Segregation of Multimodal Inputs Into Discrete Midbrain Compartments During an Early Critical Period.

The lateral cortex of the inferior colliculus (LCIC) is a multimodal subdivision of the midbrain inferior colliculus (IC) that plays a key role in sensory integration. The LCIC is compartmentally-organized, exhibiting a series of discontinuous patches or modules surrounded by an extramodular matrix. In adult mice, somatosensory afferents target LCIC modular zones, while auditory afferents terminate throughout the encompassing matrix. Recently, we defined an early LCIC critical period (birth: postnatal day 0 to P12) based upon the concurrent emergence of its neurochemical compartments (modules: glutamic acid decarboxylase, GAD+; matrix: calretinin, CR+), matching Eph-ephrin guidance patterns, and specificity of auditory inputs for its matrix. Currently lacking are analogous experiments that address somatosensory afferent shaping and the construction of discrete LCIC multisensory maps. Combining living slice tract-tracing and immunocytochemical approaches in a developmental series of GAD67-GFP knock-in mice, the present study characterizes: (1) the targeting of somatosensory terminals for emerging LCIC modular fields; and (2) the relative separation of somatosensory and auditory inputs over the course of its established critical period. Results indicate a similar time course and progression of LCIC projection shaping for both somatosensory (corticocollicular) and auditory (intracollicular) inputs. While somewhat sparse and intermingling at birth, modality-specific projection patterns soon emerge (P4-P8), coincident with peak guidance expression and the appearance of LCIC compartments. By P12, an adult-like arrangement is in place, with fully segregated multimodal afferent arrays. Quantitative measures confirm increasingly distinct input maps, exhibiting less projection overlap with age. Potential mechanisms whereby multisensory LCIC afferent systems recognize and interface with its emerging modular-matrix framework are discussed.

Intrinsic and Synaptic Dynamics Contribute to Adaptation in the Core of the Avian Central Nucleus of the Inferior Colliculus.

The reduction of neuronal responses to repeated stimulus presentation occurs in many sensory neurons, also in the inferior colliculus of birds. The cellular mechanisms that cause response adaptation are not well described. Adaptation must be explicable by changes in the activity of input neurons, short-term synaptic plasticity of the incoming connections, excitability changes of the neuron under consideration or influences of inhibitory or modulatory network connections. Using whole-cell recordings in acute brain slices of the embryonic chicken brain we wanted to understand the intrinsic and synaptic contributions to adaptation in the core of the central nucleus of the inferior colliculus (ICCc). We described two neuron types in the chicken ICCc based on their action potential firing patterns: Phasic/onset neurons showed strong intrinsic adaptation but recovered more rapidly. Tonic/sustained firing neurons had weaker adaptation but often had additional slow components of recovery from adaptation. Morphological analysis suggested two neuron classes, but no physiological parameter aligned with this classification. Chicken ICCc neurons received mostly mixed AMPA- and NMDA-type glutamatergic synaptic inputs. In the majority of ICCc neurons the input synapses underwent short-term depression. With a simulation of the putative population output activity of the chicken ICCc we showed that the different adaptation profiles of the neuron classes could shift the emphasize of stimulus encoding from transients at long intervals to ongoing parts at short intervals. Thus, we report here that description of biophysical and synaptic properties can help to explain adaptive phenomena in central auditory neurons.

Cytoarchitectural and functional abnormalities of the inferior colliculus in sudden unexplained perinatal death.

