FM signals produce robust paradoxical latency shifts in the bat’s inferior colliculus
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- Wang, X., Galazyuk, A.V. & Feng, A.S. J Comp Physiol A (2007) 193: 13. doi:10.1007/s00359-006-0167-9
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Previous studies in echolocating bats, Myotis lucifugus, showed that paradoxical latency shift (PLS) is essential for neural computation of target range and that a number of neurons in the inferior colliculus (IC) exhibit unit-specific PLS (characterized by longer first-spike latency at higher sound levels) in response to tone pulses at the unit’s best frequency. The present study investigated whether or not frequency-modulated (FM) pulses that mimic the bat’s echolocation sonar signals were equally effective in eliciting PLS. For two-thirds of PLS neurons in the IC, both FM and tone pulses could elicit PLS, but only FM pulses consistently produced unit-specific PLS. For the remainder of PLS neurons, only FM pulses effectively elicited PLS; these cells showed either no PLS or no response, to tone pulses. PLS neurons generally showed more pronounced PLS in response to narrow-band FM (each sweeping 20 kHz in 2 ms) pulse that contained the unit’s best frequency. In addition, almost all PLS neurons showed duration-independent PLS to FM pulses, but the same units exhibited duration-dependent PLS to tone pulses. Taken together, when compared to tone pulses, FM stimuli can provide more reliable estimates of target range.
KeywordsEcholocationTarget rangingFrequency modulationOscillationFirst-spike latency
Microchiropteran bats use a sonar system to orient and navigate in 3D space and to detect, discriminate, and locate prey through analysis of echoes of their emitted sonar signals. Target ranging is particularly important for localizing and tracking a moving prey and has been extensively studied behaviorally and physiologically. Behavioral studies by Simmons (1973, 1979) have demonstrated that the most important cue for ranging is the echo delay, the time between bat’s sonar emission and its echo. Physiological studies have shown that a substantial population of neurons in the central auditory system of echolocating bats is tuned to echo delays, exhibiting response facilitation to pulse–echo pairs at specific echo delays (Feng et al. 1978; O’Neill and Suga 1979; Sullivan 1982a, b; Mittmann and Wenstrup 1995; O’Neill 1995). Bats’ echolocation signals are highly adaptive but two major classes are distinguishable depending on the species and foraging behavior (Neuweiler 1984): FM bats primarily emit brief frequency-modulated (FM) sonar signals, whereas CF/FM bats emit longer constant frequency (CF) sonar pulses ending with a downward FM.
Suga and colleagues (Suga 1983) showed that, in the mustached bat (a CF/FM bat), delay-tuned cortical neurons are sensitive to the delay of the “FM” (and not the CF) component of bat’s echolocation signal. This suggests that FM plays a more important role in target ranging and processing of echo delays than does the CF component. For little brown bats (a FM species), Sullivan (1982a, b) proposed that delay-tuned responses in the auditory cortex are attributed to a coincidence detection mechanism, and neurons exhibiting paradoxical latency shift (PLS, i.e., longer response latencies for more intense sounds than for weaker sounds) play a critical role in the construction of delay-tuned responses. For the majority of cortical neurons showing PLS, the magnitude of the latency shift for each neuron corresponds to the echo delay to which the neuron is best tuned (Sullivan 1982b; Berkowitz and Suga 1989). The phenomenon of PLS has since been observed in other parts of the bat’s central auditory system, primarily at the level of the inferior colliculus (IC) and above, as well as in the IC of non-echolocating vertebrates (Olsen and Suga 1991a, b; Covey 1993; Klug et al. 2000; Galazyuk and Feng 2001; Galazyuk et al. 2005). Some of the above studies used FM and others utilized CF as sound stimuli. The relative efficacy of FM versus CF for eliciting PLS is unknown.
Galazyuk et al. (2005) recently showed that, in the IC of little brown bats, high-threshold leading inhibition of a unit’s oscillatory discharge is a building block for PLS; for PLS neurons, application of bicuculline abolishes PLS and unmasks the unit’s periodical firing pattern. Furthermore, the periodical firing pattern of IC neurons is unit-specific, as is the units’ PLS. However, it is unclear whether or not the cell-specific PLS (and oscillatory discharge) is stimulus dependent, and in particular whether FM stimuli can also elicit it. In the present study, we compared the effectiveness of FM stimuli and tone bursts in the generation of PLS.
Surgical and recording procedures
Experimental subjects comprised 20 little brown bats, Myotis lucifugus. Details of experimental methods are given in Galazyuk and Feng (2001). Briefly, for surgery, the animal was anesthetized via halothane inhalation (4% halothane administered by a precision vaporizer). After incision of the skin and clearing of the tissues above the skull, a small metal rod was glued to the skull using glass ionomer cement. Following the surgery, animals were allowed to recover for 2–4 days in individual holding cages.
