Classification of insects by echolocating greater horseshoe bats

Summary Echolocating greater horseshoe bats (Rhinolophus ferrumequinum) detect insects by concentrating on the characteristic amplitude- and frequency modulation pattern fluttering insects impose on the returning echoes. This study shows that horseshoe bats can also further analyse insect echoes and thus recognize and categorize the kind of insect they are echolocating.Four greater horseshoe bats were trained in a twoalternative forced-choice procedure to choose the echo of one particular insect species turning its side towards the bat (Fig. 1). The bats were able to discriminate with over 90% correct choices between the reward-positive echo and the echoes of other insect species all fluttering with exactly the same wingbeat rate (Fig. 4).When the angular orientation of the reward-positive insect was changed (Fig. 2), the bats still preferred these unknown echoes over echoes from other insect species (Fig. 5) without any further training. Because the untrained bats did not show any prey preference, this indicates that the bats were able to perform an aspect-anglein-dependent classification of insects.Finally we tested what parameters in the echo were responsible for species recognition. It turned out that the bats especially used the small echo-modulations in between glints as a source of information (Fig. 7). Neither the amplitudenor the frequencymodulation of the echoes alone was sufficient for recognition of the insect species (Fig. 8). Bats performed a pattern recognition task based on complex computations of several acoustic parameters, an ability which might be termed cognitive.Abbreviations AM amplitude modulation - CF constant frequency - FM frequency modulation - S+ positive stimulus - S- negative stimulus     

Electromotility of outer hair cells from the cochlea of the echolocating bat,Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 m long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/m) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 m were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.Abbreviations BM basilar membrane - FFT fast Fourier Transfer - IHC inner hair cell - OHC outer hair cell     

环境噪声对蝙蝠回声定位叫声及飞行活动的影响

环境噪声影响动物的活动及其叫声特性,已成为动物面对的一种重要选择压力。为应对噪声的干扰,多数动物类群会远离噪声区域和改变其叫声的频谱时间结构,如延长叫声持续时间、提高叫声频率等,但有些动物的活动和叫声频谱时间结构并不受环境噪声的影响。本研究在自然条件下,研究不同环境噪声强度对蝙蝠活动和回声定位声波的影响。选取噪声强度有差异的12个样点,分别录制各样点大卫鼠耳蝠、西南鼠耳蝠、亚洲长翼蝠及未知蝙蝠的回声定位声波,分析其持续时间、起始频率、峰频、终止频率和带宽,统计蝙蝠通过次数。回归分析结果显示：环境噪声强度与大卫鼠耳蝠、西南鼠耳蝠、亚洲长翼蝠及未知蝙蝠的活动无显著相关性P > 0.05）,与回声定位声波的脉冲持续时间、起始频率、峰频、终止频率及带宽均不显著相关（P > 0.05）。暗示低频低强度（< 20 kHz, < 67.5 dB）的环境噪声可能对高频回声定位蝙蝠的叫声及活动没有显著影响。     

降雨噪声对菲菊头蝠出飞行为的影响

降雨噪声属于常见的自然噪声,由雨滴撞击物体表面产生。目前,有关降雨噪声对动物的潜在影响被普遍忽视。回声定位蝙蝠主要利用声信号在黑暗环境导航空间、探测猎物及社群交流,是开展降雨噪声影响研究的理想类群。本研究选择菲菊头蝠 (Rhinolophus pusillus)作为研究对象,检验降雨噪声是否影响蝙蝠出飞行为。我们在集群栖息地外,播放强降雨噪声、空白对照和种内回声定位声波,开展野外回放实验。利用单因素方差分析及其事后检验,评价菲菊头蝠对不同回放刺激的反应差异。研究发现,相比空白对照,强降雨噪声导致菲菊头蝠的通勤数量百分比平均降低2.82倍,回声定位脉冲数量平均减少4.86倍,集群出飞时间延长3.75 min。相比空白对照,同种回声定位声波对菲菊头蝠出飞行为的影响并不显著。研究结果证实强降雨噪声抑制菲菊头蝠的出飞行为。本研究表明,降雨引起 的噪声干扰可能是导致蝙蝠躲避降雨的重要因素,为野生蝙蝠物种保育与管理提供启示。     

