A neurobehavioral model for rapid actions of corticosterone on sensorimotor integration.

Stress-induced corticosterone (CORT) secretion that causes a rapid blockade of courtship clasping by male roughskin newts (Taricha granulosa) is mediated by a specific neuronal membrane receptor for CORT. Amplectic clasping, which can be triggered by pressure on the ventral body surface and cloaca, is controlled by the influence of medullary neurons on the spinal cord. Using clasping as a simple neurobehavioral model, we have focused our analysis of CORT effects on clasping by examining the steroid's effects on neurophysiological properties of medullary neurons, especially medullary reticulospinal neurons, the principal output cells from the brain to the spinal cord. Systemic CORT caused, within 3 min of injection, diverse reductions in reticulospinal neuron excitability. Another rapid CORT effect on medullary neurons was to depress responsiveness to pressure on the cloaca. Experiments with chronically implanted, freely moving newts revealed that the rapid CORT effects are quite specific to neural processes related to clasping. CORT injections rapidly blocked clasping in response to cloacal stimuli and concurrently depressed neuronal responses to cloacal pressure and firing associated with clasping. Activity of reticulospinal neurons was often associated with nonclasping movements and this activity was rarely altered by CORT. Thus, CORT mainly affected aspects of neuronal function related to clasping. In other neurophysiological experiments, we found that the neuropeptides vasotocin and corticotropin-releasing hormone modified the neural effects of CORT. Prior exposure of medullary neurons to either of these neuropeptides caused systemic CORT administration to rapidly potentiate neuronal responses to cloacal stimuli, indicating that the direction and potency of CORT effects depend critically on the prevailing neuroendocrine state of the brain.     

Rapid, Corticosterone-Induced Disruption of Medullary Sensorimotor Integration Related to Suppression of Amplectic Clasping in Behaving Roughskin Newts (Taricha granulosa)

Endogenously secreted or injected corticosterone (CORT) rapidly suppresses courtship clasping in male roughskin newts (Taricha granulosa) by an action on a specific neuronal membrane receptor. Previous studies, using immobilized newts, showed that CORT administration rapidly depresses excitability of reticulospinal neurons and attenuates medullary neuronal responsiveness to clasp-triggering sensory stimuli. The present study used freely moving newts to examine clasping responses and concurrently record sensorimotor properties of 67 antidromically identified reticulospinal and other medullary reticular neurons before and after CORT injection. Before CORT, reticulospinal neurons fired in close association with onset and offset of clasps elicited by cloacal pressure. Reticulospinal neurons also showed firing correlates of nonclasping motor events, especially locomotion. Neuronal activity was typically reduced during clasping and elevated during locomotion. Medullary neurons that were not antidromically invaded (unidentified neurons) usually showed sensorimotor properties that resembled those of reticulospinal neurons. Intraperitoneal CORT (but not vehicle) reduced the probability and quality of hindlimb clasping in response to cloacal pressure, especially within 5–25 min of injection. Simultaneously, responses of reticulospinal and unidentified neurons to cloacal pressure and occurrence of clasping-related activity were attenuated or eliminated. CORT effects were relatively selective, altering clasping-related neuronal activity more strongly than activity associated with nonclasping motor events. The properties of CORT effects indicate that the hormone impairs clasping by depressing processing of clasp-triggering afferent activity and by disrupting the medullary control of clasping normally mediated by reticulospinal neurons. The rapid onset of these CORT effects implicates a neuronal membrane receptor rather than genomic action of the steroid.     

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.     

Androgens and male mating behavior in rough-skinned newts, Taricha granulosa

Cyproterone acetate was administered either orally or intraperitoneally to intact, adult male newts, Taricha granulosa. The number of males that exhibited the courtship behavior of clasping when tested with nuptial females was not altered by the antiandrogen treatments. In males which were unresponsive to nuptial females, the occurrence of clasping was not evoked by injections for 4 days of testosterone, dihydrotestosterone, or 11-ketotestosterone. Further, the incidence of clasping was not significantly elevated by injections of prolactin and/or testosterone for 30 days. The effect of sexual activity on testosterone and dihydrotestosterone levels in male newts was determined by radioimmunoassay of plasma collected from males which were: (1) isolated from females; (2) allowed to clasp a female for 2 min; or (3) allowed to clasp a female for 1 hr. The testosterone and dihydrotestosterone levels were unchanged during this period of clasping. In February and again in June, plasma androgen concentrations were measured in males which differed in their propensity to initiate courtship when paired with females. Androgen levels were similar for males that clasped a female and males that never attempted to clasp a female. Plasma androgen levels in the male newt are apparently not correlated with sexual responsiveness.     

