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Key points The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb-/- mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb-/- mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (Po ) in mature OHC is ∼50% in endolymphatic [Ca2+ ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting Po in vivo is also high in Tecta/Tectb-/- mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET-dependent, non-linear cochlear amplification in OHCs, fail to exhibit long-lasting adaptation to repetitive stimulation in Tecta/Tectb-/- mice. We conclude that during prolonged, sound-induced stimulation of the cochlea the TM may determine the extracellular Ca2+ concentration near the OHC's MET channels. Abstract The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb-/- double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild-type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild-type and Tecta/Tectb-/- mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb-/- mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic-like Ca2+ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near -40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET-dependent otoacoustic emissions in vivo in the Tecta/Tectb-/- mice. Therefore, the TM is likely to contribute to the regulation of Ca2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation.

Cochlear function depends on the operation of a coupled feedback loop, incorporating outer hair cells (OHCs), and structured to assure that inner hair cells (IHCs) convey frequency specific acoustic information to the brain, even at very low sound levels. Although our knowledge of OHC function and its contribution to cochlear amplification has expanded, the importance of the tectorial membrane (TM) to the processing of mechanical inputs has not been fully elucidated. In addition, there are a surprising number of genetic mutations that affect TM structure and that produce hearing loss in humans. By synthesizing old and new results obtained on several mouse mutants, we learned that animals with abnormal TMs are prone to generate spontaneous otoacoustic emissions (SOAE), which are uncommon in most wildtype laboratory animals. Because SOAEs are not produced in TM mutants or in humans when threshold shifts exceed approximately 25 dB, some degree of cochlear amplification is required. However, amplification by itself is not sufficient because normal mice are rarely spontaneous emitters. Since SOAEs reflect active cochlear operation, TM mutants are valuable for studying the oscillatory nature of the amplification process and the structures associated with its stabilization. Inasmuch as the mouse models were selected to mirror human auditory disorders, using SOAEs as a noninvasive clinical tool may assist the classification of individuals with genetic defects that influence the active mechanisms responsible for sensitivity and frequency selectivity, the hallmarks of mammalian hearing.

Vertigo is the leading symptom of vestibular disorders and a major risk factor for falls. In a genome-wide association study of vertigo (Ncases = 48,072, Ncontrols = 894,541), we uncovered an association with six common sequence variants in individuals of European ancestry, including missense variants in ZNF91, OTOG, OTOGL, and TECTA, and a cis-eQTL for ARMC9. The association of variants in ZNF91, OTOGL, and OTOP1 was driven by an association with benign paroxysmal positional vertigo. Using previous reports of sequence variants associating with age-related hearing impairment and motion sickness, we found eight additional variants that associate with vertigo. Although disorders of the auditory and the vestibular system may co-occur, none of the six genome-wide significant vertigo variants were associated with hearing loss and only one was associated with age-related hearing impairment. Our results uncovered sequence variants associating with vertigo in a genome-wide association study and implicated genes with known roles in inner ear development, maintenance, and disease., Astros Skuladottir et al. conducted a genome-wide association study on 48,072 vertigo cases and 894,541 controls from four populations with European ancestries. They identified six common variants associated with vertigo, thereby providing further insight into the etiology of vestibular disorders.

