Defining characteristics of immersion carbon dioxide gas for successful euthanasia of neonatal and young broilers.

This study investigated how the carbon dioxide (CO2) concentration within a chamber affects the efficacy of CO2 euthanasia and how the efficacy of CO2 induction methods changes as birds age. In experiment 1, pairs of broiler chicks (n = 192; 0, 3, and 6 D of age) were immersed into a chamber prefilled with 70, 80, 90, or 100% CO2. For experiment 2, 3- and 6-day-old broiler chicks (n = 88) were immersed in pairs into 100% CO2 or exposed to CO2 gradual fill in a chamber with a displacement rate of 28% chamber volume per minute. Latency to performance of headshaking (HS) and gasping (GS) as potential indicators of distress, loss of posture indicative of insensibility, and the cessation of rhythmic breathing (CRB) and cessation of movement (COM) as the indicators of death were monitored (live focal sampling/video recordings). The duration and frequency of HS and GS were assessed. For both experiments, behavior data were analyzed for CO2 method and age (4 × 3 factorial). Age and CO2 concentration interacted for latency to CRB and COM, with longer latencies for 0-day-old chicks immersed into 70% CO2 than other concentrations and ages. CO2 concentration did not affect latency to HS, GS, or loss of posture but affected CRB and COM, with latencies longest for 70% and shortest for 90 and 100% CO2. Newly hatched chicks had a longer latency to CRB and COM and longer duration and frequency of distress behaviors than older chicks. At all ages, initiation of all behaviors occurred later with gradual fill compared to immersion. There was an increased duration and frequency of distress behaviors with gradual induction compared with immersion. Overall, immersion into 90 to 100% CO2 resulted in the shortest time to insensibility and death, with a decreased duration and frequency of distress behaviors. Chicks immersed into 70% CO2 had the longest duration of GS and time to death. Age affects the efficacy of CO2 euthanasia, with increasing age decreasing time to death and the duration and frequency of distress behaviors.

Evaluation of carbon dioxide induction methods for the euthanasia of day-old cull broiler chicks.

This study was conducted to evaluate the efficacy of 5 different CO2 euthanasia induction techniques for day-old cull chicks in minimizing distress and inducing a rapid loss of sensibility and death. Each induction treatment was characterized for concentration change over time, maximum concentration, and time to reach maximum. Sixteen chicks were euthanized with the gradual treatments to establish validity of treatment. Then, all 5 treatments were evaluated for effect on distress, insensibility, and death. Day-of-hatch cull chicks (n = 110) were euthanized in pairs by either immersion into 100% CO2 or gradual induction to 100% CO2 at displacement rates of 7, 14, 21, or 28% of chamber volume added per min (% vol/min). CO2 concentration was measured at chick level. Live focal observations and video recordings were used to assess latency to behavioral responses: head shaking (HS) and gasping (GS) as indicators of distress; loss of posture (LOP) as an indicator of insensibility; and cessation of rhythmic breathing (CRB) and movement (COM), indicating death. All behaviors occurred at the earliest with immersion compared to gradual treatments, and time between first signs of distress and LOP was shorter for immersion than gradual treatments. Gradual treatments showed a linear decrease in latency to HS, GS, and LOP as displacement rate increased. Latency to CRB decreased quadratically with increasing displacement rate, while COM decreased linearly. Within gradual treatments, HS and GS occurred at CO2 concentrations between 0.43 and 1.14%, LOP between 11.1 and 17.5%, while CRB and COM occurred between 61.8 and 78.4%. Overall, immersion induced distress, insensibility, and death significantly faster and with the shortest interval between distress and insensibility. For gradual treatment, insensibility and death occurred faster with increasing displacement rates. Behavioral signs of distress were observed with all treatments, and occurred at concentrations lower than those causing insensibility. In conclusion, immersion into 100% CO2 environment resulted in the shortest time of distress and fastest time to death compared to gradual displacement rates of any speed measured.

Differences in arginine vasotocin gene transcripts and cortisol secretion in trout with high or low endogenous melanin-concentrating hormone secretion.