Corticosteroid actions from neuronal membrane to behavior: neurophysiological mechanisms underlying rapid behavioral effects of corticosterone.

Investigation of the rapid suppression of male courtship clasping behavior by corticosterone in roughskin newts (Taricha granulosa) has led to the identification of a specific neuronal membrane receptor for this stress steroid. This paper describes studies of the neurophysiological effects of the rapid, membrane receptor mediated action of corticosterone on neurons that are involved in the control of clasping. In freely behaving newts, medullary neurons, including reticulospinal neurons, process clasp-triggering sensory signals and participate in control of clasping movements. Corticosterone injection causes these brainstem neurons to show selective depression of clasping-related sensorimotor function. These corticosterone effects appear in 3-10 min and are closely associated with the simultaneous depression of clasping. In addition to these functionally specific effects, corticosterone simultaneously causes widespread, primarily depressive effects on neuronal activity and excitability in the medulla and elsewhere in the brain. Thus, the membrane actions of corticosterone lead to diverse neural effects, including changes in membrane excitability as well as specific, network-level actions that are apparent only during behavior. These rapid corticosterone effects strongly interact with actions of the neuropeptides vasotocin and corticotropin-releasing factor, such that the form and magnitude of the steroid's effects depend on the prevailing neuroendocrine state of the brain.     

Sex steroids and vasotocin interact in a female amphibian (Taricha granulosa) to elicit female-like egg-laying behavior or male-like courtship.

Female egg-laying behaviors and male amplectic clasping behaviors in the rough-skinned newt (Taricha granulosa) are similar in that animals clasp an object. In the case of egg-laying, females clasp submerged inanimate objects, whereas in amplexus, males clasp conspecific females. Considering these behavioral similarities and differences, we investigated the possibility that gonadal steroids and vasotocin (AVT) interact to control egg-laying behaviors, as has been shown for the control of amplexus in Taricha males. Intact, gravid T. granulosa females injected ip with AVT, unlike those injected with saline, exhibited egg-laying behaviors and oviposition. In ovariectomy-steroid-implant studies, no saline-injected female exhibited egg-laying behaviors, whereas AVT-injected ovariectomized females exhibited egg-laying behaviors if implanted with estradiol (E2), testosterone, or dihydrotestosterone (DHT), and not if implanted with empty capsules. When given a choice between clasping aquatic vegetation or other females (amplectic clasping), following an AVT injection, unoperated and sham-operated control females and ovariectomized females with E2 implants did not preferentially clasp aquatic vegetation over other females. In contrast, AVT-injected ovariectomized females with DHT implants preferentially clasped other females. Thus, exposure of Taricha females to estrogens or androgens appears to determine whether the AVT-induced clasping is egg-laying or amplectic clasping.     

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/0.1 mL 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.     

Pheromones enhance somatosensory processing in newt brains through a vasotocin-dependent mechanism

We tested whether the sex pheromones that stimulate courtship clasping in male roughskin newts do so, at least in part, by amplifying the somatosensory signals that directly trigger the motor pattern associated with clasping and, if so, whether that amplification is dependent on endogenous vasotocin (VT). Female olfactory stimuli increased the number of action potentials recorded in the medulla of males in response to tactile stimulation of the cloaca, which triggers the clasp motor reflex, as well as to tactile stimulation of the snout and hindlimb. That enhancement was blocked by exposing the medulla to a V1a receptor antagonist before pheromone exposure. However, the antagonist did not affect medullary responses to tactile stimuli in the absence of pheromone exposure, suggesting that pheromones amplify somatosensory signals by inducing endogenous VT release. The ability of VT to couple sensory systems together in response to social stimulation could allow this peptide to induce variable behavioural outcomes, depending on the immediate context of the social interaction and thus on the nature of the associated stimuli that are amplified. If widespread in vertebrates, this mechanism could account for some of the behavioural variability associated with this and related peptides both within and across species.     

