lateral ven-tricular recess (LVR-MCH) with fibers projecting dorsally into the thalamus. Usingin situ hybridization, this study examines MCH gene expression in both loci in rain-bow trout (Oncorhynchus mykiss) exposed to increasing strengths of sea water.

MATERIALS AND METHODS

Rainbow trout in groups of 8–12 fish were kept in 270-L fiberglass tanks withrunning fresh water and good aeration, a temperature of 11°C and a photoperiod of16.5 hours light and 7.5 hours dark. Fish were fed once daily with approximately 1%body weight commercial trout pellets. In experimental tanks, the fresh water was re-placed at intervals by pumping in appropriate quantities of full-strength sea water(Ocean Life). The salinity was increased from 50% (day 2) to 80% (day 7) and fi-nally 100% (day 14). Six trout were sampled after 24 hours and/or 6 days in eachsea-water strength.

At autopsy, blood samples were taken for the determination of plasma osmoticpressure (freezing point depression) and cortisol concentration (RIA). Paraformal-dehyde-fixed brains were embedded in wax and 10-µm sections were incubatedovernight at 37°C with 35S-labeled oligonucleotide probe specific for the MCH2gene, using a previously established method.5 Hybridized sections were apposed toautoradiography film and the resulting signals quantified using a computer-linkedimage scanning system. The Mann-Whitney U test and Student’s t test were used forstatistical analysis.

398 ANNALS NEW YORK ACADEMY OF SCIENCES

RESULTS AND DISCUSSION

A 24-hour exposure to 50% sea water had no effect on MCH transcripts (FIG. 1)but after 24 hours in 80% sea water they were significantly increased in NLT (159%increase; p <0.01) but not LVR neurons. However, following transfer to 100% seawater, MCH mRNA was reduced in both the NLT (62% controls; p <0.001) andLVR (33% controls; p <0.001) neurons. These responses were transient because af-ter 6 days of exposure to each salinity the MCH message had returned to control val-ues, suggesting the operation of a feedback mechanism. Plasma osmotic pressuresand cortisol concentrations increased linearly with successive sea water changes, thesharpest rise occurring after 24 hours in 100% sea water.

In 50% sea water, the lack of response in MCH neurons was accompanied by lowplasma cortisol and osmotic pressure, indicating functional osmoregulation. In 80%sea water, both cortisol (12 ng ml−1) and osmotic pressure (345 mOsm kg−1) weresignificantly raised but were within a normal range for sea water-adapted salmonids.In this salinity, only the NLT MCH neurons were stimulated. In 100% sea water, thecortisol (28.5 ng ml−1) and osmotic pressure (383 mOsm kg−1) had increased to lev-els suggesting inadequate osmoregulatory ability, and this was accompanied by adramatic depression of MCH mRNA in both NLT and LVR-MCH neurons.

The biphasic response of the NLT MCH cells may reflect activation of the hypo-thalmo-pituitary-interrenal (HPI) stress axis because it has been shown that mildstress stimulates preproMCH synthesis, whereas more severe stress is inhibitory.6

Alternatively, the increase in osmotic pressure may have stimulated the NLT neu-rons, whose axons in the pituitary neural lobe may exert neuromodulatory effects onthe release of other neurohypophysial hormones, such as arginine vasotocin. Inas-much as exposure to 100% sea water was likely to have been stressful to the fish, the

FIGURE 1. MCH2 hybridization signal in trout brain following sea water challenge.* p <0.01 and ** p <0.001.

399FRANCIS et al.: SALINITY AND MCH GENE EXPRESSION

subsequent high levels of cortisol may have exerted negative effects on MCHmRNA synthesis that negated the earlier osmotic stimulus.

REFERENCES

1. FELLMANN D., RISOLD P.Y., BAHJAOUI M., COMPAGNONE J.L., CLAVEQUIN M.C.,CARDOT J., GOUGET A., LENYS D., & BUGNON C. 1993. Morphofunctional studieson the neurons producing melanin-concentrating hormone. Ann. NY Acad. Sci.680: 511–516.

2. PRESSE F. & NAHON J.-L. 1993. Differential regulation of melanin-concentratinghormone gene expression in distinct hypothalamic areas under osmotic stimulationin the rat. Neuroscience 55: 709–720.

3. PARKES D. 1996. Diuretic and natriuretic actions of melanin-concentrating hormonein conscious sheep. J. Neurendocrinol. 8: 57–63.

4. BAKER B.I., LEVY A., HALL L. & LIGHTMAN S.L. 1995. Cloning and expression ofmelanin-concentrating hormone genes in the rainbow trout brain. Mol. Neuroendo-crinol. 61: 67–76.

5. SUZUKI M., NARNAWARE Y.K., BAKER B.I., & LEVY A. 1995. Influence of environ-mental colour and diurnal phase on MCH gene expression in the trout. J. Neuroen-docrinol. 7: 319–328.

6. BAKER B.I. & BIRD D.J. 1992. The biosynthesis of melanin-concentrating hormonein trout kept under different conditions of background colour and stress as deter-mined by an in vitro method. J. Neuroendocrinol. 4: 673–679.