Previous studies on trout suggest that melanin-concentrating hormone (MCH) acts at both hypothalamic and pituitary levels to restrain the release of adrenocorticotropic hormone and hence cortisol during stress. Using in situ hybridization, the present work examined whether high rates of MCH secretion were associated with changes in the synthesis of arginine vasotocin (AVT), one of the corticotropin secretogogues. It also examined whether high endogenous MCH secretion restrains cortisol secretion during intense as well as mild stress, and how exogenous MCH affects the rise in plasma cortisol following injection stress. Trout were reared in black- or white-coloured tanks for 1 year or more to achieve maximal differences in MCH secretion. Following a mild stress, cortisol secretion was greater in black-reared fish with low MCH secretion which is in line with previous findings but, following a more severe stress, plasma cortisol concentrations were similar in the two groups. Injection of MCH into black-adapted fish restrained the stress-induced rise in plasma cortisol concentration during the first hour but did not affect final cortisol values. In two separate experiments, AVT mRNA levels were significantly lower in the hypothalamus of black-reared fish. Possible explanations for this include a greater negative-feedback restraint by cortisol, which is likely to rise higher in black-adapted fish during the moderate, daily stresses of aquarium life; or the possibility that exposure to a white background may be psychologically stressful, stimulating AVT transcription. The possibility that MCH directly stimulates AVT transcription cannot be excluded but seems less likely. The results suggest that while MCH may restrain the release of hypothalamo-pituitary stress hormones under moderately stressful conditions, it does not restrain AVT synthesis.

The distribution of melanin-concentrating hormone in the lamprey brain.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

The effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone.

The adipose hormone, leptin, not only restrains appetite, but also influences energy expenditure. One such influence is to promote sexual maturation and fertility. The neuromodulatory circuits that mediate this effect are not well known but the present study suggests that one mediator could be melanin-concentrating hormone (MCH). We show that the long-form receptor (Ob-Rb) is expressed in the zona incerta of the rat and that administration of leptin (both 0.5 microg and 1.0 microg/side) into this area of ovariectomized, oestrogen-primed rats stimulated the release of luteinizing hormone (LH) within 1 h, the effect enduring for a further 1 h. Injections of leptin into the arcuate nucleus induced a smaller, transient rise in LH while injections into the paraventricular and ventromedial nuclei were without effect. MCH neurones are present in the zona incerta and administration of this hormone into the medial preoptic area (mPOA) stimulates LH release, therefore we investigated the possibility that MCH might mediate this effect of leptin. An injection of MCH antiserum into mPOA prevented the rise in LH normally induced by leptin injected into the zona incerta. In addition, melanocortin receptor antagonists ([D-Arg8]ACTH(4-10) and [Ala6]ACTH(4-10)), previously shown to inhibit the stimulatory effect of MCH on LH release, also inhibited the effect of leptin. We propose that one route by which leptin may promote reproductive activity is by enhancing MCH release from fibres within the mPOA. Speculative mechanisms for the action of MCH include the following possibilities: MCH may be acting on the specific MCH receptor which in turn interacts with a melanocortin or melanocortin-like receptor; MCH may bind directly to one of the melanocortin receptors; or melanocortin antagonists may interact with the MCH receptor.

The effects of acute and chronic stresses on vasotocin gene transcripts in the brain of the rainbow trout (Oncorhynchus mykiss).

Secretion of adrenocorticotropic hormone (ACTH) from the fish pituitary, which occurs in times of stress, is stimulated by several hypothalamic neuropeptides, one of which is arginine vasotocin (AVT). This study investigates whether gene expression for AVT is up-regulated during acute or chronic stress. Rainbow trout (Oncorhynchus mykiss) were subjected to one of two forms of acute stress-either 2 h confinement followed by 2 h recovery, or capture and transfer to low water for 2 min followed by 4 h recovery in their home tank before autopsy. In other experiments, these stresses were repeated daily for 5 or 6 days (chronic stress). Quantification of AVT transcript prevalence in the parvocellular and magnocellular neurones of the preoptic nucleus after in situ hybridization was used as a monitor of the AVT gene response to stress. The results showed that acute confinement, but apparently not brief low-water stress, significantly increased AVT transcript prevalence in a group of parvocellular perikarya. When applied repeatedly, both forms of stress caused habituation, such that the AVT hybridization signal remained at control or even lower levels despite elevated pro-opiomelanocortin transcripts in the corticotropes and raised plasma cortisol concentrations. The AVT hybridization signal in the magnocellular perikarya showed no significant response to either acute or chronic stress. The results support the idea that these parvocellular AVT neurones are involved in ACTH stimulation during acute stress, and that the system habituates to chronic stresses.