Spontaneous otoacoustic emissions (SOAEs) recorded from the ear canal in the absence of sound reflect cochlear amplification, an outer hair cell (OHC) process required for the extraordinary sensitivity and frequency selectivity of mammalian hearing. Although wild-type mice rarely emit, those with mutations that influence the tectorial membrane (TM) show an incidence of SOAEs similar to that in humans. In this report, we characterized mice with a missense mutation inTecta,a gene required for the formation of the striated-sheet matrix within the core of the TM. Mice heterozygous for the Y1870C mutation (TectaY1870C/+) are prolific emitters, despite a moderate hearing loss. Additionally, Kimura’s membrane, into which the OHC stereocilia insert, separates from the main body of the TM, except at apical cochlear locations. Multimodal SOAEs are also observed inTectaY1870C/+mice where energy is present at frequencies that are integer multiples of a lower-frequency SOAE (the primary). Second-harmonic SOAEs, at twice the frequency of a lower-frequency primary, are the most frequently observed. These secondary SOAEs are found in spatial regions where stimulus-evoked OAEs are small or in the noise floor. Introduction of high-level suppressors just above the primary SOAE frequency reduce or eliminate both primary and second-harmonic SOAEs. In contrast, second-harmonic SOAEs are not affected by suppressors, either above or below the second-harmonic SOAE frequency, even when they are much larger in amplitude. Hence, second-harmonic SOAEs do not appear to be spatially separated from their primaries, a finding that has implications for cochlear mechanics and the consequences of changes to TM structure.

Spontaneous otoacoustic emissions (SOAEs) are a signature of cochlear amplification, a process associated with outer hair cell (OHC) function and required for the sensitivity and frequency selectivity of cochlear responses. Although normal mice rarely exhibit these signals, those with mutations that influence the structure of the tectorial membrane (TM) show a greater incidence of these phenomena. In this report, we study mouse models with mutations affecting the striated-sheet matrix that forms the body of this accessory structure. In addition to CEACAM16, the matrix is composed of both α- and β-tectorin (TECTA and TECTB respectively) and these latter two noncollagenous proteins are the focus of this study. Of all the mutants tested to date, mice heterozygous (het) for a missense mutation (c.5609A>G, p.Tyr1870Cys) in Tecta (TectaY1870C/+ mice) are prolific emitters with an average of ∼7 SOAEs per cochlea, which is higher than in the very few normal mice with SOAEs where the average is 2-3 per cochlea. A small number of homozygous Tectb−/- mice lacking TECTB are also emitters, but they produce only one SOAE in any given ear. Although both mouse mutants have hearing loss, SOAE frequencies coincide with frequency regions where some degree of amplification is retained. The larger number of SOAEs in TectaY1870C/+ mice appears to correlate with an increase in porosity (1), which controls the spread of excitation of tectorial membrane traveling waves. Sellon and colleagues reported that this change in the material properties of the TM was associated with the larger size of nanoscale pores linked to the Y1870C missense mutation in TECTA. In mice lacking Tectb, where porosity is wild-type like, the number of SOAEs per cochlea is small and only a few of these animals present with this phenomenon. Characterization of traveling-wave properties in other emitting TM mutants will be required to know if the correlation between increased porosity and increased numbers of SOAEs reliably causes the change in phenotype.

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease., Summary: The tethering of zebrafish otoliths, which are important for balance and hearing in fish, occurs in two stages that are dependent on genes that have been implicated in human deafness.

The tectorial membrane (TM) connects to the stereociliary bundles of outer hair cells (OHCs). Humans with an autosomal dominant C1509G mutation in alpha-tectorin, a protein constituent of the TM, are born with a partial hearing loss that worsens over time. The Tecta(C1509/+) transgenic mouse with the same point mutation has partial hearing loss secondary to a shortened TM that only contacts the first row of OHCs. As well, Tecta(C1509G/+) mice have increased expression of the OHC electromotility protein, prestin. We sought to determine whether these changes impact OHC survival. Distortion product otoacoustic emission thresholds in a quiet environment did not change to 6 months of age. However, noise exposure produced acute threshold shifts that fully recovered in Tecta (+/+) mice but only partially recovered in Tecta(C1509G/+) mice. While Tecta(+/+) mice lost OHCs primarily at the base and within all three rows, Tecta(C1509G/+) mice lost most of their OHCs in a more apical region of the cochlea and nearly completely within the first row. In order to estimate the impact of a shorter TM on the forces faced by the stereocilia within the first OHC row, both the wild type and the heterozygous conditions were simulated in a computational model. These analyses predicted that the shear force on the stereocilia is ~50% higher in the heterozygous condition. We then measured electrically induced movements of the reticular lamina in situ and found that while they decreased to the noise floor in prestin null mice, they were increased by 4.58 dB in Tecta(C1509G/+) mice compared to Tecta(+/+) mice. The increased movements were associated with a fourfold increase in OHC death as measured by vital dye staining. Together, these findings indicate that uncoupling the TM from some OHCs leads to partial hearing loss and places the remaining coupled OHCs at higher risk. Both the mechanics of the malformed TM and the increased prestin-related movements of the organ of Corti contribute to this higher risk profile.