The inferior colliculus is a mesencephalic structure endowed with serotonergic fibers that plays an important role in the processing of acoustic information. The implication of the neuromodulator serotonin also in the aetiology of sudden unexplained fetal and infant death syndromes and the demonstration in these pathologies of developmental alterations of the superior olivary complex (SOC), a group of pontine nuclei likewise involved in hearing, prompted us to investigate whether the inferior colliculus may somehow contribute to the pathogenetic mechanism of unexplained perinatal death. Therefore, we performed in a wide set of fetuses and infants, aged from 33 gestational weeks to 7 postnatal months and died of both known and unknown cause, an in-depth anatomopathological analysis of the brainstem, particularly of the midbrain. Peculiar neuroanatomical and functional abnormalities of the inferior colliculus, such as hypoplasia/structural disarrangement and immunonegativity or poor positivity of serotonin, were exclusively found in sudden death victims, and not in controls. In addition, these alterations were frequently related to dysgenesis of connected structures, precisely the raphé nuclei and the superior olivary complex, and to nicotine absorption in pregnancy. We propose, on the basis of these results, the involvement of the inferior colliculus in more important functions than those related to hearing, as breathing and, more extensively, all the vital activities, and then in pathological conditions underlying a sudden death in vulnerable periods of the autonomic nervous system development, particularly associated to harmful risk factors as cigarette smoking.

The gene regulatory networks underlying formation of the auditory hindbrain.

Development and evolution of auditory hindbrain nuclei are two major unsolved issues in hearing research. Recent characterization of transgenic mice identified the rhombomeric origins of mammalian auditory nuclei and unraveled genes involved in their formation. Here, we provide an overview on these data by assembling them into rhombomere-specific gene regulatory networks (GRNs), as they underlie developmental and evolutionary processes. To explore evolutionary mechanisms, we compare the GRNs operating in the mammalian auditory hindbrain with data available from the inner ear and other vertebrate groups. Finally, we propose that the availability of genomic sequences from all major vertebrate taxa and novel genetic techniques for non-model organisms provide an unprecedented opportunity to investigate development and evolution of the auditory hindbrain by comparative molecular approaches. The dissection of the molecular mechanisms leading to auditory structures will also provide an important framework for auditory processing disorders, a clinical problem difficult to tackle so far. These data will, therefore, foster basic and clinical hearing research alike.

AP-2δ is a crucial transcriptional regulator of the posterior midbrain.

Ap-2 transcription factors comprise a family of 5 closely related sequence-specific DNA binding proteins that play pivotal and non-redundant roles in embryonic organogenesis. To investigate the function of Ap-2δ, wδe analyzed its expression during embryogenesis and generated Ap-2δ-deficient mice. In line with the specific expression pattern of Ap-2δ in the mesencephalic tectum and the dorsal midbrain, Ap-2δ-deficient mice failed to maintain the colliculus inferior, a derivative of the dorsal midbrain, as a consequence of increased apoptotic cell death. To identify specific Ap-2δ target genes in cells of the developing dorsal midbrain, we performed whole genome analysis of cDNA expression levels. This approach identified a set of 12 putative target genes being expressed in the developing midbrain, including the transcription factors Pitx2, Mef2c, Bhlhb4 and Pou4f3. Using chromatin immunoprecipitation (CHIP) we showed that some of these genes are direct targets of Ap-2δ. Consistently, we demonstrate that Ap-2δ occupies and activates the Pou4f3 and Bhlhb4 promoters. In addition, known Pou4f3 target genes were downregulated in the posterior midbrain of Ap-2δ-deficient mice. Despite the absence of a central part of the auditory pathway, the presence of neuronal responses to sounds in the neocortex of Ap-2δ-deficient mice indicates that auditory information from the brainstem still reaches the neocortex. In summary, our data define Ap-2δ as an important transcription factor, specifying gene expression patterns required for the development of the posterior midbrain.

Temporal distribution of mRNA expression levels of various genes in the developing human inferior colliculus.

