Oscillatory discharges in the auditory midbrain of the big brown bat contribute to coding of echo delay.

Subsequent to his breakthrough discovery of delay-tuned neurons in the bat's auditory midbrain and cortex, Albert Feng proposed that neural computations for echo delay involve intrinsic oscillatory discharges generated in the inferior colliculus (IC). To explore further the presence of these neural oscillations, we recorded multiple unit activity with a novel annular low impedance electrode from the IC of anesthetized big brown bats and Seba's short-tailed fruit bats. In both species, responses to tones, noise bursts, and FM sweeps contain long latency components, extending up to 60 ms post-stimulus onset, organized in periodic, oscillatory-like patterns at frequencies of 360-740 Hz. Latencies of this oscillatory activity resemble the wide distributions of single neuron response latencies in the IC. In big brown bats, oscillations lasting up to 30 ms after pulse onset emerge in response to single FM pulse-echo pairs, at particular pulse-echo delays. Oscillatory responses to pulses and evoked responses to echoes overlap extensively at short echo delays (5-7 ms), creating interference-like patterns. At longer echo delays, responses are separately evident to both pulses and echoes, with less overlap. These results extend Feng's reports of IC oscillations, and point to different processing mechanisms underlying perception of short vs long echo delays.

How frequency hopping suppresses pulse-echo ambiguity in bat biosonar.

Big brown bats transmit wideband FM biosonar sounds that sweep from 55 to 25 kHz (first harmonic, FM1) and from 110 to 50 kHz (second harmonic, FM2). FM1 is required to perceive echo delay for target ranging; FM2 contributes only if corresponding FM1 frequencies are present. We show that echoes need only the lowest FM1 broadcast frequencies of 25 to 30 kHz for delay perception. If these frequencies are removed, no delay is perceived. Bats begin echo processing at the lowest frequencies and accumulate perceptual acuity over successively higher frequencies, but they cannot proceed without the low-frequency starting point in their broadcasts. This reveals a solution to pulse-echo ambiguity, a serious problem for radar or sonar. In dense, extended biosonar scenes, bats have to emit sounds rapidly to avoid collisions with near objects. But if a new broadcast is emitted when echoes of the previous broadcast still are arriving, echoes from both broadcasts intermingle, creating ambiguity about which echo corresponds to which broadcast. Frequency hopping by several kilohertz from one broadcast to the next can segregate overlapping narrowband echo streams, but wideband FM echoes ordinarily do not segregate because their spectra still overlap. By starting echo processing at the lowest frequencies in frequency-hopped broadcasts, echoes of the higher hopped broadcast are prevented from being accepted by lower hopped broadcasts, and ambiguity is avoided. The bat-inspired spectrogram correlation and transformation (SCAT) model also begins at the lowest frequencies; echoes that lack them are eliminated from processing of delay and no longer cause ambiguity.

Temporal coherence of harmonic frequencies affects echo detection in the big brown bat, Eptesicus fuscus.

Echolocating big brown bats (Eptesicus fuscus) broadcast frequency modulated (FM) ultrasonic pulses containing two prominent harmonic sweeps (FM1, FM2). Both harmonics typically return as echoes at the same absolute time delay following the broadcast, making them coherent. Electronically splitting FM1 and FM2 allows their time delays to be controlled separately, making them non-coherent. Earlier work shows that big brown bats discriminate coherent from split harmonic, non-coherent echoes and that disruptions of harmonic coherence produce blurry acoustic images. A psychophysical experiment on two trained big brown bats tested the hypothesis that detection thresholds for split harmonic, non-coherent echoes are higher than those for coherent echoes. Thresholds of the two bats for detecting 1-glint echoes with coherent harmonics were around 35 and 36 dB sound pressure level, respectively, while thresholds for split harmonic echoes were about 10 dB higher. When the delay of FM2 in split harmonic echoes is shortened by 75 µs to offset neural amplitude-latency trading and restore coherence in the auditory representation, thresholds decreased back down to those estimated for coherent echoes. These results show that echo detection is affected by loss of harmonic coherence, consistent with the proposed broader role of coherence across frequencies for auditory perception.

Non-invasive auditory brainstem responses to FM sweeps in awake big brown bats.

