Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa.

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [(3)H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT)>OT≈vasotocin (VT)>vasopressin (VP)>isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT>OT>VT≈VP>IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT>VP>MT≈OT>IT. For both receptors, stimulation of IP accumulation was blocked by d(CH(2))(5)[Tyr(Me)(2)]AVP (Manning compound) and d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC(50) of Manning compound was lower for the tMTR (27nM±13) than the tVTR-1a (586nM±166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

Fluoxetine potentiates the effects of corticotropin-releasing factor on locomotor activity and serotonergic systems in the roughskin newt, Taricha granulosa.

The anxiety- and stress-related neuropeptide corticotropin-releasing factor (CRF) elicits behavioral changes in vertebrates including increases in behavioral arousal and locomotor activity. Intracerebroventricular injections of CRF in an amphibian, the roughskin newt (Taricha granulosa), induces rapid increases in locomotor activity in both intact and hypophysectomized animals. We hypothesized that this CRF-induced increase in locomotor activity involves a central effect of CRF on serotonergic neurons, based on known stimulatory actions of serotonin (5-hydroxytryptamine, 5-HT) on spinal motor neurons and the central pattern generator for locomotor activity in vertebrates. In Experiment 1, we found that neither intracerebroventricular injections of low doses of CRF (25 ng) nor the selective serotonin reuptake inhibitor fluoxetine (10, 100 ng), by themselves, altered locomotor activity. In contrast, newts treated concurrently with CRF and fluoxetine responded with marked increases in locomotor activity. In Experiment 2, we found that increases in locomotor activity following co-administration of CRF (25 ng) and fluoxetine (100 ng) were associated with decreased 5-HT concentrations in a number of forebrain structures involved in regulation of emotional behavior and emotional states, including the ventral striatum, amygdala pars lateralis, and dorsal hypothalamus, measured 37 min after treatment. These results are consistent with the hypothesis that CRF stimulates locomotor activity through activation of serotonergic systems.

Identification of mesotocin and vasotocin nucleotide sequences in two species of urodele amphibian.

We amplified and identified, for the first time in urodele amphibians, cDNA sequences that encode preprovasotocin (preproVT) and prepromesotocin (preproMT) from two distinct urodelian species, Taricha granulosa (the rough-skinned newt) and Plethodon shermanii (the spotted salamander). Each of these cDNA sequences encoded proteins that contained the characteristics of known neurohypophysial peptide precursors; each sequence consisting of (1) a signal peptide, (2) VT- or MT-like peptides, (3) neurophysin, and for the preproVTs, (4) copeptin. In T. granulosa, cDNA sequences encoded for the nine amino acids that define VT or MT. In P. shermani, cDNA sequences encoded for the VT peptide and a previously unidentified isoform of MT, ([Val(4)]-MT).

Endocannabinoids mediate the effects of acute stress and corticosterone on sex behavior.

For animals in the wild, survival depends on being able to detect and respond rapidly to danger by switching from risky (e.g. conspicuous courtship) to survival-oriented behaviors. Very little is known about the hormonal or neuroendocrine mechanisms that control the rapid switch in behavioral state that occurs when an animal detects threats or other stressors. Prior studies with rough-skinned newts (Taricha granulosa), an amphibian model, found that stress-induced suppression of male sexual behaviors (amplectic clasping) involves corticosterone (CORT) and that this steroid hormone uses a novel membrane receptor and modulates the responsiveness of medullary neurons in clasp-controlling neural circuits. We provide evidence that this rapid suppression of male sex behaviors, when induced by either acute stress or CORT administration, involves activation of endocannabinoids signaling in the hindbrain. In a series of behavioral studies, administration of a cannabinoid antagonist, AM281, blocked the suppressive effects of exposure to acute stress or an injection of CORT on the performance of clasping behaviors in sexually active males. Similarly, in electrophysiological studies, prior treatment with AM281 blocked CORT-induced suppression of spontaneous neuronal activity and sensory responsiveness of hindbrain neurons in clasp-controlling neural circuits. These data suggest that, in response to acute stress, elevated CORT concentration increases endocannabinoid signaling in the hindbrain and alters sexual behaviors by modulating the excitability of medullary circuits.

Neuroanatomical distribution of cannabinoid receptor gene expression in the brain of the rough-skinned newt, Taricha granulosa.

