Probiotic Treatment Enhances Pre-feeding Larval Development and Early Survival in Zebrafish Danio rerio.

The growth and development of healthy culture subjects are essential in increasing productivity in the aquaculture industry. A primary determinant of aquatic animal productivity is the ambient microbial population. If an aquatic animal's microbiome is diverse, with bacteria favoring beneficial over pathogenic species, the health and growth of the animal (i.e., fish or crustacean) can be substantially improved. Embryonic and newly hatched Zebrafish Danio rerio larvae were reared in the presence of (1) water from the broodstock culture tank as a control, (2) a probiotic solution containing 19 strains of live lactic acid bacteria (LAB), or (3) an antibiotic (AB) solution with amoxycillin. Developmental parameters were monitored until 10 d postfertilization. Bacteria present in the water and larvae were cultured and identified by sequencing the V4 hypervariable region of bacterial 16S ribosomal RNA. Probiotic-treated larvae showed significant increases in every measured morphological parameter and in survival compared to the controls and AB-treated larvae, including TL, eye development, and swim bladder development before first feeding. Staining with DASPEI (2-(4-[dimethylamino]styryl)-N-ethylpyridinium iodide) produced fluorescence, revealing increased mitochondrial activity in the gastrointestinal tracts of probiotic-treated larvae and reflecting advancement of initial metabolic function. Probiotic-treated larvae showed accelerated yolk absorption, resulting in increased nutrient mobilization and growth. Microbial analyses revealed a greater concentration of bacteria in larvae in response to the probiotic treatment compared to the other two treatments. Species identified in all three treatments included Pseudomonas spp. and Aeromonas spp. (Proteobacteria). The second most diverse and abundant microbiome was seen in controls, whereas AB-treated larvae had the least diverse microbiome. All treatments revealed the presence of proteobacteria, but an AB-resistant pathogenic bacterium (Stenotrophomonas maltophilia) was identified in the AB group. These results reveal that the presence of LAB and other bacteria favorably influenced early larval growth, development, digestive function, and survival in Zebrafish even before the onset of feeding.

Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of one melanocortin receptor (MCR) agonist and two antagonists on food intake in juvenile rainbow trout. Baseline food intake was established prior to 1 microl intracerebroventricular injection (ICV) of the non-specific agonist MTII, the MC4R antagonist HS024 and the MC3/4R antagonist SHU9119 at concentrations of 0.3, 1 or 3 nM. Saline-injected fish and untreated fish served as controls. Changes in food intake were observed 1h after the ICV injections. Our results showed that treatment with MTII significantly decreased food intake at 3 nM compared to control, HS024 significantly increased food intake at 3 nM compared to control and saline-treated fish, and SHU9119 significantly increased food intake at 3 nM compared to saline-treated fish. In conclusion, our study provides further evidence, and hence strengthens the hypothesis, that MC4R participates in the control of energy balance in fish in the same manner as in mammals. Our findings that HS024 is more potent than SHU9119 in increasing food intake suggest that the effects of melanocortin on energy balance in rainbow trout are mainly regulated by activation of MC4R. Hence, HS024 seems an excellent tool as a MC4R antagonist in rainbow trout.

Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes.

BACKGROUND: The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. RESULTS: We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. CONCLUSION: This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.

Cloning and sequence analysis of the neuropeptide Y receptors Y5 and Y6 in the coelacanth Latimeria chalumnae.

Two coelacanth species, Latimeria chalumnae and Latimeria menadoensis, the recently discovered second species, have a key evolutionary position at the divergence of bony fishes and tetrapods. Together with lungfishes, they are the only living species separating the species-rich tetrapods from the other major group of vertebrates, the ray-finned fishes. The coelacanth is therefore of great importance for comparisons of gene families that differ between these two groups, such as the neuropeptide Y (NPY) receptor family. In this work we have sequenced the full-length genes for two NPY receptors in Latimeria chalumnae. Phylogenetic analysis indicated that the two sequences are orthologs of the mammalian Y5 and Y6 receptors. The Y5 gene has been implicated in appetite stimulation in mammals but is absent from teleost fishes. The presence of the Y5 receptor in Latimeria together with phylogenetic analysis shows that Y5 existed before the separation of bony fishes and tetrapods. The Latimeria receptor has about 62% identity to tetrapod Y5 sequences and contains the extended third intracellular loop with several highly conserved motifs that may be involved in signal transduction. The Latimeria Y6 receptor has about 60% identity to tetrapod Y6 sequences. The functional role of Y6 is unclear as the gene is seemingly functional in some mammals but is non-functional in others. The Y6 receptor is also missing in teleost fishes. Our results confirm an early vertebrate origin for all NPY receptor subtypes presently found in mammals followed by differential gene loss in the different classes of vertebrates.

Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish.

Agonistic interactions are present throughout the animal kingdom as well as in humans. In this report, we present a model system to study neurological correlates of dominant-subordinate relationships. Zebrafish, Danio rerio, has been used as a model system for developmental biology for decades. We propose here that it is also an excellent model for studying social behavior. Adult male zebrafish were separated for 5 days and then pairs were formed and allowed to interact for 5 days. Under these conditions, aggression is prevalent and dominant-subordinate relationships are quickly established. Dominant behavior is characterized by a repeated pattern of chasing and biting, whereas subordinates engage in retreats. By day 5, the dominant-subordinate relationship was firmly established and there were differences in behavior over time. Chases, bites and retreats were all less frequent on day 5 of the social interaction than on day 1. Arginine vasotocin is the teleostean homologue of arginine vasopressin, a neuropeptide whose expression has been linked to aggression and social position in mammals. Immunohistochemistry indicated differences in vasotocin staining between dominant and subordinate individuals. Dominant individuals express vasotocin in one to three pairs of large cells in the magnocellular preoptic area whereas subordinate individuals express vasotocin in 7-11 pairs of small cells in the parvocellular preoptic area. These results suggest that the vasotocinergic system may play a role in shaping dominant-subordinate relationships and agonistic behavior in this model organism.

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss.

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.

Does serotonin influence aggression? comparing regional activity before and during social interaction.

Serotonin is widely believed to exert inhibitory control over aggressive behavior and intent. In addition, a number of studies of fish, reptiles, and mammals, including the lizard Anolis carolinensis, have demonstrated that serotonergic activity is stimulated by aggressive social interaction in both dominant and subordinate males. As serotonergic activity does not appear to inhibit agonistic behavior during combative social interaction, we investigated the possibility that the negative correlation between serotonergic activity and aggression exists before aggressive behavior begins. To do this, putatively dominant and more aggressive males were determined by their speed overcoming stress (latency to feeding after capture) and their celerity to court females. Serotonergic activities before aggression are differentiated by social rank in a region-specific manner. Among aggressive males baseline serotonergic activity is lower in the septum, nucleus accumbens, striatum, medial amygdala, anterior hypothalamus, raphe, and locus ceruleus but not in the hippocampus, lateral amygdala, preoptic area, substantia nigra, or ventral tegmental area. However, in regions such as the nucleus accumbens, where low serotonergic activity may help promote aggression, agonistic behavior also stimulates the greatest rise in serotonergic activity among the most aggressive males, most likely as a result of the stress associated with social interaction.

Serotonin, but not melatonin, plays a role in shaping dominant-subordinate relationships and aggression in rainbow trout.

The aim of this study was to clarify to what extent the effects of elevated dietary L-tryptophan (Trp) on aggressive behavior and stress responsiveness in rainbow trout are mediated by circulating melatonin and central serotonin (5-HT), respectively. Isolated rainbow trout were paired for 1h a day for 7 days in order to create fish with experience of being dominant and subordinate. Following this week, the fish were tested for aggressive behavior using a resident-intruder test after which they were subjected to one of four treatments: (1) tryptophan, (2) the selective serotonin reuptake inhibitor (SSRI) citalopram, (3) melatonin, and (4) no treatment (controls). After 7 days of treatment, the fish were subjected to a second resident-intruder test. Trp-supplemented feed resulted in a suppression of aggressive behavior in fish with experience of being dominant. Moreover, fish fed Trp-supplemented feed, regardless of social experience, also displayed lower plasma cortisol levels than controls. These effects of elevated dietary Trp were closely mimicked by citalopram treatment, whereas exogenous melatonin had no effect on either aggressive behavior or plasma cortisol. Thus, the effect of elevated dietary Trp on aggressive behavior and stress responses does not appear to be mediated by melatonin even though elevated dietary intake of Trp resulted in an increase in plasma melatonin concentrations.

Effects of a teleost tetraploidization on neuropeptide Y receptor gene repertoire in ray-finned fishes.

The ancestral vertebrate repertoire for neuropeptide Y receptor genes of the Y1 subfamily probably included four subtypes: Y1, Y4, Y6, and Y8. There was probably a single gene in the Y5 category. Both Y1 and Y5 stimulate food intake in mammals. As the genome seems to have duplicated during the evolution of ray-finned fishes, we have investigated the gene repertoire in species that diverged prior to the appearance of teleosts, as well as a basal teleost and a shark. Our results show that the genes Y1, Y5, and Y6, which are missing in many teleosts, are present in basal actinopterygians. These dramatic alterations of the teleost receptor repertoire may be related to the tetraploidization in a teleost ancestor.

Tryptophan affects both gastrointestinal melatonin production and interrenal activity in stressed and nonstressed rainbow trout.