Intracranial administration of corticotropin-like peptides increases incidence of amphibian reproductive behavior

Male rough-skinned newts (Taricha granulosa) exhibit an increase in sexual behavior (amplectic clasping) following intracerebroventricular (ICV) injection of adrenocorticotropin (ACTH 1–39), ACTH 4–10, or melanocyte-stimulating hormone (αMSH). In contrast, intraperitoneal (IP) administration of ACTH 1–39 or corticosterone significantly decreases the incidence of sexual behavior in male newts. These results suggest that a corticotropin-like peptide acts centrally to enhance sexual behavior and that systemic ACTH acts on the interrenal tissue to inhibit sexual behavior by stimulating the release of corticosterone.     

Duration of androgen treatment modifies behavioral response to arginine vasotocin in Taricha granulosa

Five and eighteen days after surgery, injection of arginine 8-vasotocin (AVT) stimulated clasping behavior in castrated controls (unimplanted or cholesterol-implanted) but not in androgen-implanted, castrated newts (Taricha granulosa). Conversely, AVT injected 33 days after castration increased the incidence of courtship behavior of androgen-implanted males, but not of unimplanted castrates. Plasma androgen concentration of androgen-implanted castrates (as determined by radioimmunoassay) was maintained at a level typical of intact males at the peak of the breeding season. Because the AVT-androgen interaction could occur at the level of the pituitary gland, castrated, androgen-implanted newts were hypophysectomized and injected with AVT. Hypophysectomy did not abolish the behavioral response to AVT.     

Male-Male Clasping May Be Part of an Alternative Reproductive Tactic in Xenopus laevis

Male Xenopus laevis frogs have been observed to clasp other males in a sustained, amplectant position, the purpose of which is unknown. We examined three possible hypotheses for this counter-intuitive behavior: 1) clasping males fail to discriminate the sex of the frogs they clasp; 2) male-male clasping is an aggressive or dominant behavior; or 3) that males clasp other males to gain proximity to breeding events and possibly engage in sperm competition. Our data, gathered through a series of behavioral experiments in the laboratory, refute the first two hypotheses. We found that males did not clasp indiscriminately, but showed a sex preference, with most males preferentially clasping a female, but a proportion preferentially clasping another male. Males that clasped another male when there was no female present were less likely to “win” reproductive access in a male-male-female triad, indicating that they did not establish dominance through clasping. However, those males did gain proximity to oviposition by continued male-male clasping in the presence of the female. Thus, our findings are consistent with, but cannot confirm, the third hypothesis of male-male clasping as an alternative reproductive tactic.     

The effects of sex steroids and vasotocin on behavioral responses to visual and olfactory sexual stimuli in ovariectomized female roughskin newts

Previous studies have found that vasotocin (AVT) administration to male roughskin newts (Taricha granulosa) enhances courtship clasping as well as appetitive responses to specific sexual stimuli and that treating female newts with androgens plus AVT induces the expression of male-typical courtship clasping (the selective clasping of females). However, the unique and/or interactive effects of sex steroids and AVT on appetitive responses to specific sexual stimuli have not yet been determined. To first identify male-typical, sexually dimorphic appetitive responses to female sexual stimuli, we tested intact newts during the breeding season and found that males, but not females, are attracted to female visual and pheromonal sexual stimuli. We then used ovariectomized (ovx) females implanted with empty silastic capsules (Blk) or with capsules containing testosterone (T), dihydrotestosterone (DHT), or estradiol (E2) and then injected with either saline or AVT to determine the effects of steroids and AVT, alone or in combination with each other, on male-typical behavioral responses to those stimuli. E2 treatment depressed responses toward female visual stimuli independently of AVT. On the other hand, only T-implanted, AVT-injected females displayed male-typical behavioral responses toward female olfactory stimuli, preferring to spend more time in proximity to female-scented than unscented newt models and selectively clasping the female-scented models. Together, these results support the conclusion that sex steroids and AVT influence behavioral responses to sexual stimuli via sensory-specific mechanisms. Furthermore, they suggest that T and AVT interact within the brain to influence sensorimotor processing in the pathways that integrate olfactory sexual stimuli into male-typical courtship behaviors.     