Recordings were made from awake bats in a sound-attenuating chamber. The metal rod on the bat’s head was secured to a small holder for restraining the animal’s head atraumatically, leaving the ears unobstructed for free-field acoustic stimulation. A small hole (∼50 μm) was then made in the skull overlying the IC through which a recording electrode was inserted to reach the IC. Throughout the recording session, the animal was offered drinking water periodically and monitored for signs of discomfort. After a recording session of 6–8 h, the exposed skull was covered with sterile bone wax, and the animal was returned to its holding cage. Such experiments proceeded every 2–3 days for a maximum of 3 weeks.
Extracellular single-unit recordings were made with glass micropipettes (10–20 MΩ, 2–3 μm tip) filled with horseradish peroxidase (5% in 0.2 M potassium acetate in Tris buffer). Such electrodes produce stable single-unit recordings over long periods and thus are preferred over other types of recording electrodes. The electrode was positioned above the IC. The relative position of each electrode was monitored from the readouts of digital micrometers using a common reference on the skull. Vertical advancement of the electrode was made by a precision piezoelectric microdrive from outside the sound-attenuating chamber. Spikes were amplified (Dagan 2400), band-pass filtered (0.3–10 kHz, Krohn-Hite 3700), stored on the computer hard drive, and processed off-line using BrainWare (Tucker-Davis Technologies).
Our experimental protocols for bats are in compliance with the “Guide for the Care and Use of Laboratory Animals” (publication no. 86–23 of the National Institutes of Health) and with the Animal Welfare Act of 1966 and its amendments of 1970 and 1976. These were reviewed and approved by the University of Illinois Lab Animal Use and Care Committee.
Two standard acoustic stimuli were used in the present study: tone pulses at the unit’s best frequency (BF) and FM pulses with linear downward frequency sweep from 80 to 20 kHz, i.e., FM80–20 that approximated bat’s echolocation pulse during the search phase—unless specified otherwise these stimuli had a duration of 2 ms and a rise–fall time of 0.4 ms. In addition to the standard FM pulses above, three narrow-band FM stimuli with a frequency sweep of 20 kHz were also used in some experiments: FM80–60, FM60–40, and FM40–20. Depending on the unit’s absolute BF, one of these narrow FM stimuli encompassed the BF and the remainder outside the BF. Typically we presented 20 tone and FM pulses at each sound level.
Acoustic stimuli were delivered to the bat from a free-field ultrasonic loudspeaker located 60 cm in front of the bat. Sound pulses of a wide range of sound levels, from 10 to 90 dB SPL, were presented at a rate of 1 s−1, in increments of 5 dB. The range of sound level was extended to lower values if the unit’s response threshold was below 10 dB SPL. The absolute sound pressure level (dB re: 20 μPa) was measured with a 1/4 in. condenser microphone (Brüel & Kjær 4135) and a RMS-measuring amplifier (Brüel & Kjær 2610).
Tone pulses were used to determine a unit’s frequency-tuning curve, the units’ BF, and its rate-level function to BF (from which we determined the unit’s temporal discharge patterns and response latencies at different sound levels). Then, the standard FM pulses were presented to generate a separate rate-level function; the unit’s temporal discharge patterns and response latencies to the tone and FM pulses were later compared. Time permitting, the unit’s rate-level functions to narrow FM pulses were made to extend the comparison further.
Dot raster histograms were used to depict a unit’s temporal discharge pattern at each level. Each dot in a raster histogram indicated a spike at the relative time instant with respect to the stimulus onset. Dot raster histograms from different sound levels were combined to create a unit’s composite dot histogram for visualization of how the response latency and the firing pattern changed globally with sound level. To quantify the response latency, we determined the mean first-spike latencies over 20 presentations within the “response window” (defined as the window wherein the spike count is >25% above the background spontaneous firing). The spontaneous firing rate was the average response over a 50 ms window before each stimulus epoch; IC neurons generally showed < 1 spike over this window. To assess the unit’s temporal discharge patterns to different stimuli, for each stimulus we constructed either a composite peri-stimulus-time (PST) histogram across all sound levels or a separate PST histogram and/or inter-spike interval (ISI) histogram at each level.
The majority of PLS neurons (25/39) exhibited latency shifts to both FM and tone pulses. For example, unit 20R31 in Fig. 1a, b showed an average latency of 8.5 ms in response to FM pulses at 10–55 dB SPL, and the response latency was shifted to 12.7 ms in a quantum step when the sound level was increased to 60 dB SPL and to 17.1 ms at ≥ 65 dB SPL. In response to tone pulses at the unit’s BF, its first-spike latency showed a shift from 9.5 to 14.3 ms at 50 dB SPL (Fig. 1b). In both cases, the shift in the first-spike latency was quantal (∼4.2–4.5 ms). Unit 19L51 in Fig. 1c, d exemplified neurons showing differential PLS for FM and tone pulses. For FM stimuli, its first-spike latency shifted quantally from 11 to 16 and 21 ms when the sound level was elevated to 80 and 90 dB SPL, respectively. In contrast, in response to tone pulses, its first-spike latency showed a progressive increase from 10 to 17 ms when the sound level was increased above 60 dB SPL. In nature, the consequence of the PLS is that the intense bat’s sonar signal (the “pulse”) can be expected to elicit relatively long response latencies, whereas the returning echo (the “echo”) is relatively weak and thus would generate relatively short latencies. The differential effects of intense versus weaker signal allow the auditory system to evaluate the elapse time between the pulse and the echo.
