Orientation by environmental geometry and feature cues in the green and black poison frog (Dendrobates auratus).

The ability to use environmental geometry when orienting in space reflects an animal's ability to use a global, allocentric framework. Therefore, understanding when and how animal's use geometry relative to other types of cues in the environment has interested comparative cognition researchers for decades. Yet, only two amphibians have been tested to date. We trained the poison frog Dendrobates auratus to find goal shelters in a rectangular arena, in the presence and absence of a feature cue, and assessed the relative influence of the two types of cues using probe trials. We chose D. auratus because the species has complex interactions with their physical and social environments, including parental care that requires navigating to and from distant locations. We found that, like many vertebrates, D. auratus are capable of using geometric information to relocate goals. In addition, the frogs preferentially used the more reliable feature cue when the location of the feature conflicted with the geometry of the arena. The frogs were equally successful at using the feature cue when it was proximal or distal to the goal shelter, consistent with prior studies that found that D. auratus can use distal cues in a flexible manner. Our results provide further evidence that amphibians can use environmental geometry during orientation. Future studies that examine when and how amphibians use geometry relative to other types of cues will contribute to a more complete picture of spatial cognition in this important, yet understudied, group.

Quantity discrimination in a spontaneous task in a poison frog.

The use of quantitative information underlies a range of animal behaviors. There are thought to be two parallel systems for judging quantity: a precise representation of small numbers of objects, typically less than 4, that can be tracked visually (object tracking system) and an imprecise system for larger quantities (approximate number system) governed by Weber's law. Using a spontaneous discrimination task with live prey, we examined the ability of the poison frog Dendrobates auratus to discriminate quantities of low (1-4) or high (4-16) numerosity over a range of ratio contrasts (0.33, 0.5, 0.67, 0.75). Similar to a previous study in treefrogs, we found that the poison frogs chose the larger quantity of flies when choosing between 1 and 3 and between 1 and 2. However, their performance was near chance when choosing between 2 and 3 and below chance when choosing between 3 and 4. When the numerosity of flies was higher, they did not discriminate between the larger and smaller quantity. Our findings are consistent with the ability of poison frogs to discriminate small quantities of objects using an object tracking system, but could also reflect a singular vs. plural discrimination. We did not find evidence of an approximate number system governed by Weber's law, nor evidence of a speed-accuracy tradeoff. However, total set size was associated with lower accuracy and longer latencies to choose. Future studies should explore quantity discrimination in additional contexts to better understand the limits of these abilities in poison frogs.

Ecology, Cognition, and the Hippocampus: A Tale of Two Frogs.

The underlying hypothesis that motivates research into the relationship between ecology, cognition, and the hippocampus is that selection to solve problems in nature shapes cognition through changes in the hippocampus. This hypothesis has been explored almost exclusively in mammals and birds. However, if one is interested in the principles that shape the evolution of vertebrate cognition, work in amphibians is essential. To address this gap, we have developed a research program contrasting cognitive abilities and hippocampal neurobiology in two species of frog with distinct social and spatial ecologies. We have found that the poison frog Dendrobates auratus, a diurnal species whose interactions with the physical and social environment are complex, is more adept and flexible at spatial learning and learned inhibition than the túngara frog, a nocturnal species that lacks complex interactions with the spatial and social environment. Because spatial learning and learned inhibition are closely associated with hippocampal function in other vertebrates, we used RNA sequencing to characterize molecular differences in the hippocampus of the two species. We have found that D. auratus has greater levels of expression of genes associated with neurogenesis, synaptic plasticity, and cellular activity, and lower levels of expression of genes associated with apoptosis, compared to the túngara frog. Our studies are consistent with the idea that D. auratus, with their more complex social and spatial ecology, have enhanced hippocampally dependent cognitive abilities compared to túngara frogs. Further characterization of the features of hippocampal neurobiology that confer distinctive cognitive abilities will help elucidate the neural features that are necessary for the evolution of enhanced hippocampally dependent cognition.

Differential encoding of signals and preferences by noradrenaline in the anuran brain.