The inferior colliculus (IC) is a major binaural integration center in the auditory pathway. Interestingly, studies on the prenatal development of the human IC are lacking. During development of the nervous system a large repertoire of proteins is involved in transforming simple neuroblast cells into functional elements of the adult neural circuits. The present study reports on the mRNA levels produced by 12 genes involved in pre- [12-29 weeks of gestation (WG)], postnatal [40 postnatal days (PND) as well as 2 and 5 postnatal months (PNM)] developing human IC. The mRNA expression levels of nestin, vimentin, GFAP and DCX during 12-24 WG indicate the stages of neurogenesis, migration and differentiation of the human fetal IC. A decrease in the GAP-43 mRNA levels along with an increase in synaptophysin and PSD-95 mRNA levels during late gestational ages (24-29 WG) suggests the formation of primitive contacts by neurons with their targets and the onset of synapse formation. Expression levels of EGAD mRNA were transient with an increase in the early gestational ages, whereas that of GAD-67 mRNA increased in late gestational ages, indicating the changing role of GABA from a trophic factor to that of a neurotransmitter. High levels of BDNF, NT-3 and MBP mRNA in the late gestational ages reveal that the human IC undergoes neuronal maturation, synaptogenesis and myelination by 29 WG. Therefore, it may be suggested that the morphological maturation of the human IC occurs between 22 and 29 WG and that this period appears to be critical in the shaping of adult-like physiological attributes.

Stereological investigation and expression of calcium-binding proteins in developing human inferior colliculus.

The mammalian inferior colliculus (IC) is a major relay nucleus in the auditory pathway. Prenatal development of the human IC has been inadequately studied. The present study reports the morphometric development and maturation of the human IC using unbiased stereology, in 18 aborted fetuses of various gestational ages (12-29 weeks) and two babies aged 40 postnatal days (PND) and 5 months (that died of postoperative complications). It also demonstrates the functional maturation of the IC by examining the expression of calcium-binding proteins--parvalbumin (PV) and calbindin (CB). There was a significant increase in the total number of neurons and glia from 18 weeks of gestation (WG). The glia and neuron volume increased significantly from 16 WG to 22 WG, respectively. The total volume of IC also increased significantly from 18 WG onwards. On the other hand, the number and volume of undifferentiated cell bodies across all ages decreased significantly. Expression of CB was concentrated in the dorsal cortex while that of PV was mainly confined to the central nucleus of the IC, possibly indicating an early segregation of parallel processing of information in the auditory pathways. Intense staining for CB in the soma and dendrites appeared earlier than that of the PV. The morphological maturation appeared to overlap the onset of functional maturation suggesting an activity-dependent mechanism in the development of IC.

Barhl1 regulatory sequences required for cell-specific gene expression and autoregulation in the inner ear and central nervous system.

The development of the nervous system requires the concerted actions of multiple transcription factors, yet the molecular events leading to their expression remain poorly understood. Barhl1, a mammalian homeodomain transcription factor of the BarH class, is expressed by developing inner ear hair cells, cerebellar granule cells, precerebellar neurons, and collicular neurons. Targeted gene inactivation has demonstrated a crucial role for Barhl1 in the survival and/or migration of these sensory cells and neurons. Here we report the regulatory sequences of Barhl1 necessary for directing its proper spatiotemporal expression pattern in the inner ear and central nervous system (CNS). Using a transgenic approach, we have found that high-level and cell-specific expression of Barhl1 within the inner ear and CNS depends on both its 5' promoter and 3' enhancer sequences. Further transcriptional, binding, and mutational analyses of the 5' promoter have identified two homeoprotein binding motifs that can be occupied and activated by Barhl1. Moreover, proper Barhl1 expression in inner ear hair cells and cerebellar and precerebellar neurons requires the presence of Atoh1. Together, these data delineate useful Barhl1 regulatory sequences that direct strong and specific gene expression to inner ear hair cells and CNS sensory neurons, establish a role for autoregulation in the maintenance of Barhl1 expression, and identify Atoh1 as a key upstream regulator.

Differences in neurogenesis differentiate between core and shell regions of auditory nuclei in the turtle (Pelodiscus sinensis): evolutionary implications.

