Image-free recognition of moderate ROP from mild with machine learning algorithm on plasma Raman spectrum.

The retinopathy of prematurity (ROP) can cause serious clinical consequences and, fortunately, it is remediable while the time window for treatment is relatively narrow. Therefore, it is urgent to screen all premature infants and diagnose ROP degree timely, which has become a large workload for pediatric ophthalmologists. We developed a retinal image-free procedure using small amount of blood samples based on the plasma Raman spectrum with the machine learning model to automatically classify ROP cases before medical intervention was performed. Statistical differences in infrared Raman spectra of plasma samples were found among the control, mild (ZIIIS1), moderate (ZIIIS2 & ZIIS1), and advanced (ZIIS2) ROP groups. With the different wave points of Raman spectra as the inputs, the outputs of our support vector machine showed that the area under the curves in the receiver operating characteristic (AUC) were 0.763 for the pair comparisons of the control with the mild groups, 0.821 between moderate and advanced groups (ZIIS2), while more than 90% in comparisons of the other four pairs: control vs. moderate (0.981), control vs. advanced (0.963), mild vs. moderate (0.936), and mild vs. advanced (0.953), respectively. Our study could advance principally the ROP diagnosis in two dimensions: the moderate ROPs have been classified remarkably from the mild ones, which leaves more time for the medical treatments, and the procedure of Raman spectrum with a machine learning model based on blood samples can be conveniently promoted to those hospitals lacking of the pediatric ophthalmologists with experience in reading retinal images.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

The Screening of Therapeutic Peptides for Anti-Inflammation through Phage Display Technology.

For the treatment of inflammatory illnesses such as rheumatoid arthritis and carditis, as well as cancer, several anti-inflammatory medications have been created over the years to lower the concentrations of inflammatory mediators in the body. Peptides are a class of medication with the advantages of weak immunogenicity and strong activity, and the phage display technique is an effective method for screening various therapeutic peptides, with a high affinity and selectivity, including anti-inflammation peptides. It enables the selection of high-affinity target-binding peptides from a complex pool of billions of peptides displayed on phages in a combinatorial library. In this review, we will discuss the regular process of using phage display technology to screen therapeutic peptides, and the peptides screened for anti-inflammation properties in recent years according to the target. We will describe how these peptides were screened and how they worked in vitro and in vivo. We will also discuss the current challenges and future outlook of using phage display to obtain anti-inflammatory therapeutic peptides.

Noise constrains the evolution of call frequency contours in flowing water frogs: a comparative analysis in two clades.

BACKGROUND: The acoustic adaptation hypothesis (AAH) states that signals should evolve towards an optimal transmission of the intended information from senders to intended receivers given the environmental constraints of the medium that they traverse. To date, most AAH studies have focused on the effect of stratified vegetation on signal propagation. These studies, based on the AAH, predict that acoustic signals should experience less attenuation and degradation where habitats are less acoustically complex. Here, we explored this effect by including an environmental noise dimension to test some AAH predictions in two clades of widespread amphibians (Bufonidae and Ranidae) that actively use acoustic signals for communication. By using data from 106 species in these clades, we focused on the characterization of the differences in dominant frequency (DF) and frequency contour (i.e., frequency modulation [FM] and harmonic performances) of mating calls and compared them between species that inhabit flowing-water or still-water environments. RESULTS: After including temperature, body size, habitat type and phylogenetic relationships, we found that DF differences among species were explained mostly by body size and habitat structure. We also showed that species living in lentic habitats tend to have advertisement calls characterized by well-defined FM and harmonics. Likewise, our results suggest that flowing-water habitats can constrain the evolutionary trajectories of the frequency-contour traits of advertisement calls in these anurans. CONCLUSIONS: Our results may support AAH predictions in frogs that vocalize in noisy habitats because flowing-water environments often produce persistent ambient noise. For instance, these anurans tend to generate vocalizations with less well-defined FM and harmonic traits. These findings may help us understand how noise in the environment can influence natural selection as it shapes acoustic signals in affected species.

Multisensory modalities increase working memory for mating signals in a treefrog.

