Central control of mating call production and spawning in the tree frog Hyla arborea savignyi (Audouin): Results of electrical stimulation of the brain

Electrical stimulation of the anterior preoptic nucleus elicited the production of mating calls by male Hyla arborea savignyi. Such calls are composed of pulses resembling those of natural mating calls both in their duration and in their sequential arrangement. As a rule, the amplitude of successive pulses within a group increases more rapidly in the electrically evoked calls than in natural calls. The frogs could be brought into a state of readiness to call, which did not lead to actual calling activity, by stimulation in the anterior preoptic, posterior preoptic, and magnocellular nuclei as well as in the hypothalamus. Stimulation of the anterior preoptic nucleus of one female elicited repeated vocalizations, which may be interpretable as mating calls. The posture adopted by the female for spawning can be elicited in males as well as in females by electrical stimulation of the anterior preoptic nucleus.     

Anuran mating calling circuits: inhibition by prostaglandin

Male American toads (Bufo americanus) were induced to mating call in response to electronically simulated, conspecific mating calls. The injection of prostaglandin (PG) F2 alpha caused suppression of mating call answering. Neural correlates of mating calling were triggered by electrical stimulation of the anterior preoptic nucleus in the isolated brainstem of male, Northern leopard frogs (Rana p. pipiens). The addition of PGF2 alpha to the bath completely abolished the correlates of mating calling without changing the correlates of pulmonary respiration. The suppression of mating calling shown here, along with the suppression of release signalling described by Diakow and Nemiroff (1981), supports the hypothesis of a close interrelation between the neural circuits of these two calls. The suppression of the neural correlates of mating calling in an isolated preparation shows a central site of action of the PG. The retention of normal correlates of pulmonary respiration, even after suppression of mating calling correlates, suggests that the generation of mating calling patterns involves the extension and pulsing of the expiratory phase of breathing.     

Mating call phonotaxis in female American toad: lesions of anterior preoptic nucleus

Lesions of the preoptic area and immediately adjacent septal area were made in female American toads, Bufo americanus. The ability to show mating call phonotaxis (MCP) was then tested for. The intensity of MCP was found to decrease as the degree of damage to the anterior tip of the anterior preoptic nucleus (APON) increased. The APON is known to concentrate androgens and to be essential for mating calling by male anurans. Perhaps the APON is involved also in the detection of the elevated levels of prostaglandin associated with MCP. Even in lesioned toads in which phonotaxis was nearly absent, there was an occasional MCP response that seemed entirely normal. Therefore, the APON is not involved in generating or guiding the motor mechanisms of phonotaxis.     

Response of ERalpha-IR and ERbeta-IR cells in the forebrain of female rats to mating stimuli

Sexual behavior in female rats depends on the action of estradiol on estrogen receptors (ERs) found in particular brain regions. While hormonal regulation of female sexual behavior requires ERalpha, the possible functions of ERbeta remain to be clarified. Mating stimulation has several behavioral and physiological consequences and induces Fos expression in many brain areas involved in the regulation of reproductive behavior and physiology. In addition, some cells in which mating induces Fos expression coexpress ERalpha. To determine whether cells in which Fos is induced by a particular mating stimulus coexpress ERalpha, ERbeta, or both, we used a triple-label immunofluorescent technique to visualize ERalpha-, ERbeta-, and mating-induced Fos-immunoreactivity (Fos-ir) in neurons in which mating stimulation reliably increases Fos expression. Ovariectomized, hormone-primed rats were either unmated, received 15 mounts, or received 15 intromissions. In the rostral medial preoptic area, Fos-ir was induced by mounts alone primarily in cells coexpressing ERalpha-ir, while Fos-ir was induced by intromissions mainly in cells coexpressing both ERalpha-ir and ERbeta-ir (ERalpha/ERbeta-ir). In the dorsal part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions in cells coexpressing ERalpha-ir and ERalpha/ERbeta-ir. However, in the ventral part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions primarily in cells coexpressing only ERbeta-ir. These data suggest that qualitatively different sexual stimuli may be integrated through distinct ER-containing circuits in the rostral medial preoptic area and posterodorsal medial amygdala. The diversity in coexpression of type of ER in cells in different brain areas after various mating stimuli suggests a role for both ERalpha and ERbeta in the integration of hormonal information and information related to mating stimuli.     