There is a clear core-versus-shell distinction in cytoarchitecture, electrophysiological properties and neural connections in the mesencephalic and diencephalic auditory nuclei of amniotes. Determining whether the embryogenesis of auditory nuclei shows a similar organization is helpful for further understanding the constituent organization and evolution of auditory nuclei. Therefore in the present study, we injected [(3)H]-thymidine into turtle embryos (Pelodiscus sinensis) at various stages of development. Upon hatching, [(3)H]-thymidine labeling was examined in both the core and shell auditory regions in the midbrain, diencephalon and dorsal ventricular ridge. Met-enkephalin and substance P immunohistochemistry was used to distinguish the core and shell regions. In the mesencephalic auditory nucleus, the occurrence of heavily labeled neurons in the nucleus centralis of the torus semicircularis reached its peak at embryonic day 9, one day later than the surrounding shell. In the diencephalic auditory nucleus, the production of heavily labeled neurons in the central region of the reuniens (Re) was highest at embryonic day (E) 8, one day later than that in the shell region of reuniens. In the region of the dorsal ventricular ridge that received inputs from the central region of Re, the appearance of heavily labeled neurons also reached a peak one day later than that in the area receiving inputs from the shell region of reuniens. Thus, there is a core-versus-shell organization of neuronal generation in reptilian auditory areas.

Time course of embryonic midbrain and thalamic auditory connection development in mice as revealed by carbocyanine dye tracing.

Central auditory connections develop in mice before the onset of hearing, around postnatal day 7. Two previous studies have investigated the development of auditory nuclei projections and lateral lemniscal nuclear projections in embryonic rats, respectively. Here, we provide detail for the first time of the initiation and progression of projections from the inferior colliculus (IC) to the medial geniculate body (MGB) and from the MGB to the auditory cortex (AC). Overall, the developmental progression of projections follows that of terminal mitoses in various nuclei, suggesting the consistent use of a developmental timetable at a given nucleus, independent of that of other nuclei. Our data further suggest that neurons project specifically and reciprocally from the MGB to the AC as early as embryonic day 14.5. These projections develop approximately a day before the reciprocal connections between the MGB and IC and before development of projections from the auditory nuclei to the IC. The development of IC projections is prolonged and progresses from rostral to caudal areas. Brainstem nuclear projections to the IC arrive first from the lateral lemniscus nuclei then the superior olive and finally the cochlear nuclei. Overall, the auditory connection development strongly suggests that most of the overall specificity of nuclear connections is set up at least 2 weeks before the onset of sound-mediated cochlea responses in mice and, thus, is likely governed predominantly by molecular genetic clues.

Mammalian brain morphogenesis and midline axon guidance require heparan sulfate.

Heparan sulfate (HS) is required for morphogen signaling during Drosophila pattern formation, but little is known about its physiological importance in mammalian development. To define the developmental role of HS in mammalian species, we conditionally disrupted the HS-polymerizing enzyme EXT1 in the embryonic mouse brain. The EXT1-null brain exhibited patterning defects that are composites of those caused by mutations of multiple HS-binding morphogens. Furthermore, the EXT1-null brain displayed severe guidance errors in major commissural tracts, revealing a pivotal role of HS in midline axon guidance. These findings demonstrate that HS is essential for mammalian brain development.

Development of output connections from the inferior colliculus to the optic tectum in barn owls.

We studied the development of the projection from the external nucleus of the inferior colliculus (ICX) to the optic tectum (OT) in the barn owl. The projection was labeled by tracer application in vitro to either the OT or the ICX, or by staining ICX cells intracellularly with biocytin. The axons of ICX neurons bifurcated into an ascending branch that projected toward the OT and a descending branch that coursed caudally to an unknown target in the brainstem. Axons of the ICX were observed to grow into the OT from embryonic day 16 (E16) on. From E22 on, side branches of the axonal projections could be found within the OT. At the day of hatching (E32), the projection displayed a dorsoventral topography comparable to the adult owl; however, atopically projecting cells remained. The complexity of the axonal arborization in the adult barn owl was found to be slightly increased compared with the hatchling. The terminal area of individual ICX cells in the OT of the adult barn owl was still broad, a finding that had not been expected from the sharply defined physiological response properties of the bimodal neurons in the space map of the OT. However, the width of the termination zone was in accordance with the large dendritic tree of the adult ICX cells, because both spanned comparable angles in their respective maps. Our data suggest that a coarse projection from the ICX to the OT can develop without coherent sensory input and may, therefore, be innately determined.