Animal choruses, such as those found in insects and frogs, are often intermittent. Thus, females sampling males in the chorus might have to remember the location of the potential mates' calls during periods of silence. Although a number of studies have shown that frogs use and prefer multimodal mating signals, usually acoustic plus visual, it is not clear why they do so. Here we tested the hypothesis that preference for multimodal signals over unimodal signals might be due to multimodal signals instantiating longer memories than unimodal signals, particularly during the inter-chorus intervals. We tested this hypothesis in serrate-legged small treefrogs Kurixalus odontotarsus whose males produce advertisement calls accompanied by conspicuous vocal sac inflation. Females were tested with acoustic and acoustic + visual (video of inflating-deflating vocal sac) mating calls. We found that females prefer multimodal calls over unimodal, audio-only calls. Furthermore, multimodal calls are still preferred after a silent period of up to 30 s, a time that spans the average silent period of the chorus. This was not true of unimodal calls. Our results demonstrate that a multimodal signal can engage longer working memory than a unimodal signal, and thus female memory might favour the evolution of multimodal signals in males through sexual selection. Selection might also favour female preference for multimodal signals if longer memory facilitates mate searching and assessment. Our study does not allow us to elucidate the sequence of evolution of this trait and preference.

Metabolomic changes of blood plasma associated with two phases of rat OIR.

Although acute hyperoxia/hypoxia alternation can shift sharply physiological processes of vessel development, e.g. oxygen induced retinopathy (OIR), very little is known of metabolic products resulted from the neovascularization disorder. In this study, the influence of abnormal oxygen exposures on the plasma metabolomic profiles of rats with OIR was investigated by the gas chromatography mass spectrometry (GC-MS). Rat pups were divided into four groups, each with 12 individuals: (i) reared in room air and sampled at P12 (CT1); (ii) exposed to high oxygen for 5 days and sampled at P12 (HO1, simulating the vaso-obeliteration process (phase I)); (iii) reared in room air and sampled at P17 (CT2); (iv) exposed to high oxygen for 5 days then followed by room air for 5 days and sampled at P17 (HO2, simulating the neovasculization one (phase II)). Plasma samples were analyzed with GC-MS, resulted in 122 metabolite species. Distinct differences in the plasma metabolome were found between groups of CT1 vs. HO1, and HO1 vs. HO2, by using univariate and multivariate analyses. Alternating hyperoxia/hypoxia conditions induced significant changes of richness of proline, ornithine and glutamine, that were important components of 'arginine and proline metabolism' pathway. These metabolites contributed largely to plasma sample classification, determined with receiver operating characteristic curve analysis and were involved profoundly in the proline-dependent production of reactive oxygen species (ROS) related to the cellular redox reactions. Our results from the rat OIR model suggest proline and 'arginine and proline metabolism' pathway as the potential biomarkers for human retinopathy of prematurity (ROP) diagnosis.

Sex differences in vocalization are reflected by event-related potential components in the music frog.

Sex differences in vocalization have been commonly found in vocal animals. It remains unclear, however, how animals perceive and discriminate these differences. The amplitudes and latencies of event-related potentials (ERP) components can reflect the auditory processing efficiency and time course. We investigated the neural mechanisms of auditory processing in the Emei music frog (Nidirana daunchina) using an Oddball paradigm with ERP. We recorded and analyzed eletroencephalogram (EEG) signals from the forebrain and midbrain when the subjects listened to white noise (WN) and conspecific sex-specific vocalizations. We found that (1) both amplitudes and latencies of some ERP components evoked by conspecific calls were significantly higher than those by WN, suggesting the music frogs can discriminate conspecific vocalizations from background noise; (2) both amplitudes and latencies of most ERP components evoked by female calls were significantly higher or longer than those by male calls, implying that the ERP components can reflect sex differences in vocalization; and (3) there were significant differences in ERP amplitudes between male and female subjects, suggesting a sexual dimorphism in auditory perception. Together, the present results indicate that the music frog could discriminate conspecific calls from noise, male's calls from female's ones, and sexual dimorphism of auditory perception existed in this species.