The tectorial membrane (TM) is an extracellular matrix of the cochlea whose prominent role in hearing has been demonstrated through mutation studies. The C1509G mutation of the Tecta gene, which encodes for the α-tectorin protein, leads to hearing loss. The heterozygote TM only attaches to the first row of outer hair cells (OHCs), and the homozygote TM does not attach to any OHCs. Here we measured the morphology and mechanical properties of wild-type, heterozygous, and homozygous Tecta TMs. Morphological analyses conducted with second- and third-harmonic imaging, scanning electron microscopy, and immunolabeling revealed marked changes in the collagen architecture and stereocilin-labeling patterns of the mutant TMs. The mechanical properties of the mutant TM were measured by force spectroscopy. Whereas the axial Young's modulus of the low-frequency (apical) region of Tecta mutant TM samples was similar to that of wild-type TMs, it significantly decreased in the basal region to a value approaching that found at the apex. Modeling simulations suggest that a reduced TM Young's modulus is likely to reduce OHC stereociliary deflection. These findings argue that the heterozygote C1509G mutation results in a lack of attachment of the TM to the OHCs, which in turn reduces both the overall number of OHCs that are involved in mechanotransduction and the degree of mechanotransduction exhibited by the OHCs that remain attached to the TM.

The inner ear is composed of a cochlear duct and five vestibular organs in which mechanosensory hair cells play critical roles in receiving and relaying sound and balance signals to the brain. To identify novel genes associated with hair cell differentiation or function, we analyzed an archived gene expression dataset from embryonic mouse inner ear tissues. Since atonal homolog 1a (Atoh1) is a well known factor required for hair cell differentiation, we searched for genes expressed in a similar pattern with Atoh1 during inner ear development. The list from our analysis includes many genes previously reported to be involved in hair cell differentiation such as Myo6, Tecta, Myo7a, Cdh23, Atp6v1b1, and Gfi1. In addition, we identified many other genes that have not been associated with hair cell differentiation, including Tekt2, Spag6, Smpx, Lmod1, Myh7b, Kif9, Ttyh1, Scn11a and Cnga2. We examined expression patterns of some of the newly identified genes using real-time polymerase chain reaction and in situ hybridization. For example, Smpx and Tekt2, which are regulators for cytoskeletal dynamics, were shown specifically expressed in the hair cells, suggesting a possible role in hair cell differentiation or function. Here, by reanalyzing archived genetic profiling data, we identified a list of novel genes possibly involved in hair cell differentiation.

Electrically evoked otoacoustic emissions are sounds emitted from the inner ear when alternating current is injected into the cochlea. Their temporal structure consists of short- and long-delay components and they have been attributed to the motile responses of the sensory-motor outer hair cells of the cochlea. The nature of these motile responses is unresolved and may depend on either somatic motility, hair bundle motility, or both. The short-delay component persists after almost complete elimination of outer hair cells. Outer hair cells are thus not the sole generators of electrically evoked otoacoustic emissions. We used prestin knockout mice, in which the motor protein prestin is absent from the lateral walls of outer hair cells, and Tecta ΔENT/ΔENT mice, in which the tectorial membrane, a structure with which the hair bundles of outer hair cells normally interact, is vestigial and completely detached from the organ of Corti. The amplitudes and delay spectra of electrically evoked otoacoustic emissions from Tecta ΔENT/ΔENT and Tecta +/+ mice are very similar. In comparison with prestin +/+ mice, however, the short-delay component of the emission in prestin −/− mice is dramatically reduced and the long-delay component is completely absent. Emissions are completely suppressed in wild-type and Tecta ΔENT/ΔENT mice at low stimulus levels, when prestin-based motility is blocked by salicylate. We conclude that near threshold, the emissions are generated by prestin-based somatic motility.

