Mark L. Gabriele

Development of afferent patterns in the inferior colliculus of the rat: Projection from the dorsal nucleus of the lateral lemniscus

The inferior colliculus (IC) receives a variety of layered afferent projections. The purpose of the present study was to determine the development of the projection from the dorsal nucleus of the lateral lemniscus (DNLL) to the IC in rat prior to the onset of hearing (postnatal day 12/13). Lipophilic carbocyanine dye, DiI (1,1′‐dioctodecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate), was used to trace the crossed inhibitory projection of DNLL in a developmental series of rat embryos and pups between ages embryonic day 15 (E15) and postnatal day 12 (P12). Dye‐coated pins were positioned in paraformaldehyde‐fixed brains either unilaterally in DNLL (embryonic cases), or in the commissure of Probst where DNLL fibers cross the midline (postnatal cases). By E15, pioneer fibers have left DNLL and crossed the midline. A few fibers have nearly reached the contralateral IC by E19. At birth (E22 = P0), the projection has invaded ventromedial, high‐frequency layers of the IC. The vast majority of DNLL axons parallel the presumptive IC layers by P4, and by P8 the projection has segregated into a pattern of bands (afferent dense) and interband (afferent sparse) spaces that encompasses the entire frequency axis of the IC. Adult‐like patches, regions along afferent bands that exhibit the heaviest labeling, develop by P12. These results indicate that some mature projection patterns are in place prior to the onset of hearing. Such findings suggest that evoked activity may not be required for the initial organization of patterned projections in the ascending auditory pathway. J. Comp. Neurol. 416:368‐382, 2000.

Organization of the disynaptic pathway from the anteroventral cochlear nucleus to the lateral superior olivary nucleus in the ferret

 The medial nucleus of the trapezoid body (MNTB) is one of three major nuclei of the superior olivary complex and provides an important inhibitory input from the contralateral ear to the lateral superior olivary nucleus (LSO) in the initial binaural pathway for coding interaural intensity differences. The major input to the MNTB from the contralateral anteroventral cochlear nucleus (AVCN) involves giant, calyx-like endings that have a one-to-one relationship with cells in the MNTB as confirmed in the ferret in this study. The main objective of the present study was to define the subsequent organization of projections from cells receiving these calyx-like endings. Several anatomical tracers (Phaseolus vulgaris leucoagglutinin, dextran-biotin, and biocytin) were used that are transported both anterogradely and retrogradely within neuronal projections in order to define the organization of MNTB connections with the LSO in the adult ferret. Analysis focused on determining the topography in both the transverse and longitudinal planes of the projections. Focal tracer injections in the LSO resulted in retrograde labeling of a long, narrow column of cells in the MNTB. The orientation and location of labeled cells was dependent on the medial-lateral position of the injection site. In the rostral-caudal dimension of MNTB, there was no such topographic relation between the injection site and the position of labeled cells. Labeled cells in the MNTB were distributed more or less evenly in a longitudinal column regardless of whether the injection site was restricted to the rostral, middle or caudal part of the LSO. In keeping with this pattern, tracer injections in the MNTB resulted in bands of labeled axons that distributed endings throughout the rostral-caudal axis of the LSO. These bands or sheets varied in medial-lateral position relative to the location of the injection site, but lacked any such rostral-caudal gradient. Thus, overall the MNTB-LSO projections have a convergent-divergent pattern of organization. While MNTB cells receive singular calyx-like endings from the AVCN, LSO cells receive projections from a long column of cells in the MNTB. Implications for processing interaural intensity differences are discussed.