Social preferences enable animals to selectively interact with some individuals over others. One influential idea for the evolution of social preferences is that preferred signals evolve because they elicit greater neural responses from sensory systems. However, in juvenile plains spadefoot toad (Spea bombifrons), a species with condition-dependent mating preferences, responses of the preoptic area, but not of the auditory midbrain, mirror adult social preferences. To examine whether this separation of signal representation from signal valuation generalizes to other anurans, we compared the relative contributions of noradrenergic signalling in the preoptic area and auditory midbrain of S. bombifrons and its close relative Spea multiplicata We manipulated body condition in juvenile toads by controlling diet and used high pressure liquid chromatography to compare call-induced levels of noradrenaline and its metabolite MHPG in the auditory midbrain and preoptic area of the two species. We found that calls from the two species induced different levels of noradrenaline and MHPG in the auditory system, with higher levels measured in both species for the more energetic S. bombifrons call. In contrast, noradrenaline levels in the preoptic area mirrored patterns of social preferences in both S. bombifrons and S. multiplicata That is, noradrenaline levels were higher in response to the preferred calls within each species and were modified by diet in S. bombifrons (with condition-dependent preferences) but not S. multiplicata (with condition-independent preferences). Our results are consistent with a potentially important role for preoptic noradrenaline in the development of social preferences and indicate that it could be a target of selection in the evolution of condition-dependent social preferences.

A cognitive map in a poison frog.

A fundamental question in cognitive science is whether an animal can use a cognitive map. A cognitive map is a mental representation of the external world, and knowledge of one's place in this world, that can be used to determine efficient routes to any destination. Many birds and mammals are known to employ a cognitive map, but whether other vertebrates can create a cognitive map is less clear. Amphibians are capable of using beacons, gradients and landmarks when navigating, and many are proficient at homing. Yet only one prior study directly tested for a cognitive map in amphibians, with negative results. Poison frogs exhibit unusually complex social and spatial behaviors and are capable of long-distance homing after displacement, suggesting that they may be using complex spatial navigation strategies in nature. Here, we trained the poison frog Dendrobates auratus in a modified Morris water maze that was designed to suppress thigmotaxis to the maze wall, promoting exploration of the arena. In our moat maze, the poison frogs were able to use a configuration of visual cues to find the hidden platform. Moreover, we demonstrate that they chose direct paths to the goal from multiple random initial positions, a hallmark of a cognitive map. The performance of the frogs in the maze was qualitatively similar to that of rodents, suggesting that the potential to evolve a cognitive map is an evolutionarily conserved trait of vertebrates.

Monoaminergic integration of diet and social signals in the brains of juvenile spadefoot toads.

Social behavior often includes the production of species-specific signals (e.g. mating calls or visual displays) that evoke context-dependent behavioral responses from conspecifics. Monoamines are important neuromodulators that have been implicated in context-dependent social behavior, yet we know little about the development of monoaminergic systems and whether they mediate the effects of early life experiences on adult behavior. We examined the effects of diet and social signals on monoamines early in development in the plains spadefoot toad (Spea bombifrons), a species in which diet affects the developmental emergence of species recognition and body condition affects the expression of adult mating preferences. To do so, we manipulated the diet of juveniles for 6 weeks following metamorphosis and collected their brains 40 min following the presentation of either a conspecific or a heterospecific call. We measured levels of monoamines and their metabolites using high pressure liquid chromatography from tissue punches of the auditory midbrain (i.e. torus semicircularis), hypothalamus and preoptic area. We found that call type affected dopamine and noradrenaline signaling in the auditory midbrain and that diet affected dopamine and serotonin in the hypothalamus. In the preoptic area, we detected an interaction between diet and call type, indicating that diet modulates how the preoptic area integrates social information. Our results suggest that the responsiveness of monoamine systems varies across the brain and highlight preoptic dopamine and noradrenaline as candidates for mediating effects of early diet experience on later expression of social preferences.

Effects of estradiol on neural responses to social signals in female túngara frogs.

Estradiol plays an important role in mediating changes in female sexual behavior across reproductive cycles. In the túngara frog [Physalaemus (=Engystomops) pustulosus], the relationship between gonadal activity and female sexual behavior, as expressed by phonotaxis, is mediated primarily by estradiol. Estradiol receptors are expressed in auditory and motivational brain areas and the hormone could serve as an important modulator of neural responses to conspecific calls. To better understand how estradiol modifies neural responses to conspecific social signals, we manipulated estradiol levels and measured expression of the immediate early gene egr-1 in the auditory midbrain, thalamus and limbic forebrain in response to conspecific or heterospecific calls. We found that estradiol and conspecific calls increased egr-1 expression in the auditory midbrain and limbic forebrain, but in the thalamus, only conspecific calls were effective. In the preoptic area, estradiol enhanced the effect of the conspecific call on egr-1 expression, suggesting that the preoptic area could act as a hormonal gatekeeper to phonotaxis. Overall, the results suggest that estradiol has broad influences on the neural circuit involved in female reproduction, particularly those implicated in phonotaxis.

