Electrophysiological responses to conspecific odorants in Xenopus laevis show potential for chemical signaling.

The fully aquatic African clawed frog, Xenopus laevis, has an unusual and highly adapted nose that allows it to separately sample both airborne and waterborne stimuli. The function of the adult water nose has received little study, despite the fact that it is quite likely to receive information about conspecifics through secretions released into the water and could aid the frog in making decisions about social and reproductive behaviors. To assess the potential for chemical communication in this species, we developed an in situ electroolfactogram preparation and tested the olfactory responses of adult males to cloacal fluids and skin secretions from male and female conspecifics. We found robust olfactory responses to all conspecific stimuli, with greatest sensitivity to female cloacal fluids. These results open the door to further testing to identify compounds within cloacal fluids and skin secretions that are driving these responses and examine behavioral responses to those compounds. Understanding the role of chemical communication in social and reproductive behaviors may add to our rich understanding of vocal communication to create a more complete picture of social behavior in this species.

Electrophysiological and Motor Responses to Chemosensory Stimuli in Isolated Cephalopod Arms.

While there is behavioral and anatomical evidence that coleoid cephalopods use their arms to "taste" substances in the environment, the neurophysiology of chemosensation has been largely unexamined. The range and sensitivity of detectable chemosensory stimuli, and the processing of chemosensory information, are unknown. To begin to address these issues, we developed a technique for recording neurophysiological responses from isolated arms, allowing us to test responses to biologically relevant stimuli. We tested arms from both a pelagic species (Doryteuthis pealeii) and a benthic species (Octopus bimaculoides) by attaching a suction electrode to the axial nerve cord to record neural activity in response to chemical stimuli. Doryteuthis pealeii arms showed anecdotal responses to some stimuli but generally did not tolerate the preparation; tissue was nonviable within minutes ex vivo. Octopus bimaculoides arms were used successfully, with tissue remaining healthy and responsive for several hours. Arms responded strongly to fish skin extract, glycine, methionine, and conspecific skin extract but not to cephalopod ink or seawater controls. Motor responses were also observed in response to detected stimuli. These results suggest that chemosensory receptor cells on O. bimaculoides arms were able to detect environmentally relevant chemicals and drive local motor responses within the arm. Further exploration of potential chemical stimuli for O. bimaculoides arms, as well as investigations into the neural processing within the arm, could enhance our understanding of how this species uses its arms to explore its environment. While not successful in D. pealeii, this technique could be attempted with other cephalopod species, as comparative questions remain of interest.

Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication.

In many species, vocal communication is essential for coordinating social behaviors including courtship, mating, parenting, rivalry, and alarm signaling. Effective communication requires accurate production, detection, and classification of signals, as well as selection of socially appropriate responses. Understanding how signals are generated and how acoustic signals are perceived is key to understanding the neurobiology of social behaviors. Here we review our long-standing research program focused on Xenopus, a frog genus which has provided valuable insights into the mechanisms and evolution of vertebrate social behaviors. In Xenopus laevis, vocal signals differ between the sexes, through development, and across the genus, reflecting evolutionary divergence in sensory and motor circuits that can be interrogated mechanistically. Using two ex vivo preparations, the isolated brain and vocal organ, we have identified essential components of the vocal production system: the sexually differentiated larynx at the periphery, and the hindbrain vocal central pattern generator (CPG) centrally, that produce sex- and species-characteristic sound pulse frequencies and temporal patterns, respectively. Within the hindbrain, we have described how intrinsic membrane properties of neurons in the vocal CPG generate species-specific vocal patterns, how vocal nuclei are connected to generate vocal patterns, as well as the roles of neurotransmitters and neuromodulators in activating the circuit. For sensorimotor integration, we identified a key forebrain node that links auditory and vocal production circuits to match socially appropriate vocal responses to acoustic features of male and female calls. The availability of a well supported phylogeny as well as reference genomes from several species now support analysis of the genetic architecture and the evolutionary divergence of neural circuits for vocal communication. Xenopus thus provides a vertebrate model in which to study vocal communication at many levels, from physiology, to behavior, and from development to evolution. As one of the most comprehensively studied phylogenetic groups within vertebrate vocal communication systems, Xenopus provides insights that can inform social communication across phyla.

Probing forebrain to hindbrain circuit functions in Xenopus.

The vertebrate hindbrain includes neural circuits that govern essential functions including breathing, blood pressure and heart rate. Hindbrain circuits also participate in generating rhythmic motor patterns for vocalization. In most tetrapods, sound production is powered by expiration and the circuitry underlying vocalization and respiration must be linked. Perception and arousal are also linked; acoustic features of social communication sounds-for example, a baby's cry-can drive autonomic responses. The close links between autonomic functions that are essential for life and vocal expression have been a major in vivo experimental challenge. Xenopus provides an opportunity to address this challenge using an ex vivo preparation: an isolated brain that generates vocal and breathing patterns. The isolated brain allows identification and manipulation of hindbrain vocal circuits as well as their activation by forebrain circuits that receive sensory input, initiate motor patterns and control arousal. Advances in imaging technologies, coupled to the production of Xenopus lines expressing genetically encoded calcium sensors, provide powerful tools for imaging neuronal patterns in the entire fictively behaving brain, a goal of the BRAIN Initiative. Comparisons of neural circuit activity across species (comparative neuromics) with distinctive vocal patterns can identify conserved features, and thereby reveal essential functional components.