Preference of spectral features in auditory processing for advertisement calls in the music frogs.

BACKGROUND: Animal vocal signals encode very important information for communication during which the importance of temporal and spectral characteristics of vocalizations is always asymmetrical and species-specific. However, it is still unknown how auditory system represents this asymmetrical and species-specific patterns. In this study, auditory event related potential (ERP) changes were evaluated in the Emei music frog (Babina daunchina) to assess the differences in eliciting neural responses of both temporal and spectral features for the telencephalon, diencephalon and mesencephalon respectively. To do this, an acoustic playback experiment using an oddball paradigm design was conducted, in which an original advertisement call (OC), its spectral feature preserved version (SC) and temporal feature preserved version (TC) were used as deviant stimuli with synthesized white noise as standard stimulus. RESULTS: The present results show that 1) compared with TC, more similar ERP components were evoked by OC and SC; and 2) the P3a amplitudes in the forebrain evoked by OC were significantly higher in males than in females. CONCLUSIONS: Together, the results provide evidence for suggesting neural processing for conspecific vocalization may prefer to the spectral features in the music frog, prompting speculation that the spectral features may play more important roles in auditory object perception or vocal communication in this species. In addition, the neural processing for auditory perception is sexually dimorphic.

Auditory sensitivity exhibits sexual dimorphism and seasonal plasticity in music frogs.

Seasonal changes in the structure and function of the vertebrate brain have been described in many species, particularly in seasonal breeders. However, it remains unclear whether sexual dimorphism varies between breeding seasons for specific brain regions. Auditory event-related potential (ERP) changes were evaluated in the Emei music frog (Babina daunchina) to assess sexual dimorphism and seasonal variations in auditory sensitivity. An acoustic playback experiment using an oddball paradigm design was conducted, in which two conspecific call types were used as deviant stimuli with synthesized white noise as standard stimulus. ERP components were analyzed for the telencephalon and mesencephalon of both sexes in the non-reproductive and reproductive states. Results show that auditory sensitivity is modulated by reproductive status, implying that seasonal plasticity is involved in auditory perception. Moreover, the amplitude of the N1 ERP component (mean amplitudes during the interval occurring 30-130 ms after stimulus onset) is higher in females for the telencephalon and higher in males for the mesencephalon, regardless of reproductive status and acoustic stimulus type. These results show that auditory ERP responses for specific brain regions exhibit sexual dimorphism in the absence of exogenous sexual stimulation during both the two reproductive states in the music frog.

Auditory neural networks involved in attention modulation prefer biologically significant sounds and exhibit sexual dimorphism in anurans.

Allocating attention to biologically relevant stimuli in a complex environment is critically important for survival and reproductive success. In humans, attention modulation is regulated by the frontal cortex, and is often reflected by changes in specific components of the event-related potential (ERP). Although brain networks for attention modulation have been widely studied in primates and avian species, little is known about attention modulation in amphibians. The present study aimed to investigate the attention modulation networks in an anuran species, the Emei music frog (Babina daunchina). Male music frogs produce advertisement calls from within underground nest burrows that modify the acoustic features of the calls, and both males and females prefer calls produced from inside burrows. We broadcast call stimuli to male and female music frogs while simultaneously recording electroencephalographic (EEG) signals from the telencephalon and mesencephalon. Granger causal connectivity analysis was used to elucidate functional brain networks within the time window of ERP components. The results show that calls produced from inside nests which are highly sexually attractive result in the strongest brain connections; both ascending and descending connections involving the left telencephalon were stronger in males while those in females were stronger with the right telencephalon. Our findings indicate that the frog brain allocates neural attention resources to highly attractive sounds within the window of early components of ERP, and that such processing is sexually dimorphic, presumably reflecting the different reproductive strategies of males and females.

The thermal background determines how the infrared and visual systems interact in pit vipers.