For each PLS neuron, we compared the difference in the average first-spike latencies at 90 and at ∼50 dB SPL for each stimulus. We found that the average differences in response latencies for tone and standard FM pulses were 3.5 and 4.8 ms, respectively; these were statistically significant (paired Student’s t test, P < 0.005).
The FM is a prominent feature in the sonar signals of echolocating bats. As described earlier, it is an important sound component for target ranging, for both FM bats and CF/FM bats. Interestingly, in spite of its importance in coding of target range, the underlying reason has not been determined physiologically. In theory, any sound pulse with a clear-cut onset and offset (i.e., having distinct amplitude modulations) can be used to estimate the elapse time between the pulse and its echo, whether the sound is a constant frequency tone or a broadband FM stimulus. However, behavioral evidence indicates that FM signal is better suited for temporal processing than pure tone because it is broadband and thus is processed by an array of neurons tuned to different narrow frequency bands (Simmons 1979; Roverud 1993)—these studies showed that bats do not monitor echo onset and instead they make use of information contained in the internal structure of the signal.
Results of the present study support the behavioral studies showing that FM stimuli are well suited for encoding echo delay. We found that FM stimuli produce oscillatory discharges and PLS more consistently and reliably than CF stimuli (Figs. 2, 3). Specifically, over one-third of the 39 PLS neurons in the IC exhibited oscillatory discharges and PLS to FM stimuli only; these cells either did not respond to tone pulses or showed no PLS in response to tone pulses. For little brown bats, previous studies showed that neurons exhibiting oscillatory discharges comprise a building block for PLS (Galazyuk and Feng 2001; Galazyuk et al. 2005), and that in the cortex PLS is involved in coincident detection of echo delay and the creation of delay-tuned responses (Sullivan 1982b). Taken together, FM signal not only permits multiple estimations of target range by a large network of neurons, but also more reliable estimates of target range (see below).
Our study also showed that the PLS resulting from FM stimuli was largely independent of stimulus duration (Fig. 4). By comparison, the PLS to tone pulses was duration dependent. During a target pursuit, an echolocating bat typically increases its sonar emission rate and concomitantly it progressively decreases the signal duration (Surlykke and Moss 2000; Wadsworth and Moss 2000; Ghose and Moss 2003). The consistency of PLS for FM stimuli across a wide range of stimulus durations can offer an invariant estimate of target range over the entire target pursuit sequence. Our results support the suggestion of Sanderson and Simmons (2005) that the response selectivity of neurons in the bat IC to tone duration (Pinheiro et al. 1991; Casseday et al. 1994; Ehrlich et al. 1997) probably plays little role in guiding the bat’s target pursuit.
At this time, the mechanisms that underlie differential dynamic responses to broadband FM and narrow-band tone pulses are unclear. Various studies have investigated the response selectivity of central auditory neurons to FM sounds (see review by Langner 1992). Along the ascending auditory pathways, neurons become increasingly more selective to FM sounds. In particular, many IC neurons are tuned to narrow ranges of FM (Suga 1965, 1968, 1969; Schuller 1979; Bodenhamer and Pollak 1981; Rees and Moller 1983; Suga et al. 1983; Langner and Schreiner 1988; Fitzpatrick et al. 1991; Casseday and Covey 1992; Poon et al. 1992; Fuzessery 1994; Casseday et al. 1994, 1997; Gordon and O’Neill 2000; O’Neill and Brimijoin 2002), and this response selectivity is abolished when GABAergic inhibition is blocked (Fuzessery and Hall 1996). Although many of these studies investigated the relative efficacy of FM versus tone stimuli for eliciting neural responses at several stimulus levels, the investigations focused on the spike count metric, thereby overlooking the units’ temporal discharge patterns. As such, further studies, and particularly intracellular studies, are needed to elucidate the mechanisms underlying the differential PLS responses to FM and tone bursts.
Wong and colleagues (Shannon-Hartman et al. 1992; Maekawa et al. 1992) previously showed that, in the primary auditory cortex of the little brown bat, most neurons are FM sensitive and display delay-tuned responses to paired-FM sweep containing the units’ BF (but not to paired tone pulses). Results of the present study in the IC of little brown bats are consistent with their findings. Specifically, we found that a number of IC neurons gave robust oscillatory discharges and PLS in response to FM stimuli that contained the unit’s BF (Fig. 3). As oscillatory discharges serve as a building block for PLS, and the latter is the foundation for delay-tuned responses (Sullivan 1982b; Galazyuk and Feng 2001; Galazyuk et al. 2005), these neurons likely contribute to the delay-tuned responses to paired-FM sweep in the cortex.
This research is supported by a grant from the National Institute on Deafness and Communication Disorders of the NIH (R01DC04998). We thank Wenyu Lin and Karla Melendez for their comments on earlier versions of this manuscript.