Seth S. Horowitz

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 Barringtons 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. J. Comp. Neurol. 474:227–245, 2004.

Versatility of biosonar in the big brown bat, Eptesicus fuscus

Infrared cameras and ultrasonic microphones were used to record big brown bats (Eptesicus fuscus) flying in natural conditions at night while they hunted for insects. As expected, bats avoided obstacles while flying through vegetation and intercepted flying prey in the open. But bats also appeared to capture insects near and possibly on the ground and near or in vegetation, flew low over water to drink, and pursued each other in aerial “dogfights.” In less than a minute, the same bat often used echolocation for several different tasks, showing a wider repertoire of sonar-guided behavior than revealed by previous observations limited to seeing bats flying against the evening sky or being photographed in fixed fields-of-view.

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. J. Comp. Neurol. 487:127–146, 2005.

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 3H-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.

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.

Metamorphic development of the bronchial columella of the larval bullfrog (Rana catesbeiana)

Histological and immunohistochemical analyses of head and respiratory structures in bullfrog (Rana catesbeiana) tadpoles were undertaken to address the hypothesis that the bronchial columella (BC) is the primary sound conduction pathway in these larval anurans. In postembryonic tadpoles, the BC is composed of fibroblasts surrounded by a Type I collagen matrix, with Type II collagen located in basement membranes at the distal ends. It provides a highly flexible tendon-like attachment between the round window and the membranous sac of the primary bronchus of the ipsilateral lung. As the animals approach metamorphic climax stages, the fibroblasts decrease in number and the BC becomes almost exclusively collagenous. During metamorphic climax, the BC degenerates and is completely resorbed by the time the animal becomes a postmetamorphic froglet. At all larval stages examined, the BC is structurally and immunohistochemically different from both the opercularis muscle of tadpoles and the tympanic columella (stapes homolog) of postmetamorphic animals. These observations suggest that the BC may not be rigid enough to provide an effective coupling between the lungs and the round window. An alternative hypothesis for the function of the BC, based on its structure, is presented.

Plasticity in the Auditory System across Metamorphosis

Many species of anuran amphibians undergo a developmental process called metamorphosis during which free-living, herbivorous, nonreproductive larvae (tadpoles) transform into partly terrestrial, carnivorous, reproductively active adults. Metamorphosis in anurans is a period of rapid morphological and physiological change affecting all sensory, motor, and vegetative systems. The pattern of larval development and the extent of change during metamorphosis vary (McDiarmid and Altig 1999); some species (e.g., Eleutherodactylus) undergo direct development, whereas others (such as the bullfrog, Rana catesbeiana) have relatively lengthy tadpole periods. Pipids, such as the African clawed frog, Xenopus laevis, remain aquatic as adults, and metamorphic change is not as extensive as that observed in anuran species which become partly terrestrial. Development in anurans thus encompasses diverse patterns of change and is a rich resource for analysis of theories and mechanisms of plasticity, offering a vertebrate system in which neural and anatomical development can be examined in nonfetal animals. Metamorphosis features regression of structures important only in larval forms, transformation of larval into adult structures, and development of new structures necessary for the adult (Fritzsch et al. 1988). The metamorphic transition involves considerable alteration in external morphology (Gosner 1960; Nieuwkoop and Faber 1994); in behaviors such as respiration, locomotion, and feeding (Etkin 1964; Stehouwer 1988; Burggren and Infantino 1994); and in peripheral and central nervous system structure and functioning (Fritzsch et al. 1988; Lannoo 1999). Metamorphosis also involves changes in neural and hormonal foundations allowing later emergence of reproductive behaviors. All sensory systems undergo some sort of modification or reorganization during the larval period, and at different time courses (Spaeti 1978). In particular, the metamorphic shift from aquatic tadpole to amphibious frog imposes substantial changes in the type of auditory input available to the organism, based on the different acoustic properties of underwater and terrestrial environments (Bass and Clark 2002). Adult anurans rely on vocalizations for mate attraction and territorial defense (Gerhardt

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.

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 μm. 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.

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 β 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.