Jeffrey J. Wenstrup

Development of social vocalizations in mice.

Adult mice are highly vocal animals, with both males and females vocalizing in same sex and cross sex social encounters. Mouse pups are also highly vocal, producing isolation vocalizations when they are cold or removed from the nest. This study examined patterns in the development of pup isolation vocalizations, and compared these to adult vocalizations. In three litters of CBA/CaJ mice, we recorded isolation vocalizations at ages postnatal day 5 (p5), p7, p9, p11, and p13. Adult vocalizations were obtained in a variety of social situations. Altogether, 28,384 discrete vocal signals were recorded using high-frequency-sensitive equipment and analyzed for syllable type, spectral and temporal features, and the temporal sequencing within bouts. We found that pups produced all but one of the 11 syllable types recorded from adults. The proportions of syllable types changed developmentally, but even the youngest pups produced complex syllables with frequency-time variations. When all syllable types were pooled together for analysis, changes in the peak frequency or the duration of syllables were small, although significant, from p5 through p13. However, individual syllable types showed different, large patterns of change over development, requiring analysis of each syllable type separately. Most adult syllables were substantially lower in frequency and shorter in duration. As pups aged, the complexity of vocal bouts increased, with a greater tendency to switch between syllable types. Vocal bouts from older animals, p13 and adult, had significantly more sequential structure than those from younger mice. Overall, these results demonstrate substantial changes in social vocalizations with age. Future studies are required to identify whether these changes result from developmental processes affecting the vocal tract or control of vocalization, or from vocal learning. To provide a tool for further research, we developed a MATLAB program that generates bouts of vocalizations that correspond to mice of different ages.

Responses to Combinations of Tones in the Nuclei of the Lateral Lemniscus

Combination-sensitive neurons integrate specific spectral and temporal elements in biologically important sounds, and they may underlie the analysis of biosonar and social vocalizations. Combination-sensitive neurons are found in the forebrain of a variety of vertebrates. In the mustached bat, they also occur in the central nucleus of the inferior colliculus (ICC). However, it is not known where combination-sensitive response properties emerge. To address this question, we used a two-tone paradigm to examine responses of single units to combination stimuli in a brainstem structure, the nuclei of the lateral lemniscus (NLL). We recorded and histologically localized 101 single units in the NLL. The majority (82%) of units had a single excitatory frequency tuning curve. Seven units had two separate excitatory frequency tuning curves but displayed no combinatorial interaction. Twelve units were combination-sensitive. Of these, three units were facilitated by the combination of two separate frequency bands and nine units were inhibited by combinatorial stimuli. The three facilitatory neurons had excitatory responses tuned to the second harmonic constant frequency (CF2, 57-60 kHz) component of the biosonar signal and were facilitated by a second signal within the first harmonic (Hl, 24-30 kHz) of the biosonar call. Most of the inhibitory interactions occurred between signals in the frequency bands associated with the frequency-modulated (FM) components of the biosonar call. The strongest combinatorial effects (facilitatory and inhibitory) were elicited by simultaneous onset of the two signals (i.e., 0 ms delay). All combination-sensitive units were in the intermediate nucleus of the NLL (INLL), which in bats is a hypertrophied structure that projects strongly to combination-sensitive neurons in the ICC. Thus, the combination-sensitive neurons in the INLL may impart their response properties onto ICC neurons. However, the small number of facilitatory combination-sensitive neurons in the NLL suggests that the majority of these combinatorial responses originate in the ICC.