The present experiments were designed to test the hypothesis that elevated dietary levels of l-tryptophan (Trp) result in elevated plasma levels of melatonin and that this increase in plasma melatonin concentration is caused by elevated melatonin production and secretion by the gastro-intestinal-tract (GIT). Feeding juvenile rainbow trout (Oncorhynchus mykiss) Trp-supplemented feed for 7 days resulted in elevated daytime plasma levels of melatonin and reduced poststress plasma cortisol concentrations. Nighttime plasma melatonin concentrations were, however, not affected by elevated dietary Trp. Moreover, stress caused a reduction in daytime plasma levels of melatonin in fish fed Trp-supplemented feed, an effect that was counteracted by treatment with an alpha-receptor antagonist. These results clearly suggest that elevated dietary intake of Trp results in an increase in the GIT production of melatonin in rainbow trout. A suggestion that was further supported by the results from an in vitro experiment demonstrating that addition of Trp to the incubation medium stimulates melatonin production and release by incubated rainbow trout GIT. The results from this study led us to suggest a possible mechanism for melatonin in mediating the effects of elevated dietary Trp on poststress plasma cortisol concentrations and aggressive behavior in rainbow trout.

Expression of engrailed in the developing brain and appendages of the onychophoran euperipatoides kanangrensis (Reid).

We have cloned an engrailed-class gene in the onychophoran Euperipatoides kanangrensis and investigated its expression using in situ hybridisation. The expression pattern was found to differ drastically from that previously described for another onychophoran species. In the present investigation, engrailed transcripts were detected in a subset of developing neurons in the brain anlage, and in the mesoderm as well as ectoderm of the developing limb buds. The engrailed positive cells of the brain are of differing developmental maturity, ranging from subepidermal neuronal precursors to neurons located basally in the embryo with developing axons. The lack of the traditional expression in the posterior compartment of segments reported earlier in onychophorans is discussed, and we suggest that onychophorans may have acquired two copies of engrailed with different functions.

Dynamics and mechanics of social rank reversal.

Stable social relationships are rearranged over time as resources such as favored territorial positions change. We test the hypotheses that social rank relationships are relatively stable, and although social signals influence aggression and rank, they are not as important as memory of an opponent. In addition, we hypothesize that eyespots, aggression and corticosterone influence serotonin and N-methyl-D: -aspartate (NMDA) systems in limbic structures involved in learning and memory. In stable adult dominant-subordinate relationships in the lizard Anolis carolinensis, social rank can be reversed by pharmacological elevation of limbic serotonergic activity. Any pair of specific experiences: behaving aggressively, viewing aggression or perceiving sign stimuli indicative of dominant rank also elevate serotonergic activity. Differences in the extent of serotonergic activation may be a discriminating and consolidating factor in attaining superior rank. For instance, socially aggressive encounters lead to increases in plasma corticosterone that stimulate both serotonergic activity and expression of the NMDA receptor subunit 2B (NR(2B)) within the CA(3) region of the lizard hippocampus. Integration of these systems will regulate opponent recognition and memory, motivation to attack or retreat, and behavioral and physiological reactions to stressful social interactions. Contextually appropriate social responses provide a modifiable basis for coping with the flexibility of social relationships.

Social stress affects circulating melatonin levels in rainbow trout.

In salmonid fishes there are indications that socially subordinate individuals avoid competition with larger, dominant fish by adjusting daily feeding and activity cycles. As in other vertebrates, the pineal organ and its hormone melatonin act as synchronizers of daily rhythms to the external light/dark cycle in salmonids. Social defeat may act as a potent stressor; inducing elevated glucocorticoid secretion and a general behavioral inhibition. Here, we show that social stress also affects circulating melatonin levels in rainbow trout, a species known to display strong dominance hierarchies both in the wild and under captive rearing. Subordinate individuals had significantly higher nighttime melatonin levels than dominant fish or controls. There was no effect of social rank on the much lower melatonin levels observed in animals sampled during the day. Correlations between circulating glucocorticoids and melatonin depended on circadian cycles as well as social context. This study suggests that altered melatonin production contributes to the physiological and behavioral profile of subordinate animals. Social status, and other determinants of the stress level of experimental animals, therefore should be taken into consideration as potential factors influencing the results from in vivo research on this hormone.

Behavioral and neuroendocrine correlates of displaced aggression in trout.