Opioid kappa-receptor agonists suppress sexual behaviors in male rough-skinned newts (Taricha granulosa)

Four experiments were performed to evaluate a possible opioid involvement in the regulation of sexual behavior (amplectic clasping of a female) in intact adult male rough-skinned newts (Taricha granulosa) during the breeding season. It was found that an ip injection of bremazocine, a kappa-receptor opiate agonist, can markedly reduce sexual activity and that an ip injection of naloxone can reverse this inhibition in a dose-dependent fashion. In contrast, in male newts that were sexually inactive before treatment, injections of naloxone failed to induce sexual behavior, suggesting that opioid mechanisms do not normally exert a tonic inhibition of amphibian sexual behavior. In addition, an injection of ethylketocyclazocine (another kappa-receptor agonist), but not morphine (a mu-receptor agonist) suppressed sexual behaviors of male newts. These results indicate that opioid mechanisms that include kappa-type opioid receptors may contribute to the regulation of sexual behavior in nonmammalian vertebrates.     

T5 spinal cord transection increases susceptibility to reperfusion-induced ventricular tachycardia by enhancing sympathetic activity in conscious rats

We recently documented that paraplegia (T(5) spinal cord transection) alters cardiac electrophysiology and increases the susceptibility to ventricular tachyarrhythmias induced by programmed electrical stimulation. However, coronary artery occlusion is the leading cause of death in industrially developed countries and will be the major cause of death in the world by the year 2020. The majority of these deaths result from tachyarrhythmias that culminate in ventricular fibrillation. beta-Adrenergic receptor antagonists have been shown to reduce the incidence of sudden cardiac death. Therefore, we tested the hypothesis that chronic T(5) spinal cord transection increases the susceptibility to clinically relevant ischemia-reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity. Intact and chronic (4 wk after transection) T(5) spinal cord-transected (T(5)X) male rats were instrumented to record arterial pressure, body temperature, and ECG. In addition, a snare was placed around the left main coronary artery. The susceptibility to sustained ventricular tachycardia produced by 2.5 min of occlusion and reperfusion of the left main coronary artery was determined in conscious rats by pulling on the snare. Reperfusion culminated in sustained ventricular tachycardia in 100% of T(5)X rats (susceptible T(5)X, 10 of 10) and 0% of intact rats [susceptible intact, 0 of 10 (P < 0.05, T(5)X vs. intact)]. Beta-adrenergic receptor blockade prevented reperfusion-induced sustained ventricular tachycardia in T(5)X rats [susceptible T(5)X 0 of 8, 0% (P < 0.05)]. Thus paraplegia increases the susceptibility to reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity.     

Locomotion induced by epidural stimulation in a decerebrated cat after spinal cord injury

The effect of partial and complete spinal cord transection (Th7–Th8) on locomotor activity evoked in decerebrated cats by electrical epidural stimulation (segment L5, 80–100 μA, 0.5 ms at 5 Hz) has been investigated. Transection of dorsal columns did not substantially influence the locomotion. Disruption of the ventral spinal quadrant resulted in deterioration and instability of the locomotor rhythm. Injury to lateral or medial descending motor systems led to redistribution of the tone in antagonist muscles. Locomotion could be evoked by epidural stimulation within 20 h after complete transection of the spinal cord. The restoration of polysynaptic components in EMG responses correlated with recovery of the stepping function. The data obtained confirm that initiation of locomotion under epidural stimulation is caused by direct action on intraspinal systems responsible for locomotor regulation. With intact or partially injured spinal cord, this effect is under the influence of supraspinal motor systems correcting and stabilizing the evoked locomotor pattern.     

Locomotion induced by spinal cord stimulation in the neonate rat in vitro.