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.

Transformation of receptive field properties from lateral geniculate nucleus to superficial V1 in the tree shrew.

Tree shrew primary visual cortex (V1) exhibits a pronounced laminar segregation of inputs from different classes of relay neurons in the lateral geniculate nucleus (LGN). We examined how several receptive field (RF) properties were transformed from LGN to V1 layer 4 to V1 layer 2/3. The progression of RF properties across these stages differed markedly from that found in the cat. V1 layer 4 cells are largely similar to the the LGN cells that provide their input, being dominated by a single sign (ON or OFF) and being strongly modulated by sinusoidal gratings. Some layer 4 neurons, notably those near the edges of layer 4, exhibited increased orientation selectivity, and most layer 4 neurons exhibited a preference for lower temporal frequencies. Neurons in cortical layer 2/3 differ significantly from those in the LGN; most exhibited strong orientation tuning and both ON and OFF responses. The strength of orientation selectivity exhibited a notable sublaminar organization, with the strongest orientation tuned neurons in the most superficial parts of layer 2/3. Modulation indexes provide evidence for simple and complex cells in both layer 4 and layer 2/3. However, neurons with high modulation indexes were heterogenous in the spatial organization of ON and OFF responses, with many of them exhibiting unbalanced ON and OFF responses rather than well-segregated ON and OFF subunits. When compared to the laminar organization of V1 in other mammals, these data show that the process of natural selection can result in significantly altered structure/function relationships in homologous cortical circuits.

NMDAR-dependent control of call duration in Xenopus laevis.

Many rhythmic behaviors, such as locomotion and vocalization, involve temporally dynamic patterns. How does the brain generate temporal complexity? Here, we use the vocal central pattern generator (CPG) of Xenopus laevis to address this question. Isolated brains can elicit fictive vocalizations, allowing us to study the CPG in vitro. The X. laevis advertisement call is temporally modulated; calls consist of rhythmic click trills that alternate between fast (approximately 60 Hz) and slow (approximately 30 Hz) rates. We investigated the role of two CPG nuclei--the laryngeal motor nucleus (n.IX-X) and the dorsal tegmental area of the medulla (DTAM)--in setting rhythm frequency and call durations. We discovered a local field potential wave in DTAM that coincides with fictive fast trills and phasic activity that coincides with fictive clicks. After disrupting n.IX-X connections, the wave persists, whereas phasic activity disappears. Wave duration was temperature dependent and correlated with fictive fast trills. This correlation persisted when wave duration was modified by temperature manipulations. Selectively cooling DTAM, but not n.IX-X, lengthened fictive call and fast trill durations, whereas cooling either nucleus decelerated the fictive click rate. The N-methyl-d-aspartate receptor (NMDAR) antagonist dAPV blocked waves and fictive fast trills, suggesting that the wave controls fast trill activation and, consequently, call duration. We conclude that two functionally distinct CPG circuits exist: 1) a pattern generator in DTAM that determines call duration and 2) a rhythm generator (spanning DTAM and n.IX-X) that determines click rates. The newly identified DTAM pattern generator provides an excellent model for understanding NDMAR-dependent rhythmic circuits.

Xenopus vocalizations are controlled by a sexually differentiated hindbrain central pattern generator.

Male and female African clawed frogs (Xenopus laevis) produce rhythmic, sexually distinct vocalizations as part of courtship and mating. We found that Xenopus vocal behavior is governed by a sexually dimorphic central pattern generator (CPG) and that fictive vocalizations can be elicited from an in vitro brain preparation by application of serotonin or by electrical stimulation of a premotor nucleus. Male brains produced fictive vocal patterns representing two calls commonly produced by males in vivo (advertisement and amplectant call), as well as one call pattern (release call) that is common for juvenile males and females in vivo but rare for adult males. Female brains also produced fictive release call. The production of male calls is androgen dependent in Xenopus; to test the effects of androgens on the CPG, we examined fictive calling in the brains of testosterone-treated females. Both fictive male advertisement call and release call were produced. This suggests that all Xenopus possess a sexually undifferentiated pattern generator for release call. Androgen exposure leads to a gain-of-function, allowing the production of male-specific call types without prohibiting the production of the undifferentiated call pattern. We also demonstrate that the CPG is located in the brainstem and seems to rely on the same nuclei in both males and females. Finally, we identified endogenous serotonergic inputs to both the premotor and motor nuclei in the brainstem that may regulate vocal activity in vivo.