We introduce two EEG techniques, one based on conventional monopolar electrodes and one based on a novel tripolar electrode, to record for the first time auditory brainstem responses (ABRs) from the scalp of unanesthetized, unrestrained big brown bats. Stimuli were frequency-modulated (FM) sweeps varying in sweep direction, sweep duration, and harmonic structure. As expected from previous invasive ABR recordings, upward-sweeping FM signals evoked larger amplitude responses (peak-to-trough amplitude in the latency range of 3-5 ms post-stimulus onset) than downward-sweeping FM signals. Scalp-recorded responses displayed amplitude-latency trading effects as expected from invasive recordings. These two findings validate the reliability of our noninvasive recording techniques. The feasibility of recording noninvasively in unanesthetized, unrestrained bats will energize future research uncovering electrophysiological signatures of perceptual and cognitive processing of biosonar signals in these animals, and allows for better comparison with ABR data from echolocating cetaceans, where invasive experiments are heavily restricted.

A comprehensive computational model of animal biosonar signal processing.

Computational models of animal biosonar seek to identify critical aspects of echo processing responsible for the superior, real-time performance of echolocating bats and dolphins in target tracking and clutter rejection. The Spectrogram Correlation and Transformation (SCAT) model replicates aspects of biosonar imaging in both species by processing wideband biosonar sounds and echoes with auditory mechanisms identified from experiments with bats. The model acquires broadband biosonar broadcasts and echoes, represents them as time-frequency spectrograms using parallel bandpass filters, translates the filtered signals into ten parallel amplitude threshold levels, and then operates on the resulting time-of-occurrence values at each frequency to estimate overall echo range delay. It uses the structure of the echo spectrum by depicting it as a series of local frequency nulls arranged regularly along the frequency axis of the spectrograms after dechirping them relative to the broadcast. Computations take place entirely on the timing of threshold-crossing events for each echo relative to threshold-events for the broadcast. Threshold-crossing times take into account amplitude-latency trading, a physiological feature absent from conventional digital signal processing. Amplitude-latency trading transposes the profile of amplitudes across frequencies into a profile of time-registrations across frequencies. Target shape is extracted from the spacing of the object's individual acoustic reflecting points, or glints, using the mutual interference pattern of peaks and nulls in the echo spectrum. These are merged with the overall range-delay estimate to produce a delay-based reconstruction of the object's distance as well as its glints. Clutter echoes indiscriminately activate multiple parts in the null-detecting system, which then produces the equivalent glint-delay spacings in images, thus blurring the overall echo-delay estimates by adding spurious glint delays to the image. Blurring acts as an anticorrelation process that rejects clutter intrusion into perceptions.

Amplitude discrimination is predictably affected by echo frequency filtering in wideband echolocating bats.

Big brown bats echolocate using wideband frequency-modulated (FM) ultrasonic pulses, perceiving target range from echo delay and target size from echo amplitude. Echolocation pulses contain two prominent down-sweeping harmonics (FM1, ∼55-22 kHz; FM2, ∼100-55 kHz), which are affected differently by propagation to the target and back to the bat. Previous work demonstrates that big brown bats utilize the low frequencies in FM1 for target ranging, while FM2 only contributes if FM1 is also present. The present experiments test the hypothesis that the bat's ability to discriminate echo amplitude is also affected by selectively attenuating FM1 or FM2 in target or nontarget echoes. Bats were trained to perform an amplitude discrimination task with virtual echo targets located 83 cm away. Echo delay was fixed and echo amplitude was varied, while either FM1 or FM2 was attenuated by highpass or lowpass filtering. Bats' performance decreased when lower frequencies were attenuated in target echoes and when higher frequencies were attenuated in nontarget echoes. Performance was reversed in the opposite filtering conditions. The bat's ability to distinguish between virtual targets varying in amplitude at the same simulated range indicates a high level of focused attention for perceptual isolation of target from non-target echoes.

Population registration of echo flow in the big brown bat's auditory midbrain.

Echolocating big brown bats (Eptesicus fuscus) perceive their surroundings by broadcasting frequency-modulated (FM) ultrasonic pulses and processing returning echoes. Bats echolocate in acoustically cluttered environments containing multiple objects, where each broadcast is followed by multiple echoes at varying time delays. The bat must decipher this complex echo cascade to form a coherent picture of the entire acoustic scene. Neurons in the bat's inferior colliculus (IC) are selective for specific acoustic features of echoes and time delays between broadcasts and echoes. Because of this selectivity, different subpopulations of neurons are activated as the bat flies through its environment, while the physical scene itself remains unchanging. We asked how a neural representation based on variable single-neuron responses could underlie a cohesive perceptual representation of a complex scene. We recorded local field potentials from the IC of big brown bats to examine population coding of echo cascades similar to what the bat might encounter when flying alongside vegetation. We found that the temporal patterning of a simulated broadcast followed by an echo cascade is faithfully reproduced in the population response at multiple stimulus amplitudes and echo delays. Local field potentials to broadcasts and echo cascades undergo amplitude-latency trading consistent with single-neuron data but rarely show paradoxical latency shifts. Population responses to the entire echo cascade move as a unit coherently in time as broadcast-echo cascade delay changes, suggesting that these responses serve as an index for the formation of a cohesive perceptual representation of an acoustic scene.NEW & NOTEWORTHY Echolocating bats navigate through cluttered environments that return cascades of echoes in response to the bat's broadcasts. We show that local field potentials from the big brown bat's auditory midbrain have consistent responses to a simulated echo cascade varying across echo delays and stimulus amplitudes, despite different underlying individual neuronal selectivities. These results suggest that population activity in the midbrain can build a cohesive percept of an auditory scene by aggregating activity over neuronal subpopulations.