Axon guidance in the auditory system: Multiple functions of Eph receptors

The neural pathways of the auditory system underlie our ability to detect sounds and to transform amplitude and frequency information into rich and meaningful perception. While it shares some organizational features with other sensory systems, the auditory system has some unique functions that impose special demands on precision in circuit assembly. In particular, the cochlear epithelium creates a frequency map rather than a space map, and specialized pathways extract information on interaural time and intensity differences to permit sound source localization. The assembly of auditory circuitry requires the coordinated function of multiple molecular cues. Eph receptors and their ephrin ligands constitute a large family of axon guidance molecules with developmentally regulated expression throughout the auditory system. Functional studies of Eph/ephrin signaling have revealed important roles at multiple levels of the auditory pathway, from the cochlea to the auditory cortex. These proteins provide graded cues used in establishing tonotopically ordered connections between auditory areas, as well as discrete cues that enable axons to form connections with appropriate postsynaptic partners within a target area. Throughout the auditory system, Eph proteins help to establish patterning in neural pathways during early development. This early targeting, which is further refined with neuronal activity, establishes the precision needed for auditory perception.

Distribution of the calcium-binding proteins calbindin D-28K and parvalbumin in the superior colliculus of adult and neonatal cat and rhesus monkey.

Abstract. The distribution of the calcium-binding proteins calbindin D-28K and parvalbumin was examined in newborn and adult superior colliculus of cat and rhesus monkey using immunohistochemical techniques. In adult animals of both species, calbindin-immunoreactive neurons had a three-tiered arrangement: one band was present in the upper aspects of the superficial laminae, a second in the intermediate laminae, and a third in the deep laminae. The intermediate tier was less obvious in the monkey, whereas the deep tier was less pronounced in the cat. Parvalbumin-immunoreactive neurons had a complementary distribution to calbindin-immunoreactive neurons within these laminae in both species, although the segregation of calbindin immunoreactivity and parvalbumin immunoreactivity in the superficial laminae was not as precise in the monkey as it was in the cat. At birth, calbindin immunoreactivity in the newborns of both species was remarkably mature, with its three-tiered distribution clearly evident. By contrast, parvalbumin immunoreactivity was distinctly different in the newborn cat than in the newborn monkey: whereas parvalbumin immunoreactivity in the newborn monkey was already very similar to its adult-like pattern, the pattern in the newborn cat was quite immature. The superficial laminae of the newborn cat were virtually devoid of parvalbumin immunoreactivity, and, although the intermediate laminae displayed robust parvalbumin-immunoreactive neuropil, comparatively fewer parvalbumin-immunoreactive neurons were observed. Conspicuously few in number were the large multipolar neurons in the intermediate laminae, which give rise to the descending efferents to the brainstem. However, parvalbumin-immunoreactive neurons were present within the deep laminae, suggesting a ventral-to-dorsal maturational gradient in parvalbumin expression that parallels the ventral-to-dorsal gradient of neurogenesis. The differences in parvalbumin immunoreactivity observed between these two species at parturition are consistent with the advanced visual and visuomotor capabilities of the newborn monkey and the absence of visually related behaviors in the newborn cat.

EphA4 and ephrin-B2 expression patterns during inferior colliculus projection shaping prior to experience.

Central processing of complex auditory tasks requires elaborate circuitry. The auditory midbrain, or inferior colliculus (IC), epitomizes such precise organization, where converging inputs form discrete, tonotopically‐arranged axonal layers. Previously in rat, we established that shaping of multiple afferent patterns in the IC central nucleus (CNIC) occurs prior to experience. This study implicates an Eph receptor tyrosine kinase and a corresponding ephrin ligand in signaling this early topographic registry. We report that EphA4 and ephrin‐B2 expression patterns in the neonatal rat and mouse IC correlate temporally and spatially with that of developing axonal layers. DiI‐labeling confirms projections arising from the lateral superior olive (LSO) form frequency‐specific layers within the ipsilateral and contralateral mouse CNIC, as has been described in other species. Immunohistochemistry (EphA4 and ephrin‐B2) and ephrin‐B2 lacZ histochemistry reveal clear gradients in expression across the tonotopic axis, with most concentrated labeling observed in high‐frequency, ventromedial aspects of the CNIC. Discrete patches of labeling were also discernible in the external cortex of the IC (ECIC; EphA4 patches in rat, ephrin‐B2 patches in mouse). Observed gradients in the CNIC and compartmentalized ECIC expression persisted through the first postnatal week, before becoming less intense and more homogeneously distributed by the functional onset of hearing. EphA4 and ephrin‐B2‐positive neurons were evident in several auditory brainstem nuclei known to send patterened inputs to the IC. These findings suggest the involvement of cell–cell EphA4 and ephrin‐B2 signaling in establishing order in the developing IC.

