Pose analysis in free-swimming adult zebrafish, Danio rerio : "fishy" origins of movement design.

Movement requires maneuvers that generate thrust to either make turns or move the body forward in physical space. The computational space for perpetually controlling the relative position of every point on the body surface can be vast. We hypothesize the evolution of efficient design for movement that minimizes active (neural) control by leveraging the passive (reactive) forces between the body and the surrounding medium at play. To test our hypothesis, we investigate the presence of stereotypical postures during free-swimming in adult zebrafish, Danio rerio . We perform markerless tracking using DeepLabCut, a deep learning pose estimation toolkit, to track geometric relationships between body parts. To identify putative clusters of postural configurations obtained from twelve freely behaving zebrafish, we use unsupervised multivariate time-series analysis (B-SOiD machine learning software). When applied to single individuals, this method reveals a best-fit for 36 to 50 clusters in contrast 86 clusters for data pooled from all 12 animals. The centroids of each cluster obtained over 14,000 sequential frames recorded for a single fish represent an apriori classification into relatively stable "target body postures" and inter-pose "transitional postures" that lead to and away from a target pose. We use multidimensional scaling of mean parameter values for each cluster to map cluster-centroids within two dimensions of postural space. From a post-priori visual analysis, we condense neighboring postural variants into 15 superclusters or core body configurations. We develop a nomenclature specifying the anteroposterior level/s (upper, mid and lower) and degree of bending. Our results suggest that constraining bends to mainly three levels in adult zebrafish preempts the neck, fore- and hindlimb design for maneuverability in land vertebrates.

NemoTrainer: Automated Conditioning for Stimulus-Directed Navigation and Decision Making in Free-Swimming Zebrafish.

Current methods for associative conditioning in animals involve human intervention that is labor intensive, stressful to animals, and introduces experimenter bias in the data. Here, we describe a simple apparatus and a flexible, microcontroller-based conditioning paradigm that minimizes human intervention. Our methodology exploits directed movement towards a target that depends on spatial working memory, including processing of sensory inputs, motivational drive, and attentional mechanisms. Within a stimulus-driven conditioning paradigm designed to train zebrafish, we present a localized pulse of light via LEDs and/or sounds via an underwater transducer. A webcam placed below a glass tank records fish-swimming behavior. For classical conditioning, animals simply associate a sound or light with an unconditioned stimulus, such as a small food reward presented at a fixed location, and swim towards that location to obtain a few grains of food dispensed automatically via a sensor-triggered, stepper motor. During operant conditioning, a fish must first approach a proximity sensor at a remote location and then swim to the reward location. For both types of conditioning, a timing-gated interrupt activates stepper motors via custom software embedded within a microcontroller (Arduino). "Ardulink", a Java facility, implements Arduino-computer communication protocols. In this way, a Java-based user interface running on a host computer can provide full experimental control. Alternatively, a similar level of control is achieved via an Arduino script communicating with an event-driven application controller running on the host computer. Either approach can enable precise, multi-day scheduling of training, including timing, location, and intensity of stimulus parameters; and the feeder. Learning can be tracked by monitoring turning, location, response times, and directional swimming of individual fish. This facilitates the comparison of performance within and across a cohort of animals. Our scheduling and control software and apparatus ("NemoTrainer") can be used to study multiple aspects of species-specific behaviors as well as the effects on them of various interventions.

Hemispheric and Sex Differences in Mustached Bat Primary Auditory Cortex Revealed by Neural Responses to Slow Frequency Modulations.