Frequency tuning in the cochlea is determined by the passive mechanical properties of the basilar membrane and active feedback from the outer hair cells, sensory-effector cells that detect and amplify sound-induced basilar membrane motions. The sensory hair bundles of the outer hair cells are imbedded in the tectorial membrane, a sheet of extracellular matrix that overlies the cochlea's sensory epithelium. The tectorial membrane contains radially organized collagen fibrils that are imbedded in an unusual striated-sheet matrix formed by two glycoproteins, alpha-tectorin (Tecta) and beta-tectorin (Tectb). In Tectb(-/-) mice the structure of the striated-sheet matrix is disrupted. Although these mice have a low-frequency hearing loss, basilar-membrane and neural tuning are both significantly enhanced in the high-frequency regions of the cochlea, with little loss in sensitivity. These findings can be attributed to a reduction in the acting mass of the tectorial membrane and reveal a new function for this structure in controlling interactions along the cochlea.

Synaptic organization and spatiotemporal pattern of the tectal response to electrical stimulation of pretectal (Lpd/P) nuclei were examined in the bullfrog (Rana catesbeiana) by means of intracellular study and a current source density (CSD) analysis. The pretectal stimulation elicited mainly two types of responses (EI; I type) in the ipsilateral tectum, only one type of response (I type) in the contralateral tectum. EPSPs with short latencies were concluded to be monosynaptic. Most IPSPs were generated through di- or polysynaptic paths, but monosynaptic IPSPs were also recorded in both optic tecta. The antidromically invaded tecto-pretectal projecting cells were recorded in both tecta, which revealed reciprocal connections between the tectum and particular pretectal nuclei. In all CSD profiles, only one type of spatiotemporal pattern composed of one sink with short latency was observed. The short-latency sinks appear to be associated with monosynaptic activity generated by the pretectal inputs. The CSD analysis results show good accordance with the intracellular study.

Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal hearing. Its regulatory mechanisms are unknown. Several mouse models of hearing loss demonstrate increased prestin, inspiring us to investigate how hearing loss might feedback onto OHCs. To test whether centrally mediated feedback regulates prestin, we developed a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) into adult CBA mice produced significant loss of inner hair cells without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to receive normal auditory mechanical stimulation and having normal function. Patch-clamp experiments demonstrated no change in OHC prestin, indicating that loss of information transfer centrally did not alter prestin expression. To test whether local mechanical feedback regulates prestin, we used TectaC1509Gmice, where the tectorial membrane is malformed and only some OHCs are stimulated. OHCs connected to the tectorial membrane had normal prestin levels, whereas OHCs not connected to the tectorial membrane had elevated prestin levels, supporting an activity-dependent model. To test whether the endocochlear potential was necessary for prestin regulation, we studied TectaC1509Gmice at different developmental ages. OHCs not connected to the tectorial membrane had lower than normal prestin levels before the onset of the endocochlear potential and higher than normal prestin levels after the onset of the endocochlear potential. Taken together, these data indicate that OHC prestin levels are regulated through local feedback that requires mechanoelectrical transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment.

Alpha-tectorin (encoded by Tecta) is a component of the tectorial membrane, an extracellular matrix of the cochlea. In humans, the Y1870C missense mutation in TECTA causes a 50- to 80-dB hearing loss. In transgenic mice with the Y1870C mutation in Tecta, the tectorial membrane's matrix structure is disrupted, and its adhesion zone is reduced in thickness. These abnormalities do not seriously influence the tectorial membrane's known role in ensuring that cochlear feedback is optimal, because the sensitivity and frequency tuning of the mechanical responses of the cochlea are little changed. However, neural thresholds are elevated, neural tuning is broadened, and a sharp decrease in sensitivity is seen at the tip of the neural tuning curve. Thus, using Tecta(Y1870C/+) mice, we have genetically isolated a second major role for the tectorial membrane in hearing: it enables the motion of the basilar membrane to optimally drive the inner hair cells at their best frequency.