Frequency tuning, latencies, and responses to frequency-modulated sweeps in the inferior colliculus of the echolocating bat, Eptesicus fuscus

Neurons in the inferior colliculus (IC) of the awake big brown bat, Eptesicus fuscus, were examined for joint frequency and latency response properties which could register the timing of the bat's frequency-modulated (FM) biosonar echoes. Best frequencies (BFs) range from 10 kHz to 100 kHz with 50% tuning widths mostly from 1 kHz to 8 kHz. Neurons respond with one discharge per 2-ms tone burst or FM stimulus at a characteristic latency in the range of 3–45 ms, with latency variability (SD) of 50 μs to 4–6 ms or more. BF distribution is related to biosonar signal structure. As observed previously, on a linear frequency scale BFs appear biased to lower frequencies, with 20–40 kHz overrepresented. However, on a hyperbolic frequency (linear period) scale BFs appear more uniformly distributed, with little overrepresentation. The cumulative proportion of BFs in FM₁ and FM₂ bands reconstructs a scaled version of the spectrogram of FM broadcasts. Correcting FM latencies for absolute BF latencies and BF time-in-sweep reveals a subset of IC cells which respond dynamically to the timing of their BFs in FM sweeps. Behaviorally, Eptesicus perceives echo delay and phase with microsecond or even submicrosecond accuracy and resolution, but even with use of phase-locked FM and tone-burst stimuli the cell-by-cell precision of IC time-frequency registration seems inadequate by itself to account for the temporal acuity exhibited by the bat. Accepted: 21 June 1997     

Distributions of the endocrine cells in the gastrointestinal tract of nectarivorous and sanguivorous bats: A comparative immunocytochemical study

The present study was conducted to clarify the influence of feeding habits on regional distribution and relative frequency of endocrine cells secreting cholecystokinin (CCK), gastrin (GAS), serotonin (5-HT) and enteroglucagon (GLUC) in the nectarivorous Anoura geoffroyi and Glossophaga soricina and the sanguivorous Desmodus rotundus bats of the Phyllostomidae family, by specific immunohistochemical methods. The regional distribution and frequency of the different types of endocrine cells varied according to their location in the GIT. 5-HT immunoreactive cells (IR), detected throughout the GIT of three bats, were the most predominant gastrointestinal endocrine cells. GAS-IR cells in A. geoffroyi were found at the base of the pyloric gland, while in G. soricina they could also be observed in the middle to basal portions of the gland. GLUC-IR cells were located in the fundic region of A. geoffroyi, G. soricina and D. rotundus. These endocrine cells were more abundant in the sanguivorous bat. In nectarivorous bats were compared to sanguivorous bat, which differ in dietary habits, difference in the distribution and relative frequency of gut endocrine cells would be predicted. The absence of some, and decrease in frequency of other, gastrointestinal endocrine cells may reflect, in part, its interspecies differences or dietary habits.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Delayed mitochondrial dysfunction in apoptotic hair cells in chinchilla cochleae following exposure to impulse noise

Apoptotic death of hair cells (HCs) in the cochlea has been found following exposure to intense noise. The current study was designed to examine the mitochondrial energetic function of HCs during the course of noise-induced apoptosis. Two aspects of the mitochondrial energetic function, succinate dehydrogenase (SDH) activity and mitochondrial membrane potential (MMP), were examined in HCs of chinchilla cochleae following exposure to a series of 75 pairs of impulse noises at 155 dB pSPL. The results showed that nuclear condensation and uptake of propidium iodide or trypan blue appeared at 10 min after the noise exposure, indicating a rapid progression of HC apoptosis. However, SDH activity was preserved at this time point. As the time elapsed (1 hr or 24 hrs) after the noise exposure, all newly-generated apoptotic HCs showed strong SDH activity, indicating the preservation of SDH activity during the course of apoptosis. Examination of MMP with rhodamine 123 staining revealed that MMP was sustained in the apoptotic HCs having mild nuclear condensation, even after the occurrence of cell membrane leakage. MMP was reduced with further progression of nuclear condensation. These results suggest the presence of a delayed mitochondrial dysfunction in apoptotic HCs following exposure to intense noise. Research was supported by the Grant NIDCD 1R03 DC006181-01A1.     

Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium

Vestibular hair cells (V–HCs) in the inner ear have important roles and various functions. When V–HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V–HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V–HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V–HCs.     

Cochlear stem/progenitor cells from a postnatal cochlea respond to Jagged1 and demonstrate that notch signaling promotes sphere formation and sensory potential

Hair cells and supporting cells of the mammalian cochlea terminally differentiate during development. Recent in vitro evidence suggests the presence of hair cell progenitors in the postnatal cochlea. Phenotypic properties of these cells and factors that promote their ability to generate spheres in aggregate cultures have not been reported. We define an in vitro system that allows stem/progenitor cells harvested from the early postnatal cochlea to develop into spheres. These spheres contain Abcg2, Jagged1 and Notch1 positive progenitor cells that can divide and generate new hair cell-like cells, i.e. immunopositive for specific hair cell markers, including Myosin VI, Myosin VIIa, Math1 and ability to uptake FM1-43.We demonstrate that reducing Notch signaling with a gamma secretase inhibitor decreases the number of spheres generated following treatment of the stem/progenitor cell cultures. Additionally, activation of Notch by an exogenous soluble form of a Notch ligand, i.e. Jagged1 protein, promotes sphere formation and the sensory potential of cochlear stem/progenitor cells. Our findings suggest that Notch1/Jagged1 signaling plays a role in maintaining a population of Abcg2 sensory stem/progenitor cells in the postnatal cochlea.     

The responses of primary and secondary sensory hair cells in the squid statocyst to mechanical stimulation

Summary Intracellular recordings were obtained from primary and secondary sensory hair cells in the anterior transverse crista segment of the squid (Alloteuthis subulata) statocyst during imposed displacements of the overlying cupula. The secondary sensory hair cells were depolarized by ventral movements of the cupula and hyperpolarized by dorsal cupula movements. The displacement/response curve was asymmetric around the zero position and sigmoidal in shape, similar to that already described for vertebrate hair cells. The cells are estimated to have a sensitivity of at least 0.5 mV per degree angle of cilia displacement. The responses showed pronounced adaptation and could be blocked by bath applied alcohols, such as heptanol or octanol, or by high concentrations of aminoglycosides.The primary sensory hair cells were depolarized by dorsal movements of the cupula, usually responding with a burst of action potentials. The displacement/response curve was also sigmoidal in shape and the firing pattern showed strong adaptation to maintained displacements of the cupula.The cupula itself appeared to be irregular in shape, extending much further into the statocyst cavity in its central part than at its edges. This is likely to result in differences in the responses of the underlying hair cells along the length of the crista ridge.     

Intercellular cross-linkages between the stereociliary bundles of adjacent hair cells in the guinea pig cochlea

Summary Hair cells of the guinea pig organ of Corti have been examined using high resolution scanning electron microscopy. In addition to the extensive array of cross-links between the stereocilia of individual hair cells which have been reported previously, we have seen examples of attachments between the stereocilia of both adjacent inner and adjacent outer hair cells. The implications of these observations are discussed.     

Light- and electron microscopy of isolated vestibular hair cells from the guinea pig

Summary Cells isolated from the guinea-pig vestibular sensory epithelia were studied using light- and electron-microscopic techniques. The cells maintained their characteristic shapes when they had been separated. Mammalian vestibular cells are traditionally divided into two classes, type-I and type-II hair cells. It was, however, found that the population of isolated cells consisted of hair cells with a striking variability in shape and size. This was most conspicuous for the type-I hair cells. Isolated hair cells processed for electron microscopy showed that the isolation process caused minor ultrastructural damage but that the separation often was incomplete in that the large calyx-like nerve endings were still attached to type-I cells. The results suggest that the distinction of only two classes might be insufficient to describe mammalian vestibular hair cells.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Applying genomics to the avian inner ear: development of subtractive cDNA resources for exploring sensory function and hair cell regeneration

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.