Development of tectal connectivity across metamorphosis in the bullfrog (Rana catesbeiana).

In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit ß and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors.

Multiple mechanosensory modalities influence development of auditory function.

Sensory development can be dependent on input from multiple modalities. During metamorphic development, ranid frogs exhibit rapid reorganization of pathways mediating auditory, vestibular, and lateral line modalities as the animal transforms from an aquatic to an amphibious form. Here we show that neural sensitivity to the underwater particle motion component of sound follows a different developmental trajectory than that of the pressure component. Throughout larval stages, cells in the medial vestibular nucleus show best frequencies to particle motion in the range from 15 to 65 Hz, with displacement thresholds of <10 mum. During metamorphic climax, best frequencies significantly increase, and sensitivity to lower-frequency (<25 Hz) stimuli tends to decline. These findings suggest that continued sensitivity to particle motion may compensate for the considerable loss of sensitivity to pressure waves observed during the developmental deaf period. Transport of a lipophilic dye from peripheral end organs to the dorsal medulla shows that fibers from the saccule in the inner ear and from the anterior lateral line both terminate in the medial vestibular nucleus. Saccular projections remain stable across larval development, whereas lateral line projections degenerate during metamorphic climax. Sensitivity to particle motion may be based on multimodal input early in development and on saccular input alone during the transition to amphibious life.

Neuronal connexin expression in the cochlear nucleus of big brown bats.

We present immunohistochemical data describing the presence and distribution of connexins, structural component of gap junctions, in the cochlear nuclei of adult big brown bats (Eptesicus fuscus). Echolocating big brown bats show microsecond scale echo-delay sensitivity that requires accurate synchronization of neuronal responses to the timing of echoes. Midbrain and auditory cortical neuronal response timing is similar to that observed in other non-echolocating mammals, suggesting that lower auditory processing nuclei may have specialized mechanisms for obtaining the required temporal hyperacuity. Our data shows that connexin 36, a gap junction protein specific to neurons, is most densely expressed in the bat's cochlear nuclear complex, the medullary region that receives and processes first-order afferents from the auditory nerve. Cx36 expression is absent in the cochlear nucleus of normal mice, which have high-frequency hearing sensitivity similar to big brown bats. Glial connexins, Cx26 and Cx43, expressed in astrocytes and several inner ear structures, are also found in the bat cochlear nucleus complex, associated with major fiber tracts in and around the cochlear nuclei. The extensive presence of neuronally-associated Cx36 in brainstem auditory structures of adult bats suggests a possible role for gap junctions in mediating echo-delay hyperacuity.

Cell proliferation in the forebrain and midbrain of the adult bullfrog, Rana catesbeiana.

The distribution of proliferating cells in the midbrain, thalamus, and telencephalon of adult bullfrogs (Rana catesbeiana) was examined using immunohistochemistry for the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) and DNA dot-blotting. At all time points examined (2 to 28 days post-injection), BrdU-labeled cells were located in ventricular zones at all levels of the neuraxis, but with relatively more label around the telencephalic ventricles. Labeled cells, some showing profiles indicative of dividing and migrating cells, were present in brain parenchyma from 7 to 28 days post-injection. These labeled cells were particularly numerous in the dorsal and ventral hypothalamus, preoptic area, optic tectum, and laminar and principal nuclei of the torus semicircularis, with label also present, but at qualitatively reduced levels, in thalamic and telencephalic nuclei. Double-label immunohistochemistry using glial and early neural markers indicated that gliogenesis and neurogenesis both occurred, with new neurons observed particularly in the hypothalamus, optic tectum, and torus semicircularis. In all brain areas, many cells not labeled with BrdU were nonetheless labeled with the early neural marker TOAD-64, indicating that these cells were postmitotic. Incorporation of DNA measured by dot-blotting confirms the presence of DNA synthesis in the forebrain and brainstem at all time points measured. The pattern of BrdU label confirms previous experiments based on labeling with (3)H-thymidine and proliferating cell nuclear antigen showing cell proliferation in the adult ranid brain, particularly in hypothalamic nuclei. The consistent appearance of new cells in the hypothalamus of adult frogs suggests that proliferative activity may be important in mediating reproductive behaviors in these animals.

Developmental and regional patterns of GAP-43 immunoreactivity in a metamorphosing brain.