α-Tectorin (TECTA), β-tectorin (TECTB), and carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM) are secreted glycoproteins that are present in the tectorial membrane (TM), an extracellular structure overlying the hearing organ of the inner ear, the organ of Corti. Previous studies have shown that TECTA and TECTB are both required for formation of the striated-sheet matrix within which collagen fibrils of the TM are imbedded and that CEACAM16 interacts with TECTA. To learn more about the structural and functional significance of CEACAM16, we created a Ceacam16-null mutant mouse. In the absence of CEACAM16, TECTB levels are reduced, a clearly defined striated-sheet matrix does not develop, and Hensen's stripe, a prominent feature in the basal two-thirds of the TM in WT mice, is absent. CEACAM16 is also shown to interact with TECTB, indicating that it may stabilize interactions between TECTA and TECTB. Although brain-stem evoked responses and distortion product otoacoustic emissions are, for most frequencies, normal in young mice lacking CEACAM16, stimulus-frequency and transiently evoked emissions are larger. We also observed spontaneous otoacoustic emissions (SOAEs) in 70% of the homozygous mice. This incidence is remarkable considering that 15 kHz correlates with the loss of Hensen's stripe. Results from mice lacking CEACAM16 are consistent with the idea that the organ of Corti evolved to maximize the gain of the cochlear amplifier while preventing large oscillations. Changes in TM structure appear to influence the balance between energy generation and dissipation such that the system becomes unstable.

The vertebrate optic tectum is a functionally coupled bilateral structure which plays a major role in the generation of motor commands for orienting responses. However, the characteristics of the tectotectal connectivity are unknown in fish, and have been reported only to a limited extent in other vertebrates. The purpose of the present study was to determine the anatomical basis underlying the functional coupling between tecta in goldfish, and to identify both similarities and differences to those features reported in other vertebrate species. The present experiments used the bidirectional tracer biotinylated dextran amine to map the distribution of labeled cells and synaptic boutons in the contralateral tectum following injections into identified tectal sites. Fibers that interconnect both tecta coursed through the tectal commissure. The cells of origin of these fibers, the tectotectal cells, and their synaptic endings were located in the deep layers, mainly in the strata periventricular and griseum central, respectively. Corresponding sites throughout the two tecta were interconnected in a symmetrical point-to-point fashion. The tectal commissure was composed of at least two distinct bundles of axons, which differed in their dorsoventral location, fiber diameter, and projection targets. The dorsal axons were tectotectal axons, they were thinner in diameter and profusely branched, and gave off en passant and terminal boutons in the deep layers of the contralateral tectum. The ventral axons were thicker in diameter, and formed the contralateral tectofugal-descending tract. Such fibers had few axon collaterals and boutons in the contralateral tectum. Boutons adjacent to retrogradely labeled tectotectal cells were very scarce. The data are discussed in terms of the coupling between tecta generating the motor commands required for orienting movements.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.

