Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis.

In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT-OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 µg/0.1mL amphibian Ringers). The brains were collected 30 min post-VT-OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT-OG, sum intensity of VT-OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses.

Brainstem neuronal and behavioral activation by corticotropin-releasing hormone depend on the behavioral state of the animal.

Central administration of corticotropin-releasing hormone (CRH) is known to enhance locomotion across a wide range of vertebrates, including the roughskin newt, Taricha granulosa. The present study aimed to identify the CRH effects on locomotor-controlling medullary neurons that underlie the peptide's behavioral stimulating actions. Single neurons were recorded from the rostral medullary reticular formation before and after intraventricular infusion of CRH in freely behaving newts and newts paralyzed with a myoneural blocking agent. In behaving newts, most medullary neurons showed increased firing 3-23 min after CRH infusion. Decreases in firing were less common. Of particular importance was the finding that in behaving newts, medullary neurons showed a cyclic firing pattern that was strongly associated with an increase in the incidence of walking bouts, an effect blocked by pretreatment with the CRH antagonist, alpha-helical CRH and not seen following vehicle administration. In contrast, the majority of medullary neurons sampled in immobilized newts lacked temporal cyclicity in their firing patterns following intraventricular infusion of CRH. That is, there was no evidence for a fictive locomotor activity pattern. Our results indicate that the actual expression of locomotion is a critical factor in regulating the behavior-activating effects of CRH and underscore the importance of using an awake, unrestrained animal for analysis of a hormone's neurobehavioral actions.

Brainstem reticulospinal neurons are targets for corticotropin-releasing factor-Induced locomotion in roughskin newts.

Stress-induced release or central administration of corticotropin-releasing factor (CRF) enhances locomotion in a wide range of vertebrates, including the roughskin newt, Taricha granulosa. Although CRF's stimulatory actions on locomotor behavior are well established, the target neurons through which CRF exerts this effect remain unknown. To identify these target neurons, we utilized a fluorescent conjugate of CRF (CRF-TAMRA 1) to track this peptide's internalization into reticulospinal and other neurons in the medullary reticular formation (MRF), a region critically involved in regulating locomotion. Epifluorescent and confocal microscopy revealed that CRF-TAMRA 1 was internalized by diverse MRF neurons, including reticulospinal neurons retrogradely labeled with Cascade Blue dextran. In addition, we immunohistochemically identified a distinct subset of serotonin-containing neurons, located throughout the medullary raphé, that also internalized the fluorescent CRF-TAMRA 1 conjugate. Chronic single-unit recordings obtained from microwire electrodes in behaving newts revealed that intracerebroventricular (icv) administration of CRF-TAMRA 1 increased medullary neuronal firing and that appearance of this firing was associated with, and strongly predictive of, episodes of CRF-induced locomotion. Furthermore, icv administered CRF-TAMRA 1 produced behavioral and neurophysiological effects identical to equimolar doses of unlabeled CRF. Collectively, these findings provide the first evidence that CRF directly targets reticulospinal and serotonergic neurons in the MRF and indicate that CRF may enhance locomotion via direct effects on the hindbrain, including the reticulospinal system.

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.

Anthropomorphism and 'mental welfare' of fishes.

Anthropomorphism, the use of human characteristics as a foundation for interpreting behavior and mental capacities of animals, is a bias undermining our understanding of other species, especially species as evolutionarily distant from humans as fishes. Anthropomorphism is not justified by allusions to evolutionary continuity among vertebrates, because no living vertebrate was ever a descendant of humans, so none could have inherited human traits. Nonetheless, it has recently been claimed that fishes are capable of conscious experiences of pain and emotional feelings and that mental welfare is an important issue for fishes. This paper shows that the evidence supporting claims for experiences of pain or conscious emotions by fishes is conceptually and methodologically flawed. In addition, the paper shows that the natural history and behavior of diverse fish species is inconsistent with a presumption of human-like awareness. This behavioral evidence is in accord with neurobiological observations showing that fishes are very different from us and are unlikely to have a capacity for awareness of pain or emotional feelings that meaningfully resemble our own. The factors that are detrimental to fish welfare have been well delineated by valid, objective indicators of physiological and behavioral well-being. This knowledge should guide welfare decisions. An empirical and non-anthropomorphic examination of diverse fishes and their adaptations should be the foundation for welfare decisions that would be truly beneficial to fishes and humans alike.

