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Sperm morphology is considered the best indicator of male fertility. In Neotropical bats, important aspects of sperm morphology have been scantly studied. The aim of the present study was to characterize and compare the sperm morphology and morphometry of Artibeus planirostris and Sturnira erythromos. A total of 11 specimens were analyzed from the Colección de Mamíferos Lillo: five A. planirostris and six S. erythromos. The fixed epididymis were extracted and macerated in Farmer's solution, followed by the routine cytological procedure with different stains. To carry out the description and morphometric analysis, microphotographs were taken under an optical, epifluorescence and scanning electron microscope. A total of 50 sperm from each individual were measured for morphometric analysis. The percentage of normal/abnormal spermatozoa was estimated and the sperm abnormalities were classified. Both species showed morphologically simple spermatozoa with a spatulate head, a short neck, a helical midpiece and a tail that tapers at the final end, similar to other species of Phyllostomidae. The differences observed were: apex of the head was conical in A. planirostris and was oval in S. erythromos; longer head and midpiece in S. erythomos and longer sperm in A. planirostris. Both species showed a high percentage of sperm with normal appearance: 65% for A. planirostris and 72% for S. erythromos. The main sperm abnormalities were: scattered tails and heads, coiled tails, folded midpieces and presence of cytoplasmic droplets. The present work will improve the understanding of their reproductive biology. RESEARCH HIGHLIGHTS: Morphological descriptions and morphometric analyses of the sperm of Artibeus planirostris and Sturnira erythromos were carried out with optical, epifluorescence and scanning electron microscopy., (© 2024 Wiley Periodicals LLC.)

Bats have the ability to fly without eye application in the darkness. In this study, we aimed to characterize the functional and structural acclimations of the lenses of two common bats with a various lifestyle in the Egyptian environment: the insectivorous bat (IB) (Pipistrellus kuhlii) and Egyptian fruit bat (FB) (Rousettus aegyptiacus). From each species, seven lenses were extracted from adult eyes. The scanning electron microscopic (SEM) and light microscopic examination of the lens were carried out. FB lenses were made up primarily of fiber cells and sheets, which were encapsulated by a thin collagenous capsule and covered by single epithelial layer anteriorly. On the other hand, the IB lens had two poles and was visibly oval shaped. Both lenses had epithelial cells of the same cuboidal form that were subjected to continuous division and differentiation into new fiber cells at the center. SEM revealed that the normal FB lens had regularly organized shells of fiber cells of intact lens fibers which were connected by membrane interdigitations with different shapes mainly ball-and-socket junctions through the superficial cortical fiber cells. The IB lens was composed of parallel, evenly spaced fibers with various types of interdigitations between fibers that can be seen and increased close to the middle region revealing tiny bumps along the scrubby portions and sockets and balls in the center of the wide portions. Near the center of both lenses, there were large interlocking paddles with little and lengthy protrusions along their short sides. In conclusion, our study discovered several ultrastructural and structural variations among the investigated species. The detection of specialized membrane interdigitations with different shapes protruding from the lens fiber sheets is considered the most characteristic of the FB lens. RESEARCH HIGHLIGHTS: FB lens has more organized sheets of fibers parallel to each other than IB lens. Different shapes of interdigitations protruded from the FB lens have been detected. Interlocking paddles, balls, and sockets with tongue-like fiber flabs are characteristic to FB lens., (© 2024 Wiley Periodicals LLC.)

Sperm morphology is considered a species-specific character and has been used as a tool in the classification of numerous mammalian taxa. Neotropical bats have been poorly studied, and important aspects on sperm morphology have not been elucidated. The aim of the present study was to describe and compare the sperm morphology and morphometry of Molossus molossus and Molossops temminckii. A total of 14 adults specimens were analyzed from the Colección Mamíferos Lillo, Universidad Nacional de Tucumán: five M. molossus and nine M. temminckii. The epididymis were extracted and macerated in Farmer's solution, followed by a coloration with different stains. To carry out the description and morphometric analysis, microphotographs were taken under an optical, epifluorescence, and scanning electron microscope (SEM). A total of 50 sperm from each individual were measured for morphometric analysis. The length and width of the head, midpiece and tail were taken as variables. Sperm from M. molossus and M. temminckii were practically identical, both morphologically and morphometrically. In both species, a distal bulge was observed at the end of the intermediate piece in a percentage greater than 85%. The main characteristics shared between the species were: presence of acrosomal blebs in the upper half of the head of the spermatozoa; cephalic equatorial segment with filiform ornamentations; intermembrane space of head apex wedge-shaped; helical middle piece and annulus at the end of middle piece. In the present study, SEM allowed us to visualize structures, such as acrosomal vesicles, that were not detected with other types of microscopy. RESEARCH HIGHLIGHTS: The similarities in the sperm morphology between M. molossus and M. temminckii were observed with three types of microscopy: optical, epifluorescence and scanning electron, and supported by morphometric and statistical analyses., (© 2023 Wiley Periodicals LLC.)

Haeckel's recapitulation theory has been a controversial topic in evolutionary biology. However, we have seen some recent cases applying Haeckel's view to interpret the interspecific variation of prenatal ontogeny. To revisit the validity of Haeckel's recapitulation theory, we take bats that have undergone drastic morphological changes and possess a characteristic ecology as a case study. All members of Rhinolophoidea and Yangochiroptera can generate an ultrasonic pulse from the larynx to interpret surrounding objects (laryngeal echolocation) whereas Pteropodidae lacks such ability. It is known that the petrosal bone is particularly derived in shape and expanded in laryngeal echolocators. If Haeckel's recapitulation theory holds, the formation of this derived trait should occur later than those of other bones. Therefore, we compared the prenatal ossification timing of the petrosal in 15 bat species and five outgroup species. We found that the ossification of the petrosal is accelerated in laryngeal echolocators while it is the last bone to ossify in non-laryngeal echolocating bats and non-volant mammals, which runs counter to the prediction generated by Haeckel's recapitulation theory. We point out the evolutionarily labile nature of trait developmental timing and emphasize that Haeckel's recapitulation theory does not hold in many cases. We caution that generating predictions on ancestral conditions and evolutionary history leading from Haeckel's recapitulation theory is not well supported., (© 2021 Wiley Periodicals LLC.)

Small echolocating bats are set apart from most other mammals by their relatively large cerebellum, a feature that has been associated to echolocation, as it is presumed to indicate a relatively enlarged number of neurons in the cerebellum in comparison to other brain structures. Here we quantify the neuronal composition of the cerebral cortex, cerebellum and remaining brain structures of seven species of large Pteropodid bats (formerly classified as megachiropterans), one of which echolocates, and six species of small bats (formerly classified as microchiropterans), all of which echolocate. This chiropteran data is compared to 60 mammalian species in our dataset to determine whether the relatively large cerebellum of the small echolocating bats, and possibly that of the echolocating Pteropodid, contains a relatively enlarged number of neurons. We find no evidence that the distribution of neurons differs between microchiropterans and megachiropterans, but our data indicate that microchiropterans, like the smallest shrew in our dataset, have diminutive cerebral cortices, which makes the cerebellum appear relatively large. We propose that, in agreement with the diminutive brain size of the earliest fossil mammals, this is a plesiomorphic, not a derived, feature of microchiropteran brains. The results of this study also reveal important neural characteristics related to the phylogenetic affinities and relationships of the chiropterans., (© 2020 Wiley Periodicals LLC.)

Bats are the only mammals that can fly in the dark without eye usage. This study was conducted to describe the structural and functional adaptations of the retina of two bats very common in the Egyptian fauna having a different lifestyle: the Egyptian fruit bat (Rousettus aegyptiacus) and insectivorous bat (Pipistrellus kuhlii). Seven eyes were collected from adult individuals of each species. Examination of the retina using a light microscope and a transmission electron microscope was carried out. The retina of P. kuhlii was thicker than that of R. aegyptiacus, which had many projections extended from the choroid layer into retina forming papillae. Despite rods being dominant in retinae of both species, cone photoreceptors were encountered in both retinae. The outer plexiform layer of R. aegyptiacus was arranged into islets between the outer nuclear layer produced differences in its thickness. However, the retina of P. kuhlii showed a normal arrangement of retinal structure. The retinal pigment epithelium of both bat species consists of a single layer of the cuboidal cells with a round to oval vesicular nuclei, which showed a lack of pigmentation in R. aegyptiacus and poor pigmentation in the P. kuhlii. In conclusion, our investigation detected many structural and ultrastructural differences between the two bat species. The presence of many projections protruded from the choroid layer of R. aegyptiacus retina is considered the most characteristic difference between the retinae of R. aegyptiacus and P. kuhlii., (© 2020 Wiley Periodicals LLC.)