The mustached bat (Pteronotus parnellii) is a mammalian model of cortical hemispheric asymmetry. In this species, complex social vocalizations are processed preferentially in the left Doppler-shifted constant frequency (DSCF) subregion of primary auditory cortex. Like hemispheric specializations for speech and music, this bat brain asymmetry differs between sexes (i.e., males>females) and is linked to spectrotemporal processing based on selectivities to frequency modulations (FMs) with rapid rates (>0.5 kHz/ms). Analyzing responses to the long-duration (>10 ms), slow-rate (<0.5 kHz/ms) FMs to which most DSCF neurons respond may reveal additional neural substrates underlying this asymmetry. Here, we bilaterally recorded responses from 176 DSCF neurons in male and female bats that were elicited by upward and downward FMs fixed at 0.04 kHz/ms and presented at 0-90 dB SPL. In females, we found inter-hemispheric latency differences consistent with applying different temporal windows to precisely integrate spectrotemporal information. In males, we found a substrate for asymmetry less related to spectrotemporal processing than to acoustic energy (i.e., amplitude). These results suggest that in the DSCF area, (1) hemispheric differences in spectrotemporal processing manifest differently between sexes, and (2) cortical asymmetry for social communication is driven by spectrotemporal processing differences and neural selectivities for amplitude.

Bats increase vocal amplitude and decrease vocal complexity to mitigate noise interference during social communication.

Natural background noises are common in the acoustic environments in which most organisms have evolved. Therefore, the vocalization and sound perception systems of vocal animals are inherently equipped to overcome natural background noise. Human-generated noises, however, pose new challenges that can hamper audiovocal communication. The mechanisms animals use to cope with anthropogenic noise disturbances have been extensively explored in a variety of taxa. Bats emit echolocation pulses primarily to orient, locate and navigate, while social calls are used to communicate with conspecifics. Previous studies have shown that bats alter echolocation pulse parameters in response to background noise interference. In contrast to high-frequency echolocation pulses, relatively low-frequency components within bat social calls overlap broadly with ambient noise frequencies. However, how bats structure their social calls in the presence of anthropogenic noise is not known. Here, we hypothesized that bats leverage vocal plasticity to facilitate vocal exchanges within a noisy environment. To test this hypothesis, we subjected the Asian particolored bat, Vespertilio sinensis, to prerecorded traffic noise. We observed a significant decrease in vocal complexity (i.e., an increased frequency of monosyllabic calls) in response to traffic noise. However, an increase in the duration and frequency of social calls, as have been observed in other species, was not evident. This suggests that signal simplification may increase communication efficacy in noisy environments. Moreover, V. sinensis also increased call amplitude in response to increased traffic noise, consistent with the predictions of the Lombard effect.

Great Himalayan leaf-nosed bats modify vocalizations to communicate threat escalation during agonistic interactions.

Bats vocalize extensively within various social contexts. Nevertheless, studies of agonistic interactions, associating vocalizations signalling the emotional state of a caller with individual signatures during aggressive vocalizations remain scarce. Here, we examined whether male Great Himalayan leaf-nosed bats (Hipposideros armiger) modify their aggressive vocalizations during agonistic interactions depending on the level of aggression, and whether these vocalizations encode individual signatures. We applied a cost-benefit analysis to audio-video recordings of 50 dyadic agonistic interactions to categorize displays into two levels of aggression intensity (low aggression: bared teeth, slightly pulled up body and/or wings; high aggression: rapidly flapping wings, punching, biting). Male H. armiger used graded visual agonistic displays accompanied by bent upward frequency modulation (bUFM) vocalizations to defend their roosting territories. At high aggression intensities, males decreased the minimum frequency of aggressive calls and increased the frequency bandwidth. Males also transferred energy from the second harmonic to the first harmonic as the threat escalated. These systematic modifications of acoustic parameters as aggressive intensity fluctuated corresponded to prosodic modifications in human speech, indicating that emotion-related acoustic cues are a common feature of acoustic communication in mammals. In addition, we found that the aggressive calls of eight adult males encoded discriminable signatures, and that males could discriminate among individuals based on these aggressive calls. Such discrimination is probably useful for individual identification among rival neighbours.

A three-dimensional digital neurological atlas of the mustached bat (Pteronotus parnellii).