Type I cannabinoid receptor (CB1) is a G-protein coupled receptor with a widespread distribution in the central nervous system in mammals. In a urodele amphibian, the rough-skinned newt (Taricha granulosa), recent evidence indicates that endogenous cannabinoids (endocannabinoids) mediate behavioral responses to acute stress and electrophysiological responses to corticosterone. To identify possible sites of action for endocannabinoids, in situ hybridization using a gene and species specific cRNA probe was used to label CB1 mRNA in brains of male T. granulosa. Labeling of CB1 mRNA in the telencephalon was observed in the olfactory bulb and all areas of the pallium, as well as the bed nucleus of the stria terminalis and nucleus amygdalae dorsolateralis. The labeling of CB1 mRNA was also found in regions of the preoptic area, thalamus, midbrain tegmentum and tectum, cerebellum, and the stratum griseum of the hindbrain. A notable difference in CB1 labeling between this amphibian and mammals is the abundance of labeling in areas associated with olfaction (anterior olfactory nuclei, nucleus amygdalae dorsolateralis, and lateral pallium), which hints that endocannabinoids might modulate responses to odors as well as pheromones. This widespread distribution of CB1 labeling, particularly in sensory and motor control centers, fits with prior results showing that endocannabinoids modulate sensorimotor processing and behavioral output in this species. The distribution of CB1 in the brain of T. granulosa was in many of the same sites previously observed in the brain of the anuran amphibian, Xenopus laevis, as well as those of different species of mammals, suggesting that endocannabinoid signaling pathways are conserved.

Regulation of behavioral responses by corticotropin-releasing factor.

In the wild, animals survive by responding to perceived threats with adaptive and appropriate changes in their behaviors and physiological states. The exact nature of these responses depends on species-specific factors plus the external context and internal physiological states associated with the stressful condition. The neuroendocrine mechanisms that control context-dependent stress responses are poorly understood for most animals, but some progress has been made recently. Corticotropin-releasing factor (CRF) plays an important role in mediating neuroendocrine, autonomic, and behavioral responses to stress. Across many vertebrate taxa, CRF not only stimulates the HPA axis by increasing the secretion of ACTH and glucocorticoid hormones, but also acts centrally by modifying neurotransmitter systems and behaviors. CRF or one of several CRF-related neuropeptides acts to stimulate locomotor activity during periods of acute stress. This behavioral activation consists of anxiety-related non-ambulatory motor activity, ambulatory locomotion, or swimming depending on the species and context. CRF-related neuropeptides increase swimming behaviors in amphibians and fish, apparently by activating brainstem serotonergic systems because the administration of fluoxetine (a selective serotonin re-uptake inhibitor) greatly enhances CRF-induced locomotor activity. Thus, our working model is that CRF, in part via interactions with brainstem serotonergic systems, modulates context-dependent behavioral responses to perceived threats, including both anxiety-related risk assessment behaviors and fight-or-flight locomotor responses.

Delta and mu opioid receptors from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa: cloning, heterologous expression, and pharmacological characterization.

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

Historical perspective: Hormonal regulation of behaviors in amphibians.

This review focuses on research into the hormonal control of behaviors in amphibians that was conducted prior to the 21st century. Most advances in this field come from studies of a limited number of species and investigations into the hormonal mechanisms that regulate reproductive behaviors in male frogs and salamanders. From this earlier research, we highlight five main generalizations or conclusions. (1) Based on studies of vocalization behaviors in anurans, testicular androgens induce developmental changes in cartilage and muscles fibers in the larynx and thereby masculinize peripheral structures that influence the properties of advertisement calls by males. (2) Gonadal steroid hormones act to enhance reproductive behaviors in adult amphibians, but causal relationships are not as well established in amphibians as in birds and mammals. Research into the relationships between testicular androgens and male behaviors, mainly using castration/steroid treatment studies, generally supports the conclusion that androgens are necessary but not sufficient to enhance male behaviors. (3) Prolactin acts synergistically with androgens and induces reproductive development, sexual behaviors, and pheromone production. This interaction between prolactin and gonadal steroids helps to explain why androgens alone sometimes fail to stimulate amphibian behaviors. (4) Vasotocin also plays an important role and enhances specific types of behaviors in amphibians (frog calling, receptivity in female frogs, amplectic clasping in newts, and non-clasping courtship behaviors). Gonadal steroids typically act to maintain behavioral responses to vasotocin. Vasotocin modulates behavioral responses, at least in part, by acting within the brain on sensory pathways that detect sexual stimuli and on motor pathways that control behavioral responses. (5) Corticosterone acts as a potent and rapid suppressor of reproductive behaviors during periods of acute stress. These rapid stress-induced changes in behaviors use non-genomic mechanisms and membrane-associated corticosterone receptors.