The thermal infrared (IR) sensing system of pit vipers is believed to complement vision and provide a substitute imaging system in dark environments. Theoretically, the IR system would best image a scene consisting of a homothermal target in cold surroundings as a bright spot on a dark background. To test this hypothesis, we evaluated how the pit viper (Gloydius brevicaudus) discriminates and strikes prey when the background temperature is either higher or lower than that of the prey (approximately 32-33°C) in different parts of the scene. Snakes were tested in a modified predation cage in which background temperatures were set to 26°C on one side and either 33 or 40°C on the opposite side when the eyes, the pit organs or neither sensory system was occluded. When the eyes were blocked, snakes preferred to strike prey on the 26°C side rather than on the 33°C side but showed no bias in the other conditions. Snakes showed no preference for 26 versus 40°C background temperature, although more missed strikes occurred when the eyes were occluded. The results thus revealed that the pit viper IR system can accomplish a 'brightness constancy' computation reflecting the difference between the target and background temperatures, much as the visual system compares the luminance of a figure and the background. Furthermore, the results show that the IR system performs less well for locating prey when the background is warmer than the target.

Positive selection acted on the extracellular transmembrane linkers of heat receptors during evolution.

All organisms must maintain body temperature within a suitable range and be able to sense the environmental temperature variations. However, it remains largely unknown how thermal sensing systems have evolved in animals. The transient receptor potential cation channel (TRP) protein family acts as warm/heat or cool/cold receptors by changing the probability of channel opening in response to thermal stimulation. Here, we examined the selective pressures acting on the transmembrane region of six segments (S1~S6) of thermo-TRP family members. Our results showed that there exist positive selection sites in heat receptors, but not in cold receptors. When all sequences of thermal TRP channels were pooled together, more significant selection pressures were found in the linker region between the transmembrane segments at the external side of the cellular membrane. Moreover, the P-loop region between S5 and S6 contains the most selected sites, indicating their importance in the thermal sense. Our study suggests that the heat receptor is more evolutionarily diverse than the cold receptor. This is consistent with the idea that hot environments usually have high heterogeneity, and that it is of great biological importance for animals to choosewarm basking places or escape harsh environments which are hot and dangerous such as forest fires.

The biological significance of acoustic stimuli determines ear preference in the music frog.

Behavioral and neurophysiological studies support the idea that right ear advantage (REA) exists for perception of conspecific vocal signals in birds and mammals. Nevertheless, few studies have focused on anuran species that typically communicate through vocalization. The present study examined the direction and latencies of orientation behaviors in Emei music frogs (Babina daunchina) produced in response to six auditory stimuli emitted by a speaker placed directly behind the subjects. The stimuli included male advertisement calls produced from within burrow nests, which have been shown to be highly sexually attractive (HSA), calls produced from outside burrows, which are of low sexual attractiveness (LSA), screech calls produced when frogs are attacked by snakes, white noise, thunder and silence. For all sound stimuli except the screech, the frogs preferentially turned to the right. Right ear preference was strongest for HSA calls. For the screech and thunder stimuli, there was an increased tendency for subjects to move further from the speaker rather than turning. These results support the idea that in anurans, right ear preference is associated with perception of positive or neutral signals such as the conspecific advertisement call and white noise, while a left ear preference is associated with perception of negative signals such as predatory attack.

Sound Classification and Call Discrimination Are Decoded in Order as Revealed by Event-Related Potential Components in Frogs.

Species that use communication sounds to coordinate social and reproductive behavior must be able to distinguish vocalizations from nonvocal sounds as well as to identify individual vocalization types. In this study we sought to identify the neural localization of the processes involved and the temporal order in which they occur in an anuran species, the music frog Babina daunchina. To do this we measured telencephalic and mesencephalic event-related potentials (ERPs) elicited by synthesized white noise (WN), highly sexually attractive (HSA) calls produced by males from inside nests and male calls of low sexual attractiveness (LSA) produced outside of nests. Each stimulus possessed similar temporal structures. The results showed the following: (1) the amplitudes of the first negative ERP component (N1) at ∼ 100 ms differed significantly between WN and conspecific calls but not between HSA and LSA calls, indicating that discrimination between conspecific calls and nonvocal sounds occurs in ∼ 100 ms, (2) the amplitudes of the second positive ERP component (P2) at ∼ 200 ms in the difference waves between HSA calls and WN were significantly higher than between LSA calls and WN in the right telencephalon, implying that call characteristic identification occurs in ∼ 200 ms and (3) WN evoked a larger third positive ERP component (P3) at ∼ 300 ms than conspecific calls, suggesting the frogs had classified the conspecific calls into one category and perceived WN as novel. Thus, both the detection of sounds and the identification of call characteristics are accomplished quickly in a specific temporal order, as reflected by ERP components. In addition, the most dynamic ERP patterns appeared in the left mesencephalon and the right telencephalon, indicating the two brain regions might play key roles in anuran vocal communication.