Glycinergic “Inhibition” Mediates Selective Excitatory Responses to Combinations of Sounds

In the mustached bats inferior colliculus (IC), combination-sensitive neurons display time-sensitive facilitatory interactions between inputs tuned to distinct spectral elements in sonar or social vocalizations. Here we compare roles of ionotropic receptors to glutamate (iGluRs), glycine (GlyRs), and GABA (GABAARs) in facilitatory combination-sensitive interactions. Facilitatory responses to 36 single IC neurons were recorded before, during, and after local application of antagonists to these receptors. The NMDA receptor antagonist CPP [(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid], alone (n = 14) or combined with AMPA receptor antagonist NBQX (n = 22), significantly reduced or eliminated responses to best frequency (BF) sounds across a broad range of sound levels, but did not eliminate combination-sensitive facilitation. In a subset of neurons, GABAAR blockers bicuculline or gabazine were applied in addition to iGluR blockers. GABAAR blockers did not “uncover” residual iGluR-mediated excitation, and only rarely eliminated facilitation. In nearly all neurons for which the GlyR antagonist strychnine was applied in addition to iGluR blockade (22 of 23 neurons, with or without GABAAR blockade), facilitatory interactions were eliminated. Thus, neither glutamate nor GABA neurotransmission are required for facilitatory combination-sensitive interactions in IC. Instead, facilitation may depend entirely on glycinergic inputs that are presumed to be inhibitory. We propose that glycinergic inputs tuned to two distinct spectral elements in vocal signals each activate postinhibitory rebound excitation. When rebound excitations from two spectral elements coincide, the neuron discharges. Excitation from glutamatergic inputs, tuned to the BF of the neuron, is superimposed onto this facilitatory interaction, presumably mediating responses to a broader range of acoustic signals.

Topographical distribution of delay-tuned responses in the mustached bat inferior colliculus.

In the mustached bat, delay-tuned neurons respond best to specific delays between the first harmonic frequency modulated (FM) component (FM1; 24-29 kHz) of the emitted biosonar pulse and a higher harmonic FM component in returning echoes (e.g. FM3, 72-89 kHz). These delay-tuned, combinatorial responses predominate in the inferior colliculus (IC) of the mustached bat. This study examined the topographical distribution of delay-tuned neurons in the 72-89 kHz frequency representation of the IC. We recorded and histologically localized 163 single units. Ninety units were facilitated and 41 were inhibited by the combination of two frequencies in the 24-29 kHz and 72-89 kHz ranges. The facilitatory responses were selective for delays up to 20 ms between the two signals. To determine if delay-tuned neurons were topographically organized, we plotted the dorsomedio-ventrolateral and caudo-rostral positions of each unit versus its best delay. Best delay was not correlated with either location. Response latency to best frequency tones was topographically organized, but was not correlated with best delay. This indicates that the latency axis in the IC is unrelated to the delay tuning of these combinatorial neurons. Because delay-tuned neurons are not topographically organized in the IC but are in the auditory cortex, our findings suggest that the creation and organization of delay-tuned neurons occur at different stages in the ascending auditory system.

Excitatory and facilitatory frequency response areas in the inferior colliculus of the mustached bat

In the mustached bats central nucleus of the inferior colliculus (ICC), many neurons display facilitatory or inhibitory responses when presented with two tones of distinctly different frequencies. Our previous studies have focused on spectral interactions between specific frequency bands contained in the bats sonar vocalization. In this study, we describe excitatory and facilitatory frequency response areas across all frequencies in the mustached bats audible range. We show that many neurons in the ICC have more extensive frequency interactions than previously documented. We recorded responses of 96 single units to single tones and combinations of two tones. Best frequencies of the units ranged from 59-15 kHz. Forty-one units had a single, excitatory frequency response area. The rest of the units had more complex frequency tuning that included multiple excitatory frequency response areas and facilitatory frequency response areas. Some of the facilitatory frequency interactions were between one sound with energy in a sonar frequency band and a second sound with energy in a non-sonar frequency band. We also found that neurons could be facilitated by more than one additional frequency band. Our findings of extensive frequency interactions in the ICC of the mustached bat suggest that some neurons may be well suited for the analysis of complex sounds, possibly including social communication sounds.