Dissection of the cellular and molecular events that position cerebellar Purkinje cells: a study of the math1 null-mutant mouse.

Granule cell precursors in the external germinal layer (EGL) of the cerebellum have been proposed to be a major player in the migration and positioning of Purkinje cells through the expression of the Netrin-like receptor Unc5h3 and the extracellular matrix molecule Reelin. To explore the role of the EGL on these processes, we made use of the math1 null-mutant mouse in which the EGL does not form. In the absence of the EGL, we find three populations of ectopic Purkinje cells. First, we find 1% of all Purkinje cells in a supracerebellar position at the dorsal midline. Second, we find 7% of all Purkinje cells in the inferior colliculus, similar to what is seen in the Unc5h3 mutant. Our finding that Unc5h3 expression is not disrupted in these cells supports the proposed role of EGL granule cell precursors in establishing the anterior cerebellar boundary through the expression of Unc5h3. Third, we find 20% of all Purkinje cells positioned deep to the cerebellar cortex as seen in the reeler mutant. However, unlike the reeler mutant, where 5% of the Purkinje cells migrate successfully, we find that in the math1 null that 72% of the Purkinje cells migrate successfully. This finding demonstrates that Purkinje cell migration is not solely dependent on Reelin signaling from the EGL and is likely caused by Reelin signals emanating from the nuclear transitory zone or the ventricular zone, or both.

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.

Development of calretinin immunoreactivity in the brainstem auditory nuclei of the barn owl (Tyto alba).

The early development of calretinin immunoreactivity (CR-IR) was described in the auditory nuclei of the brainstem of the barn owl. CR-IR was first observed in the auditory hindbrain at embryonic day (E17) and a day later (E18) in the inferior colliculus. In each of the auditory nuclei studied, CR-IR did not develop homogeneously, but began in the regions that map high best frequencies in the adult barn owl. In the hindbrain, CR-IR was first observed in the rostromedial regions of the cochlear nucleus magnocellularis and the nucleus laminaris, and in the dorsal regions of the nucleus angularis and in the nucleus of the lateral lemniscus. In the inferior colliculus, CR-IR began in the ventral region of the central core. The edge of these gradients moved along the future tonotopic axes during the development of all nuclei studied, until adult patterns of CR-IR were achieved about a week after hatching.

Transient CD15 expression reflects stages of differentiation and maturation in the human subcortical central auditory pathway.

The expression of the terminal saccharide determinant CD15 (3[a1-3]-fucosyl-N-acetyl-lactosamine) was evaluated in the central auditory system of the human developing brain by using monoclonal antibodies against this epitope. CD15 immunoreactivity was first observed in the ventral cochlear nucleus at 10 weeks of gestation, whereas the dorsal cochlear nucleus became positive from 13 weeks of gestation. In both nuclei, the intensity of immunoreactivity increased until 16 weeks of gestation and lasted until 25 weeks of gestation. In the inferior colliculi, CD15 was poorly expressed in the central nucleus from 13 to 23 weeks of gestation and later with moderate levels until birth. Within the medial geniculate nucleus, a biphasic pattern of expression was observed with peaks around 14-17 and 21-24 weeks of gestation. Heterogeneous expression in the medial geniculate nucleus, which was associated either with neurons or the neuropil, allowed distinction of subnuclei. In many of the auditory pathway structures (e.g., ventral cochlear nucleus and central nucleus of the inferior colliculus), a heterogeneous pattern of CD15 expression in the form of repeating parallel bands, possibly related to tonotopic organization, became transiently apparent around 23 weeks of gestation, whereas in the magnocellular part of the medial geniculate nucleus, a striking modular or compartmental arrangement of immunoreactive structures (which could also be associated with tonotopic organization) was also noted at about 23 weeks of gestation. We propose that the initiation of CD15 expression in each nucleus heralds the appearance of functional contacts and that high levels of neuropil labeling are related to the formation of nonstabilized synaptic contacts. Thus, transient CD15 expression in the central auditory system is possibly correlated with phases of functional plasticity in this pathway.