Substantial knowledge of auditory processing within mammalian nervous systems emerged from neurophysiological studies of the mustached bat (Pteronotus parnellii). This highly social and vocal species retrieves precise information about the velocity and range of its targets through echolocation. Such high acoustic processing demands were likely the evolutionary pressures driving the over-development at peripheral (cochlea), metencephalic (cochlear nucleus), mesencephalic (inferior colliculus), diencephalic (medial geniculate body of the thalamus), and telencephalic (auditory cortex) auditory processing levels in this species. Auditory researchers stand to benefit from a three dimensional brain atlas of this species, due to its considerable contribution to auditory neuroscience. Our MRI-based atlas was generated from 2 sets of image data of an ex-vivo male mustached bat's brain: a detailed 3D-T2-weighted-RARE scan [(59 × 63 x 85) µm3] and track density images based on super resolution diffusion tensor images [(78) µm3] reconstructed from a set of low resolution diffusion weighted images using Super-Resolution-Reconstruction (SRR). By surface-rendering these delineations and extrapolating from cortical landmarks and data from previous studies, we generated overlays that estimate the locations of classic functional subregions within mustached bat auditory cortex. This atlas is freely available from our website and can simplify future electrophysiological, microinjection, and neuroimaging studies in this and related species.

Acoustically diverse vocalization repertoire in the Himalayan leaf-nosed bat, a widely distributed Hipposideros species.

Insectivorous bats vocalize to both communicate with conspecifics and to echolocate. The communicative vocalizations or "calls" of bats either consist of or are constructed from discrete acoustic units, termed "syllables." This study examined syllable diversity in the Himalayan leaf-nosed bat, Hipposideros armiger, a species that is widely distributed across Southeast Asia. This social species' vocalizations were hypothesized to consist of a wide variety of syllables facilitating its social interactions. To test this hypothesis, multiple acoustic parameters were measured from recorded vocalizations to map the acoustic boundaries of syllables. Spectrographic signatures were used to classify all recorded sounds into 35 distinct syllable types-18 as simple syllables and 17 as composites. K-means clustering independently provided an optimal fit of simple syllables into 18 clusters with a good correspondence to 15 spectrographically assigned syllable types. Discriminant analysis further confirmed the spectrographic classification of constant frequency syllables (0% misclassification) and revealed a low (<15%) misclassification of spectrograms for all examples of frequency modulation syllables. Multidimensional scaling of mean values of multiple parameters provided a spectrographically constrained relational mapping of syllable types within two dimensions. These data suggest that H. armiger has a complex, well organized syllabic repertoire despite simple syllables being rarely emitted in isolation.

Using sounds for making decisions: greater tube-nosed bats prefer antagonistic calls over non-communicative sounds when feeding.

Bats vocalize extensively within different social contexts. The type and extent of information conveyed via their vocalizations and their perceptual significance, however, remains controversial and difficult to assess. Greater tube-nosed bats, Murina leucogaster, emit calls consisting of long rectangular broadband noise burst (rBNBl) syllables during aggression between males. To experimentally test the behavioral impact of these sounds for feeding, we deployed an approach and place-preference paradigm. Two food trays were placed on opposite sides and within different acoustic microenvironments, created by sound playback, within a specially constructed tent. Specifically, we tested whether the presence of rBNBl sounds at a food source effectively deters the approach of male bats in comparison to echolocation sounds and white noise. In each case, contrary to our expectation, males preferred to feed at a location where rBNBl sounds were present. We propose that the species-specific rBNBl provides contextual information, not present within non-communicative sounds, to facilitate approach towards a food source.

Situational and Age-Dependent Decision Making during Life Threatening Distress in Myotis macrodactylus.