Optic nerve activity helps determine the placement of retinal ganglion cell terminals in the optic tectum of the frog. We investigated whether the presence of this nerve might also influence a characteristic of its target structure, neurotransmitter biosynthesis. We performed unilateral optic nerve transections on adult animals and assayed the percent and intensity of substance P- and serotoninlike immunoreactive (SP-ir and 5-HT-ir, respectively) cells in the deafferented and afferented tectal lobes. Regeneration of the optic nerve was prevented. The percent of SP-ir cells in the afferented tectal lobes was significantly less than that in the deafferented ones either 6 weeks or 5 months following optic nerve lesion. Comparison to normal animals indicated that the change in SP-ir expression was due to a decrease in the percent of immunoreactive cells in the afferented tecta ipsilateral to the optic nerve lesion. The serotoninlike immunoreactivity of tectal cells was also significantly different in the two lobes following optic nerve lesions. This difference resulted from an increase in the percent of 5-HT-ir cells in the deafferented tectum. In addition, the intensity of 5-HT-ir cells in the deafferented lobe was significantly greater than in the afferented one. The staining intensity of SP-ir cells underwent only a transient, relative decrease in the deafferented tectum. We conclude that the optic nerve does regulate substance P and serotonin expression in the tectum, but that this regulation likely occurs through different pathways.

1. The optic tectum of the barn owl contains a physiological map of interaural level difference (ILD) that underlies, in part, its map of auditory space. Monaural occlusion shifts the range of ILDs experienced by an animal and alters the correspondence of ILDs with source locations. Chronic monaural occlusion during development induces an adaptive shift in the tectal ILD map that compensates for the effects of the earplug. The data presented in this study indicate that one site of plasticity underlying this adaptive adjustment is in the posterior division of the ventral nucleus of the lateral lemniscus (VLVp), the first site of ILD comparison in the auditory pathway. 2. Single and multiple unit sites were recorded in the optic tecta and VLVps of ketamine-anesthetized owls. The owls were raised from 4 wk of age with one ear occluded with an earplug. Auditory testing, using digitally synthesized dichotic stimuli, was carried out 8-16 wk later with the earplug removed. The adaptive adjustment in ILD coding in each bird was quantified as the shift from normal ILD tuning measured in the optic tectum. Evidence of adaptive adjustment in the VLVp was based on statistical differences between the VLVp's ipsilateral and contralateral to the occluded ear in the sensitivity of units to excitatory-ear and inhibitory-ear stimulation. 3. The balance of excitatory to inhibitory influences on VLVp units was shifted in the adaptive direction in six out of eight owls. In three of these owls, adaptive differences in inhibition, but not in excitation, were found. For this group of owls, the patterns of response properties across the two VLVps can only be accounted for by plasticity in the VLVp. For the other three owls, the possibility that the difference between the two VLVps resulted from damage to one of the VLVps could not be eliminated, and for one of these, plasticity at a more peripheral site (in the cochlea or cochlear nucleus) could also explain the data. In the remaining two owls, there was no evidence of adaptive adjustment in the VLVp despite large adaptive adjustments in the optic tectum. 4. The adjustment of ILD coding in the VLVp was always substantially smaller than expected based on the adjustment of ILD tuning in the optic tectum measured in the same animals. This indicates the involvement of at least one additional site of adaptive plasticity in the auditory pathway above the level of the VLVp.(ABSTRACT TRUNCATED AT 400 WORDS)

The nature of the expansion of the visual field projection was studied in goldfish in which size disparities were created between the retina and the tectum. After removal of one-half of the retina, the remaining retinal ganglion cells expand their projections so that the entire contralateral optic tectum is encompassed (Schmidt et al.1978). We wished to determine whether this expansion is accompanied by increased arborization including proliferation of synaptic terminals by the spared retinal ganglion axons or whether field expansion is accomplished by increased arborization without changes in synaptic number. Portions of the retina were ablated and the animals were allowed to survive for at least 5 months, the time at which expansion can be demonstrated, before sacrifice. We mapped retinotectal projections to determine the extent of the expanded visual fields and used stereological and morphometric analyses of synaptic contacts in the retinal target lamina, the stratum fibrosum et griseum superficialis (SFGS), in the optic tectum to estimate synaptic number. Numbers of synaptic terminals in the SFGS contralateral to the lesioned retina were not different from numbers in the comparable portion of control tecta. These observations indicate that the surviving retinal axons increased the number of synaptic contacts on tectal target cells in response to removal of other retinal ganglion cells.