Electroencephalographic signals synchronize with behaviors and are sexually dimorphic during the light-dark cycle in reproductive frogs.

Male frogs behave differently from females during the breeding season, particularly with respect to courtship displays and in response to mating signals. In search of physiological correlates of these differences, the present study measured changes in baseline electroencephalogram (EEG) power output within four frequency bands in the telencephalon and mesencephalon, together with changes in locomotor activity as a function of the light-dark cycle in male and female Emei music frogs (Babina daunchina) at the reproductive stage. Previous studies have shown that male vocal activity varies both seasonally and daily in this species and that females use male advertisement calls to locate and select mates. The present results show that both EEG and locomotor activity exhibit highly correlated circadian patterns with peaks around light onset and offset. Importantly, during the reproductive stage, statistically significant sex differences in EEG output across brain regions during the light and dark phases were found indicating that sexual dimorphism exists for EEG activity which may underlie sexually specific information processing and behavioral activities.

Male vocal competition is dynamic and strongly affected by social contexts in music frogs.

Male-male vocal competition in anuran species is critical for mating success; however, it is also highly time-consuming, energetically demanding and likely to increase predation risks. Thus, we hypothesized that changes in the social context would cause male vocal competition to change in real time in order to minimize the costs and maximize the benefits of competition. To test this hypothesis, we assessed the effect of repeating playbacks of either white noise (WN) or male advertisement calls on male call production in the Emei music frog (Babina daunchina), a species in which males build mud-retuse burrows and call from within these nests. Previous studies have shown that calls produced from inside burrows are highly sexually attractive (HSA) to females while those produced outside nests are of low sexual attractiveness (LSA). Results showed that most subjects called responsively after the end of WN playbacks but before the onset of conspecific call stimuli although call numbers were similar, indicating that while males adjusted competitive patterns according to the biological significance of signals, their competitive motivation did not change. Furthermore, these data indicate that the frogs had evolved the ability of interval timing. Moreover, when the inter-stimulus interval (ISI) between playbacks was varied, the subjects preferentially competed with HSA calls when the ISI was short (<4 s) but responded equally to HSA and LSA calls if the ISI was long (≥4 s), suggesting that males allocate competitive efforts depending on both the perceived sexual attractiveness of rivals and the time available for calling. Notably, approximately two-thirds of male calls occurred in response to HSA calls, a preference rate comparable to that previously found for females in phonotaxis experiments and consistent with the idea that the mechanisms underlying both the male's competitive responses to rivals and the female's preferences toward potential mates coevolved under the same selective pressure.

Screening TLR4 Binding Peptide from Naja atra Venom Glands Based on Phage Display.

Toll-like receptor 4 (TLR4) is a crucial inflammatory signaling pathway that can serve as a potential treatment target for various disorders. A number of inhibitors have been developed for the TLR4 pathway, and although no inhibitors have been approved for clinical use, most have been screened against the TLR4-MD2 conformation. The venom gland is the organ of venomous snakes that secretes substances that are toxic to other animals. The level of gene transcription in venom glands is different from that in other tissues, includes a large number of biologically active ingredients, and is an important natural resource for the development of new drugs. We constructed a T7 phage display library using the cobra (Naja atra) venom gland from the Guangdong Snake Breeding Plant and performed three rounds of screening with TLR4 as the target, randomly selecting monoclonal phage spots for PCR followed by Sanger sequencing. The obtained sequences were subjected to length analysis, molecular docking, solubility prediction, and stability prediction, and a peptide containing 39 amino acids (NA39) was finally screened out. The BLAST results indicated that NA39 was a sequence in RPL19 (Ribosomal Protein L19). After peptide synthesis, the binding ability of NA39 to TLR4 was verified by the surface plasmon resonance (SPR) technique. In this study, a new peptide that can specifically bind TLR4 was successfully screened from the cobra venom gland cDNA library, further demonstrating the effectiveness of phage display technology in the field of drug discovery.