Echolocation and audiovocal communication have been studied extensively in bats. The manner in which these abilities are incorporated within escape behaviors during life-threatening distress is largely unknown. Here we tested the hypothesis that behavioral response profiles expressed during distress are relatively stereotypic given their evolutionary adaptations to avoid predators. We subjected juvenile and adult big-footed myotis (Myotis macrodactylus) to a sequence of three types of life threatening distress: 1) trapping them in a mist-net (environmental threat), 2) approaching them when trapped (predator threat), and 3) partially restraining their freedom to move (arrest), and recorded their escape behavior in each of the three conditions. Response profiles differed across individuals and with the context in which they were expressed. During environmental and predator threat, bats displayed significantly more biting and wing-flapping behaviors and emitted more echolocation pulses than during arrest. Response profiles also varied with age. During arrest, juveniles were more likely than adults to emit distress calls and vice-versa for biting and wing flapping during environmental and predator threat. Overall, individualized response profiles were classified into ten clusters that were aligned along two divergent response trajectories when viewed within two-dimensional, multifactorial decision space. Juvenile behaviors tended to follow a predominantly "social-dependence" trajectory, whereas adult behaviors were mostly aligned along a "self-reliance" trajectory. We conclude that bats modify their vocal behavior and make age-appropriate and contextually adaptive decisions when distressed. This decision-making ability is consistent with observations in other social species, including humans.

Social and vocal behavior in adult greater tube-nosed bats (Murina leucogaster).

Many studies have revealed the significant influence of the social nature and ecological niche of a species on the design and complexity of their communication sounds. The knowledge of communication sounds and particularly of the flexibility in their use among mammals, however, remains patchy. Being highly vocal and social, bats are well suited for investigating vocal plasticity as well as vocal diversity. Thus, the overall aim of this study was to test the presence of structural overlap between calls used in social communication and echolocation pulses emitted during foraging in greater tube-nosed bats (Murina leucogaster). Acoustic analysis and spectrotemporal decomposition of calls revealed a rich communication repertoire comprising 12 simple syllables and 5 composites with harmonics in the ultrasonic range. Simultaneous recording of vocal and social behavior in the same species yielded a strong correspondence between distinct behaviors and specific call types in support of Morton's motivation-structure hypothesis. Spectrographic analysis of call types also revealed the presence of modified components of echolocation pulses embedded within social calls. Altogether, the data suggest that bats can parse complex sounds into structurally simpler components that are recombined within behaviorally meaningful and multifunctional contexts.

From student to steward: the Interdisciplinary Program in Neuroscience at Georgetown University as a case study in professional development during doctoral training.

A key facet of professional development is the formation of professional identity. At its most basic level, professional identity for a scientist centers on mastery of a discipline and the development of research skills during doctoral training. To develop a broader understanding of professional identity in the context of doctoral training, the Carnegie Initiative on the Doctorate (CID) ran a multi-institutional study from 2001 to 2005. A key outcome of the CID was the development of the concept of 'stewards of the discipline'. The Interdisciplinary Program in Neuroscience (IPN) at Georgetown University participated in CID from 2003 to 2005. Here, we describe the IPN and highlight the programmatic developments resulting from participation in the CID. In particular, we emphasize programmatic activities that are designed to promote professional skills in parallel with scientific development. We describe activities in the domains of leadership, communication, teaching, public outreach, ethics, collaboration, and mentorship. Finally, we provide data that demonstrate that traditional metrics of academic success are not adversely affected by the inclusion of professional development activities in the curricula. By incorporating these seven 'professional development' activities into the required coursework and dissertation research experience, the IPN motivates students to become stewards of the discipline.

Stimulation of the basal and central amygdala in the mustached bat triggers echolocation and agonistic vocalizations within multimodal output.