Cloning, heterologous expression and pharmacological characterization of a kappa opioid receptor from the brain of the rough-skinned newt, Taricha granulosa.

A full-length cDNA that encodes a kappa (kappa) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt kappa receptor has 75% sequence identity to kappa opioid receptors cloned from mammals, and 66% sequence identity to the kappa opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt kappa receptor possessed a single, high-affinity (Kd = 1.5 nM) binding site for the kappa-selective agonist U69593. In competition binding assays, the expressed newt kappa receptor displayed high affinity for the kappa-selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the kappa-selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt kappa receptor. The expressed receptor displayed essentially no affinity for the delta-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the mu-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the kappa opioid receptor is expressed in amphibians and that the pharmacology of the newt kappa receptor closely matches mammalian kappa opioid receptors. However, the functional dichotomy between the classic opioid receptors (kappa, delta, mu) and ORL1 appears less strict in amphibians than in mammals.

Cloning proenkephalin from the brain of a urodele amphibian (Taricha granulosa) using a DOR-specific primer in a 3'RACE reaction.

A large cDNA fragment that codes for proenkephalin (PENK) was cloned from the rough-skinned newt, Taricha granulosa (GenBank Accession: AY817670). This 1299-bp PENK cDNA extends from the poly(A) sequence on the 3' end into the 5'-UTR (221bp) upstream of an open reading frame that codes for 264 amino acids and a stop codon. Within the precursor are five Met-enkephalin sequences and two C-terminally extended forms of Met-enkephalin (YGGFMRGV and YGGFMRY). The organization of the opioid core sequences within the newt PENK closely resembles that reported for other vertebrates. In this urodele amphibian, as in anurans, PENK does not contain the penultimate Leu-enkephalin opioid sequence found in mammals, and instead has in this position Met-enkephalin. PENK cDNA was amplified from newt brain in a RACE PCR targeting the 3' end of the newt delta opioid receptor (DOR). It remains to be determined whether generating the cDNA for the newt PENK while cloning its receptor was serendipitous or the result of a meaningful coincidence between the DOR and PENK sequences.

Cloning, pharmacological characterization and tissue distribution of an ORL1 opioid receptor from an amphibian, the rough-skinned newt Taricha granulosa.

We have cloned and characterized an opioid receptor-like (ORL1; also referred to as NOP) receptor from a urodele amphibian, the rough-skinned newt Taricha granulosa The cDNA clone encodes a protein of 368 amino acids that contains the seven hydrophobic domains characteristic of G-protein-coupled receptors, and has the highest sequence identity to the frog (Rana pipiens) nociceptin-like and human ORL1 opioid receptors (79.6 and 68.4%, respectively). Saturation binding assays on membranes from COS-7 cells transiently expressing the newt ORL1 (nORL) receptor revealed a single, high-affinity (estimated Kd, 0.1974 nM) binding site for the ORL1-specific agonist [3H]orphanin FQ analog ([3H]oFQ). In competition binding assays, the [3H]oFQ-binding site, like the mammalian ORL1 receptor, had no affinity for the non-selective opioid receptor antagonist naloxone, the kappa-selective agonists U69593 and U50488, or the mu- and delta-selective opioid receptor agonists DAMGO and DPDPE, respectively. However, the nORL receptor displayed higher affinities for the kappa-selective agonists dynorphin A (1-13), dynorphin B, and dynorphin A (1-8) (Ki values, 2.8, 151.8, and 183.0 nM, respectively) than its mammalian homologue. The tissue distribution of the nORL receptor, as determined by reverse transcriptase PCR, was also found to differ from reports on the mammalian ORL1 receptor, with mRNA detected in brain, spinal cord, and lung, but not detected in a number of other peripheral tissues reported to express the receptor in mammals. This is the first report describing the expression and characterization of an amphibian ORL1 receptor, and contributes to our understanding of the evolution of the opioid system.

Neuroanatomical distribution of vasotocin and mesotocin in two urodele amphibians (Plethodon shermani and Taricha granulosa) based on in situ hybridization histochemistry.