The claustrum is a gray-matter structure that underlies neocortex and reciprocates connections with cortical and subcortical targets. In lower mammals, the claustrum is directly adjacent to neocortex, making the definition of claustral boundaries challenging. Latexin, an endogenous inhibitor of metallocarboxypeptidases, localizes to claustral cells, enabling a clear delineation of claustrum. Given its proportionately large claustrum, we hypothesized that the short-tailed fruit bat, Carollia perspicillata, can be a useful model for claustral structure-function relations. We used latexin immunohistochemistry to identify claustral boundaries and intrinsic structure and multielectrode recordings from brain slices to explore intrinsic excitatory connectivity of the claustrum. Carollia's claustrum contains cells whose intrinsic connectivity and alignment permit the generation of spontaneous, synchronous population events and mirror their pattern of spread in disinhibited brain slices over millimeters. Carollia shows cellular alignment and spontaneous population-activity spread along both horizontal and dorsoventral axes. Carollia claustrum possesses intrinsic excitatory connectivity sufficient to: 1) generate single, spontaneous, synchronized burst discharges, 2) support activity spread along axes where claustral cells are aligned, and 3), because of multiple axes for cell alignment, support activity spread along both rostrocaudal and dorsoventral axes. The smaller event sizes in bat claustrum compared with rat claustrum are consistent with events occurring in population subsets rather than the full claustral cell population. The overall size of claustrum, its pronounced vascularity, and its more complex intrinsic connectivity than rat suggest that the bat is an animal model for claustral structure and function that will permit unique access to claustrum's processing capabilities. J. Comp. Neurol. 525:1459-1474, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Megachiropteran bats (megabats) show remarkable diversity in microhabitat occupation and trophic specializations, but information on how vision relates to their behavioral ecology is scarce. Using stereology and retinal wholemounts, we measured the topographic distribution of retinal ganglion cells and determined the spatial resolution of eight African megachiropterans with distinct roosting and feeding ecologies. We found that species roosting in open microhabitats have a pronounced streak of high retinal ganglion cell density, whereas those favoring more enclosed microhabitats have a less pronounced streak (or its absence in Hypsignathus monstrosus). An exception is the cave-dwelling Rousettus aegyptiacus, which has a pronounced horizontal streak that potentially correlates with its occurrence in more open environments during foraging. In all species, we found a temporal area with maximum retinal ganglion cell density (∼5,000-7,000 cells/mm 2 ) that affords enhanced resolution in the frontal visual field. Our estimates of spatial resolution based on peak retinal ganglion cell density and eye size (∼6-12 mm in axial length) range between ∼2 and 4 cycles/degree. Species that occur in more enclosed microhabitats and feed on plant material have lower spatial resolution (∼2 cycles/degree) compared with those that roost in open and semiopen areas (∼3-3.8 cycles/degree). We suggest that the larger eye and concomitant higher spatial resolution (∼4 cycles/degree) in H. monstrosus may have facilitated the carnivorous aspect of its diet. In conclusion, variations in the topographic organization and magnitude of retinal ganglion density reflect the specific ecological needs to detect food/predators and the structural complexity of the environments. J. Comp. Neurol. 525:186-203, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

To provide a modern description of the Chiropteran visual system, the subcortical retinal projections were studied in the short-tailed fruit bat, Carollia perspicillata, using the anterograde transport of eye-injected cholera toxin B subunit, supplemented by the silver-impregnation of anterograde degeneration following eye removal, and compared with the retinal projections of the mouse. The retinal projections were heavily labeled by the transported toxin in both species. Almost all components of the murine retinal projection are present in Carollia in varying degrees of prominence and laterality. The projections: to the superior colliculus, accessory optic nuclei, and nucleus of the optic tract are predominantly or exclusively contralateral; to the dorsal lateral geniculate nucleus and posterior pretectal nucleus are predominantly contralateral; to the ventral lateral geniculate nucleus, intergeniculate leaflet, and olivary pretectal nucleus have a substantial ipsilateral component; and to the suprachiasmatic nucleus are symmetrically bilateral. The retinal projection in Carollia is surprisingly reduced at the anterior end of the dorsal lateral geniculate and superior colliculus, suggestive of a paucity of the relevant ganglion cells in the ventrotemporal retina. In the superior colliculus, in which the superficial gray layer is very thin, the projection is patchy in places where the layer is locally absent. Except for a posteriorly located lateral terminal nucleus, the other accessory optic nuclei are diminutive in Carollia, as is the nucleus of the optic tract. In both species the cholera toxin labeled sparse groups of apparently terminating axons in numerous regions not listed above. A question of their significance is discussed., (© 2015 Wiley Periodicals, Inc.)

We studied the retinal cone bipolar cells of Carollia perspicillata, a microchiropteran bat of the phyllostomid family. Microchiroptera are strongly nocturnal, with small eyes and rod-dominated retinae. However, they also possess a significant cone population (2-4%) comprising two spectral types, which are hence the basis for daylight and color vision. We used antibodies against the calcium-binding protein recoverin and the carbohydrate epitope 15 (CD15) as reliable markers for certain cone bipolar cells. Dye injections of recoverin- or CD15-prelabeled cone bipolar cells in vertical slices revealed the morphology of the axon terminal system of individual bipolar cells. Seven distinct cone bipolar cell types were identified. They differed in the morphology and stratification level of their axon terminal system in the inner plexiform layer and in immunoreactivity for recoverin and/or CD15. Additional immunocytochemical markers were used to assess the functional ON/OFF subdivision of the inner plexiform layer. In line with the extended thickness of the ON sublayer of the inner plexiform layer in the microbat retina, more ON than OFF cone bipolar cell types were found, namely, four versus three. Most likely, in the bats' predominantly dark environment, ON signals have greater importance for contrast perception. We conclude that the microbat retina conforms to the general mammalian blueprint, in which light signals of intensities above rod sensitivity are detected by cones and transmitted to various types of ON and OFF cone bipolar cells., (© 2014 Wiley Periodicals, Inc.)

The pallid bat (Antrozous pallidus) listens to prey-generated noise to localize and hunt terrestrial prey while reserving echolocation to avoid obstacles. The thalamocortical connections in the pallid bat are organized as parallel pathways that may serve echolocation and prey localization behaviors. Thalamic inputs to the cortical echolocation call- and noise-selective regions originate primarily in the suprageniculate nucleus (SG) and ventral division of medial geniculate body (MGBv), respectively. Here we examined the distribution of parvalbumin (PV) and calbindin (CB) expression in cortical regions and thalamic nuclei of these pathways. Electrophysiology was used to identify cortical regions selective for echolocation calls and noise. Immunohistochemistry was used to stain for PV and CB in the auditory cortex and MGB. A higher percentage (relative to Nissl-stained cells) of PV(+) cells compared with CB(+) cells was found in both echolocation call- and noise-selective regions. This was due to differences in cortical layers V-VI, but not layers I-IV. In the MGB, CB(+) cells were present across all divisions of the MGB, with a higher percentage in the MGBv than the SG. Perhaps the most surprising result was the virtual absence of PV staining in the MGBv. PV staining was present only in the SG. Even in the SG, the staining was mostly diffuse in the neuropil. These data support the notion that calcium binding proteins are differentially distributed in different processing streams. Our comparative data, however, do not support a general mammalian pattern of PV/CB staining that distinguishes lemniscal and nonlemniscal pathways., (Copyright © 2014 Wiley Periodicals, Inc.)

Myotis nigricans is an endemic species of vespertilionid bat, from the Neotropical region, that resembles temperate zone bats in their reproductive cycle; presenting an annual reproductive cycle with two periods of testicular regression, which are not linked to the apoptotic process and seems to be not directly linked to any seasonal abiotic variation. Thus, this study aimed to ultrastructurally evaluate their reproductive cycle. The process of testicular regression could be divided into four periods: active; regressing; regressed and recrudescence; with all presenting distinct characteristics. The active period was similar to that of other bats, presenting the complete occurrence of spermatogenesis, with three main types of spermatogonia (A(d), A(p), and B) and 12 steps in spermatid differentiation; however, it differed in having the outer dense fibers 1, 5, 6, and 9 larger than the others. These three types of spermatogonia undergo considerable morphologic changes from regressing to the regressed period, and in the recrudescence, they return to the basic morphology, which reactivates spermatogenesis. In conclusion, our study described the process of spermatogenesis, the ultrastructure of the spermatozoa and the distinct morphologic variations in the ultrastructure of the testicular cells of M. nigricans during the four different periods of its annual reproductive cycle., (Copyright © 2013 Wiley Periodicals, Inc.)

Environmental factors can influence the reproductive rates in bats, and since morphometric information of bats testis is scarce, we aimed to compare the organization and quantification of the intertubular components in the testes of the bat Molossus molossus, collected in different seasons. Testicular histological sections were evaluated using light and electron microscopy. The intertubular compartment occupied an average 10% of the testes, being predominately constituted of Leydig cells (LC). The percentages of the testes occupied by the intertubular compartment and by LC were significantly higher in summer, while the other intertubular components did not vary significantly among the seasons. As suspected under light microscopy, the ultrastructural analysis confirmed the existence of multinucleated LC during winter. The increase in the nuclear percentage of LC in winter seems to have caused the decrease of the cytoplasmatic measurements in that season, as well as in the volume of LC. The highest cytoplasmatic values and volume of LC registered in the spring, summer, and fall can be related to greater activity of this cell in these seasons. The higher investment in intertubular tissue and in LC observed in summer, compared to winter; suggest an increase in the steroidogenic capacity of this bat during summer. The analyses correlating testicular morphometry and abiotic environmental factors in this study confirm the influence of climatic factors on the reproduction of M. molossus., (Copyright © 2012 Wiley Periodicals, Inc.)