The present studies employed the neonate rat brain stem-spinal cord preparation to determine whether electrical stimulation of the lumbosacral enlargement (LE) of the spinal cord itself can be used to elicit locomotion, and whether or not such stimulation persists in inducing locomotion following midthoracic spinal cord transection or hindlimb deafferentation. Results suggest that (1) stimulation of the dorsal columns or ventral funiculus of the LE is effective in inducing airstepping in the neonatal rat brain stem-spinal cord limb-attached preparation; (2) central disconnection by midthoracic spinal cord transection does not alter LE-stimulation-induced airstepping and may lead to an increase in stepping frequency if suprathreshold stimulation is used; and (3) dorsal root section also leads to an increase in the frequency of suprathreshold LE-stimulation-induced locomotion, but there is not further increase in frequency if a spinal cord transection is performed in addition to dorsal rhizotomy.     

Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone

In addition to the classic genomic effects, increasing evidence suggests that GC can generate multiple rapid effects on many tissues and cells through nongenomic pathway. In the present study, the effects of corticosterone (CORT) on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord astrocytes were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of [Ca²⁺]i. CORT (0.01–10 μM) caused a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured dorsal spinal cord astrocytes. The action of CORT on astrocytic [Ca²⁺]i was blocked by pertussis toxin (a blocker of G protein activation, 100 ng/ml), but was unaffected by RU38486 (glucocorticoid receptor antagonist, 10 μM). In addition, cycloheximide (protein-synthesis inhibitor, 10 μg/ml) pretreatment could not impair the CORT-evoked [Ca²⁺]i elevation. Furthermore, Ca²⁺ mobilization induced by CORT was abolished by chelerythrine chloride (protein kinase C inhibitor, 10 μM), but was not impaired by H89 (protein kinase A inhibitor, 10 μM). These observations suggest that a nongenomic pathways might be involved in the effect of CORT on [Ca²⁺]i in cultured dorsal spinal cord astrocytes. In addition, our results also raise a possibility that a putative pertussis toxin-sensitive mGCR (G-protein-coupled membrane-bound glucocorticoid receptor) and the downstream activation of protein kinase C may be responsible for CORT-induced Ca²⁺ mobilization in cultured dorsal spinal cord astrocytes.     

Brain arginine vasotocin concentrations related to sexual behaviors and hydromineral balance in an amphibian

Arginine vasotocin (AVT), a potent stimulator of sexual behaviors and regulator of hydromineral balance in male rough-skinned newts (Taricha granulosa), was measured in 11 brain areas using microdissection and radioimmunoassay procedures. A 60-min test for sexual behaviors was used to segregate males into two groups: sexually responsive (initiated amplectic clasping behaviors) and sexually unresponsive (exhibited no sexual behaviors). Compared to sexually unresponsive males, sexually responsive males had significantly higher concentrations of immunoreactive (ir) AVT in the dorsal preoptic area, optic tectum, ventral infundibular nucleus, and cerebrospinal fluid. These results provide evidence for a behavioral action of endogenous AVT in T. granulosa. In another study, irAVT was measured in normal males (control newts maintained in water) and males that were dehydrated for 6 hr. Compared to normal males, dehydrated males had significantly lower concentrations of irAVT in the ventral preoptic area, but not in the other 10 areas of the brain. That different brain areas are associated with sexual behaviors and hydromineral balance suggests that there are some neuroanatomical separations between the behavioral and hydromineral aspects of the vasotocinergic system in this amphibian.     

Increased protein kinase C activity in the central nervous system of the newt during limb regeneration.

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.     

Stage-dependent restoration of sensory dorsal columns following spinal cord transection in anuran tadpoles

In frogs sensory axons from the lumbar dorsal roots ascend in the dorsal column of the spinal cord to terminate in the medulla and cerebellum. The response of these axons to complete transection of the thoracic spinal cord has been analysed in Rana temporaria tadpoles at different stages of development. The presence and position of dorsal column axons were assessed by using the anterograde transport of horseradish peroxidase or by electrophysiological methods. Before developmental stage VIII, dorsal column axons can grow across the transection and reach their normal areas of termination in the brainstem. Axons that do cross the transection follow their normal pathways. From stage VIII onwards this capacity for growth is largely lost. These results are discussed in terms of the relation between neurogenesis, axon growth and axonal regeneration.