The neural substrate for the perception of vocalizations is relatively well described, but how their timing and specificity are tightly coupled with accompanying physiological changes and context-appropriate behaviors remains unresolved. We hypothesized that temporally integrated vocal and emotive responses, especially the expression of fear, vigilance and aggression, originate within the amygdala. To test this hypothesis, we performed electrical microstimulation at 461 highly restricted loci within the basal and central amygdala in awake mustached bats. At a subset of these sites, high frequency stimulation with weak constant current pulses presented at near-threshold levels triggered vocalization of either echolocation pulses or social calls. At the vast majority of locations, microstimulation produced a constellation of changes in autonomic and somatomotor outputs. These changes included widespread co-activation of significant tachycardia and hyperventilation and/or rhythmic ear pinna movements (PMs). In a few locations, responses were constrained to vocalization and/or PMs despite increases in the intensity of stimulation. The probability of eliciting echolocation pulses vs. social calls decreased in a medial-posterior to anterolateral direction within the centrobasal amygdala. Microinjections of kainic acid (KA) at stimulation sites confirmed the contribution of cellular activity rather than fibers-of-passage in the control of multimodal outputs. The results suggest that localized clusters of neurons may simultaneously modulate the activity of multiple central pattern generators (CPGs) present within the brainstem.

Tectal etiology for irrepressible saccades: a case study in a Rhesus monkey.

Brain circuits controlling eye movements are widely distributed and complex. The etiology of irrepressible square wave saccades is not fully understood and is likely different for different neuropathologies. In a previous study, spontaneously occurring irrepressible saccades were noted after a cerebrovascular accident that damaged the rostral superior colliculus (SC) and its commissure in a Rhesus monkey. Here, we tracked and quantified the development of similar symptoms in a Rhesus monkey caused by a lesion in the rostromedial SC and its commissure. We documented the changes in these saccadic intrusions while the monkey attempted fixation of a target on three consecutive days post-onset. On the first day, eye jerk amplitude was ~10 degrees and the direction was ~30 degrees above the left horizontal meridian. On the second day, the amplitude decreased to 6.5 degrees and the direction shifted towards vertical, ~20 degrees to the left of the vertical meridian. Size, but not direction, of the eye jerks continued to decrease until intrusions dissipated within one month. Histological examination after ~6 months from the first appearance of the intrusions revealed a lesion in the commissure of the SC. Results from this and the previous study confirm the involvement of the commissure of the SC as the common target for triggering this neuropathy. Our data suggest that commissural fibers play an important role in maintaining normal visual stability. Interrupting the commissure between the two superior colliculi causes saccadic intrusions in the form of irrepressible jerking of the eyes, probably by disrupting inhibitory signals transmitted through the commissure. Furthermore, disappearance of the symptoms suggests that inhibitory fields within the SC are plastic and can expand, possibly via inputs from inter-collicular and nigrotectal pathways.

A flexible user-interface for audiovisual presentation and interactive control in neurobehavioral experiments.

A major problem facing behavioral neuroscientists is a lack of unified, vendor-distributed data acquisition systems that allow stimulus presentation and behavioral monitoring while recording neural activity. Numerous systems perform one of these tasks well independently, but to our knowledge, a useful package with a straightforward user interface does not exist. Here we describe the development of a flexible, script-based user interface that enables customization for real-time stimulus presentation, behavioral monitoring and data acquisition. The experimental design can also incorporate neural microstimulation paradigms. We used this interface to deliver multimodal, auditory and visual (images or video) stimuli to a nonhuman primate and acquire single-unit data. Our design is cost-effective and works well with commercially available hardware and software. Our design incorporates a script, providing high-level control of data acquisition via a sequencer running on a digital signal processor to enable behaviorally triggered control of the presentation of visual and auditory stimuli. Our experiments were conducted in combination with eye-tracking hardware. The script, however, is designed to be broadly useful to neuroscientists who may want to deliver stimuli of different modalities using any animal model.

Simple syllabic calls accompany discrete behavior patterns in captive Pteronotus parnellii: an illustration of the motivation-structure hypothesis.