Social signals increase monoamine levels in the tegmentum of juvenile Mexican spadefoot toads (Spea multiplicata).

Monoamines are important neuromodulators that respond to social cues and that can, in turn, modify social responses. Yet we know very little about the ontogeny of monoaminergic systems and whether they contribute to the development of social behavior. Anurans are an excellent model for studying the development of social behavior because one of its primary components, phonotaxis, is expressed early in life. To examine the effect of social signals on monoamines early in ontogeny, we presented juvenile Mexican spadefoot toads (Spea multiplicata) with a male mating call or no sound and measured norepinephrine, epinephrine, dopamine, serotonin, and a serotonin metabolite, across the brain using high-pressure liquid chromatography. Our results demonstrate that adult-like monoaminergic systems are in place shortly after metamorphosis. Perhaps more interestingly, we found that mating calls increased the level of monoamines in the juvenile tegmentum, a midbrain region involved in sensory-motor integration and that contributes to brain arousal and attention. We saw no such increase in the auditory midbrain or in forebrain regions. We suggest that changes in monoamine levels in the juvenile tegmentum may reflect the effects of social signals on arousal state and could contribute to context-dependent modulation of social behavior.

Diet alters species recognition in juvenile toads.

Whether environmental effects during juvenile development can alter the ontogeny of adult mating behaviour remains largely unexplored. We evaluated the effect of diet on the early expression of conspecific recognition in spadefoot toads, Spea bombifrons. We found that juvenile toads display phonotaxis behaviour six weeks post-metamorphosis. However, preference for conspecifics versus heterospecifics emerged later and was diet dependent. Thus, the environment can affect the early development of species recognition in a way that might alter adult behaviour. Evaluating such effects is important for understanding variation in hybridization between species and the nature of species boundaries.

Ultrasound-evoked immediate early gene expression in the brainstem of the Chinese torrent frog, Odorrana tormota.

The concave-eared torrent frog, Odorrana tormota, has evolved the extraordinary ability to communicate ultrasonically (i.e., using frequencies > 20 kHz), and electrophysiological experiments have demonstrated that neurons in the frog's midbrain (torus semicircularis) respond to frequencies up to 34 kHz. However, at this time, it is unclear which region(s) of the torus and what other brainstem nuclei are involved in the detection of ultrasound. To gain insight into the anatomical substrate of ultrasound detection, we mapped expression of the activity-dependent gene, egr-1, in the brain in response to a full-spectrum mating call, a filtered, ultrasound-only call, and no sound. We found that the ultrasound-only call elicited egr-1 expression in the superior olivary and principal nucleus of the torus semicircularis. In sampled areas of the principal nucleus, the ultrasound-only call tended to evoke higher egr-1 expression than the full-spectrum call and, in the center of the nucleus, induced significantly higher egr-1 levels than the no-sound control. In the superior olivary nucleus, the full-spectrum and ultrasound-only calls evoked similar levels of expression that were significantly greater than the control, and egr-1 induction in the laminar nucleus showed no evidence of acoustic modulation. These data suggest that the sampled areas of the principal nucleus are among the regions sensitive to ultrasound in this species.

Auditory selectivity for acoustic features that confer species recognition in the tungara frog.

In anurans, recognition of species-specific acoustic signals is essential to finding a mate. In many species, behavioral tests have elucidated which acoustic features contribute to species recognition, but the mechanisms by which the brain encodes these species-specific signal components are less well understood. The túngara frog produces a `whine' mating call that is characterized by a descending frequency sweep. However, much of the signal is unnecessary for recognition, as recognition behavior can be triggered by a descending two-tone step that mimics the frequency change in a portion of the whine. To identify the brain regions that contribute to species recognition in the túngara frog, we exposed females to a full-spectrum whine, a descending two-tone step that elicits recognition, the reversed two-tone step that does not elicit recognition, or no sound, and we measured expression of the neural activity-dependent gene egr-1 in the auditory brainstem and thalamus. We found that the behavioral relevance of the stimuli was the best predictor of egr-1 expression in the laminar nucleus of the torus semicircularis but not elsewhere. That is, the laminar nucleus responded more to the whine and the two-tone step that elicits recognition than to the reversed two-tone step. In contrast, in other brainstem and thalamic nuclei, whines induced egr-1 expression but tones did not. These data demonstrate that neural responses in the laminar nucleus correspond to behavioral responses of females and they suggest that the laminar nucleus may act as a feature detector for the descending frequencies characteristic of conspecific calls.

Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of túngara frogs.

Sex steroid hormones are potent regulators of behavior and they exert their effects through influences on sensory, motor, and motivational systems. To elucidate where androgens and estrogens can act to regulate sex-typical behaviors in the túngara frog (Physalaemus pustulosus), we quantified expression of the androgen receptor (AR), estrogen receptor alpha (ERalpha), and estrogen receptor beta (ERbeta) genes in the brains of male and females. To do so, we cloned túngara-specific sequences for AR, ERalpha, and ERbeta, determined their distribution in the brain, and then quantified their expression in areas that are important in sexual communication. We found that AR, ERalpha, and ERbeta were expressed in the pallium, limbic forebrain (preoptic area, hypothalamus, nucleus accumbens, amygdala, septum, striatum), parts of the thalamus, and the auditory midbrain (torus semicircularis). Males and females had a similar distribution of AR and ER expression, but expression levels differed in some brain regions. In the auditory midbrain, females had higher ERalpha and ERbeta expression than males, whereas males had higher AR expression than females. In the forebrain, females had higher AR expression than males in the ventral hypothalamus and medial pallium (homolog to hippocampus), whereas males had higher ERalpha expression in the medial pallium. In the preoptic area, striatum, and septum, males and females had similar levels of AR and ER expression. Our results suggest that sex steroid hormones have sexually dimorphic effects on auditory processing, sexual motivation, and possibly memory and, therefore, have important implications for sexual communication in this system.

Integrative Comparative Cognition: Can Neurobiology and Neurogenomics Inform Comparative Analyses of Cognitive Phenotype?

A long-standing question in animal behavior is what are the patterns and processes that shape the evolution of cognition? One effective way to address this question is to study cognitive abilities in a broad spectrum of animals. While comparative psychologists have traditionally focused on a narrow range of organisms, today they may work with any number of species, from frogs to birds or bees. This broader range of study species has greatly enriched our understanding of the diversity of cognitive processes among animals. Yet, this diversity has highlighted the fundamental challenge of comparing cognitive processes across animals. An analysis of the neural and molecular mechanisms of cognition may be necessary to solve this problem. The goal of our symposium was to bring together speakers studying a range of species to gain a broadly integrative perspective on cognition while at the same time considering the potentially important role of neurobiology and genomics in addressing the difficult problem of comparing cognition across species. For example, work by MaBouDi et al. indicates that neural constraints on computing power may impact the cognitive processes underlying numerical discrimination in bees. A presentation by Lara LaDage demonstrated how neurobiology can be used to better understand cognition and its evolution in reptiles while Edwards et al. identify the cerebellum as potentially important in the performance of the complex process of nest building. We see that molecular approaches highlight the contributions of the prefrontal cortex and hippocampus to cognitive phenotype across vertebrates while, at the same time, identifying the genes and cellular processes that may contribute to evolution of cognition. The potentially important role of neurogenesis and synaptic plasticity emerge clearly from such studies. Still unanswered is the question of whether molecular tools will contribute to our ability to discriminate convergent/parallel evolution from homology in the evolution of cognitive phenotype.

Cognitive Phenotype and Differential Gene Expression in a Hippocampal Homologue in Two Species of Frog.