Social Context Influences Androgenic Effects on Calling in the Green Treefrog (Hyla cinerea)

Courtship behavior in frogs is an ideal model for investigating the relationships among social experience, gonadal steroids, and behavior. Reception of mating calls causes an increase in androgen levels in listening males, and calling, in turn, depends on the presence of androgens. However, previous studies found that androgen replacement does not always restore calling to intact levels, and the relationship between androgens and calling may be context dependent. We examined the influence of androgens on calling behavior in the presence and the absence of social signals in male green treefrogs (Hyla cinerea). We categorized calling during an acoustic stimulus (mating chorus or tones) as evoked and calling in the absence of a stimulus as spontaneous. Intact males received a cholesterol implant, castrated males were castrated and received a cholesterol implant, and T-implanted males were castrated and received a testosterone implant. The androgen levels (mean +/- SE ng/ml of plasma) achieved by the implants were as follows: castrated males, 1.2 +/- 0.2; intact males 21.9 +/- 7.0; T-implanted males, 254.6 +/- 39.5. As in other frogs, calling depends on the presence of androgens, as castration abolished and T replacement maintained calling. However, among intact and T-implanted males, the influence of androgens on calling differed between spontaneous and evoked calling. There was a positive effect of androgen treatment on spontaneous call rate and a positive correlation between spontaneous call rate and androgen levels. The influence of androgen levels on evoked call rate was more complex and interacted with acoustic treatment. Surprisingly, T implants suppressed the chorus-specific increase in calling that is evident in intact males. In addition, in response to the chorus, T-implanted males called less than did intact males, in spite of higher androgen levels. Furthermore, variation in androgens did not explain variation in evoked call rate. These data indicate that androgens influence the motivation to call, but that, when socially stimulated, androgens are necessary but insufficient for calling.     

Neural correlates of frog calling

Summary A tissue bath incorporating a screen for support of the specimen and an air-lift pump to circulate saline across the screen was designed to provide maximum exposure of isolated frog brainstems ofRana pipiens pipiens to oxygenated saline (Fig. 1). Normal neural correlates of electrically-evoked mating calling were recorded from the region of the pretrigeminal nucleus and the laryngeal nerve in the isolated brainstem (Fig. 3A) and isolated hemi-brainstem (Fig. 2) of the Northern leopard frog. Conspicuous slow-wave activity in the region of the pretrigeminal nucleus supports the possibility that this may be an important integrative area for calling. It appears that the pretrigeminal region is not able, independently, to generate the pulses of the vocal phase of calling. Synchronizing and reinforcing inter-connections between the calling mechanisms of the two sides were identified. The data are summarized in a revised model of mating calling (Fig. 7).This work was supported by NINDS grant NS-06673. The electronic equipment was set up and maintained by Mr. Wayne R. Hudson. I am grateful to Dr. William Van Meter for suggesting the Sylgard for the pinning block and to Dr. Patricia Gallagher for suggesting the saline solution of Phillis and Tebcis.     

Neuroendocrine Mechanisms of Environmental Integration

SYNOPSIS. In the dwarf Siberian hamster, Phodopus sungorus,the photoperiodic response can be modified by numerous environmentalstimuli, including social interactions, dietary, and climaticchanges. Photoperiodic information is processed in both themedial basal hypothalamus and the preoptic area. Transfer ofanimals from a long summer photoperiod to a short winter photoperiodresults in decreases in the concentration of both norepinephrineand dopamine in both of these brain areas. Results from thesestudies indicate that both dietary supplements and social interactionscan override the effects of day length on changes in brain neurotransmitters.Specifically, social interactions can override the decreasesin norepinephrine and dopamine in the medial basal hypothalamusbut not the preoptic area. Conversely, dietary manipulationsoverride the decreases in the preoptic area but not in the medialbasal hypothalamus. These results suggest that photoperiod isa general stimulus that depresses neurotransmitter activityin multiple brain areas including the medial basal hypothalamus,and preoptic area. Fine tuning information, such as dietaryand social cues, is then processed in very specific areas ofthe brain and can override the photoperiod induced changes inthese specific areas     