Transverse boundaries divide the mammalian cerebellar cortex into transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. This topography is highly conserved across the Mammalia. Bats have a remarkable cerebellum with presumed adaptations to flight and to echolocation, but nothing is known of its compartmentation. We have therefore used two Purkinje cell compartmentation antigens, zebrin II/aldolase C and phospholipase Cbeta4, to reveal the topography of the cerebellum in microchiropteran bats. Three species of bat were studied, Lasiurus cinereus, Lasionycteris noctivagans, and Eptesicus fuscus. A reproducible pattern of zones and stripes was revealed that is similar across the three species. The architecture of the bat cerebellum conforms to the ground plan of other mammals. However, two exceptions to the highly conserved mammalian architectural plan were revealed. First, many Purkinje cells in lobule I express zebrin II. A zebrin II-immunopositive lobule I has not been seen previously in mammals but is characteristic of the avian cerebellum. Second, lobules VI-VII comprise the large central zone. Within the central zone two subdomains are evident, a small anterior subdomain (lobule VI) in which Purkinje cells are predominantly zebrin II-immunopositive/PLCbeta4-immunonegative, as in other mammals, and a posterior subdomain (lobule VII), in which alternating zebrin II/phospholipase Cbeta4 stripes are prominent.

The auditory thalamus is the major target of the inferior colliculus and connects in turn with the auditory cortex. In the mustached bat, biosonar information is represented according to frequency in the central nucleus of the inferior colliculus (ICc) but according to response type in the cortex. In addition, the cortex has multiple areas with neurons of similar response type compared to the single tonotopic representation in the ICc. To investigate whether these transformations occur at the level of the thalamus, we injected anatomical tracers into physiologically defined locations in the mustached bat's auditory cortex. Injections in areas used for target ranging labeled contiguous regions of the auditory thalamus rather than separate patches corresponding to regions that respond to the different harmonic frequencies used for ranging. Injections in the two largest ranging areas produced labeling in separate locations. These results indicate that the thalamus is organized according to response type rather than frequency and that multiple mappings of response types exist. Injections in areas used for target detection labeled thalamic regions that were largely separate from those that interconnect with ranging areas. However, injections in an area used for determining target velocity overlapped with the areas connected to ranging areas and areas involved in target detection. Thus, separation by functional type and multiplication of areas with similar response type occurs by the thalamic level, but connections with the cortex segregate the functional types more completely than occurs in the thalamus., ((c) 2006 Wiley-Liss, Inc.)

We present evidence for parallel auditory thalamocortical pathways that serve two different behaviors. The pallid bat listens for prey-generated noise (5-35 kHz) to localize prey, while reserving echolocation [downward frequency-modulated (FM) sweeps, 60-30 kHz] for obstacle avoidance. Its auditory cortex contains a tonotopic map representing frequencies from 6 to 70 kHz. The high-frequency (BF > 30 kHz) representation is dominated by FM sweep-selective neurons, whereas most neurons tuned to lower frequencies prefer noise. Retrograde tracer injections into these physiologically distinct cortical regions revealed that the high-frequency region receives input from the suprageniculate (SG) nucleus, but not the ventral division of the medial geniculate body (MGBv), in all experiments (n = 9). In contrast, the low-frequency region receives tonotopically organized input from the MGBv in all experiments (n = 16). Labeling in the SG was observed in only two of these experiments. Both cortical regions also receive sparse inputs from medial (MGBm) and parts of the dorsal division (MGBd) outside the SG. These results show that the low- and high-frequency regions of a single tonotopic map receive dominant inputs from different thalamic divisions. Within the low-frequency region, most neurons are binaurally inhibited, and an orderly map of interaural intensity difference (IID) sensitivity is present. We show that the input to the IID map arises from topographically organized projections from the MGBv. As observed in other species, a frequency-dependent organization is observed in the lateromedial direction in the MGBv. These data demonstrate that MGBv-to-auditory cortex connections are organized with respect to both frequency and binaural selectivity., ((c) 2006 Wiley-Liss, Inc.)

The wide range of dietary niches filled by modern mammals is reflected in morphological diversity of the feeding apparatus. Despite volumes of data on the biomechanics of feeding, the extent to which the shape of mammal skulls reflects stresses generated by feeding is still unknown. In addition to the feeding apparatus, the skull accommodates the structural needs of the sensory systems and brain. We turned to bats as a model system for separating optimization for masticatory loads from optimization for other functions. Because the energetic cost of flight increases with body mass, it is reasonable to suggest that bats have experienced selective pressure over evolutionary time to minimize mass. Therefore, the skulls of bats are likely to be optimized to meet functional demands. We investigate the hypothesis that there is a biomechanical link between biting style and craniofacial morphology by combining biting behavior and bite force data gathered in the field with finite-element (FE) analysis. Our FE experiments compared patterns of stress in the craniofacial skeletons within and between two species of bats (Artibeus jamaicensis and Cynopterus brachyotis) under routine and atypical loading conditions. For both species, routine loading produced low stresses in most of the skull. However, the skull of Artibeus was most resistant to loads applied via its typical biting style, suggesting a mechanical link between routine loading and skull form. The same was not true of Cynopterus, where factors other than feeding appear to have had a more significant impact on craniofacial morphology.

We immunohistochemically examined the organization of the main olfactory bulbs (MOBs) in seven mammalian species, including moles, hedgehogs, tree shrews, bats, and mice as well as laboratory musk shrews and rats. We focused our investigation on two points: 1) whether nidi, particular spheroidal synaptic regions subjacent to glomeruli, which we previously reported for the laboratory musk shrew MOBs, are also present in other animals and 2) whether the compartmental organization of glomeruli and two types of periglomerular cells we proposed for the rat MOBs are general in other animals. The general laminar pattern was similar among these seven species, but discrete nidi and the nidal layer were recognized only in two insectivores, namely, the mole and laboratory musk shrew. Olfactory marker protein-immunoreactive (OMP-IR) axons extended beyond the limits of the glomerular layer (GL) into the superficial region of the external plexiform layer (EPL) or the nidal layer in the laboratory musk shrew, mole, hedgehog, and tree shrew but not in bat, mouse, and rat. We observed, in nidi and the nidal layer in the mole and laboratory musk shrew MOBs, only a few OMP-IR axons. In the hedgehog, another insectivore, OMP-IR processes extending from the glomeruli were scattered and intermingled with calbindin D28k-IR cells at the border between the GL and the EPL. In the superficial region of the EPL of the tree shrew MOBs, there were a small number of tiny glomerulus-like spheroidal structures where OMP-IR axons protruding from glomeruli were intermingled with dendritic branches of surrounding calbindin D28k-IR cells. Furthermore, we recognized the compartmental organization of glomeruli and two types of periglomerular cells in the MOBs of all of the mammals we examined. These structural features are therefore considered to be common and important organizational principles of the MOBs., (Copyright 2004 Wiley-Liss, Inc.)

The topography and structure of the follicular cells and the follicular cavity of the hypophyseal pars tuberalis (PT) were studied in adult hibernating bats (Pipistrellus pipistrellus and Rhinolophus ferrumequinum) of both sexes, during the annual seasonal cycle and the reproductive cycle. The follicular cells were found to be organized around a central cavity. They showed a polyhedral shape and apical microvilli protruding into central cavities. During hibernation, the follicular cells showed active cytoplasmic organelles, clusters of glycogen particles, and lipid droplets. In the supranuclear cytoplasm, 9+2 type cilia, some dense bodies, microvesicular vacuoles, and thin actin-like filaments (rather scarce during autumn) were detected. The contents of the follicular cavity showed well-defined ultrastructural seasonal characteristics, with a colloid-like aspect during awakening and a strongly granular aspect during autumn oestrus and mating. Positive staining for PAS and paraldehyde fuchsin, and a marked reaction to lectins PHA-L4, MAM, and RCA 60 suggested the presence of sialo-glycoproteins in the follicular cavities. Both follicular and endocrine PT-specific cells appeared to mark the boundary of follicular cavities. This finding suggests that the follicular cavity contents are comprised of both types of cells, rather than by cell fragmentation or degeneration products., (Copyright 2003 Wiley-Liss, Inc.)

Voltage-gated potassium channels play an important role in shaping membrane properties that underlie neurons' discharge patterns and the ways in which they transform their input. In the auditory system, low threshold potassium currents such as those created by Kv1.1 subunits contribute to precise phaselocking and to transient onset responses that provide time markers for temporal features of sounds. The purpose of the present study was to compare information about the distribution of neurons expressing the KV 1.1 in the brainstem auditory nuclei with the distribution of neurons with known functional properties in the auditory system of the big brown bat, Eptesicus fuscus. We used immunocytochemistry and light microscopy to look at the distribution of Kv1.1 subunits in the brainstem auditory nuclei. There was prominent expression in cell types known to contain high levels of Kv1.1 in other species and known to respond to auditory signals with high temporal precision. These included octopus cells and spherical bushy cells of the cochlear nucleus and principal neurons of the medial nucleus of the trapezoid body. In addition, we found high levels of Kv1.1 in neurons of the columnar subdivision of the ventral nucleus of the lateral lemniscus and in ventral periolivary cell groups. Neurons with high levels of Kv1.1 were differentially distributed in the intermediate nucleus of the lateral lemniscus and in the inferior colliculus, suggesting that these structures contain functionally distinct cell populations, some of which may be involved in high-precision temporal processing., (Copyright 2003 Wiley-Liss, Inc.)