Patterning of multiple layered projections to the auditory midbrain prior to experience

The precise arrangement of patterned inputs into discrete functional domains is a common organizational feature of primary sensory structures. While the specific organization of patterned connections has been well documented in the visual and somatosensory systems, comparatively little is known about the arrangement of neighboring afferent patterns in the emerging auditory system. Here we report early projection specificity for multiple converging inputs to the rat central nucleus of the inferior colliculus (ICC). Afferents arising from the dorsal cochlear nucleus (DCN), the dorsal nucleus of the lateral lemniscus (DNLL), and the lateral superior olive (LSO) establish discernible axonal layers a week prior to experience. By hearing onset, contralateral DCN and contralateral LSO layers are clearly defined and segregated from contralateral DNLL terminal zones. Layering of the ipsilateral LSO projection, on the other hand, exhibits considerable spatial overlap with the contralateral DNLL pattern. This fine laminar structure of interdigitating and overlapping inputs likely underlies the complex signal processing performed in the auditory midbrain and may serve as a model system for examining competitive interactions between neighboring excitatory and inhibitory projections early in development.

Early segregation of layered projections from the lateral superior olivary nucleus to the central nucleus of the inferior colliculus in the neonatal cat.

The central nucleus of the inferior colliculus (IC) is a laminated structure that receives multiple converging afferent projections. These projections terminate in a layered arrangement and are aligned with dendritic arbors of the predominant disc-shaped neurons, forming fibrodendritic laminae. Within this structural framework, inputs terminate in a precise manner, establishing a mosaic of partially overlapping domains that likely define functional compartments. Although several of these patterned inputs have been described in the adult, relatively little is known about their organization prior to hearing onset. The present study used the lipophilic carbocyanine dyes DiI and DiD to examine the ipsilateral and contralateral projections from the lateral superior olivary (LSO) nucleus to the IC in a developmental series of paraformaldehyde-fixed kitten tissue. By birth, the crossed and uncrossed projections had reached the IC and were distributed across the frequency axis of the central nucleus. At this earliest postnatal stage, projections already exhibited a characteristic banded arrangement similar to that described in the adult. The heaviest terminal fields of the two inputs were always complementary in nature, with the ipsilateral input appearing slightly denser. This early arrangement of interdigitating ipsilateral and contralateral LSO axonal bands that occupy adjacent sublayers supports the idea that the initial establishment of this highly organized mosaic of inputs that defines distinct synaptic domains within the IC occurs largely in the absence of auditory experience. Potential developmental mechanisms that may shape these highly ordered inputs prior to hearing onset are discussed.

Ephrin-B2 reverse signaling is required for topography but not pattern formation of lateral superior olivary inputs to the inferior colliculus.

Graded and modular expressions of Eph‐ephrins are known to provide positional information for the formation of topographic maps and patterning in the developing nervous system. Previously we have shown that ephrin‐B2 is expressed in a continuous gradient across the tonotopic axis of the central nucleus of the inferior colliculus (CNIC), whereas patterns are discontinuous and modular in the lateral cortex of the IC (LCIC). The present study explores the involvement of ephrin‐B2 signaling in the development of projections to the CNIC and LCIC arising from the lateral superior olivary nuclei (LSO) prior to hearing onset. Anterograde and retrograde fluorescent tracing methods in neonatal fixed tissue preparations were used to compare topographic mapping and the establishment of LSO layers/modules in wild‐type and ephrin‐B2lacZ/+ mice (severely compromised reverse signaling). At birth, pioneer LSO axons occupy the ipsilateral IC in both groups but are delayed contralaterally in ephrin‐B2lacZ/+ mutants. By the onset of hearing, both wild‐type and mutant projections form discernible layers bilaterally in the CNIC and modular arrangements within the ipsilateral LCIC. In contrast, ephrin‐B2lacZ/+ mice lack a reliable topography in LSO‐IC projections, suggesting that fully functional ephrin‐B2 reverse signaling is required for normal projection mapping. Taken together, these ephrin‐B2 findings paired with known coexpression of EphA4 suggest the importance of these signaling proteins in establishing functional auditory circuits prior to experience. J. Comp. Neurol. 521:1585–1597, 2013.