Proteomic Analysis of the Maternal Preoptic Area in Rats

The behavior of female rats changes profoundly as they become mothers. The brain region that plays a central role in this regulation is the preoptic area, and lesions in this area eliminates maternal behaviors in rodents. The molecular background of the behavioral changes has not been established yet; therefore, in the present study, we applied proteomics to compare protein level changes associated with maternal care in the rat preoptic area. Using 2-dimensional fluorescence gel electrophoresis followed by identification of altered spots with mass spectrometry, 12 proteins were found to be significantly increased, and 6 proteins showed a significantly reduced level in mothers. These results show some similarities with a previous proteomics study of the maternal medial prefrontal cortex and genomics approaches applied to the preoptic area. Gene ontological analysis suggested that most altered proteins are involved in glucose metabolism and neuroplasticity. These proteins may support the maintenance of increased neuronal activity in the preoptic area, and morphological changes in preoptic neuronal circuits are known to take place in mothers. An increase in the level of alpha-crystallin B chain (Cryab) was confirmed by Western blotting. This small heat shock protein may also contribute to maintaining the increased activity of preoptic neurons by stabilizing protein structures. Common regulator and target analysis of the altered proteins suggested a role of prolactin in the molecular changes in the preoptic area. These results first identified the protein level changes in the maternal preoptic area. The altered proteins contribute to the maintenance of maternal behaviors and may also be relevant to postpartum depression, which can occur as a molecular level maladaptation to motherhood.

     

Preoptic hypnogenic area and reticular activating system

The stimulation, in the encéphale isolé cat, of the basal preoptic hypnogenic area be brief electrical pulses evokes bilaterally an extracellular positive (P) field potential of 20 to 60 msec duration in the brain stem and thalamic activating ascending reticular system. The properties of this P wave have led to consider it as the extracellular and abbreviated counterpart of an hyperpolarizing postsynaptic inhibitory process which, by the functional depression it exerts on the arousal system, would be instrumental in the induction and maintenance of synchronized sleep. The positive field potential response of the reticular system shows the same recruiting build-up and amplitude modulation, and the same sensibility to amphetamine and to barbiturates, as the cortical potential of diffuse distribution which is evoked simultaneously. It is strychnine--and picrotoxin--resistant. Preoptic stimulation exerts, within a 100 msec interval, a strong suppressive effect on the excitatory response evoked in the n. centromedian by a mesencephalic reticular testing shock. On the other hand, the application of the latter as a conditioning stimulus results in a marked increase of the amplitude of the P wave response of the CM to a testing preoptic shock. A negative feedback interpretation of this interaction is suggested. No clear evidence of a tonic functioning of the preoptic hypnogenic structure could be found in experiments involving the production of small bilateral lesions in the basal preoptic area in the encéphale isolé cat. Reasons limiting the interpretation of this negative result are given. The functional significance, on the basis of experimental data, of the diffuse cortical synchronization produced by the low frequency stimulation of the basal preoptic area and of other hypnogenic structures is discussed.     

The role of the medial preoptic area in the organization of paradoxical sleep

Influence of electrical stimulation of the medial preoptic area of cats on characteristics of paradoxical sleep and activity of medial preoptic neurons were studied in the course of sleep-waking cycle. Low-frequency stimulation of this structure in the state of slow-wave sleep evoked short-latency electrocortical desynchronization and induced transition to paradoxical sleep or paradocical sleep-like state. The same stimulation during the whole period of paradoxical sleep results in a reduction of its duration, practically complete disappearance of tonic stage, and increase in the density of rapid eye movements in phasic stage. The vast majority of meurons in the medial preoptic area decreased their firing rates during quiet waking and slow-wave sleep and dramatically increased their activity during paradoxical sleep. More than 50% of such neurons displayed activation 20-70 s prior to the appearance of electrocorticographic correlates of paradoxical sleep. Some neurons were selectively active during paradoxical sleep. Approximately 50% of cells increased their firing rates a few seconds prior to and/or during series of rapid eye movements. The results suggest that the medial preoptic area contains the units of the executive system (network) of paradoxical sleep and are involved in the mechanisms of neocortical desynchronization.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.     

From social behavior to neural circuitry: steroid hormones rapidly modulate advertisement calling via a vocal pattern generator