The inner ear macular sensory epithelia of the Daubenton's bat were examined quantitatively to estimate the area and total number of hair cells. Ultrastructural examination of the sensory epithelium reveals two main types of hair cells: the chalice-innervated hair cell and the bouton-innervated hair cell. The existence of an intermediate type, with a nerve ending covering the lateral side of the hair cell, indicates that the chalice-innervated hair cells are derived from bouton-innervated hair cells. Thus, at least a part of the bouton-innervated hair cells forms a transitional stage. A number of immature as well as apoptotic hair cells were observed. It is suggested that a continuous production of new hair cells takes place in mature individuals, probably based on transdifferentiation of supporting cells., (Copyright 2001 Wiley-Liss, Inc.)

The superior olivary complex (SOC) is a mammalian auditory brainstem structure that contains several nuclei. Some of them are part of the ascending system projecting to higher auditory centers, others belong to the descending system projecting to the cochlear nuclei or the cochlea itself. The main nuclei of the ascending system, the lateral and medial superior olive (LSO, MSO), as well as the lateral and medial nuclei of the trapezoid body (LNTB, MNTB), have been traditionally associated with sound localization. Here we review the results of recent studies on the main SOC nuclei in echolocating bats. These studies suggest that some SOC structures and functions are highly conserved across mammals (e.g., the LSO, which is associated with interaural intensity difference processing), while others are phylogenetically highly variable in both form and function (e.g., the MSO, traditionally associated with interaural time difference processing). For the MSO, these variations indicate that we should broaden our view regarding what functions the MSO might participate in, since its function in echolocation seems to lie in the context of pattern recognition rather than sound localization. Furthermore, across bat species, variations in the form and physiology of the MSO can be linked to specific behavioral adaptations associated with different echolocation strategies. Finally, the comparative approach, including auditory specialists such as bats, helps us to reach a more comprehensive view of the functional anatomy of auditory structures that are still poorly understood, like the nucleus of the central acoustic tract (NCAT)., (Copyright 2000 Wiley-Liss, Inc.)

It has been proposed that flying foxes and echolocating bats evolved independently from early mammalian ancestors in such a way that flying foxes form one of the suborders most closely related to primates. A major piece of evidence offered in support of a flying fox-primate link is the highly developed visual system of flying foxes, which is theorized to be primate-like in several different ways. Because the calcium-binding proteins parvalbumin (PV) and calbindin (CB) show distinct and consistent distributions in the primate visual system, the distribution of these same proteins was examined in the flying fox (Pteropus poliocephalus) visual system. Standard immunocytochemical techniques reveal that PV labeling within the lateral geniculate nucleus (LGN) of the flying fox is sparse, with clearly labeled cells located only within layer 1, adjacent to the optic tract. CB labeling in the LGN is profuse, with cells labeled in all layers throughout the nucleus. Double labeling reveals that all PV+ cells also contain CB, and that these cells are among the largest in the LGN. In primary visual cortex (V1) PV and CB label different classes of non-pyramidal neurons. PV+ cells are found in all cortical layers, although labeled cells are found only rarely in layer I. CB+ cells are found primarily in layers II and III. The density of PV+ neuropil correlates with the density of cytochrome oxidase staining; however, no CO+ or PV+ or CB+ patches or blobs are found in V1. These results show that the distribution of calcium-binding proteins in the flying fox LGN is unlike that found in primates, in which antibodies for PV and CB label specific separate populations of relay cells that exist in different layers. Indeed, the pattern of calcium-binding protein distribution in the flying fox LGN is different from that reported in any other terrestrial mammal. Within V1 no PV+ patches, CO blobs, or patchy distribution of CB+ neuropil that might reveal interblobs characteristic of primate V1 are found; however, PV and CB are found in separate populations of non-pyramidal neurons. The types of V1 cells labeled with antibodies to PV and CB in all mammals examined including the flying fox suggest that the similarities in the cellular distribution of these proteins in cortex reflect the fact that this feature is common to all mammals.

The organisation of extrastriate cortex was studied in anaesthetised flying foxes (Pteropus poliocephalus) by using multiunit recording techniques. Based on the visuotopic organisation and response characteristics, the cortex immediately rostral to the second visual area (V2) was subdivided into two fields: visual area 3 (V3) laterally and the occipitoparietal area (OP) medially. Area V3 is a 1.0-1.5 mm wide strip of cortex that represents the entire contralateral hemifield as a mirror image of the representation found in V2. The representation of the vertical meridian and the area centralis form the rostral border of V3. In area OP, receptive fields are much larger than those of V3 and form a separate visuotopic map, with the upper quadrant represented rostral to the lower quadrant. Multiunit clusters in the cortex rostral to area OP (posterior parietal area) respond to both visual and somatosensory stimuli. Farther laterally, in the cortex rostral to V3, the occipitotemporal area (OT) was found to form yet another map of the visual field. Similar to the middle temporal area in primates, area OT in the flying fox forms a first-order representation of the visual field, with the lower quadrant represented medially, the upper quadrant represented laterally, the area centralis represented caudally, and the visual field periphery represented rostrally. The cortex surrounding area OT rostrally and ventrally is also visually responsive but could not be subdivided due to the large receptive fields. Finally, visual responses were elicited from an area adjacent to the peripheral representation in the first visual area (V1) in the splenial sulcus. These results demonstrate that nearly half of the flying fox cortex is related to vision, which contrasts with that of microchiropteran bats, in which auditory areas predominate. A comparison of the flying fox with other mammals suggests that several areas, including homologues of V1, V2, V3, OT, and the splenial area, may have originated early in mammalian evolution and have been inherited by most present-day eutherians. However, studies in other species will be needed to distinguish patterns of common ancestry from parallel evolution., (Copyright 1999 Wiley-Liss, Inc.)

The interhemispheric connections of somatosensory cortex in the gray-headed flying fox (Pteropus poliocephalus) were examined. Injections of anatomical tracers were placed into five electrophysiologically identified somatosensory areas: the primary somatosensory area (SI or area 3b), the anterior parietal areas 3a and 1/2, and the lateral somatosensory areas SII (the secondary somatosensory area) and PV (pairetal ventral area). In two animals, the hemisphere opposite to that containing the injection sites was explored electrophysiologically to allow the details of the topography of interconnections to be assessed. Examination of the areal distribution of labeled cell bodies and/or axon terminals in cortex sectioned tangential to the pial surface revealed several consistent findings. First, the density of connections varied as a function of the body part representation injected. For example, the area 3b representation of the trunk and structures of the face are more densely interconnected than the representation of distal body parts (e.g., digit 1, D1). Second, callosal connections appear to be both matched and mismatched to the body part representations injected in the opposite hemisphere. For example, an injection of retrograde tracer into the trunk representation of area 3b revealed connections from the trunk representation in the opposite hemisphere, as well as from shoulder and forelimb/wing representations. Third, the same body part is differentially connected in different fields via the corpus callosum. For example, the D1 representation in area 3b in one hemisphere had no connections with the area 3b D1 representation in the opposite hemisphere, whereas the D1 representation in area 1/2 had relatively dense reciprocal connections with area 1/2 in the opposite hemisphere. Finally, there are callosal projections to fields other than the homotopic, contralateral field. For example, the D1 representation in area 1/2 projects to contralateral area 1/2, and also to area 3b and SII.

The mere appearance of a tubular, epithelially-covered, bilateral structure, no matter how minuscule, on the anteroventral nasal septum of tetrapods, is generally called the vomeronasal organ (of Jacobson). However, considering the functionality of this chemosensory structure, the presence of a non-cilated (microvillar) neuroepithelium (and not just any odd type of epithelium) encased in a variously shaped vomeronasal cartilage, along with vomeronasal nerve bundles and above all an accessory olfactory bulb connected to the limbic vomeronasal amygdala, are the absolute essential neurostructural characteristics and anatomic requirement for a functional VNO and the accessory olfactory system in any tetrapod. The distribution of the vomeronasal organ is reported here in two mammalian orders: Chiroptera and Primates. An impressive data pool on the vomeronasal organ of bats is now available, pointing to the fact that at this time bats may be the only group in which this organ system is extremely variable, ranging from total absence (even in the embryo) to spectacular development with numerous intervening stages in different chiropteran species. Of the eighteen bat families, only one family of New World leaf-nosed bats, family Phyllostomidae, exhibits functional vomeronasal organs. The vespertilionid bat Miniopterus, and the mormoopid bat Pteronotus, present exceptions to this rule. Among Primates, very few species have been rigorously studied. As a result, developmental variability of the vomeronasal organ is almost unknown; either the vomeronasal organ is well developed (such as in New World monkeys) or absent (as in Old World monkeys and great apes) in the adult. The concept whether adult humans or embryonic and fetal forms are endowed with this so-called sixth sense, is a controversial one and is under intense study in our laboratory and by others. The general phylogenetic implications based on our cladistic analysis of bats are that the vomeronasal organ complex has evolved several times. Among the prosimians and platyrrhine primates, the organ is well developed, although to a varying degree. Among catarrhine primates, its loss has occurred only once, as it is generally absent in the adult forms.