Melanin-concentrating hormone, melanocortin receptors and regulation of luteinizing hormone release.

Melanin-concentrating hormone (MCH) is a neuropeptide, identified by its ability to either mimic or antagonize the melanin-dispersing action of alpha-melanocyte stimulating hormone (alphaMSH) on skin melanophores. MCH and alphaMSH also have antagonistic actions in the brain affecting feeding behaviour, aggression, anxiety, arousal and reproductive function through the release of luteinizing hormone (LH). It is not clear, however, how they exert their opposite effects in the central nervous system (CNS). One possibility is that they act via a common receptor. In this study we have examined the effect of a number of MC receptor antagonists, with relative selectivity for the MC3, 4 and 5 subtypes, on the actions of MCH on LH release. We confirmed that bilateral administration of MCH (100 and 200 ng/side) into the medial preoptic area of oestrogen-primed (oestradiol benzoate 5 microgram) ovariectomized anaesthetized rats, stimulated the release of LH. This effect was blocked by the concomitant administration into the medial preoptic area of the MC4/5 antagonist ([D-Arg8]ACTH(4-10) and the MC3/5 antagonist ([Ala6]ACTH(4-10)-both at 500 ng/side-but not by the MC3/4 antagonist, SHU9119 (200 ng/side). Furthermore, the MC3 agonist [Nle3]-gamma2 MSH failed to affect LH release. These results indicate that the MC3 and MC4 receptors are not involved in mediating the action of MCH but are consistent with an action via the MC5 subtype. Preputial glands, which express MC5 receptors, were also stimulated by MCH which is in keeping with this idea. In HEK293 cells transfected with the MC5 receptor MCH increased the production of IP3. However, it was much less potent than alphaMSH and unlike alphaMSH, had no effect on the production of cAMP. MCH (10-10 to 10-5 M) also failed to displace I125NDP-MSH from cells transfected with MC5 receptors indicating that it was not acting as a competitive antagonist and its binding site was distinct from that of alphaMSH. Thus while MCH may function as an agonist at the MC5 receptor, its stimulation of LH release is more likely to be mediated via a specific MCH receptor that has common properties with the MC5 receptor.

The influence of gonadal steroids on pre-pro melanin-concentrating hormone mRNA in female rats.

Melanin-concentrating hormone (MCH) may have a regulatory role in the control of luteinizing hormone (LH) release. We have investigated if gonadal steroids induce changes in the expression of pre-pro MCH (ppMCH) that are associated with changes in the pattern of LH release. Using quantitative in-situ hybridization histochemistry we have determined the effect of administration of either oestradiol benzoate (5 microg/rat) or oestradiol benzoate followed 44 or 48 h later by progesterone (0.5 mg/rat) to ovariectomized rats on the expression of ppMCH in the medial and lateral zona incerta and the lateral hypothalamus. The prevalence of ppMCH transcripts in the intact female rat at 12.00 and 19.00 h on proestrus and the first day of dioestrus was also examined. Oestrogen reduced the intensity of hybridization signal for ppMCH mRNA and this was associated with both a decrease in the number of cells in which the message was detected in the medial zona incerta and a negative feedback effect on LH release in ovariectomized rats. Progesterone administration to oestradiol benzoate-primed rats did not alter the reduced expression in the medial zona incerta in spite of its positive feedback effect on LH release. We suggest that progesterone may act only on post-translational events. Expression in the MCH cell bodies of the lateral zona incerta were not affected but there was a transient decrease 4 h after progesterone treatment in the oestradiol benzoate-primed rats in expression in the lateral hypothalamus. No changes in ppMCH mRNA were seen in intact animals on proestrus or the first day of dioestrus indicating that gonadal steroids are not important in the modulation of ppMCH gene expression during the oestrous cycle. In other steroid-dependent physiological situations, however, oestrogen may influence the expression of ppMCH in a subpopulation of cell bodies in the medial zona incerta.

Rainbow trout (Oncorhynchus mykiss) urotensin-I: structural differences between urotensins-I and urocortins.