Across vertebrates, androgens are rapidly elevated within minutes in response to aggressive or reproductive stimuli, yet it is unclear what the causal relationship is between fast androgen elevation and the ongoing (minute-by-minute) expression of behavior. This study tested the hypothesis that rapid increases in plasma steroid levels induce similarly rapid increases in both vocal behavior and the neurophysiological output of a central pattern generator that governs vocal behavior. In Gulf toadfish (Opsanus beta), males call to attract females to their nesting sites, and both males and females vocalize in aggressive interactions. Previous field experiments with males showed that simulated territorial challenges produce rapid and concurrent elevations in ongoing calling behavior and circulating levels of the teleost-specific androgen 11-ketotestosterone (11kT), but not the glucocorticoid cortisol. The current field experiments showed that non-invasive (food) delivery of 11kT, but not cortisol, induced an elevation within 10 min in the ongoing calling behavior of males. Electrophysiological experiments revealed that intramuscular injections of either 11kT or cortisol, but neither testosterone nor 17-beta-estradiol, induced increases within 5 min in the output of the vocal pattern generator in males, whereas only cortisol had similarly fast effects in females. The field behavioral results support predictions generated by the challenge hypothesis and also parallel the 11kT-dependent modulation of the vocal pattern generator in males. The cortisol effect on the vocal pattern generator in both sexes predicts that glucocorticoids regulate vocalizations in non-advertisement contexts. Together, these experiments provide strong support for the hypothesis that surges in circulating steroid levels play a causal role in shaping rapid changes in social behavior (vocalizations) through non-genomic-like actions on neural (vocal motor) circuits that directly encode behavioral patterning.     

Cerebral Evoked Potential Correlates in Forced-paced Tasks

CEREBRAL evoked potentials can be monitored by averaging scalp records in subjects performing psychological tasks. The waveform is affected not only by the physical features of the sensory stimulus but also by cognitive and motivational parameters^1–7. These can be manipulated through verbal instructions given to the subject and several studies have shown that a slow positive component of about 300 ms peak latency (P₃₀₀) can be elicited by stimuli to which perceptual significance is attached^2,5–8. We have examined the evoked potential correlates of sensory overload in forced-paced auditory tasks and find that the cerebral “decision” potential can reveal intermittency in the perceptual channel.     

Acoustic modulation of neural activity in the preoptic area and ventral hypothalamus of the green treefrog (Hyla cinerea)

Summary Responses of neurons in the preoptic area and ventral hypothalamus to conspecific mating calls or white noise bursts were examined in male green treefrogs (Hyla cinerea) during different seasons. In the winter, 34.3% of preoptic neurons and 46.7% of ventral hypothalamic cells demonstrated significant changes in activity level during presentation of a conspecific mating call. In contrast, only 13.3% of preoptic units and 16.7% of ventral hypothalamic cells responded to the white noise. The percentage of preoptic and hypothalamic units responding to the advertisement call did not differ significantly during the summer breeding season. Type I units exhibited a dramatic increase in activity during acoustic stimulation followed by a rapid return to baseline activity levels after stimulus offset. Type II cells showed a robust activity increase during stimulation, but maintained an intermediate activity level after stimulus offset. In the preoptic area, a third response type exhibited suppressed activity during acoustic stimulation. Although seasonal condition did not alter the percentage of acoustically responsive units within either nucleus, the proportion of Type I units in the ventral hypothalamus was greatest during the summer.Abbreviations MC mating call - NS no stimulus - POA preoptic area - VH ventral hypothalamus - WN white noise     

Lung respiratory rhythm and pattern generation in the bullfrog: role of neurokinin-1 and μ-opioid receptors

Location of the lung respiratory rhythm generator (RRG) in the bullfrog brainstem was investigated by examining neurokinin-1 and μ-opioid receptor (NK1R, μOR) colocalization by immunohistochemistry and characterizing the role of these receptors in lung rhythm and episodic pattern generation. NK1R and μOR occurred in brainstems from all developmental stages. In juvenile bullfrogs a distinct area of colocalization was coincident with high-intensity fluorescent labeling of μOR; high-intensity labeling of μOR was not distinctly and consistently localized in tadpole brainstems. NK1R labeling intensity did not change with development. Similarity in colocalization is consistent with similarity in responses to substance P (SP, NK1R agonist) and DAMGO (μOR agonist) when bath applied to bullfrog brainstems of different developmental stages. In early stage tadpoles and juvenile bullfrogs, SP increased and DAMGO decreased lung burst frequency. In juvenile bullfrogs, SP increased lung burst frequency, episode frequency, but decreased number of lung bursts per episode and lung burst duration. In contrast, DAMGO decreased lung burst frequency and burst cycle frequency, episode frequency, and number of lung bursts per episode but increased all other lung burst parameters. Based on these results, we hypothesize that NK1R and μOR colocalization together with a metamorphosis-related increase in μOR intensity marks the location of the lung RRG but not necessarily the lung episodic pattern generator.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Summary Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement.These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.Supported by grants from the IBRO/MacArthur Foundation Network Grant, the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266), the National Foundation of Technical Development (OKKFT Tt 286/1986) and the Well-come Trust (14685/1.5)     