Bat biosonar signals.

The Reflections series takes a look back on historical articles from The Journal of the Acoustical Society of America that have had a significant impact on the science and practice of acoustics.

Label-free, non-contact, in vivo ophthalmic imaging using photoacoustic remote sensing microscopy.

We present, to the best of our knowledge, the first label-free, non-contact, in vivo imaging of the ocular vasculature using photoacoustic remote sensing (PARS) microscopy. Both anterior and posterior segments of a mouse eye were imaged. Vasculature of the iris, sclera, and retina tissues were clearly resolved. To the best of our knowledge, this is the first study showing non-contact photoacoustic imaging conducted on in vivo ocular tissue. We believe that PARS microscopy has the potential to advance the diagnosis and treatment of ocular diseases.

Long-latency optical responses from the dorsal inferior colliculus of Seba's fruit bat.

We used a novel microendoscope system to record simultaneously optical activity (fluorescence of a calcium indicator dye) and electrical activity (multi-unit activity and local field potentials) from the dorsal inferior colliculus of the echolocating bat, Carollia perspicillata. Optically recorded calcium responses to wide-band noise and to frequency-modulated bursts were recorded at probe depths down to 1300 µm, with the majority of active sites encountered at more shallow depths down to 800 µm. Calcium activity exhibited long latencies, within the time span of 50-100 ms after stimulus onset, significantly longer than onset latencies of either multi-unit activity or local field potentials. Latencies and amplitude/latency trading of these electrical responses were consistent with those seen in standard electrophysiological recordings, confirming that the microendoscope was able to record both neural and optical activity successfully. Optically recorded calcium responses rose and decayed slowly and were correlated in time with long-latency negative deflections in local field potentials. These data suggest that calcium-evoked responses may reflect known, sustained inhibitory interactions in the inferior colliculus.

Biosonar interpulse intervals and pulse-echo ambiguity in four species of echolocating bats.

In complex biosonar scenes, the delay of echoes represents the spatial distribution of objects in depth. To avoid overlap of echo streams from successive broadcasts, individual echolocation sounds should only be emitted after all echoes of previous sounds have returned. However, close proximity of obstacles demands rapid pulse updates for steering to avoid collisions, which often means emitting a new sound before all of the previous echoes have returned. When two echo streams overlap, there is ambiguity about assigning echoes to the corresponding broadcasts. In laboratory tests of flight in dense, cluttered scenes, four species of echolocating bats exhibited different patterns of pulse emissions to accommodate potential pulse-echo ambiguity. Miniopterus fuliginosus emitted individual FM pulses only after all echoes of previous pulses had returned, with no alternating between long and short intervals. Pipistrellus abramus and Eptesicus fuscus alternated between emitting long FM pulse intervals to receive all echoes before the next pulse, and short intervals to update the rapidly changing scene while accepting partial overlap of successive echo streams. Rhinolophus ferrumequinum nippon transmitted CF/FM pulses in alternating short and long intervals, usually two to four closely spaced sounds that produced overlapping echo streams, followed by a longer interval that separated echo streams. Rhinolophus f. nippon is a statistical outlier from the three FM species, which are more similar to each other. The repeated overlap of CF/FM echo streams suggests that CF components have a distinct role in rejection of clutter and mitigation of ambiguity.

High-frequency soundfield microphone for the analysis of bat biosonar.

Numerous bat species emit wideband frequency-modulated signals for echolocation to hunt prey and avoid obstacles. Research investigating the behavioral and physiological responses of bats to echoes typically includes analysis of acoustic signals from microphones and/or microphone arrays, using time difference of arrival between array elements or the microphones to locate flying bats (azimuth and elevation). This has provided insight into transmission adaptations such as pulse duration and duty cycle with respect to target distance, clutter, and interferers. Microphones recording transmitted signals and echoes near a stationary bat provide sound pressure as a function of time but no directional information. In this work, the authors propose a spatial audio/soundfield microphone array to both track bats in flight and pinpoint the directions of echoes received by a bat. The authors introduce an ultrasonic (20-80 kHz) tetrahedral soundfield microphone to capture bat sounds up to 80 kHz. A spatial audio decoding technique called high angular resolution planewave expansion (HARPEx) supplies angle and elevation estimates, either for a flying bat based on the bat pulses or for targets based on echoes. Experiments using the soundfield microphone and HARPEx show that the approach accurately estimates the sound direction of arrival in both scenarios.