Novel interneuronal network in the mouse posterior piriform cortex.

The neural circuits of the piriform cortex mediate field potential oscillations and complex functions related to integrating odor cues with behavior, affective states, and multisensory processing. Previous anatomical studies have established major neural pathways linking the piriform cortex to other cortical and subcortical regions and major glutamatergic and GABAergic neuronal subtypes within the piriform circuits. However, the quantitative properties of diverse piriform interneurons are unknown. Using quantitative neural anatomical analysis and electrophysiological recording applied to a GAD65-EGFP transgenic mouse expressing GFP (green fluorescent protein) under the control of the GAD65 promoter, here we report a novel inhibitory network that is composed of neurons positive for GAD65-EGFP in the posterior piriform cortex (PPC). These interneurons had stereotyped dendritic and axonal properties that were distinct from basket cells or interneurons expressing various calcium-binding proteins (parvalbumin, calbindin, and calretinin) within the PPC. The GAD65-GFP neurons are GABAergic and outnumbered any other interneurons (expressing parvalbumin, calbindin, and calretinin) we studied. The firing pattern of these interneurons was highly homogenous and is similar to the regular-spiking nonpyramidal (RSNP) interneurons reported in primary sensory and other neocortical regions. Robust dye coupling among these interneurons and expression of connexin 36 suggested that they form electrically coupled networks. The predominant targets of descending axons of these interneurons were the dendrites of Layer III principal cells. Additionally, synapses were found on dendrites and somata of deep Layer II principal neurons and Layer III basket cells. A similar interneuronal subtype was also found in GAD65-EGFP-negative mouse. The extensive dendritic bifurcation at superficial lamina IA among horizontal afferent fibers and unique axonal targeting pattern suggests that these interneurons may play a role in direct feedforward inhibitory and disinhibitory olfactory processing. We conclude that the GAD65-GFP neurons may play distinct roles in regulating information flow and olfactory-related oscillation within the PPC in vivo.

Identification of roughskin newt medullary vasotocin target neurons with a fluorescent vasotocin conjugate.

Arginine8 vasotocin (AVT), a neurohypophyseal peptide in nonmammalian vertebrates, plays a key role in the regulation of social behaviors related to reproduction. In male roughskin newts (Taricha granulosa), AVT is an important facilitator of several reproductive behaviors, including courtship clasping of females. Although AVT is known to act in certain brain regions and AVT receptors have been localized to some extent, specific target neurons for this peptide have not been identified in any species. Internalization of a receptor-specific conjugate of AVT and the fluorescent dye Oregon green was used to identify AVT target cells in the medulla of male roughskin newts. Medullary neurons are of interest because they appear to mediate facilitation of clasping by AVT. Direct application of AVT-Oregon green to the fourth ventricular surface of the medulla in vivo resulted in conjugate internalization by a widespread population of medullary neurons, particularly in the medial reticular formation and nuclei of cranial nerves V, VII, VIII, IX, and X. Some fourth-ventricle ependymal cells were also labeled. Reticulospinal neurons, which play an important role in clasping, were identified by retrograde labeling with tetramethylrhodamine dextran amine. AVT-Oregon green was internalized by 72% of these neurons. These results show that AVT can directly affect a very large and diverse medullary neuronal population, which may underlie the peptide's role in multiple neuroendocrinological processes, including autonomic and behavioral regulation. Selectivity of the AVT action may arise through interactions between AVT and steroids such as corticosterone.