Growth-associated protein-43 is typically expressed at high levels in the nervous system during development. In adult animals, its expression is lower, but still observable in brain areas showing structural or functional plasticity. We examined patterns of GAP-43 immunoreactivity in the brain of the bullfrog, an animal whose nervous system undergoes considerable reorganization across metamorphic development and retains a strong capacity for plasticity in adulthood. Immunolabeling was mostly diffuse in hatchling tadpoles, but became progressively more discrete as larval development proceeded. In many brain areas, intensity of immunolabel peaked at metamorphic climax, the time of final transition from aquatic to semi-terrestrial life. Changes in intensity of GAP-43 expression in the medial vestibular nucleus, superior olivary nucleus, and torus semicircularis appeared correlated with stage-dependent functional changes in processing auditory stimuli. Immunolabeling in the Purkinje cell layer of the cerebellum and in the cerebellar nucleus was detectable at most developmental time points. Heavy immunolabel was present from early larval stages through the end of climax in the thalamus (ventromedial, anterior, posterior, central nuclei). Immunolabel in the tadpole telencephalon was observed around the lateral ventricles, and in the medial septum and ventral striatum. In postmetamorphic animals, immunoreactivity was confined mainly to the ventricular zones and immediately adjacent cell layers. GAP-43 expression was present in olfactory, auditory and optic cranial nerves throughout larval and postmetamorphic life. The continued expression of GAP-43 in brain nuclei and in cranial nerves throughout development and into adulthood reflects the high regenerative potential of the bullfrog's central nervous system.

Dynamic visualization of the developing nervous system of the bullfrog, Rana catesbeiana.

Anuran amphibians undergo a rapid and dramatic process of metamorphosis featuring widespread structural reorganization of the central nervous system. Although morphological changes during embryonic stages of anuran development have been well documented, much less information is available describing structural changes in the brain during larval (tadpole) stages. Using still images from cresyl-violet-stained material, we present an adaptation of the digital image and video manipulation technique of morphing that allows these images to be compiled in such a manner as to highlight key periods in tadpole brain development in a dynamic fashion. We present three morphed video data sets from ranid tadpoles that facilitate the identification of developmental changes in nuclear boundaries at different levels of the neuraxis. The use of animation allows dynamic examination of anatomical changes across long developmental spans without requiring additional anatomical preparations or specialized expensive equipment.

Medial vestibular connections with the hypocretin (orexin) system.

The mammalian medial vestibular nucleus (MVe) receives input from all vestibular endorgans and provides extensive projections to the central nervous system. Recent studies have demonstrated projections from the MVe to the circadian rhythm system. In addition, there are known projections from the MVe to regions considered to be involved in sleep and arousal. In this study, afferent and efferent subcortical connectivity of the medial vestibular nucleus of the golden hamster (Mesocricetus auratus) was evaluated using cholera toxin subunit-B (retrograde), Phaseolus vulgaris leucoagglutinin (anterograde), and pseudorabies virus (transneuronal retrograde) tract-tracing techniques. The results demonstrate MVe connections with regions mediating visuomotor and postural control, as previously observed in other mammals. The data also identify extensive projections from the MVe to regions mediating arousal and sleep-related functions, most of which receive immunohistochemically identified projections from the lateral hypothalamic hypocretin (orexin) neurons. These include the locus coeruleus, dorsal and pedunculopontine tegmental nuclei, dorsal raphe, and lateral preoptic area. The MVe itself receives a projection from hypocretin cells. CTB tracing demonstrated reciprocal connections between the MVe and most brain areas receiving MVe efferents. Virus tracing confirmed and extended the MVe afferent connections identified with CTB and additionally demonstrated transneuronal connectivity with the suprachiasmatic nucleus and the medial habenular nucleus. These anatomical data indicate that the vestibular system has access to a broad array of neural functions not typically associated with visuomotor, balance, or equilibrium, and that the MVe is likely to receive information from many of the same regions to which it projects.

Intergeniculate leaflet and ventral lateral geniculate nucleus afferent connections: An anatomical substrate for functional input from the vestibulo-visuomotor system.