The tectorial membrane (TM) connects to the stereociliary bundles of outer hair cells (OHCs). Herein, we summarize key experimental data and modeling analyses that describe how biophysical alterations to these connections underlie hearing loss. The heterozygous C1509G mutation in alpha tectorin produces partial congenital hearing loss that progresses in humans. We engineered this mutation in mice, and histology revealed that the TM was shortened. DIC imaging of freshly‐dissected cochlea as well as imaging with optical coherence tomography indicated that the TM is malformed and only stimulates the first row of OHCs. Noise exposure produced acute threshold shifts that fully recovered in Tecta+/+ mice although there was some OHC loss within all three rows at the cochlear base. In contrast, threshold shifts only partially recovered in TectaC1509G/+ mice. This was associated with OHC loss more apically and nearly entirely within the first row. Young's modulus of the TM, measured using atomic force microscopy, ...

SUMMARY Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous TectaC1509G/+ mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both TectaC1509G/+ and TectaC1509G/C1509G mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

Recent observations have changed our understanding of tectorial membrane function. Transgenic mice have shown that the tectorial membrane is a structure that can influence the sensitivity and tuning properties of the cochlea in several ways. It ensures that the gain and timing of cochlear feedback are optimal; that the hair bundles of the inner hair cells are driven efficiently by the outer hair cells, and it may influence the extent to which different elements are coupled along the length of the cochlea.

The frog nucleus isthmi (homolog of the mammalian parabigeminal nucleus) is a visually responsive tegmental structure that is reciprocally connected with the ipsilateral optic tectum; cells in nucleus isthmi also project to the contralateral optic tectum. We investigated the location of the isthmotectal cells that project ipsilaterally and contralaterally using three retrograde fluorescent label solutions: Alexa Fluor 488 10,000 mw dextran conjugate; Rhodamine B isothiocyanate; and Nuclear Yellow. Dye solutions were pressure-injected into separate sites in the superficial optic tectum. Following a 6-day survival, brains were fixed, sectioned, and then photographed. Injection of the different labels at separate, discrete locations in the optic tectum result in retrograde filling of singly labeled clusters of cells in both the ipsilateral and contralateral nucleus isthmi. Generally, ipsilaterally projecting cells are dorsal to the contralaterally projecting cells, but there is a slight overlap between the two sets of cells. Nonetheless, when different retrograde labels are injected into opposite tecta, there is no indication that individual cells project to both tecta. The set of cells that project to the ipsilateral tectum and the set of cells that project to the contralateral tectum form a visuotopic map in a roughly vertical, transverse slab. Our results suggest that nucleus isthmi can be separated into two regions with cells in the dorsolateral portion projecting primarily to the ipsilateral optic tectum and cells in the ventrolateral nucleus isthmi projecting primarily to the contralateral optic tectum. J. Comp. Neurol. 502:38–54, 2007. © 2007 Wiley-Liss, Inc.

Contains fulltext : 53245.pdf (Publisher’s version ) (Closed access) In DFNA8/12, an autosomal dominantly inherited type of nonsyndromic hearing impairment, the TECTA gene mutation causes a defect in the structure of the tectorial membrane in the inner ear. Because DFNA8/12 affects the tectorial membrane, patients with DFNA8/12 may show specific audiometric characteristics. In this study, five selected members of a Dutch DFNA8/12 family with a TECTA sensorineural hearing impairment were evaluated with pure-tone audiometry, loudness scaling, speech perception in quiet and noise, difference limen for frequency, acoustic reflexes, otoacoustic emissions, and gap detection. Four out of five subjects showed an elevation of pure-tone thresholds, acoustic reflex thresholds, and loudness discomfort levels. Loudness growth curves are parallel to those found in normal-hearing individuals. Suprathreshold measures such as difference limen for frequency modulated pure tones, gap detection, and particularly speech perception in noise are within the normal range. Distortion otoacoustic emissions are present at the higher stimulus level. These results are similar to those previously obtained from a Dutch DFNA13 family with midfrequency sensorineural hearing impairment. It seems that a defect in the tectorial membrane results primarily in an attenuation of sound, whereas suprathreshold measures, such as otoacoustic emissions and speech perception in noise, are preserved rather well. The main effect of the defects is a shift in the operation point of the outer hair cells with near intact functioning at high levels. As most test results reflect those found in middle-ear conductive loss in both families, the sensorineural hearing impairment may be characterized as a cochlear conductive hearing impairment.