The fork head transcription factor Fkh5/Mf3 is a developmental marker gene for superior colliculus layers and derivatives of the hindbrain somatic afferent zone.

Fork head-5 (Fkh5; also known as Mf3 and TWH) is a transcription factor of the winged helix family. As part of an extended project to understand the function of this protein in the developing mouse brain, in the present work we have used Fkh5/Mf3 expression as a marker to study the development of the midbrain and hindbrain. In the midbrain, Fkh5/Mf3 is expressed in the superior colliculus, in the ventricular layer of the inferior colliculus and in the isthmus. In the superior colliculus, Fkh5/Mf3 is expressed by cells of layers 4a and 4c since early in development. In the hindbrain, Fkh5/Mf3 is a longitudinal marker (as opposed to a transverse or rhombomeric one), since it labels nuclei belonging to the somatic afferent zone (ventral cochlear nucleus, cuneate and external cuneate nuclei, principal and spinal nuclei of the trigeminal). In addition, Fkh5/Mf3 is expressed by the developing endopiriform nucleus and by the olivary pretectal nucleus. The results suggest that Fkh5/Mf3 has an early role in the lamination of the tectum and in the longitudinal differentiation of the hindbrain.

Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system.

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

Aberrant axonal projections in mice lacking EphA8 (Eek) tyrosine protein kinase receptors.

We have generated mice homozygous for a mutation that disrupts the gene encoding EphA8, a member of the Eph family of tyrosine protein kinase receptors, previously known as Eek. These mice develop to term, are fertile and do not display obvious anatomical or physiological defects. The mouse ephA8/eek gene is expressed primarily in a rostral to caudal gradient in the developing tectum. Axonal tracing experiments have revealed that in these mutant mice, axons from a subpopulation of tectal neurons located in the superficial layers of the superior colliculus do not reach targets located in the contralateral inferior colliculus. Moreover, ephA8/eek null animals display an aberrant ipsilateral axonal tract that projects to the ventral region of the cervical spinal cord. Retrograde labeling revealed that these abnormal projections originate from a small subpopulation of superior colliculus neurons that normally express the ephA8/eek gene. These results suggest that EphA8/Eek receptors play a role in axonal pathfinding during development of the mammalian nervous system.

Axogenesis in the human fetal auditory system, demonstrated by neurofilament immunohistochemistry.

Morphological, electrophysiological and behavioral evidence indicates that the onset of rapid, synchronized conduction of auditory impulses occurs in the human brainstem during the 28th-29th fetal weeks. This implies that axonal connections in the brainstem auditory pathway are generated prior to this time. In order to investigate the sequence of axogenic events in the human brainstem pathway, we employed immunohistochemical techniques and an antibody to neurofilament protein. Immunostaining for axonal neurofilaments in an age-graded series of fetal brains demonstrates that a small number of cochlear nerve axons have invaded the ventral cochlear nucleus by the 16th fetal week. By this same time point, a limited number of trapezoid body-lateral lemniscus axons have reached the superior olivary complex and inferior colliculus. Between gestational weeks 16 and 26, there is marked expansion and collateralization of the ascending pathway from cochlear nerve to inferior colliculus. By week 26, ascending axons have begun to form plexuses of terminal neuropil within all of the brainstem auditory nuclei. Beginning in week 22, there is development of commissural axons (dorsal commissure of the lateral lemniscus and commissure of the inferior colliculus) and descending projections (descending collicular axons and olivocochlear bundle). This early establishment of a mature pattern of axonal connections presumably forms the basis for the appearance of myelin, acousticomotor reflexes and recordable brainstem responses by fetal week 29.