Modular-extramodular organization in developing multisensory shell regions of the mouse inferior colliculus

The complex neuroanatomical connections of the inferior colliculus (IC) and its major subdivisions offer a juxtaposition of segregated processing streams with distinct organizational features. While the tonotopically layered central nucleus is well‐documented, less is known about functional compartments in the neighboring lateral cortex (LCIC). In addition to a laminar framework, LCIC afferent‐efferent patterns suggest a multimodal mosaic, consisting of a patchy modular network with surrounding extramodular domains. This study utilizes several neurochemical markers that reveal an emerging LCIC modular‐extramodular microarchitecture. In newborn and post‐hearing C57BL/6J and CBA/CaJ mice, histochemical and immunocytochemical stains were performed for acetylcholinesterase (AChE), nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d), glutamic acid decarboxylase (GAD), cytochrome oxidase (CO), and calretinin (CR). Discontinuous layer 2 modules are positive for AChE, NADPH‐d, GAD, and CO throughout the rostrocaudal LCIC. While not readily apparent at birth, discrete cell clusters emerge over the first postnatal week, yielding an identifiable modular network prior to hearing onset. Modular boundaries continue to become increasingly distinct with age, as surrounding extramodular fields remain largely negative for each marker. Alignment of modular markers in serial sections suggests each highlight the same periodic patchy network throughout the nascent LCIC. In contrast, CR patterns appear complementary, preferentially staining extramodular LCIC zones. Double‐labeling experiments confirm that NADPH‐d, the most consistent developmental modular marker, and CR label separate, nonoverlapping LCIC compartments. Determining how this emerging modularity may align with similar LCIC patch‐matrix‐like Eph/ephrin guidance patterns, and how each interface with, and potentially influence developing multimodal LCIC projection configurations is discussed.

Graded and discontinuous EphA–ephrinB expression patterns in the developing auditory brainstem

Eph-ephrin interactions guide topographic mapping and pattern formation in a variety of systems. In contrast to other sensory pathways, their precise role in the assembly of central auditory circuits remains poorly understood. The auditory midbrain, or inferior colliculus (IC) is an intriguing structure for exploring guidance of patterned projections as adjacent subdivisions exhibit distinct organizational features. The central nucleus of the IC (CNIC) and deep aspects of its neighboring lateral cortex (LCIC, Layer 3) are tonotopically-organized and receive layered inputs from primarily downstream auditory sources. While less is known about more superficial aspects of the LCIC, its inputs are multimodal, lack a clear tonotopic order, and appear discontinuous, terminating in modular, patch/matrix-like distributions. Here we utilize X-Gal staining approaches in lacZ mutant mice (ephrin-B2, -B3, and EphA4) to reveal EphA-ephrinB expression patterns in the nascent IC during the period of projection shaping that precedes hearing onset. We also report early postnatal protein expression in the cochlear nuclei, the superior olivary complex, the nuclei of the lateral lemniscus, and relevant midline structures. Continuous ephrin-B2 and EphA4 expression gradients exist along frequency axes of the CNIC and LCIC Layer 3. In contrast, more superficial LCIC localization is not graded, but confined to a series of discrete ephrin-B2 and EphA4-positive Layer 2 modules. While heavily expressed in the midline, much of the auditory brainstem is devoid of ephrin-B3, including the CNIC, LCIC Layer 2 modular fields, the dorsal nucleus of the lateral lemniscus (DNLL), as well as much of the superior olivary complex and cochlear nuclei. Ephrin-B3 LCIC expression appears complementary to that of ephrin-B2 and EphA4, with protein most concentrated in presumptive extramodular zones. Described tonotopic gradients and seemingly complementary modular/extramodular patterns suggest Eph-ephrin guidance in establishing juxtaposed continuous and discrete neural maps in the developing IC prior to experience.