Mustached bats, Pteronotus parnellii, are highly social and vocal. Individuals of this species roost in tight clusters, and emit an acoustically rich repertoire of calls whose behavioral significance is largely unknown. We recorded their social and vocal behaviors within a colony housed under semi-natural conditions. We also quantified the spatial spread of each bat's roosting location and discovered that this was relatively fixed and roughly confined to an individual's body width. The spatial precision in roosting was accompanied by an equally remarkable match between specific vocalizations and well-timed, discrete, identifiable postures/behaviors, as revealed by logistic regression analysis. The bodily behaviors included crouching, marking, yawning, nipping, flicking, fighting, kissing, inspecting, and fly-bys. Two echolocation-like calls were used to maintain spacing in the colony, two noisy broadband calls were emitted during fights, two tonal calls conveyed fear, and another tonal call signaled appeasement. Overall, the results establish that mustached bats exhibit complex social interactions common to other social mammals. The correspondence of relatively low frequency and noisy, broadband calls with aggression, and of tonal, high frequency calls with fear supports Morton's Motivation-Structure hypothesis, and establishes a link between motivation and the acoustic structure of social calls emitted by mustached bats.

Sex-dependent hemispheric asymmetries for processing frequency-modulated sounds in the primary auditory cortex of the mustached bat.

Species-specific vocalizations of mammals, including humans, contain slow and fast frequency modulations (FMs) as well as tone and noise bursts. In this study, we established sex-specific hemispheric differences in the tonal and FM response characteristics of neurons in the Doppler-shifted constant-frequency processing area in the mustached bat's primary auditory cortex (A1). We recorded single-unit cortical activity from the right and left A1 in awake bats in response to the presentation of tone bursts and linear FM sweeps that are contained within their echolocation and/or communication sounds. Peak response latencies to neurons' preferred or best FMs were significantly longer on the right compared with the left in both sexes, and in males this right-left difference was also present for the most excitatory tone burst. Based on peak response magnitudes, right hemispheric A1 neurons in males preferred low-rate, narrowband FMs, whereas those on the left were less selective, responding to FMs with a variety of rates and bandwidths. The distributions of parameters for best FMs in females were similar on the two sides. Together, our data provide the first strong physiological support of a sex-specific, spectrotemporal hemispheric asymmetry for the representation of tones and FMs in a nonhuman mammal. Specifically, our results demonstrate a left hemispheric bias in males for the representation of a diverse array of FMs differing in rate and bandwidth. We propose that these asymmetries underlie lateralized processing of communication sounds and are common to species as divergent as bats and humans.

Right-left asymmetry in the cortical processing of sounds for social communication vs. navigation in mustached bats.

In the Doppler-shifted constant frequency processing area in the primary auditory cortex of mustached bats, Pteronotus parnellii, neurons respond to both social calls and to echolocation signals. This multifunctional nature of cortical neurons creates a paradox for simultaneous processing of two behaviorally distinct categories of sound. To test the possibility of a stimulus-specific hemispheric bias, single-unit responses were obtained to both types of sounds, calls and pulse-echo tone pairs, from the right and left auditory cortex. Neurons on the left exhibited only slightly higher peak response magnitudes for their respective best calls, but they showed a significantly higher sensitivity (lower response thresholds) to calls than neurons on the right. On average, call-to-tone response ratios were significantly higher for neurons on the left than for those on the right. Neurons on the right responded significantly more strongly to pulse-echo tone pairs than those on the left. Overall, neurons in males responded to pulse-echo tone pairs with a much higher spike count compared to females, but this difference was less pronounced for calls. Multidimensional scaling of call responses yielded a segregated representation of call types only on the left. These data establish for the first time, a behaviorally directed right-left asymmetry at the level of single cortical neurons. It is proposed that a lateralized cortex emerges from multiparametric integration (e.g. combination-sensitivity) within a neuron and inhibitory interactions between neurons that come into play during the processing of complex sounds.

Basolateral amygdala responds robustly to social calls: spiking characteristics of single unit activity.