Identification of brain target neurons using a fluorescent conjugate of corticotropin-releasing factor.

Corticotropin-releasing factor (CRF) is a peptide well known for its role in coordinating various neuroendocrine, autonomic, and behavioral components of the vertebrate stress response, including rapid enhancement of locomotor activity. Although CRF's locomotor enhancing properties are well documented, the neuronal mechanisms and specific target neurons that underlie the peptide's effect on locomotor behavior remain poorly understood. In the present study, we describe the synthesis and functional characteristics of a CRF rhodamine analogue TAMRA-X conjugate mixture (CRF-TAMRA 1), to be used for tracking this peptide's internalization into target neurons in the brainstem of an amphibian, the roughskin newt (Taricha granulosa). CRF-TAMRA 1 conjugate administration into the lateral cerebral ventricle resulted in a rapid, endosomal-like internalization of fluorescence into brainstem medullary neurons. In addition, central CRF-TAMRA 1 administration produced neurobehavioral effects comparable to the native peptide, effects that were blocked by pre-treatment with the CRF receptor antagonist, alpha-helical CRF. Taken together, our results show the efficacy of CRF-TAMRA 1 as a novel tool for tracking CRF internalization into targets neurons in vivo and ultimately, aiding in elucidating the neuronal mechanisms and circuitry underlying CRF's influence on behavioral and physiological responses to stress.

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.

Rapid corticosterone-induced impairment of amplectic clasping occurs in the spinal cord of roughskin newts (taricha granulosa).

Courtship clasping, a reproductive behavior in male roughskin newts (Taricha granulosa), is rapidly blocked by an action of corticosterone (CORT) at a specific neuronal membrane receptor. The CORT-induced impairment of clasping in behaving newts appears to be mediated partly by an elimination of clasping-related activity in medullary reticulospinal neurons. Previous studies of rapid CORT actions in Taricha have focused on the brain, so existence of CORT action in the spinal cord or peripheral nervous system has not been assessed. The present study used newts with a high cervical spinal transection to examine potential spinal or peripheral CORT effects on clasping by the hindlimbs in response to pressure on the cloaca. Spinal transection causes clasps elicited by cloacal stimulation to be very sustained beyond the termination of the eliciting stimulus. In spinally transected newts, CORT caused a dose-dependent depression in the duration as well as quality of the clasp that appeared within 10 min of injection. CORT selectively impaired the usual sustained maintenance of a clasp after termination of cloacal stimulation, but not clasp elicitation during stimulation. These effects were not produced by dexamethasone, a synthetic glucocorticoid that binds poorly to the CORT membrane receptor. The CORT effect on clasp maintenance but not clasp elicitation implies selective action on an intraspinal generator for clasping but not on sensory or efferent neuromuscular aspects of the response. These results indicate the presence in the newt spinal cord of the CORT membrane receptor that exerts functional effects distinctly different from those on the brainstem.

Fluorescent vasotocin conjugate for identification of the target cells for brain actions of vasotocin.

The effects of neuropeptides on the brain are a major focus of neuroendocrine research, and little progress has been made in the identification of the target neurons for many neuropeptides. Arginine8-vasotocin (AVT) is a neurohypophyseal peptide present in nonmammalian vertebrates that controls many neural and behavioral functions. Here we describe synthesis and functional characterization of an AVT-Oregon green conjugate 1 (AVT-OG 1) that can be used in vivo to identify AVT target neurons. Application of AVT-OG 1 to the brainstem of an amphibian produces rapid, endosome-like internalization together with typical AVT-like neurophysiological effects. Thus, preparation of AVT-OG 1, which preserves the peptide's neurophysiological effects, is useful as a fluorescent marker for AVT target neurons. Consequently, AVT-OG 1 conjugate will have considerable utility for analyzing the neural actions of AVT in the intact brain.