INPUTS TO COMBINATION-SENSITIVE NEURONS OF THE INFERIOR COLLICULUS

In the mustached bat, combination‐sensitive neurons display integrative responses to combinations of acoustic elements in biosonar or social vocalizations. One type of combination‐sensitive neuron responds to multiple harmonics of the frequency‐modulated (FM) components in the sonar pulse and echo of the bat. These neurons, termed FM‐FM neurons, are sensitive to the pulse–echo delay and may encode the distance of sonar targets. FM‐FM neurons are common in high‐frequency regions of the central nucleus of the inferior colliculus (ICC) and may be created there. If so, they must receive low‐frequency inputs in addition to the expected high‐frequency inputs. We placed single deposits of a tracer at FM‐FM recording sites in the ICC and then analyzed retrograde labeling in the brainstem and midbrain. We were particularly interested in labeling patterns suggestive of low‐frequency input to these FM‐FM neurons. In most nuclei containing labeled cells, there was a single focus of labeling in regions thought to be responsive to high‐frequency sounds. More complex labeling patterns were observed in three nuclei. In the anteroventral cochlear nucleus, labeling in the anterior and marginal cell divisions occurred in regions thought to respond to low‐frequency sounds. This labeling comprised 6% of total brainstem labeled cells. Labeling in the intermediate nucleus of the lateral lemniscus and the magnocellular part of the ventral nucleus of the lateral lemniscus together comprised nearly 40% of all labeled cells. In both nuclei, multiple foci of labeling occurred. These different foci may represent groups of cells tuned to different frequency bands. Thus, one or more of these three nuclei may provide low‐frequency input to high‐frequency‐sensitive cells in the ICC, creating FM‐FM responses. We also examined whether ICC neurons responsive to lower frequencies project to high‐frequency‐sensitive ICC regions; only 0.15% of labeling originated from these lower frequency representations. If the spectral integration of FM‐FM neurons is created at the level of the ICC, these results suggest that neurons of the anteroventral cochlear nucleus or monaural nuclei of the lateral lemniscus may provide the essential low‐frequency input. In contrast, there is little evidence that the low‐frequency representation of the ICC contributes to these integrative responses. J. Comp. Neurol. 409:509–528, 1999.

Social Vocalizations of Big Brown Bats Vary with Behavioral Context

Bats are among the most gregarious and vocal mammals, with some species demonstrating a diverse repertoire of syllables under a variety of behavioral contexts. Despite extensive characterization of big brown bat (Eptesicus fuscus) biosonar signals, there have been no detailed studies of adult social vocalizations. We recorded and analyzed social vocalizations and associated behaviors of captive big brown bats under four behavioral contexts: low aggression, medium aggression, high aggression, and appeasement. Even limited to these contexts, big brown bats possess a rich repertoire of social vocalizations, with 18 distinct syllable types automatically classified using a spectrogram cross-correlation procedure. For each behavioral context, we describe vocalizations in terms of syllable acoustics, temporal emission patterns, and typical syllable sequences. Emotion-related acoustic cues are evident within the call structure by context-specific syllable types or variations in the temporal emission pattern. We designed a paradigm that could evoke aggressive vocalizations while monitoring heart rate as an objective measure of internal physiological state. Changes in the magnitude and duration of elevated heart rate scaled to the level of evoked aggression, confirming the behavioral state classifications assessed by vocalizations and behavioral displays. These results reveal a complex acoustic communication system among big brown bats in which acoustic cues and call structure signal the emotional state of a caller.

Contribution of NMDA and AMPA Receptors to Temporal Patterning of Auditory Responses in the Inferior Colliculus