Frequency-modulated up-chirps produce larger evoked responses than down-chirps in the big brown bat auditory brainstem.

In many mammals, upward-sweeping frequency-modulated (FM) sounds (up-chirps) evoke larger auditory brainstem responses than downward-sweeping sounds (down-chirps). To determine if similar effects occur in FM echolocating bats, auditory evoked responses (AERs) in big brown bats in response to up-chirps and down-chirps at different chirp durations and levels were recorded. Even though down-chirps are the biologically relevant stimulus for big brown bats, up-chirps typically evoked larger peaks in the AER, but with some exceptions at the shortest chirp durations. The up-chirp duration that produced the largest AERs and the greatest differences between up-chirps and down-chirps varied between individual bats and stimulus levels. Cross-covariance analyses using the entire AER waveform confirmed that amplitudes were typically larger to up-chirps than down-chirps at supra-threshold levels, with optimal durations around 0.5-1 ms. Changes in response latencies with stimulus levels were consistent with previous estimates of amplitude-latency trading. Latencies tended to decrease with increasing up-chirp duration and increase with increasing down-chirp duration. The effects of chirp direction on AER waveforms are generally consistent with those seen in other mammals but with small differences in response patterns that may reflect specializations for FM echolocation.

Ebolavirus Glycoprotein Directs Fusion through NPC1+ Endolysosomes.

Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca(2+) channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.

Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry.

UNLABELLED: ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+) endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env. IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed-embedded in the membrane and present at high density-on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains.

Lipid rafts in plasma membranes have emerged as possible platforms for the entry of HIV and other viruses into cells. However, little is known about how lipid phase heterogeneity contributes to viral entry because of the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T cell membranes and found that raft-like liquid-ordered (Lo-phase) lipid domains were necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion were low in homogeneous liquid-disordered (Ld-phase) and Lo-phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo-Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld-phase vesicles proceeded only to hemifusion. Thus, we propose that edges but not areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.

Big brown bats (Eptesicus fuscus) maintain hearing sensitivity after exposure to intense band-limited noise.

Thresholds to short-duration narrowband frequency-modulated (FM) sweeps were measured in six big brown bats (Eptesicus fuscus) in a two-alternative forced choice passive listening task before and after exposure to band-limited noise (lower and upper frequencies between 10 and 50 kHz, 1 h, 116-119 dB sound pressure level root mean square; sound exposure level 152 dB). At recovery time points of 2 and 5 min post-exposure, thresholds varied from -4 to +4 dB from pre-exposure threshold estimates. Thresholds after sham (control) exposures varied from -6 to +2 dB from pre-exposure estimates. The small differences in thresholds after noise and sham exposures support the hypothesis that big brown bats do not experience significant temporary threshold shifts under these experimental conditions. These results confirm earlier findings showing stability of thresholds to broadband FM sweeps at longer recovery times after exposure to broadband noise. Big brown bats may have evolved a lessened susceptibility to noise-induced hearing losses, related to the special demands of echolocation.

Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step.

UNLABELLED: Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE: Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.

Target shape perception and clutter rejection use the same mechanism in bat sonar.

Big brown bats (Eptesicus fuscus) emit frequency-modulated (FM) biosonar sounds containing two or more harmonic sweeps. Echoes from frontally located targets arrive with first and second harmonics intact, leading to focused delay images. Echoes from offside or distant objects arrive with the second harmonic relatively weaker (lowpass-filtered), leading to defocused images, which prevents their clutter interference effects (Bates et al. J Exp Biol 214:394-401, 2011). Realistic targets contain several glints at slightly different distances and reflect several echoes at correspondingly different delays. The bat registers the delay of the nearest glint's echoes in the time domain. The delays of echoes from the farther glints are registered in the frequency domain, from interference nulls in the spectrum. Lowpass-filtering of echoes directly affects the image of the nearest glint by defocusing the delay image. However, lowpass-filtering also is superimposed on the interference spectrum used to register the farther glints, which distorts the pattern of interference nulls, defocusing the farther glints inversely, in the spectral domain, before they are perceived as delays. Differences in blurring between time-domain and frequency-domain parts of images identifies separate computational paths to perceptually reconstruct objects and prevent interference from off-side or distant clutter.