The Role of Steroid Modulation of Amino Acid Transmitters in the Regulation of Female Reproduction

SYNOPSIS. The amino acid transmitters can be placed in two generalcategories, excitatory and inhibitory. This discussion focuseson the role of the inhibitory transmitter GAB A and the excitatoryamino acids aspartate and glutamate in the control of gonadotropinsecretion and reproductive behavior. GABAergic neurotransmissionin the preoptic area inhibits gonadotropin secretion via directsynaptic contact with LHRH neurons and possibly through presynapticinhibition of noradrenergic fibers that stimulate LH release.In the arcuate-median eminence, GABA acting at GABAA receptorsincreases gonadotropin release by inhibiting a currently unidentifiedinhibitory interneuron. In regard to reproductive behavior,GABA acting in the preoptic area inhibits female sexual receptivitywhereas GABA in the mediobasal hypothalamus and the midbraincentral gray facilitates this behavior. The effects of GABAon reproductive behavior do not appear to be secondary to actionson defensive or locomotor behavior. Gonadal steroids modulateactivity at the GABAA receptor in a highly complex manner andthese effects may be involved in the role GABA plays in controllinggonadotropin secretions as well as behavior. The excitatory amino acids also affect gonadotropin secretion,exerting a stimulatory effect both in the preoptic area andat the level of the median eminence. When a specific antagonistfor one of the excitatory amino acid receptors is infused intothe preoptic area or when an excitatory amino acid receptoragonist is infused into the mediobasal hypothalamus, femalesexual behavior is inhibited. There have only been limited reportsof steroid modulation of excitatory amino acid neurotransmission.     

Testosterone induction of male-typical sexual behavior is associated with increased preoptic NADPH diaphorase and citrulline production in female whiptail lizards

In rodents, male-typical copulatory behavior is generally dependent on gonadal sex steroids such as testosterone, and it is thought that the mechanism by which the hormone gates the behavior involves the gaseous neurotransmitter nitric oxide. According to one model, testosterone induces an up-regulation of nitric oxide synthase (NOS) in the preoptic area, increasing nitric oxide synthesis following exposure to a sexual stimulus. Nitric oxide in turn, possibly through its effect on catecholamine turnover, influences the way the stimulus is processed and enables the appropriate copulatory behavioral response. In whiptail lizards (genus Cnemidophorus), administration of male-typical levels of testosterone to females induces the display of male-like copulatory responses to receptive females, and we hypothesized that this radical change in behavioral phenotype would be accompanied by a large change in the expression of NOS in the preoptic area. As well as comparing NOS expression using NADPH diaphorase histochemistry between testosterone-treated females and controls, we examined citrulline immunoreactivity (a marker of recent nitric oxide production) in the two groups, following a sexual stimulus and following a nonsexual stimulus. Substantially more NADPH diaphorase-stained cells were observed in the testosterone-treated animals. Citrulline immunoreactivity was greater in testosterone-implanted animals than in blank-implanted animals, but only following exposure to a sexual stimulus. This is the first demonstration that not only is NOS up-regulated by testosterone, but NOS thus up-regulated is activated during male-typical copulatory behavior.     

Electrical stimulation of the preoptic area in Eigenmannia: evoked interruptions in the electric organ discharge

The functional role of the basal forebrain and preoptic regions in modulating the normally regular electric organ discharge was determined by focal brain stimulation in the weakly electric fish, Eigenmannia. The rostral preoptic area, which is connected with the diencephalic prepacemaker nucleus, was examined physiologically by electrical stimulation in a curarized fish. Electrical stimulation of the most rostral region of the preoptic area with trains of relatively low intensity current elicits discrete bursts of electric organ discharge interruptions in contrast to other forebrain loci. These responses were observed primarily as after-responses following the termination of the stimulus train and were relatively immune to variations in the stimulus parameters. As the duration and rate of these preoptic-evoked bursts of electric organ discharge interruptions (approximately 100 ms at 2 per s) are similar to duration and rate of natural interruptions, it is proposed that these bursts might be precursors to natural interruptions. These data suggest that the preoptic area, consistent with its role in controlling reproductive behaviors in vertebrates, may be influencing the occurrence of electric organ discharge courtship signals by either direct actions on the prepacemaker nucleus or through other regions that are connected with the diencephalic prepacemaker nucleus. Accepted: 16 October 1999