Previous research suggests that considerable species-specific variation exists in the neuroanatomical distributions of arginine vasotocin (AVT) and mesotocin (MST), non-mammalian homologues of vasopressin and oxytocin. An earlier study in rough-skinned newts (Taricha granulosa) indicated that the neuroanatomical distribution of cells labeled for AVT-immunoreactivity (ir) was greater in this urodele amphibian than in any other species. It was unknown whether the widespread distribution of AVT-ir is unique to T. granulosa or a feature common among salamanders. Using in situ hybridization (ISH) histochemistry and gene-specific riboprobes, the current study labeled AVT and MST mRNA in T. granulosa and the red-legged salamander (Plethodon shermani). In T. granulosa, AVT ISH-labeled cells were found to be widespread and localized in brain areas including the dorsal and medial pallium, lateral and medial septum, bed nucleus of the stria terminalis, amygdala, preoptic area, ventral hypothalamus, nucleus isthmus, tectum mesencephali, inferior colliculus, and hindbrain. In P. shermani, the distribution of AVT ISH-labeled neurons matched that of T. granulosa, except in the lateral septum, ventral hypothalamus, and inferior colliculus, but did however include labeled cell bodies in the lateral pallium. The distribution of MST ISH-labeled cells was more restricted than AVT ISH labeling and was limited to regions of the preoptic area and ventral thalamus, which is consistent with the limited distribution of MST/OXY in other vertebrates. These findings support the conclusion that urodele amphibians possess a well-developed vasotocin system, perhaps more extensive than other vertebrate taxa.

Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha).

The present study investigated whether the serotonergic system is involved in mediating the behavioral effects of corticotropin-releasing hormone (CRH) in juvenile spring chinook salmon, Oncorhynchus tshawytscha. An intracerebroventricular (ICV) injection of CRH induced hyperactivity. The effect of CRH was potentiated in a dose-dependent manner by the concurrent administration of the serotonin (5-HT) selective reuptake inhibitor fluoxetine. However, administration of fluoxetine alone had no effect on locomotor activity, suggesting that the locomotor-stimulating effect of CRH is mediated by the activation of the serotonergic system. Conversely, ICV injections of the 5-HT(1A) receptor antagonist NAN-190 attenuated the effect of CRH on locomotor activity when given in combination with CRH but had no effect when administered alone. These results provide the first evidence to support the hypothesis that the effect of CRH on locomotor activity in teleosts is mediated by activating the serotonergic system.

Neuroendocrinology of context-dependent stress responses: vasotocin alters the effect of corticosterone on amphibian behaviors.

The ability of an animal to respond with appropriate defensive behaviors when confronted with an immediate threat can affect its survival and reproductive success. In the roughskin newt (Taricha granulosa), exogenous corticosterone (CORT) rapidly blocks and vasotocin (VT) enhances reproductive behaviors (mainly clasping behavior). Electrophysiological studies have shown that pretreatment of male Taricha with VT counteracts the inhibitory effects of CORT on neuronal activity in the medulla. To test whether similar interactions between VT and CORT influence reproductive behaviors in Taricha, we recorded the time spent and incidence of clasping in males injected with VT or vehicle at 60 min and then CORT or vehicle at 5 min before presentation of a female. This study found that clasping behavior is suppressed in males that received vehicle and then CORT, but is not suppressed in males that received VT and then CORT. Considering these results and the possibility that the performance of clasping behaviors might cause increases in endogenous VT activity, we tested whether the suppressive effects of CORT administration on clasping behavior would occur in males that had recently clasped females. The study found that, in contrast to males that had been isolated from females, CORT administration did not suppress clasping behavior in males that had been allowed to clasp females for 60 min prior to the hormone injection. Our results suggest that, at least in this amphibian and perhaps in other animals, the neuroendocrine regulation of alternative behavioral responses to threats involves functional interactions between corticosteroids and VT-like peptides.

Behavioral neuroendocrinology of vasotocin and vasopressin and the sensorimotor processing hypothesis.

Vasotocin (AVT) and vasopressin (AVP) are potent modulators of social behaviors in diverse species of vertebrates. This review addresses questions about how and where AVT and AVP act to modulate social behaviors, focusing on research with an amphibian model (Taricha granulosa). In general, the behaviorally important AVT and AVP neurons occur in the forebrain and project to sites throughout the brain. Social behaviors are modulated by AVT and AVP acting at multiple sites in the brain and at multiple levels in the behavioral sequence. This review proposes that AVT and AVP can act on sensory pathways to modulate the responsiveness of neurons to behaviorally relevant sensory stimuli and also can act on motor pathways in the brainstem and spinal cord to modulate the neuronal output to behavior-specific pattern generators. This neurobehavioral model, in which AVT and AVP are thought to modulate social behaviors by affecting sensorimotor processing, warrants further research.