The distribution and origin of cerebrovascular nitrergic nerves were studied immunohistochemically and histochemically in the bent-winged bat. The supply of nitric oxide synthase (NOS)-immunoreactive (IR) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive nerves to the bat major cerebral arteries differs from the general mammalian pattern in that it is preferential for the vertebrobasilar system (VBS) as opposed to the internal carotid system. Interestingly, a few nerve cells with bright NOS immunofluorescence and intense NADPHd activity were localized in the walls of the vertebral artery (VA) and basilar artery (BA) from many individual bats. Cerebral perivascular NOS-IR nerves were generally immunoreactive for vasoactive intestinal polypeptide (VIP). NOS-IR neurons intrinsic to the BA and VA expressed variable degrees of VIP immunoreactivity and showed no acetylcholinesterase (AChE) activity. Most cell bodies of the microganglia (MG) in the carotid canal and tympanic cavity, and those of the cranial and cervical facial ganglia, showed both NOS and VIP immunoreactivities and were stained intensely for NADPHd. From these and other findings, it is suggested that, in the bent-winged bat at least, the BA and VA of the cerebral arterial tree are frequently dually innervated by two neurochemically defined nitrergic neurons, the cranial parasympathetic VIP-IR and AChE-positive neurons, which are derived mainly from the MG via the internal carotid artery, and the intrinsic neurons, either IR or immunonegative for VIP but negative for AChE, which form an outflow tract from some caudally located ganglia projecting to the VBS via the VA.

Cytoplasmic crystalloids are abundant in the excretory ducts of the accessory submandibular gland of the long-winged bat, Miniopterus magnator. The crystalloids, which always lack a membranous enclosure, may have an intricate silhouette. They consist of parallel linear densities with a 12.5 nm periodicity. These densities actually may be thin-walled tubules. In some crystalloids, intersecting subcrystalloids produce a complex pattern of decussate densities. In a few rare instances, continuities were detected between a crystalloid and a smooth-surfaced cisternal element. In other mammalian species, similar crystalloids connected to smooth endoplasmic reticulum play a role in steroid metabolism. We postulate that the ductular crystalloids in M. magnator might be involved in production of a factor that influences mating behavior.

This study investigates the functional organization of the superior olivary complex of the mustached bat with classical transmission electron microscopy and postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) and glycine antisera in semithin serial sections. The ultrastructure and distribution of terminal types in the lateral superior olive (LSO) and the medial nucleus of the trapezoid body (MNTB) closely resemble that of other mammals; the organization within the medial superior olive (MSO) differs significantly. The differences concern the relative proportion of putatively inhibitory boutons, which appear as symmetrical synapses with flattened vesicles on MSO somata. In the bat, inhibitory boutons comprised 75-100% of perisomatic boutons, a value identical to that observed in the LSO. These terminals most likely arise from the MNTB. In other species, putatively inhibitory terminals form a much smaller proportion of perisomatic boutons in MSO. This difference suggests that in the bat MSO excitatory input to cell somata is considerably reduced and outweighed by inhibitory input. This suggestion is corroborated by immunocytochemical data. Glycine-immunoreactive puncta encrust somata of LSO and MSO cells to a similar degree and in rather homogeneous patterns throughout these nuclei. Putatively GABAergic terminals are located mainly on distal dendrites of MSO and LSO cells. Regional variations in the density of GABA-immunoreactive puncta in LSO suggest that different tonotopic zones are under differential modulatory influence. Both the LSO and MSO of the mustached bat contain significant amounts of putatively inhibitory projection cells. Coexistence of both antigens was commonly observed in subsets of cells. Quantitative analyses of labeling patterns and comparisons with other mammals suggest that the mix of neurotransmitters in projection cells of LSO and MSO is phylogenetically flexible, and thus the details of the functions of ascending pathways are species specific. In contrast to other mammals, the bat MSO forms parallel output pathways with excitatory and inhibitory components. Data are discussed in relation to specialized physiological response features.

The nuclei of the lateral lemniscus in the echolocating bat, Eptesicus fuscus, are large and highly differentiated. In each nucleus, different characteristic response properties predominate. To determine whether the dissimilar response properties are due in part to differential ascending input, we examined the retrograde transport from small deposits of horseradish peroxidase (HRP) or HRP conjugated with wheat germ agglutinin (WGA-HRP) in the nuclei of the lateral lemniscus. The intermediate nucleus (INLL) and the two divisions of the ventral nucleus (VNLL) receive almost exclusively monaural input from the anteroventral and posteroventral cochlear nuclei and from the medial nucleus of the trapezoid body. Lesser inputs originate in the lateral nucleus of the trapezoid body and the ventral periolivary area. Although the three monaural nuclei of the lateral lemniscus all receive input from the same set of nuclei, and from the same identified cell types in the cochlear nucleus, there is a difference in the relative proportions of input from these sources. The dorsal nucleus (DNLL) receives input mostly from binaural structures, the lateral and medial superior olives and the contralateral DNLL, with only a minor projection from the cochlear nucleus. The lateral and medial superior olives project bilaterally; the bilateral projection from the medial superior olive is unusual in that it is found in only a few mammalian species. The results show a segregated pattern of binaural projections to DNLL and monaural projections to INLL and VNLL that is consistent with the binaural response properties found in DNLL and the exclusively monaural response properties found in INLL and VNLL. The differences in response properties between monaural nuclei, however, are not due to input from different nuclei or cell types but may be influenced by differing magnitudes of the constituent ascending projections.

The distribution and morphology of neurons and axonal endings (puncta) immunostained with antibodies to gamma-aminobutyric acid (GABA) and glycine (Gly) were analyzed in auditory brainstem, thalamic, and cortical centers in the mustache bat. The goals of the study were (1) to compare and contrast the location of GABAergic and glycinergic neurons and puncta, (2) to determine whether nuclei containing immunoreactive neurons likewise have a similar concentration of puncta, (3) to assess the uniformity of immunostaining within a nucleus and to consider regional differences that were related to or independent of cytoarchitecture, and (4) to compare the patterns recognized in this bat with those in other mammals. There are nine major conclusions. (1) Glycinergic immunostaining is most pronounced in the hindbrain. (2) In the forebrain, GABA alone is present. (3) Some nuclei have GABAergic or glycinergic neurons exclusively; a few have neither. (4) Although there is sometimes a close relationship between the relative number of immunopositive neurons and the density of the puncta, just as often there is no particular correlation between them; this reflects the fact that many GABAergic and glycinergic neurons project beyond their nucleus of origin. (5) Even nuclei devoid of or with few GABAergic or glycinergic neurons contain relatively abundant numbers of puncta; some neurons receive axosomatic terminals of each type. (6) In a few nuclei there are physiological subregions with specific local patterns of immunostaining. (7) The patterns of immunostaining resemble those in other mammals; the principal exceptions are in nuclei that, in the bat, are hypertrophied (such as those of the lateral lemniscus) and in the medial geniculate body. (8) Cellular colocalization of GABA and Gly is specific to only a few nuclei. (9) GABA and glutamic acid decarboxylase (GAD) immunostaining have virtually identical distributions in each nucleus. Several implications follow. First, the arrangements of GABA and Gly in the central auditory system represent all possible patterns, ranging from mutually exclusive to overlapping within a nucleus to convergence of both types of synaptic endings on single neurons. Second, although both transmitters are present in the hindbrain, glycine appears to be dominant, and it is often associated with circuitry in which precise temporal control of aspects of neuronal discharge is critical. Third, the auditory system, especially at or below the level of the midbrain, contains significant numbers of GABAergic or glycinergic projection neurons. The latter feature distinguishes it from the central visual and somatic sensory pathways.

Double staining immunohistochemistry was used to investigate the origin and projection of nerves with substance P (SP) immunoreactivity (-IR) in the walls of the major cerebral arteries in two microchiropteran species. In the greater horseshoe bat, most of the cerebral perivascular nerves with SP-IR did not exhibit calcitonin gene-related peptide (CGRP)-IR, but emitted bright immunofluorescence for vasoactive intestinal polypeptide (VIP). In this species, a large number of cell bodies with both SP- and VIP-IR were observed in many cranial ganglia along various branches of the facial and glossopharyngeal nerves. There were no cell bodies immunoreactive for either SP and VIP in the two sensory (trigeminal and upper cervical dorsal root), two sympathetic (superior cervical and stellate), or two vagal (superior and jugular) ganglia. In addition, several thick fiber bundles with both SP- and VIP-IR were present in the wall of the cerebral carotid artery, and descended progressively reaching as far as the middle part of the basilar artery (BA). These and other findings suggest that SP-immunoreactive nerves with VIP-IR but not CGRP-IR, which contribute to the rich innervation of the vertebrobasilar system in the greater horseshoe bat, originate from neurons with the same combination of peptide-IR in the major or local facial or glossopharyngeal parasympathetic ganglia, and enter the cranial cavity along the internal carotid artery. In the bent-winged bat, however, cerebral perivascular SP-immunoreactive nerves, as well as SP-immunoreactive neurons within the trigeminal and upper cervical dorsal root ganglia (uCDRG), showed neither CGRP-IR nor VIP-IR, and were mostly confined to the caudal BA and the vertebral artery (VA). These observations, in addition to the projection of this nerve type to the BA via the VA as fiber bundles, or through the meninges, indicate that the principal source of the cerebrovascular SP-immunoreactive innervation in this species is the uCDRG.