High-Frequency Local Field Potential Oscillations for Pigeons in Effective Turning.

Flexible turning behavior endows Homing Pigeons (Columba livia domestica) with high adaptability and intelligence in long-distance flight, foraging, hazard avoidance, and social interactions. The present study recorded the activity pattern of their local field potential (LFP) oscillations and explored the relationship between different bands of oscillations and turning behaviors in the formatio reticularis medialis mesencephali (FRM). The results showed that the C (13-60 Hz) and D (61-130 Hz) bands derived from FRM nuclei oscillated significantly in active turning, while the D and E (131-200 Hz) bands oscillated significantly in passive turning. Additionally, compared with lower-frequency stimulation (40 Hz and 60 Hz), 80 Hz stimulation can effectively activate the turning function of FRM nuclei. Electrical stimulation elicited stronger oscillations of neural activity, which strengthened the pigeons' turning locomotion willingness, showing an enhanced neural activation effect. These findings suggest that different band oscillations play different roles in the turning behavior; in particular, higher-frequency oscillations (D and E bands) enhance the turning behavior. These findings will help us decode the complex relationship between bird brains and behaviors and are expected to facilitate the development of neuromodulation techniques for animal robotics.

Grade-control outdoor turning flight of robo-pigeon with quantitative stimulus parameters.

Introduction: The robo-pigeon using homing pigeons as a motion carrier has great potential in search and rescue operations due to its superior weight-bearing capacity and sustained flight capabilities. However, before deploying such robo-pigeons, it is necessary to establish a safe, stable, and long-term effective neuro-electrical stimulation interface and quantify the motion responses to various stimuli. Methods: In this study, we investigated the effects of stimulation variables such as stimulation frequency (SF), stimulation duration (SD), and inter-stimulus interval (ISI) on the turning flight control of robo-pigeons outdoors, and evaluated the efficiency and accuracy of turning flight behavior accordingly. Results: The results showed that the turning angle can be significantly controlled by appropriately increasing SF and SD. Increasing ISI can significantly control the turning radius of robotic pigeons. The success rate of turning flight control decreases significantly when the stimulation parameters exceed SF > 100 Hz or SD > 5 s. Thus, the robo-pigeon's turning angle from 15 to 55° and turning radius from 25 to 135 m could be controlled in a graded manner by selecting varying stimulus variables. Discussion: These findings can be used to optimize the stimulation strategy of robo-pigeons to achieve precise control of their turning flight behavior outdoors. The results also suggest that robo-pigeons have potential for use in search and rescue operations where precise control of flight behavior is required.

Electroencephalogram bands modulated by vigilance states in an anuran species: a factor analytic approach.

Dramatic changes in neocortical electroencephalogram (EEG) rhythms are associated with the sleep-waking cycle in mammals. Although amphibians are thought to lack a neocortical homologue, changes in rest-activity states occur in these species. In the present study, EEG signals were recorded from the surface of the cerebral hemispheres and midbrain on both sides of the brain in an anuran species, Babina daunchina, using electrodes contacting the meninges in order to measure changes in mean EEG power across behavioral states. Functionally relevant frequency bands were identified using factor analysis. The results indicate that: (1) EEG power was concentrated in four frequency bands during the awake or active state and in three frequency bands during rest; (2) EEG bands in frogs differed substantially from humans, especially in the fast frequency band; (3) bursts similar to mammalian sleep spindles, which occur in non-rapid eye movement mammalian sleep, were observed when frogs were at rest suggesting sleep spindle-like EEG activity appeared prior to the evolution of mammals.