The complexity of an animal's interaction with its physical and/or social environment is thought to be associated with behavioral flexibility and cognitive phenotype, though we know little about this relationship in amphibians. We examined differences in cognitive phenotype in two species of frog with divergent natural histories. The green-and-black poison frog (Dendrobates auratus) is diurnal, displays enduring social interactions, and uses spatially distributed resources during parental care. Túngara frogs (Physalaemus=Engystomops pustulosus) are nocturnal, express only fleeting social interactions, and use ephemeral puddles to breed in a lek-type mating system. Comparing performance in identical discrimination tasks, we find that D. auratus made fewer errors when learning and displayed greater behavioral flexibility in reversal learning tasks than túngara frogs. Further, túngara frogs preferred to learn beacons that can be used in direct guidance whereas D. auratus preferred position cues that could be used to spatially orient relative to the goal. Behavioral flexibility and spatial cognition are associated with hippocampal function in mammals. Accordingly, we examined differential gene expression in the medial pallium, the amphibian homolog of the hippocampus. Our preliminary data indicate that genes related to learning and memory, synaptic plasticity, and neurogenesis were upregulated in D. auratus, while genes related to apoptosis were upregulated in túngara frogs, suggesting that these cellular processes could contribute to the differences in behavioral flexibility and spatial learning we observed between poison frogs and túngara frogs.

Estradiol induces sexual behavior in female túngara frogs.

Steroid hormones play an important role in regulating vertebrate sexual behavior. In frogs and toads, injections of exogenous gonadotropins, which stimulate steroid hormone production, are often used to induce reproductive behavior, but steroid hormones alone are not always sufficient. To determine which hormonal conditions promote sexual behavior in female túngara frogs, we assessed the effect of hormone manipulation on the probability of phonotaxis behavior toward conspecific calls in post-reproductive females. We injected females with human chorionic gonadotropin (HCG), estradiol, estradiol plus progesterone, saline, or HCG plus fadrozole (an aromatase blocker) and tested their responses to mating calls. We found that injections of HCG, estradiol, and estradiol plus progesterone all increased phonotaxis behavior, whereas injections of saline or HCG plus fadrozole did not. Since injections of estradiol alone were effective at increasing phonotaxis behavior, we concluded that estradiol is sufficient for the expression of phonotaxis behavior. Next, to determine if estradiol-injected females display the same behavioral preferences as naturally breeding females, we compared mating call preferences of naturally breeding females to those of post-reproductive females injected with estradiol. We found that, when injected with estradiol, females show similar call preferences as naturally breeding females, although they were less likely to respond across multiple phonotaxis tests. Overall, our results suggest that estradiol is sufficient for the expression of sexual responses to mating calls in túngara frogs. To our knowledge, ours is the only study to find that estradiol alone is capable of promoting phonotaxis behavior in a frog.

Cytoarchitecture of a cichlid fish telencephalon.

Although the telencephalon of ray-finned fishes has garnered considerable attention from comparative neuroanatomists, detailed descriptions of telencephalic organization are available for only a few species. This necessarily limits our understanding of telencephalic evolution, particularly in light of the extraordinary diversity of ray-finned fishes. Thus, we have charted the cyctoarchitecture of the telencephalon of the African cichlid fish, Astatotilapia (Haplochromis) burtoni. We examined tissue sectioned in the transverse plane, and categorized cell groups based on size, shape, and staining intensity of cells, the density and distribution of cells, cell-poor zones, and relationship of cell groups to the anterior commissure and external sulci. In addition, to facilitate visualization of the transitions among cell groups, we aligned and animated a series of 100 sequential brain sections. We found that the A. burtoni telencephalon was similar to other percomorphs in being highly elaborated with many distinct cell groups. In the pallium, Dm, Dl, and Dc had a large number of cell groups, whereas Dd and Dp were more uniform. Although we recognized many similarities between the pallium of A. burtoni and other teleosts, we also recognized two cell groups (Dl-g and Dm-2) that might represent specializations of cichlids. We found that the subpallium had a similar organization to that of other ray-finned fishes.

Reconsidering sex differences during place learning in túngara frogs.

In túngara frogs, female mate choice requires remembering the location and/or calls of preferred males who advertise from fixed positions within a breeding pond. A previous study found that, when solving a place discrimination task in the laboratory, female túngara frogs were able to learn a visual cue to solve the task, whereas males were not. In that task, male performance appeared to be inhibited, in part, by their attempt to use egocentric cues. We tested whether the sex difference in place learning previously reported would generalize to other training parameters with different cues available by eliminating the potential to use egocentric cues and increasing the number of trials per day. As before, frogs were given a choice between a red or yellow door, one of which led to shelters and return to their home cage. In the current testing conditions, we detected a preference for the red door; thus, we only considered frogs rewarded to the yellow door. Training was associated with an increase in correct choices and an increased preference for the yellow door. However, there was no evidence for a sex difference in learning. In summary, under the current training conditions, we found that the apparent female advantage in place learning was no longer evident. Future studies that investigate sex differences in cue preference and/or ability to switch among cues will further illuminate the conditions under which sex differences in learning are manifest in túngara frogs.