The cytoarchitectonic organization of the medial geniculate body and adjoining thalamic nuclei was analyzed in the mustached bat (Pteronotus parnellii). These subdivisions provide a reference for structural, physiological, connectional, and neurochemical work. Most nuclei recognized in other mammals exist in the mustached bat, although the relative volume of the three divisions was species specific. The ventral division contains medium-sized neurons and a few smaller cells and is well developed. Neurons in the lateral part lie in regularly aligned rows corresponding to the laminae in Golgi material; in the medial part, these laminae are obscured by fibers. The dorsal division has at least four nuclei, each with a unique cytoarchitecture and myeloarchitectonic organization. The suprageniculate nucleus is prominent and has many large radiate neurons. Cells in the superficial dorsal nucleus have weekly laminated dendrites, while dorsal nucleus neurons have spherical dendritic fields. There is a wide range of neuropil patterns within the dorsal division. The suprageniculate nucleus has thick myelinated axons, while the fibers in the superficial and dorsal nuclei are much thinner. The rostral pole nucleus becomes prominent in the anterior one-half of the auditory thalamus; its architectonic affiliation is equivocal, and connectional and immunocytochemical studies suggest that it may belong to the dorsal division. The medial division is one nucleus with many types of neurons, and it has coarse axons without laminar orientation. It is the smallest of the divisions and is present throughout the medial geniculate complex, except at the caudal tip and at the rostral pole. Many features of medial geniculate body organization evident in other mammals are recognized in the mustached bat. These include a prominent ventral division, some of whose neurons have a laminar organization, and a comparatively small medial division that is devoid of fibrodendritic laminae. Other features, such as the presence of a large rostral pole nucleus, whose homologue in other species is uncertain, or the sparse number or small cells that may participate in local circuits, set it apart from carnivores and primates and suggest that there are species specific patterns of medial geniculate body organization.

The neurons in the medial geniculate body were studied in Golgi preparations from adult mustached bats (Pteronotus parnellii). Their somatic and dendritic configurations were compared with those of cells in other, nonecholocating mammals. A second goal was to use the thalamic nuclear subdivisions derived from Golgi material to integrate the findings in parallel studies of cytoarchitecture, immunocytochemistry, and tectothalamic connections. Three primary divisions are defined. The ventral division is large and has a stereotyped neuronal organization. Medium-sized perikarya (about 10 microns in diameter) represent tufted neurons; the fibrodendritic plexus forms laminae in the lateral part along which midbrain axons terminate. A smaller, possibly intrinsic, neuron with thin, sparse dendrites is rarely impregnated. Neurons in the larger, medial part, which represents frequencies of 60 kHz and higher, have more spherical dendritic fields; their branching pattern remains tufted, and the laminar organization was less evident. The dorsal division is about equal in size, and it has many nuclei and a corresponding neuronal diversity. These neurons are medium-sized except in the suprageniculate nucleus, where many cells are larger. Four dorsal division nuclei are recognized. Each has neurons with radiate or weakly tufted dendritic arbors. Superficial dorsal nucleus neurons are oriented from medial to lateral, imparting a slightly laminated appearance to the neuropil. A few smaller, stellate neurons with modest dendritic domains are present. Suprageniculate nucleus neurons have radiating dendritic fields that project spherically; they have fewer branches than dorsal nucleus neurons. The posterior limitans nucleus is dorsomedial to the suprageniculate nucleus; it has small neurons with long, sparsely branched dendrites. The rostral pole nucleus, included in the dorsal division on cytoarchitectonic grounds, had too few neurons impregnated to reveal its neuronal architecture. The medial division, the smallest of the main parts, is one nucleus with at least six types of cells, including the magnocellular, bushy tufted, disc-shaped, medium-sized multipolar, elongated, and small stellate neurons. There is no laminar arrangement. Many of the neurons resemble those in rodent, marsupial, carnivore, and primate auditory thalamic nuclei. Despite such morphological correspondences, functional differences, such as the evolution of combination sensitivity, suggest that structurally comparable auditory thalamic neurons may subserve diverse physiological representations.

The superior olivary complex (SOC) is the first station in the ascending auditory pathway that receives binaural projections. Two of the principal nuclei, the lateral superior olive (LSO) and the medial superior olive (MSO), are major sources of ascending projections to the inferior colliculus. Whereas almost all mammals have an LSO, it has traditionally been thought that only animals that hear low frequencies have an MSO. Recent reports, however, suggest that the medial part of the SOC in bats is highly variable and that at least some bats have a well-developed MSO. Thus, the main goal of this study was to evaluate the cytoarchitecture and connections of the principal superior olivary nuclei of the Mexican free-tailed bat, with specific attention directed at the MSO. Cell and fiber stained material revealed that the LSO and the medial nucleus of the trapezoid body (MNTB) are similar to those described for other mammals. There are two medial nuclei we refer to as dorsomedial periolivary nucleus (DMPO) and MSO. Tracer experiments exhibited that the DMPO receives bilateral projections from the cochlear nucleus, and additional projections from the ipsilateral MNTB. The DMPO sends a strong projection to the ipsilateral inferior colliculus. Positive staining for acetylcholinesterase indicates that the DMPO is a part of the olivocochlear system, as it is in other animals. The MSO in the free-tailed bat meets many of the criteria that traditionally define this nucleus. These include the presence of bipolar and multipolar principal cells, bilateral innervation from the cochlear nucleus, a strong projection from the ipsilateral MNTB, and the absence of cholinergic cells. The major difference from traditional MSO features is that it projects bilaterally to the inferior colliculus. Approximately 30% of its cells provide collateral projections to the colliculi on both sides. Functional implications of the MSO for the free-tailed bat are considered in the Discussion.

The body surface representation in the gracile, cuneate, and spinal trigeminal nuclei of the little red flying fox (Pteropus scapulatus) was examined. As in other species, it was found that any single cross-section through all three nuclei contains a representation of most, or all, of the body surface. In the little red flying fox, however, this representation is arranged as a series of dorsolateral to ventromedially oriented bands, within which there are no apparent topographies. These bands are arranged in such a way that the spatial relationships between body regions in the representation do not reflect those at the periphery.

The representation of the visual field in the occipital cortex was studied by multiunit recordings in seven flying foxes (Pteropus spp.), anesthetized with thiopentone/N2O and immobilized with pancuronium bromide. On the basis of its visuotopic organization and architecture, the primary visual area (V1) was distinguished from neighboring areas. Area V1 occupies the dorsal surface of the occipital pole, as well as most of the tentorial surface of the cortex, the posterior third of the mesial surface of the brain, and the upper bank of the posterior portion of the splenial sulcus. In each hemisphere, it contains a precise, visuotopically organized representation of the entire extent of the contralateral visual hemifield. The representation of the vertical meridian, together with 8-15 degrees of ipsilateral hemifield, forms the anterior border of V1 with other visually responsive areas. The representation of the horizontal meridian runs anterolateral to posteromedial, dividing V1 so that the lower visual quadrant is represented medially, and the upper quadrant laterally. The total surface area of V1 is about 140 mm2 for P. poliocephalus, and 110 mm2 for P. scapulatus. The representation of the central visual field is greatly magnified relative to that of the periphery. The cortical magnification factor decreases with increasing eccentricity, following a negative power function. Conversely, receptive field sizes increase markedly with increasing eccentricity, and therefore the point-image size is approximately constant throughout V1. The emphasis in the representation of the area centralis in V1 is much larger than that expected on the basis of ganglion cell counts in flat-mounted retinas. Thus, a larger degree of convergence occurs at the peripheral representations in the retino-geniculo-cortical pathway, in comparison with the central representations. The marked emphasis in the representation of central vision, the wide extent of the binocular field of vision, and the relatively large surface area of V1 reflect the importance of vision in megachiropterans.