Vocalizations emitted within a social context can trigger call-specific changes in the emotional and physiological/autonomic state of the receiver. The amygdala is implicated in mediating these changes, but its role in call perception remains relatively unexplored. We examined call and pitch selectivity of single neurons within the basolateral amygdala (BLA) by recording spiking activity in response to 5 pitch variants of each of 14 species-specific calls presented to awake, head-restrained mustached bats, Pteronotus parnellii. A response-wise analysis across neurons revealed seven types of temporal response patterns based on the timing and duration of spiking. Roughly half of the responses to different call types were significantly affected by changes in call pitch. A neuron-wise analysis revealed that ∼ 12% (8/69) of the neurons preferred the same pitch across all call types. Ninety-three percent (93/100) of neurons were excited by at least one call type and 76% exhibited either complete or transient suppression to one or more call types. The majority of neurons preferred fewer than half of the 14 different simple-syllabic calls. A call-wise analysis of spiking activity revealed that call types signaling either threat or fear most consistently evoked increases in the spike rate. In contrast, calls emitted during appeasement tended to evoke spike suppression. Our data suggest that BLA neurons participate in the processing of multiple call types and exhibit a rich variety of temporal response patterns that are neither neuron nor call specific.

Fear conditioned discrimination of frequency modulated sweeps within species-specific calls of mustached bats.

Social and echolocation vocalizations of bats contain different patterns of frequency modulations. An adult bat's ability to discriminate between various FM parameters, however, is not well established. Using changes in heart rate (HR) as a quantitative measure of associative learning, we demonstrate that mustached bats (Pteronotus parnellii) can be fear conditioned to linear frequency modulated (FM) sweeps typically centered at their acoustic fovea (approximately 60 kHz). We also show that HR is sensitive to a change in the direction of the conditional frequency modulation keeping all other parameters constant. In addition, a change in either depth or duration co-varied with FM rate is reflected in the change in HR. Finally, HR increases linearly with FM rate incremented by 0.1 kHz/ms from a pure tone to a target rate of 1.0 kHz/ms of the conditional stimulus. Learning is relatively rapid, occurring after a single training session. We also observed that fear conditioning enhances local field potential activity within the basolateral amygdala. Neural response enhancement coinciding with rapid learning and a fine scale cortical representation of FM sweeps shown earlier make FMs prime candidates for discriminating between different call types and possibly communicating socially relevant information within species-specific sounds.

DSCF neurons within the primary auditory cortex of the mustached bat process frequency modulations present within social calls.

Neurons in the Doppler-shifted constant frequency processing (DSCF) area in the primary auditory cortex of mustached bats, Pteronotus parnellii, are multifunctional, responding both to echolocation and communication sounds. Simultaneous presentation of a DSCF neuron's best low and high frequencies (BF(low) and BF(high), respectively) facilitates its response. BF(low) corresponds to a frequency in the frequency-modulated (FM) component of the first harmonic in the echolocation pulse, and BF(high) corresponds to the constant frequency (CF) component in the second harmonic of the echo. We systematically varied the slopes, bandwidths, and central frequencies of FMs traversing the BF(high) region to arrive at the "best FM" for single DSCF neurons. We report that nearly half (46%) of DSCF neurons preferred linear FMs to CFs and average response magnitude to FMs was not significantly less (P = 0.08) than that to CFs at BF(high) when each test stimulus was paired with a CF at BF(low). For linear FMs ranging in slope from 0.04 to 4.0 kHz/ms and in bandwidth from 0.44 to 7.88 kHz, the majority of DSCF neurons preferred upward (55%) to downward (21%) FMs. Central frequencies of the best FMs were typically close to but did not always match a neuron's BF(high). Neurons exhibited combination-sensitivity to "call fragments" (calls that were band-pass filtered in the BF(high) region) paired with their BF(low). Our data show a close match between the modulation direction of a neuron's best FM and that of its preferred call fragment. These response properties show that DSCF neurons extract multiple parameters of FMs and are specialized for processing both FMs for communication and CFs for echolocation.