In humans and other primates, violent actions performed by victims of aggression are often directed toward an individual or object that is not the source of provocation. This psychological phenomenon is often called displaced aggression. We demonstrate that displaced aggression is either rooted in evolutionarily conserved behavioral and neuroendocrine mechanisms, or represent a convergent pattern that has arisen independently in fish and mammals. Rainbow trout that briefly encountered large, aggressive fish reacted with increased aggression toward smaller individuals. There was a strong negative correlation between received aggression and behavioral change: Individuals subjected to intense aggression were subdued, while moderate assaults induced strong agitation. Patterns of forebrain serotonin turnover and plasma cortisol suggest that the presence of socially subordinate fish had an inhibitory effect on neuroendocrine stress responses. Thus, subordinate individuals may serve as stress-reducing means of aggressive outlet, and displaced aggression toward such individuals appears to be a behavioral stress coping strategy in fishes.

Cloning, tissue distribution, pharmacology and three-dimensional modelling of melanocortin receptors 4 and 5 in rainbow trout suggest close evolutionary relationship of these subtypes.

The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas alpha-, beta- and gamma-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 microM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.

Three neuropeptide Y receptor genes in the spiny dogfish, Squalus acanthias, support en bloc duplications in early vertebrate evolution.

It has been debated whether the increase in gene number during early vertebrate evolution was due to multiple independent gene duplications or synchronous duplications of many genes. We describe here the cloning of three neuropeptide Y (NPY) receptor genes belonging to the Y1 subfamily in the spiny dogfish, Squalus acanthias, a cartilaginous fish. The three genes are orthologs of the mammalian subtypes Y1, Y4, and Y6, which are located in paralogous gene regions on different chromosomes in mammals. Thus, these genes arose by duplications of a chromosome region before the radiation of gnathostomes (jawed vertebrates). Estimates of duplication times from linearized trees together with evidence from other gene families supports two rounds of chromosome duplications or tetraploidizations early in vertebrate evolution. The anatomical distribution of mRNA was determined by reverse-transcriptase PCR and was found to differ from mammals, suggesting differential functional diversification of the new gene copies during the radiation of the vertebrate classes.

Cloning, pharmacology, and distribution of the neuropeptide Y-receptor Yb in rainbow trout.

This work describes the isolation and pharmacological characterization of a neuropeptide Y (NPY) receptor from rainbow trout (Oncorhynchus mykiss). The receptor exhibits approximately 45% amino acid sequence identity to mammalian Y1-subfamily receptors, Y1, Y4 and y6, a similar degree of identity as these subtypes display to one another. Because it displays highest sequence identity to zebrafish Yb (75%), we named it the trout Yb receptor. The receptor exhibits high binding affinity for zebrafish and human NPY and peptide YY (PYY) but not truncated forms of the peptides. Human pancreatic polypeptide (PP) also binds with high affinity. Y1 selective antagonists exhibit poor binding as is the case for Y2 and Y5 selective ligands. This binding profile supports membership in the Y1 subfamily. Sequence data also support this relationship suggesting that Yb is a fourth and separate member of the Y1 subfamily. NPY has a number of important physiological functions such as regulating food intake and reproduction. The expression of the receptor in the hypothalamus and telencephalon suggests a possible role in these processes. This and other receptors from this species have potential for improving aquaculture.

Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard anolis carolinensis.

Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events.

Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides alpha-, beta-, and gamma-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue 'back' to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.

Monoamines stimulate sex reversal in the saddleback wrasse.

Monoamine neurotransmitters (norepinephrine, dopamine, and serotonin) play an important role in reproduction and sexual behavior throughout the vertebrates. They are the first endogenous chemical signals in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. In teleosts with behavioral sex determination, much is known about behavioral cues that induce sex reversal. The cues are social, processed via the visual system and depend on the ratio of females to males in the population. The mechanisms by which these external behavioral cues are converted to an internal chemical regulatory process are largely unknown. The protogynous Hawaiian saddleback wrasse, Thalassoma duperrey, was used to investigate the biological pathway mediating the conversion of a social cue into neuroendocrine events regulating sex reversal. Because monoamines play an important role in the regulation of the HPG axis, they were selected as likely candidates for such a conversion. To determine if monoamines could affect sex reversal, drugs affecting monoamines were used in an attempt to either induce sex reversal under non-permissive conditions, or prevent sex reversal under permissive conditions. Increasing norepinephrine or blocking dopamine or serotonin lead to sex reversal in experimental animals under non-permissive conditions. Increasing serotonin blocked sex reversal under permissive conditions, while blocking dopamine or norepinephrine retarded the process. The results presented here demonstrate that monoamines contribute significantly to the control sex reversal. Norepinephrine stimulates initiation and completion of gonadal sex of reversal as well as color change perhaps directly via its effects on the HPG axis. Dopamine exercises inhibitory action on the initiation of sex reversal while 5-HT inhibits both initiation and completion of sex reversal. The serotonergic system appears to be an integral part of the pathway mediating the conversion of a social cue into a neuroendocrine event. The complex organization of neurochemical events controlling the psychosocial, physiological, and anatomical events that constitute reversal of sexual identity includes monoamine neurotransmitters.