Rapid behavioral and genomic responses to social opportunity.

From primates to bees, social status regulates reproduction. In the cichlid fish Astatotilapia (Haplochromis) burtoni, subordinate males have reduced fertility and must become dominant to reproduce. This increase in sexual capacity is orchestrated by neurons in the preoptic area, which enlarge in response to dominance and increase expression of gonadotropin-releasing hormone 1 (GnRH1), a peptide critical for reproduction. Using a novel behavioral paradigm, we show for the first time that subordinate males can become dominant within minutes of an opportunity to do so, displaying dramatic changes in body coloration and behavior. We also found that social opportunity induced expression of the immediate-early gene egr-1 in the anterior preoptic area, peaking in regions with high densities of GnRH1 neurons, and not in brain regions that express the related peptides GnRH2 and GnRH3. This genomic response did not occur in stable subordinate or stable dominant males even though stable dominants, like ascending males, displayed dominance behaviors. Moreover, egr-1 in the optic tectum and the cerebellum was similarly induced in all experimental groups, showing that egr-1 induction in the anterior preoptic area of ascending males was specific to this brain region. Because egr-1 codes for a transcription factor important in neural plasticity, induction of egr-1 in the anterior preoptic area by social opportunity could be an early trigger in the molecular cascade that culminates in enhanced fertility and other long-term physiological changes associated with dominance.

Acoustic modulation of immediate early gene expression in the auditory midbrain of female túngara frogs.

To better understand the molecular consequences of auditory processing in frogs, we investigated the acoustic modulation of two immediate early genes (IEGs), egr-1 and fos, in the auditory midbrain of female túngara frogs. Since túngara frog egr-1 had already been identified, we first isolated a túngara-specific fos clone using degenerate PCR followed by Rapid Amplification of cDNA Ends. In order to examine the temporal kinetics of acoustically modulated IEG mRNA expression, we first acoustically isolated females collected from a mating chorus and analyzed the decline in IEG expression in the torus semicircularis (homolog of the inferior colliculus). We found that IEG mRNA levels declined rapidly and reached baseline within 2 h. Next, we presented females with a 30-min recording of a mating chorus and analyzed IEG expression following different survival times. We found that IEG expression increased within 15-30 min of sound presentation but, compared to other vertebrates, in the túngara frog it took longer to reach the highest and lowest mRNA levels in response to sound and isolation, respectively. We also found that acoustic stimulation of egr-1 and fos differed in the three subdivisions of the torus semicircularis, suggesting that, as in birds, the two genes could provide largely different information when used in IEG mapping studies. While our results confirm the generality of sensory-induced IEG expression in vertebrates, whether the longer time course of IEG expression that we observed represents a species difference in the mechanisms of IEG transcription awaits further study.

Acoustically evoked immediate early gene expression in the pallium of female túngara frogs.

In anurans, much is known about the role of the auditory midbrain in processing conspecific calls, but comparatively little is known about the role of the pallium. To address this deficiency, we investigated the induction of the immediate early gene egr-1 by natural mate chorus in the medial, dorsal, lateral, and ventral pallium of female túngara frogs. We found strong acoustically evoked egr-1 expression in the dorsal medial pallium (p < 0.01) and ventral pallium (p = 0.02), with a weaker effect in the lateral pallium (p = 0.05). In the ventral pallium, acoustically induced egr-1 expression was stronger in the anterior portion. Measures of movement and olfactory activity could not explain a significant portion of acoustically evoked pallial egr-1 expression. In contrast, egr-1 expression in the auditory midbrain covaried with egr-1 expression in the dorsal medial pallium and ventral pallium, suggesting that their activity was coupled with auditory activity. Taken together, these results suggest that the acoustically evoked egr-1 expression in the dorsal medial pallium and ventral pallium were a direct result of auditory stimulation. Furthermore, although both anatomical and electrophysiological evidence demonstrate that multiple modalities overlap in the frog pallium, our results show that a multimodal stimulus is not required to activate pallial neurons. Although the functional role of the frog pallium is not known, our results demonstrate that species-specific sounds activate spatially segregated and anatomically distinct areas of the frog pallium, inviting further investigation into the role of the frog pallium in acoustic communication.