In bony fishes, both corticotropin-releasing factor (CRF) and urotensin-I play a role in the regulation of interrenal glucocorticoid release. The rainbow trout, Oncorhynchus mykiss, is a useful model for understanding the mechanisms of stress and the hypothalamo-pituitary-interrenal axis because of its phylogenetic position at the base of the euteleostei and its popularity as a food fish. Urotensin-I may act as a glucocorticoid releaser in a mechanism phylogenetically older than that of CRF. The structural and functional relationships of trout urotensin-I have been investigated. The transcript was cloned from a trout brain hypothalamic cDNA library. A single positive clone was isolated and sequenced. It possesses 3218 bases and has the longest 3' untranslated region of all urotensins-I and CRF transcripts found to date. In comparison to the other fish orthologues, it has the closest sequence identity to the mammalian urocortins. The transcript appears to be differentially processed in brain and urophysis as determined by Northern blot analysis and the presence of polyadenylation signals in the 3' untranslated region. Synthetic trout urotensin-I activated both human CRF-R1 and -R2 receptor-transfected CHO cells with a potency similar to that of white sucker (Catostomus commersoni) urotensin-I. Both fish neuropeptides possessed an order of magnitude less potency than human urocortin in CRF-R2 transfected cells.

Diurnal changes in the expression of genes encoding for arginine vasotocin and pituitary pro-opiomelanocortin in the rainbow trout (Oncorhynchus mykiss): correlation with changes in plasma hormones.

Using quantitative in-situ hybridization, this study monitored diurnal changes in the abundance of the gene transcripts of two corticotropin-releasing peptides, arginine vasotocin (AVT) and isotocin in hypothalamic neurones, and of pro-opiomelanocortin (POMC) mRNA in the pituitary of the rainbow trout (Oncorhynchus mykiss). A significant diurnal pattern of gene expression was only displayed in the hypothalamus by the parvocellular AVT neurones of the preoptic nucleus. Abundance of AVT mRNA in these neurones was low at lights on (06.00 h), increased during the morning to reach a plateau of peak values between 14.00 h and 22.00 h, and then declined during the dark phase. This pattern was the inverse of that shown by plasma cortisol values. Changes in AVT transcript abundance are also considered in terms of the reported diurnal change in circulating AVT concentration. Pituitary and hypothalamic AVT peptide content did not change. Transcripts of both POMC genes (POMC-A and POMC-B) were monitored in pituitary corticotropes and melanotropes. Only POMC-A mRNA was detected in corticotropes where it showed no diurnal change in abundance. Transcripts of both POMC genes were found in the melanotropes, although, judging from autoradiographic intensity, POMC-A mRNA predominated. Both genes showed diurnal differences in their transcription with POMC-A mRNA showing peak values at 10.00 h and a nadir at 02.00 h, while POMC-B mRNA showed an inverse pattern. The results indicate that the two POMC genes can be independently regulated.

Behavioral effects of neuropeptide E-I (NEI) in the female rat: interactions with alpha-MSH, MCH and dopamine.

The behavioral and neurochemical effects of NEI, and its interaction with alpha-MSH or MCH were investigated in the ventromedial nucleus (VMN) and medial preoptic area (MPOA) in female rats (bilateral administration, 100 ng in 0.5 microliter/side). NEI in the VMN (but not in the MPOA) stimulated exploratory behavior, increased anxiety and reduced dopamine and DOPAC release. The behavioral effects were antagonized by alpha-MSH. NEI stimulated female sexual receptivity in the MPOA. In the VMN, NEI did not have any effect on sexual activity, but partially antagonized the stimulatory effect of MCH. These results show that NEI in the hypothalamus participates in the regulation of behavior, possibly through dopaminergic mediation.

Responses of melanin-concentrating hormone mRNA to salt water challenge in the rainbow trout.