Although NMDA receptors (NMDARs) are associated with synaptic plasticity, they form an essential part of responses to sensory stimuli. We compared contributions of glutamatergic NMDARs and AMPA receptors (AMPARs) to auditory responses in the inferior colliculus (IC) of awake, adult mustached bats. We examined the magnitude and temporal pattern of responses to tonal signals in single units before, during, and after local micro-iontophoretic application of selective antagonists to AMPARs [NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide)] and NMDARs [CPP ((±)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid)]. Combined blockade of AMPARs and NMDARs eliminated excitatory responses in nearly all neurons, whereas separate blockade of each receptor was quantitatively similar, causing substantial (>50%) spike reductions in ∼75% of units. The major result was that effects of receptor blockade were most closely related to the first-spike latency of a unit. Thus, AMPAR blockade substantially reduced spikes in all short-latency units (<12 ms) but never in long-latency units (≥12 ms). NMDAR blockade had variable effects on short-latency units but reduced spikes substantially for all long-latency units. There were no distinct contributions of AMPARs and NMDARs to early and late elements of responses. Thus, AMPAR blockade reduced early (onset) spikes somewhat more effectively than NMDAR blockade in short-latency units, but NMDAR blockade reduced onset spikes more effectively in long-latency units. AMPAR and NMDAR blockade were equally effective in reducing later elements of sustained responses in short-latency units, whereas NMDAR blockade was much more effective in long-latency units. These results indicate that NMDARs play multiple roles for signal processing in adult IC neurons.

Intracellular Recordings From Combination-Sensitive Neurons in the Inferior Colliculus

In vertebrate auditory systems, specialized combination-sensitive neurons analyze complex vocal signals by integrating information across multiple frequency bands. We studied combination-sensitive interactions in neurons of the inferior colliculus (IC) of awake mustached bats, using intracellular somatic recording with sharp electrodes. Facilitated combinatorial neurons are coincidence detectors, showing maximum facilitation when excitation from low- and high-frequency stimuli coincide. Previous work showed that facilitatory interactions originate in the IC, require both low and high frequency-tuned glycinergic inputs, and are independent of glutamatergic inputs. These results suggest that glycinergic inputs evoke facilitation through either postinhibitory rebound or direct depolarizing mechanisms. However, in 35 of 36 facilitated neurons, we observed no evidence of low frequency-evoked transient hyperpolarization or depolarization that was closely related to response facilitation. Furthermore, we observed no evidence of shunting inhibition that might conceal inhibitory inputs. Since these facilitatory interactions originate in IC neurons, the results suggest that inputs underlying facilitation are electrically segregated from the soma. We also recorded inhibitory combinatorial interactions, in which low frequency sounds suppress responses to higher frequency signals. In 43% of 118 neurons, we observed low frequency-evoked hyperpolarizations associated with combinatorial inhibition. For these neurons, we conclude that low frequency-tuned inhibitory inputs terminate on neurons primarily excited by high-frequency signals; these inhibitory inputs may create or enhance inhibitory combinatorial interactions. In the remainder of inhibited combinatorial neurons (57%), we observed no evidence of low frequency-evoked hyperpolarizations, consistent with observations that inhibitory combinatorial responses may originate in lateral lemniscal nuclei.

A novel coding mechanism for social vocalizations in the lateral amygdala

The amygdala plays a central role in evaluating the significance of acoustic signals and coordinating the appropriate behavioral responses. To understand how amygdalar responses modulate auditory processing and drive emotional expression, we assessed how neurons respond to and encode information that is carried within complex acoustic stimuli. We characterized responses of single neurons in the lateral nucleus of the amygdala to social vocalizations and synthetic acoustic stimuli in awake big brown bats. Neurons typically responded to most of the social vocalizations presented (mean = nine of 11 vocalizations) but differentially modulated both firing rate and response duration. Surprisingly, response duration provided substantially more information about vocalizations than did spike rate. In most neurons, variation in response duration depended, in part, on persistent excitatory discharge that extended beyond stimulus duration. Information in persistent firing duration was significantly greater than in spike rate, and the majority of neurons displayed more information in persistent firing, which was more likely to be observed in response to aggressive vocalizations (64%) than appeasement vocalizations (25%), suggesting that persistent firing may relate to the behavioral context of vocalizations. These findings suggest that the amygdala uses a novel coding strategy for discriminating among vocalizations and underscore the importance of persistent firing in the general functioning of the amygdala.