Retinal projections and visual thalamo-cortical connections were studied in the subterranean mole rat, belonging to the superspecies Spalax ehrenbergi, by anterograde and retrograde tracing techniques. Quantitative image analysis was used to estimate the relative density and distribution of retinal input to different primary visual nuclei. The visual system of Spalax presents a mosaic of both regressive and progressive morphological features. Following intraocular injections of horseradish peroxidase conjugates, the retina was found to project bilaterally to all visual structures described as receiving retinal afferents in non-fossorial rodents. Structures involved in form analysis and visually guided behaviors are reduced in size by more than 90%, receive a sparse retinal innervation, and are cytoarchitecturally poorly differentiated. The dorsal lateral geniculate nucleus, as defined by cyto- and myelo-architecture, cytochrome oxidase, and acetylcholinesterase distribution as well as by afferent and efferent connections, consists of a narrow sheet 3-5 neurons thick, in the dorsal thalamus. Connections with visual cortex are topographically organized but multiple cortical injections result in widespread and overlapping distributions of geniculate neurons, thus indicating that the cortical map of visual space is imprecise. The superficial layers of the superior colliculus are collapsed to a single layer, and the diffuse ipsilateral distribution of retinal afferents also suggests a lack of precise retinotopic relations. In the pretectum, both the olivary pretectal nucleus and the nucleus of the optic tract could be identified as receiving ipsilateral and contralateral retinal projections. The ventral lateral geniculate nucleus is also bilaterally innervated, but distinct subdivisions of this nucleus or the intergeniculate leaflet could not be distinguished. The retina sends a sparse projection to the dorsal and lateral terminal nuclei of the accessory optic system. The medial terminal nucleus is not present. In contrast to the above, structures of the "non-image forming" visual pathway involved in photoperiodic perception are well developed in Spalax. The suprachiasmatic nucleus receives a bilateral projection from the retina and the absolute size, cytoarchitecture, density, and distribution of retinal afferents in Spalax are comparable with those of other rodents. A relatively hypertrophied retinal projection is observed in the bed nucleus of the stria terminalis. Other regions which receive sparse visual input include the lateral and anterior hypothalamic areas, the retrochiasmatic region, the sub-paraventricular zone, the paraventricular hypothalamic nucleus, the anteroventral and anterodorsal nuclei, the lateral habenula, the mediodorsal nucleus, and the basal telencephalon.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative study of the immunostain to antibodies directed against glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the ascending auditory pathway was carried out in horseshoe bats (Rhinolophus rouxi) and mustached bats (Pteronotus parnellii). In both species GAD/GABA-positive puncta (presumed axonal boutons) and GAD/GABA-positive cells were found in the cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus the inferior colliculus, and the medial geniculate body. General features of the immunostaining pattern in the auditory pathway agree with observations in other mammals. Quantitative analysis of puncta distribution shows that many auditory centers are characterized by subregional differences in puncta density and distribution. This indicates local differences in putatively inhibitory input related to connectivity and tonotopic organization. The following species characteristic features were found: 1) The dorsal non-laminated portion of the dorsal cochlear nucleus in horseshoe bats lacks the GAD/GABA-immunoreactive cells typical for the ventral laminated portion and the dorsal cochlear nucleus of other species. Clearly, a cytoarchitectonic specialization is accompanied by a loss of putatively GABAergic local inhibitory circuits. 2) The ventral division of the medial geniculate body of the mustached bat lacks GAD/GABA-immunopositive cells. Such cells are present in the horseshoe bat and other mammals. This finding implies functional differences in the organization of the medial geniculate body within the same mammalian order.

The anatomy and the spatial distribution of neurons and axonal endings (puncta) immunoreactive for glutamic acid decarboxylase (GAD) or gamma-aminobutyric acid (GABA) were studied in the medial geniculate body of the mustached bat (Pteronotus parnellii). The principal findings are that: 1) most GABAergic neurons are present in the dorsal and ventral divisions with few, if any, in the medial division; 2) only a small fraction, about 1% or less, of auditory thalamic neurons are immunopositive; 3) the density of immunoreactive puncta is independent on the number of GABAergic neurons in the thalamic divisions, with the ventral division having the largest number/unit area, the medial division about 75% of this value, and the dorsal division only about 50%; and 4) the form of the puncta was unique to each division, those in the ventral division being medium-sized and comparatively simple, those in the medial division predominantly large, coarse, and complex, while dorsal division ending were finer and more delicate. These patterns recapitulate, with some significant exceptions, those found in the rat and cat. The puncta could originate from several sources; while many may arise from intrinsic GABAergic Golgi type II local circuit neurons, these cells may not be the only or even the principal source. Thus, the dorsal division contains comparatively many immunopositive cells though fewer puncta than might be expected if the bulk of these were to arise from auditory thalamic interneurons. This suggests that other, extrinsic sources, such as the thalamic reticular nucleus, may be the source of such endings. A second point is that the form and density of the puncta is regionally specific within the medial geniculate complex. These local patterns might have a significant and regionally specific role in controlling the differential excitability of auditory thalamic neurons. The distribution of presumptive synaptic endings also has implications for the number and arrangement of glomeruli or synaptic nests. Thus, these circuit elements, which are common to the thalamic nuclei in other species, might play an important role in local synaptic circuits between different types of cells. If so, then the structural variations embodied in these patterns could subserve functional arrangements that differ among species. Such patterns might reflect concomitant physiological differences in the organization of local circuits within the microchiropteran medial geniculate body.

The organ of Corti of the echolocating horseshoe bat (Rhinolophus rouxi) was investigated with scanning electron microscopy in order to provide a comparison with non-echolocating mammals. Throughout the cochlea of horseshoe bats, each outer hair cell (OHC) possesses three rows of stereocilia and there are no morphological distinctions among the different rows of OHCs. However, there are morphological differences between different regions along the cochlea. In the lower and upper basal turn, the receptor surfaces of OHCs are characterized by extremely wide W-shaped stereocilia bundles and wingshaped cuticular plates. The cuticular plates of OHCs of the middle and outermost rows are arranged parallel to each other. Stereocilia length is only 0.8 microns and there is an exaggerated angle of inclination of the shortest row of stereocilia towards the next taller one. Stereocilia arrangements in the apex of the horseshoe bat's cochlea closely resembles those observed in the midbasal region of the rat cochlea. Inner hair cells (IHC) in the lower basal turn appear specialized. They possess only two rows of stereocilia and only 7-8 stereocilia per row. Their cuticular plates are small and oval and widely separated from one another in the longitudinal direction. IHCs at all other locations possess three and up to four rows of stereocilia and 17-20 stereocilia per row. Their cuticular plates are elongated and closely spaced. The transition from specialized to typical mammalian morphology occurs abruptly (over a distance of about 100-150 microns) at the border between the lower and the upper basal turn. This transition is not accompanied by a change in OHC morphology. In the subsurface of the tectorial membrane, throughout the cochlea, there are distinct imprints of the tallest row of stereocilia of all three rows of OHCs and of the IHCs. Data are discussed in relation to specialized aspects of the cochlear frequency map in horseshoe bats and as possible micromechanical adaptations to ultra-high frequency hearing.

The fine structure of the organ of Corti was investigated in the echolocating horseshoe bat (Rhinolophus rouxi) by transmission electron microscopy. Particular emphasis was placed on the receptor cells and their supporting cells. The receptor cells, inner hair cells (IHC) and outer hair cells (OHC), possess the typical mammalian shape, but OHCs are extremely short (length: 12-15 microns in the basal turn and up to 28-30 microns in the apical turn). The afferent innervation of both types of receptor cells and the efferent innervation of the IHC system conform to the general mammalian scheme; however, confirming earlier reports, an efferent innervation to the OHCs is absent. Throughout the cochlea, IHCs and OHCs possess a single layer of subsurface cisternae. Above the level of the nucleus of the OHCs, the arrangements of the subsurface cisternae and their connection to the lateral cell membrane via pillars are highly regular, whereas in IHCs, the cisternae are of irregular shape and the pillar system is much less distinct. In the basal turn of the cochlea, the attachment sites of the OHCs to the supporting cells possess specialized features: (a) in the reticular lamina, the contact sites of the cuticular plates of OHCs with the outer pillar cells and the Deiters cell phalanges are of exaggerated length, and (b) the cup formation of the Deiters cell body, which houses the bottom of the OHC, has a specialized shape and is packed with electron-dense material and microtubules. The results are discussed in relation to cochlear ultrastructure in other mammals and in the context of active processes in cochlear mechanics.

Little is known about the immunohistochemistry of the nervous system in bats. This is particularly true of the nervus terminalis, which exerts strong influence on the reproductive system during ontogeny and in the adult. Luteinizing hormone-releasing hormone (LHRH) was visualized immunocytochemically in the nervus terminalis and brain of juvenile and adult big brown bats (Eptesicus fuscus). The peripheral LHRH-immunoreactive (ir) cells and fibers (nervus terminalis) are dispersed along the basal surface of the forebrain from the olfactory bulbs to the prepiriform cortex and the interpeduncular fossa. A concentration of peripheral LHRH-ir perikarya and fibers was found at the caudalmost part of the olfactory bulbs, near the medioventral forebrain sulcus; obviously these cells mediate between the bulbs and the remaining forebrain. Within the central nervous system (CNS), LHRH-ir perikarya and fibers were distributed throughout the olfactory tubercle, diagonal band, preoptic area, suprachiasmatic and supraoptic nuclei, the bed nuclei of stria terminalis and stria medullaris, the anterior lateral and posterior hypothalamus, and the tuber cinereum. The highest concentration of cells was found within the arcuate nucleus. Fibers were most concentrated within the median eminence, infundibular stalk, and the medial habenula. The data obtained suggest that this distribution of LHRH immunoreactivity may be characteristic for microchiropteran (insectivorous) bats. The strong projections of LHRH-containing nuclei in the basal forebrain (including the arcuate nucleus) to the habenula, may indicate close functional contact between these brain areas via feedback loops, which could be important for the processing of thermal and other environmental stimuli correlated with hibernation.