Melanin-concentrating hormone (MCH) is a structurally conserved neuropeptide, produced in the hypothalamus of all vertebrates where it probably serves as a central neurotransmitter/neuromodulator. In teleost fish it is also a neurohypophysial hormone with peripheral effects on skin colour but its central effects are less well understood. In mammals, MCH mRNA abundance changes in response to salt-loading or dehydration, suggesting an involvement in salt or water balance. The present study has used in situ hybridization to investigate the response of the MCH neurons in the rainbow trout (Oncorhynchus mykiss) to progressive changes in ambient salinity. In trout, MCH perikarya are found in two hypothalamic sites: predominantly in the nucleus lateralis tuberis (NLT) and, to a lesser extent, in neurons above the lateral ventricular recess (LVR). Immersion in 50% salt water (SW) for 24 h had no effects on MCH transcripts, plasma osmotic pressure (OP) or cortisol concentrations, but after 24 h in 80% SW, plasma OP and cortisol were raised and MCH transcripts in the NLT were significantly increased (159% of controls, p < 0.01). LVR-MCH neurons remained unaffected. However, after 24 h in 100% SW, MCH mRNA was significantly reduced in both groups of neurons (NLT -62% of controls, p < 0.001; LVR -33% of control, p < 0.001). These responses were transient and were no longer apparent after 6 days in 100% SW, despite the fact that plasma OP and cortisol levels continued to rise. The relative importance of osmotic disturbance and stress on the differential responses of the 2 groups of MCH neurons to changing salinity is discussed, together with a consideration of the potential role of MCH in osmoregulation.

Expression of MCH and POMC genes in rainbow trout (Oncorhynchus mykiss) during ontogeny and in response to early physiological challenges.

The expression of the neuropeptide melanin-concentrating hormone (MCH) in two groups of hypothalamic neurones (NLT- and LVR-MCH neurones), and POMC in the pituitary corticotropes and melanotropes, has been examined in rainbow trout larvae using immunocytochemistry and quantitative in situ hybridization. The aim was to establish at what stage in ontogeny these cells first respond to two physiological challenges-background color and stress. Trout reared in black or white trays showed adaptive skin pigmentary changes at 10 days posthatching, when fish in a pale environment abruptly exhibited melanin aggregation from a prior dispersed state, although the pigment cells were already competent to respond to adrenalin and MCH in vitro at 3 days. Immunoreactive MCH was detectable in the neurohypophysis at hatching and MCH mRNA in the NLT-MCH neurones (which project to the pituitary) was enhanced at 7 days in the white-reared trout. Immunostainable POMC was also present in the pars intermedia at hatching but their POMC mRNA was unaffected by tank color until 28 days, when it was enhanced in the black-reared trout. It is suggested that early pigment concentration depends on neural signals from the sympathetic nervous system in conjunction with MCH from the NLT rather than on a reduction in alphaMSH secretion from the pars intermedia. MCH mRNA in the LVR-MCH neurones was increased on a pale environment only 28 days after hatching, suggesting that these cells play little role in the early adaptive pigment response. Previous studies on the ontogeny of cortisol secretion indicate the hypothalamopituitary-interrenal axis can respond to stress by about 14 days. However, the pituitary ACTH cells showed no stress-induced changes in POMC mRNA until 28 days. ACTH release may therefore be dissociated from POMC transcription in the early stages of development. The LVR- and NLT-MCH neurones were both stimulated by stress, LVR-MCH mRNA responding by 14 days and NLT-MCH mRNA by 21 days. Melanotrope POMC mRNA was reduced by stress but the physiological significance of this is not known.

Melanin-concentrating hormone (MCH) antagonizes the effects of alpha-MSH and neuropeptide E-I on grooming and locomotor activities in the rat.

The intraventricular (i.c.v.) administration of the neuropeptide melanocyte stimulating hormone (alpha-MSH) is known to elicit a series of behaviors in the rat which include excessive grooming and other motor activities. In bony fish, the pigmentary effects of alpha-MSH can be antagonized by the neuropeptide melanin-concentrating hormone (MCH). We therefore examined whether MCH or its sister peptide neuro-peptide E-I (NEI), derived from the same precursor molecule, would modulate the effect of alpha-MSH on grooming and motor activity in the rat, or perhaps elicit some responses of their own. Rats were injected i.c.v. with either artificial cerebrospinal fluid, alpha-MSH, MCH, NEI, or with two peptides together, and behavioral responses were monitored over the next 65 min. The i.c.v. injection of 1 microgram MSH significantly enhanced grooming behavior. NEI at the same dose increased grooming, rearing, and locomotor activities. MCH alone had no behavioral effects but it annulled the behavioral responses induced by either alpha-MSH or NEI. alpha-MSH also antagonized the locomotor and rearing behavior induced by NEI. The physiological significance of these observations is discussed.

Stimulatory effect of melanin-concentrating hormone on luteinising hormone release.