The intergeniculate leaflet (IGL) has widespread projections to the basal forebrain and visual midbrain, including the suprachiasmatic nucleus (SCN). Here we describe IGL-afferent connections with cells in the ventral midbrain and hindbrain. Cholera toxin B subunit (CTB) injected into the IGL retrogradely labels neurons in a set of brain nuclei most of which are known to influence visuomotor function. These include the retinorecipient medial, lateral and dorsal terminal nuclei, the nucleus of Darkschewitsch, the oculomotor central gray, the cuneiform, and the lateral dorsal, pedunculopontine, and subpeduncular pontine tegmental nuclei. Intraocular CTB labeled a retinal terminal field in the medial terminal nucleus that extends dorsally into the pararubral nucleus, a location also containing cells projecting to the IGL. Distinct clusters of IGL-afferent neurons are also located in the medial vestibular nucleus. Vestibular projections to the IGL were confirmed by using anterograde tracer injection into the medial vestibular nucleus. Other IGL-afferent neurons are evident in Barrington's nucleus, the dorsal raphe, locus coeruleus, and retrorubral nucleus. Injection of a retrograde, trans-synaptic, viral tracer into the SCN demonstrated transport to cells as far caudal as the vestibular system and, when combined with IGL injection of CTB, confirmed that some in the medial vestibular nucleus polysynaptically project to the SCN and monosynaptically to the IGL, as do cells in other brain regions. The results suggest that the IGL may be part of the circuitry governing visuomotor activity and further indicate that circadian rhythmicity might be influenced by head motion or visual stimuli that affect the vestibular system.

Interaction of vestibular, echolocation, and visual modalities guiding flight by the big brown bat, Eptesicus fuscus.

The big brown bat (Eptesicus fuscus) is an aerial-feeding insectivorous species that relies on echolocation to avoid obstacles and to detect flying insects. Spatial perception in the dark using echolocation challenges the vestibular system to function without substantial visual input for orientation. IR thermal video recordings show the complexity of bat flights in the field and suggest a highly dynamic role for the vestibular system in orientation and flight control. To examine this role, we carried out laboratory studies of flight behavior under illuminated and dark conditions in both static and rotating obstacle tests while administering heavy water (D2O) to impair vestibular inputs. Eptesicus carried out complex maneuvers through both fixed arrays of wires and a rotating obstacle array using both vision and echolocation, or when guided by echolocation alone. When treated with D2O in combination with lack of visual cues, bats showed considerable decrements in performance. These data indicate that big brown bats use both vision and echolocation to provide spatial registration for head position information generated by the vestibular system.

Plasticity of auditory medullary-midbrain connectivity across metamorphic development in the bullfrog, Rana catesbeiana.

On the basis of patterns of anterograde, retrograde, and bi-directional transport of tracers from both the superior olivary nucleus (SON) and the torus semicircularis (TS), we report anatomical changes in brainstem connectivity across metamorphic development in the bullfrog, Rana catesbeiana. In early and late stages of larval development (Gosner stages 25-37), anterograde or bi-directional tracers injected into the SON produce terminal/fiber label in the contralateral SON and in the ipsilateral TS. Between stages 38-41 (deaf period), only sparse or no terminal/fiber label is visible in these target nuclei. During metamorphic climax (stages 42-46), terminal/fiber label reappears in both the contralateral SON and in the ipsilateral TS, and now also in the contralateral TS. Injections of retrograde tracers into the SON fail to label cell bodies in the ipsilateral TS in deaf period animals, mirroring the previously-reported failure of retrograde transport from the TS to the ipsilateral SON during this developmental time. Bilateral cell body label emerges in the dorsal medullary nucleus and the lateral vestibular nucleus bilaterally as a result of SON transport during the late larval period, while cell body label in the contralateral TS emerges during climax. At all larval stages, injections into the SON produce anterograde and retrograde label in the medial vestibular nucleus bilaterally. These data show anatomical stability in some pathways and plasticity in others during larval development, with the most dramatic changes occurring during the deaf period and metamorphic climax. Animals in metamorphic climax show patterns of connectivity similar to that of froglets and adults, indicating the maturation during climax of central anatomical substrates for hearing in air.

Optical and tomographic imaging of a middle ear malformation in the bullfrog (Rana catesbeiana).

Using a combination of in vivo computerized tomography and histological staining, a middle ear anomaly in two wild-caught American bullfrogs (Rana catesbeiana) is characterized. In these animals, the tympanic membrane, extrastapes, and pars media (shaft) of the stapes are absent on one side of the head, with the other side exhibiting normal morphology. The pars interna (footplate) of the stapes and the operculum are present in their normal positions at the entrance of the otic capsule on both the affected and unaffected sides. The pattern of deformity suggests a partial failure of development of tympanic pathway tissues, but with a preservation of the opercularis pathway. While a definitive proximate cause of the condition could not be determined, the anomalies show similarities to developmental defects in mammalian middle ear formation.