Cells and axons that supply direct afferent input to the medial nucleus of the trapezoid body are described. Afferents were intracellularly labeled in brainstem tissue slices of two rodent and two bat species. The main afferents are calyciferous axons from globular bushy cells of the ventral cochlear nucleus. Calyciferous axons were highly consistent across species, projecting directly from the cochlear nucleus, across the midline in the trapezoid body, to the contralateral medial nucleus of the trapezoid body. Within the target nucleus, a typical axon turned sharply away from horizontal to form a large ending, the calyx of Held, around the soma of a single principal cell. Three groups of calyciferous axons were classified based on the path taken from bend to calyx. In subjects younger than four weeks, single axons often formed two calyces, each on a different cell. These calyx pairs were often found on adjacent or vertically aligned cells. In older animals, calyx pairs were more closely aligned, but fewer double calyx axons were seen. A secondary focus of this study was the system of thin collateral branches that characterizes calyciferous axons in all species. The projection patterns of these collaterals suggest that calyciferous axons may provide ascending input to periolivary cell groups with descending projections. In addition to calyciferous afferents, labeled cells that provide input to the medial nucleus of the trapezoid body from adjacent periolivary cell groups are described. Also described is a type of afferent that descends from the level of the lateral lemniscus to the medial nucleus of the trapezoid body.

The retinotopic organisation of the superior colliculus (SC) in the megachiropteran bat Rousettus aegyptiacus was examined with single and multi-unit recordings and by tracing the retrograde and anterograde transport of horseradish peroxidase (HRP) between the retina and the SC. The pattern of projection of the visual field onto the SC in Rousettus resembles the pattern found in most mammals. The whole of the contralateral visual field is represented and, in addition, a region of the ipsilateral visual field extending 25 degrees beyond the vertical 0 degree meridian. The ipsilateral visual field is represented binocularly in the most anterior 300-500 microns of the rostral pole of the SC. The contralateral visual field up to 25 degrees from the vertical meridian is represented through both eyes for the next 500-800 microns. The peripheral part of the contralateral visual field, 25 degrees-110 degrees from the vertical meridian is seen only by the nasal retina (the monocular crescent) of the contralateral eye and is represented in the caudal part of the SC. Following multiple injections of HRP into one SC, ganglion cells were labeled in both the nasal and temporal hemiretina of the contralateral eye. In the retina ipsilateral to the injection site, labeled cells were restricted to the temporal hemiretina. After injections of HRP into one eye, labeled terminals were found all over the contralateral SC, but in the ipsilateral SC they were restricted to a band that begins 300-500 microns caudal from the rostral pole and extends to the middle of the SC. These results suggest that in Rousettus, unlike the megachiropteran bats described by Pettigrew, Jamieson, Robson, Hall, McAnally, and Cooper (Philosophical Transactions of the Royal Society of London Series B 325:489-559, 1989), the retinotopic organisation of the SC is not primate-like, but follows the general mammalian scheme. As the retinotopic organisation of the SC is not consistent among the megachiropteran bats, the pattern of this projection may not be a useful indicator of their phylogenetic origins.

Cells in the medial nucleus of the trapezoid body were intracellularly labeled in brainstem tissue slices of two bat and two rodent species. The main cell type found in this nucleus, the principal cell, is an important link in the relay of ascending projections from the contralateral cochlear nucleus to the lateral superior olive, completing an essential pathway for sound localization. Principal cells are often viewed as a highly homogeneous group with a consistent morphology as well as a common function. Intracellular labeling has revealed a number of new axonal and dendritic features of principal cells. Some of these features vary widely from cell to cell, suggesting that the population of principal cells contains several morphologically distinct subgroups. Similar subsets of principal cells were recognized in all species examined. Five subgroups were distinguished on the basis of the position of dendritic fields. Although the dendrites of most labeled cells were confined to the medial nucleus of the trapezoid body, some principal cells had dendrites that spread outside the nucleus to one of several adjacent periolivary cell groups. Cells were also found that had dendrites that spread medially across the midline and into the contralateral medial nucleus of the trapezoid body. Axonal projections were used to distinguish two additional subgroups of principal cells. All principal cells project to the lateral superior olive and virtually all have one or more secondary projections. There are two subgroups with unusual collateral projections: one with collaterals that extended to the lateral lemniscus and one with recurrent collateral axons.

The principal and accessory submandibular glands of the common vampire bat, Desmodus rotundus, were examined by electron microscopy. The secretory endpieces of the principal gland consist of serous tubules capped at their blind ends by mucous acini. The substructure of the mucous droplets and of the serous granules varies according to the mode of specimen preparation. With ferrocyanide-reduced osmium postfixation, the mucous droplets are moderately dense and homogeneous; the serous granules often have a polygonal outline and their matrix shows clefts in which bundles of wavy filaments may be present. With conventional osmium postfixation, the mucous droplets have a finely fibrillogranular matrix; the serous granules are homogeneously dense. Mucous cells additionally contain many small, dense granules that may be small peroxisomes, as well as aggregates of 10-nm cytofilaments. Intercalated duct cells are relatively unspecialized. Striated ducts are characterized by highly folded basal membranes and vertically oriented mitochondria. Luminal surfaces of all of the secretory and duct cells have numerous microvilli, culminating in a brush borderlike affair in the striated ducts. The accessory gland has secretory endpieces consisting of mucous acini with small mucous demilunes. The acinar mucous droplets contain a large dense region; the lucent portion has punctate densities. Demilune mucous droplets lack a dense region and consist of a light matrix in which fine fibrillogranular material is suspended. A ring of junctional cells, identifiable by their complex secretory granules, separates the mucous acini from the intercalated ducts. The intercalated ducts lack specialized structure. Striated ducts resemble their counterparts in the principal gland. As in the principal gland, all luminal surfaces are covered by an array of microvilli. At least some of the features of the principal and accessory submandibular glands of the vampire bat may be structural adaptations to the exigencies posed by the exclusively sanguivorous diet of these animals and its attendant extremely high intake of sodium chloride.

The morphology of retinal ganglion cells was determined in megachiroptera, commonly known as flying foxes. Retinal ganglion cells were intracellularly injected with the fluorescent dye Lucifer yellow in fixed retinae from adult little red flying foxes (Pteropus scapulatus) captured in their natural habitat. Ganglion cells closely resembled the three main classes of cat retinal ganglion cells, and therefore were classified into alpha-, beta-, and gamma-type cells. The size of the alpha- and beta-type somas and dendritic fields increased with increasing distance from the area centralis. However, this eccentricity dependence was not as pronounced as in the cat. The gamma-type cells were sub-divided into mono-, bi-, and diffusely stratified, in accordance with the ramification of their dendrites within the inner plexiform layer. The alpha- and beta-type cells were uni-stratified in either the sublamina of the inner plexiform layer closest to the ganglion cell layer or in that closest to the inner nuclear layer. These laminae correspond to those in the cat retina which contain the dendritic ramifications of ganglion cells whose central receptive fields respond best to onset of light (the "on-centre" cells), or to ganglion cells whose centres respond optimally to light being extinguished (the "off-centre" cells). Thus the flying fox retina contains a morphological correlate of the "on"/"off" dichotomy of alpha and beta cells in the cat retina. In general the flying fox retinal ganglion cells exhibit a degree of morphological complexity reminiscent of cat retinal cells and this may reflect similar functional properties.

The ascending projections of the cochlear nucleus (CN) and the sources of descending inputs to the CN were investigated in horseshoe bats (Rhinolophus rouxi) by tracing the anterograde and retrograde transport of horseradish peroxidase (HRP or WGA-HRP) injected into the CN. The tracer was iontophoretically deposited into physiologically characterized regions of the cochlear nucleus (Feng and Vater, '85). We report the course and termination of pathways arising from the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) cochlear nucleus. The projection fields within the auditory brainstem centers (superior olivary complex [SOC]; lateral lemniscus complex [LLC]; and inferior colliculus [IC]) and their tonotopic organization according to the frequency representations at the injection sites are described. While the projection pattern is generally in accordance with other mammals, several species-characteristic features are noted: i) the lateral superior olive (LSO) receives tonotopically organized input from both the AVCN and PVCN; ii) the CN-projections to medial nuclear groups of the SOC located between the LSO and the medial nucleus of the trapezoid body do not support previously suggested homologies; iii) the ventral nucleus of the LLC can be subdivided into two divisions with distinct input patterns from the AVCN and PVCN, respectively.

To begin an investigation of the auditory pathways in the brainstem of the mustache bat, we examined the cytoarchitecture of the cochlear nuclei, superior olivary complex, nuclei of the lateral lemniscus, and inferior colliculus. Although all of these auditory centers are hypertrophied in this echolocating bat, only some areas have unusual cytoarchitectural features: 1) In the anterior part of the anteroventral cochlear nucleus we do not find the large spherical cells seen in other mammals; instead, very small spherical cells are found in this area. 2) In the posterior part of the anteroventral cochlear nucleus there is a region containing a homogeneous population of very large multipolar cells. 3) The medial superior olive is unusually large for an animal with a small head. 4) The most striking observations are seen in the lateral lemniscus. The ventral nucleus of the lateral lemniscus has a distinct columnar organization. The intermediate area of the lateral lemniscus contains a large and very distinct nucleus. Each of these cytoarchitectural features distinguishes the auditory system of this bat from that of other mammals. The results raise questions about whether or not there are unique subdivisions in the auditory pathways of echolocating bats. The results also identify these unusual nuclei as candidates to play a role in the special auditory functions related to echolocation.