Alpha-melanocyte-stimulating hormone (alpha-MSH) and melanin-concentrating hormone (MCH) are two peptide neurotransmitters widely distributed in the mammalian brain, the former originating mainly from cell bodies in the arcuate nucleus and the latter from cell bodies in the zona incerta and lateral hypothalamus. Within the hypothalamus they innervate the pre-optic area, median eminence (ME) and ventromedial nucleus (VMN). Both peptides stimulate sexual behaviour and in this report we have investigated their effect on another gonadal steroid-dependent function, luteinising hormone (LH) release. alpha-MSH, MCH or a combination of the two were injected bilaterally (100 ng/side) into either the medial pre-optic area (MPOA), ME, or VMN of anaesthetised (Saffan 3 ml/kg i.p.) rats that had previously been ovariectomised and adrenalectomised (O+A) and then primed with 5 microg/rat s.c. oestradiol benzoate (OB), 48 h before peptide administration. MCH stimulated LH release when applied to the MPOA and ME; alpha-MSH was inhibitory in the ME and in this model was ineffective in the MPOA. Neither peptide was effective in the VMN. The two peptides were then injected into the MPOA of O+A rats primed with OB followed 48 h later by 0.5 mg/rat s.c. progesterone, which normally induces an LH surge. alpha-MSH, but not MCH, inhibited this induced rise in LH. Administration of anti-MCH antiserum (0.5 microg/side neat serum) also had an inhibitory effect on LH release in this model. These results show that MCH has a stimulatory effect on LH release when administered into the ME and MPOA. In the MPOA, this may be physiologically significant since blocking endogenous MCH with an anti-MCH antiserum inhibits LH release. On the other hand, alpha-MSH has an inhibitory effect on LH release in the MPOA and ME. In the teleost skin these two peptides are functionally antagonistic; it seems that a similar antagonism exists between their effects on LH release.

In the trout, CRH and AVT synergize to stimulate ACTH release.

Anterior pituitaries of the rainbow trout (Oncorhynchus mykiss) were incubated with graded concentrations of arginine vasotocin (AVT) or synthetic rat corticotrophin-releasing hormone (rCRH-41), alone or in combination, and the ACTH secreted into the medium was measured by a sensitive cytochemical bioassay. The aim was to determine the relative potencies of the two secretogogues and whether, in this fish species, they act synergistically. Rat CRF-41 and AVT both produced concentration-dependent increases in ACTH release. The minimum effective concentration for both peptides was approximately 1 nM but, at higher concentrations, the efficacy of CRF-41 was greater than that of AVT. Clear evidence of synergy between the two peptides was obtained. The response of the trout thus falls in line with observations in mammals and contrasts with findings for the goldfish.

Evidence that cortisol may protect against the immediate effects of stress on circulating leukocytes in the trout.

Rainbow trout stressed by an intraperitonal injection of saline displayed reduced phagocytic activity of their spleen and head-kidney macrophages within 3 hr. Phagocytic activity was similarly depressed by injecting noradrenalin, but was maintained in fish injected with the adrenergic blocking agent phentolamine, suggesting that endogenous catecholamines are involved in this stress response. Since stress may increase the number of circulating granulocytes, it is proposed that noradrenalin, released during stress, causes the liberation of active macrophages from the lymphocytic tissue, the remaining macrophages therefore showing a lowered phagocytic index. Cortisol injection, like phentolamine, prevented the depressive effect of stress on the phagocytic index but did not antagonize the depressive effect of exogenous noradrenalin. It is suggested that the stress-induced release of endogenous catecholamines may be prevented by cortisol. Injection stress caused a decline in the number of circulating lymphocytes/thrombocytes, indicating their retrafficking into some other tissue. This was opposed by cortisol and by high doses of noradrenalin. It is proposed that cortisol or noradrenalin may oppose, directly or indirectly, the expression of adhesion molecules which are normally induced after stress.

Developmental changes in melanin-concentrating hormone in Rana temporaria.

Melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic neurons. In bony fish, such as trout, MCH acts as a neurohypophysial hormone which, once released into the circulation, acts on pigmented skin cells with the result that the fish turns pale to camouflage itself against a light colored background. In other animals the role of MCH is not clearly established but it appears to be a neuromodulator/transmitter within the central nervous system rather than a hormone. The present study examines MCH function in the grass frog, Rana temporaria. Using immunocytochemistry the location and morphology of irMCH neurons were followed, from tadpole to adult frog. In adult R. temporaria a group of MCH neurons appeared to comprise small and large-celled populations located in the ventral and dorsal infundibular regions, respectively. A group of MCH neurons in the preoptic area is proposed, although the perikarya were rarely immunostainable. Immunoreactive fibers were seen in various areas of the brain, including the olfactory lobes, optic tecta, habenular nucleus, and spinal cord. Immunoreactive MCH cells were only visible in midmetamorphic climax stages, and cellular morphology suggested low secretory activity until the animal first emerged onto land at which time nuclear size and granulation increased significantly. No such increase was observed in equivalent stages of the South African clawed toad, Xenopus laevis, an animal which is fully aquatic throughout its life in contrast to R. temporaria which is terrestrial.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of environmental colour and diurnal phase on MCH gene expression in the trout.

Melanin-concentrating hormone (MCH) gene expression in the brain of rainbow trout, reared and maintained in either pale or black-coloured tanks, was studied using in situ hybridization histochemistry. MCH transcripts were most prevalent in the magnocellular neurones of the nucleus lateralis tuberis (NLT), which project to the pituitary gland. They were also present, although at much lower levels, in dorsally projecting parvocellular neurones, sited more posteriorly above the lateral ventricular recess (LVR). In the NLT the most intense hybridization signal was seen over the pituitary stalk; above the LVR, the most active neurones were located caudally. In both the NLT and above the LVR, MCH hybridization signal was 4-fold stronger in white-reared fish than in black-reared fish. There was also a marked diurnal variation in MCH expression in both sites, with high levels at 16.00 h and lower levels at 04.00 h. The results show that gene activity in both hormonal (NLT) and neuromodulator/neurotransmitter (LVR) MCH neurones is induced by pale environmental colour and that MCH gene activity is subject to pronounced diurnal variation.

Seasonal and photoperiod-induced changes in the secretion of alpha-melanocyte-stimulating hormone in Soay sheep: temporal relationships with changes in beta-endorphin, prolactin, follicle-stimulating hormone, activity of the gonads and growth of wool and horns.

Blood plasma concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH), beta-endorphin (beta-END), prolactin and follicle-stimulating hormone (FSH), and associated changes in the size of the testes, and growth of the horns and pelage were measured in male (n = 8), castrated male (n = 5) and female (n = 9) Soay sheep. The animals were born in April and kept outdoors near Edinburgh (56 degrees N) during the first two years of life. In all groups there was a close association between the weekly changes in the plasma concentrations of alpha-MSH and beta-END; the molar ratio in mean concentrations was close to 1:1. The blood plasma concentrations of both hormones varied markedly with season with a 3- to 10-fold increase in concentrations from the minimum in winter to the maximum in autumn. The seasonal peak occurred in September in the first year of life as juveniles, and between July (males) and September (females) in the second year when the animals were sexually mature. The plasma concentrations of ACTH did not vary in parallel with the seasonal changes in the concentrations of alpha-MSH (measured only in males); the molar ratio for the concentrations of alpha-MSH:ACTH was 1:0.12. The seasonal increase in the concentrations of alpha-MSH occurred 1-3 months after the seasonal increase in the concentrations of prolactin and the associated growth in horns and pelage, and slightly before, or coincident with the seasonal increase in the concentrations of FSH and the growth in the testes. In a second experiment, the same parameters were measured in a group of adult male Soay sheep (n = 8) housed indoors under an artificial lighting regimen of alternating 16-week periods of long (16 h light: 8 h darkness) and short days (8 h light: 16 h darkness). In this situation, there was a clearly defined photoperiod-induced cycle in the plasma concentrations of alpha-MSH with a 25-fold increase from a minimum under long days to a maximum under short days. The concentrations of beta-END varied in close parallel with the changes in alpha-MSH, and the temporal associations with the changes in the other pituitary hormones were similar to those observed in animals housed outdoors. Overall, the results support the view that alpha-MSH is co-secreted with beta-END from the melanotrophs in the pars intermedia of the pituitary gland, and that the secretory activity of the melanotrophs changes markedly with season, increasing in summer and autumn, and decreasing in winter and spring.(ABSTRACT TRUNCATED AT 400 WORDS)