Tecta, Coch, Eya4 and Strc are mouse orthologs of four human deafness-associated genes. Their expression is markedly restricted to specific cell types in cochleae. Cochleae were dissected on embryonic day 15 and cultured in vitro. Relative messenger RNA abundance of each gene was quantified by RT-PCR and compared in-vivo cochleae of equivalent embryonic age. After 48 h in culture, in-vivo and explant Strc expression levels were equivalent, Eya4 level reduced in explanted tissues, and expression of Tecta and Coch did not show the expected temporal rise. Expression of these genes was detectable even after 96 h. These results suggest that it is feasible to test the expression of inner ear specific genes in explanted cochleae.

Mature chick optic tecta consist of 16 laminae and receive retinal fiber projections in a precise retinotopic manner. Retinal axons arborize in laminae a-f of the SGFS, but do not cross the border between lamina f and g. In order to elucidate molecular mechanisms of tectal laminar formation, we first looked at the migration of tectal postmitotic cells. We found that the migration pattern of postmitotic cells changes around E5 and that late migratory cells intervened laminae that were formed by early migratory cells. The coincident appearance of Grg4 expression in the tectal ventricular layer and the change in migration pattern suggested an important role for Grg4. Clonal misexpression of Grg4 resulted in cells migrating to laminae h-j of the SGFS. Massive misexpression of Grg4 resulted in disruption of laminae that were formed by early migratory cells, in particular lamina g of the SGFS. Application of Grg4 morpholino antisense oligonucleotide or the misexpression of a dominant-negative form of Grg4 exerted the opposite effect. We concluded that Grg4 may direct tectal postmitotic cells to follow a late migratory pathway.

The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. Tecta(C1509G) transgenic mice have hearing loss, and surprisingly have increased OHC prestin levels. We hypothesized, therefore, that prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9-12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional prestin levels. We then measured prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR studies demonstrated increased prestin expression after noise exposure. Finally, we examined the effect of the prestin increase in vivo following noise damage. Immediately after noise exposure, ABR and DPOAE thresholds were elevated by 30-40 dB. While most of the temporary threshold shifts recovered within 3 days, there were additional improvements over the next month. However, DPOAE magnitudes, basilar membrane vibration, and CAP tuning curve measurements from the 9-12 kHz cochlear region demonstrated no differences between noise-exposed mice and control mice. Taken together, these data indicate that prestin is up-regulated by 32-58% in residual OHCs after noise exposure and that the prestin is functional. These findings are consistent with the notion that prestin increases in an attempt to partially compensate for reduced force production because of missing OHCs. However, in regions where there is no OHC loss, the cochlea is able to compensate for the excess prestin in order to maintain stable auditory thresholds and frequency discrimination.

The barn owl's optic tectum contains a map of auditory space that is based, in part, on a map of interaural time difference (ITD). Previous studies have shown that this ITD map is shaped by auditory experience. In this study, we investigated whether the plasticity responsible for experience-induced changes in ITD tuning in the tectum occurs within the tectum itself or at an earlier stage in the auditory pathway.We altered auditory experience in young owls by implanting an acoustic filtering device in one ear that caused frequency-dependent changes in sound timing and level. We analyzed the representation of ITD in normal and device-reared owls in two nuclei in the ascending pathway: the external nucleus of the inferior colliculus (ICX), the primary source of ascending auditory input to the tectum, and the lateral shell of the central nucleus of the inferior colliculus (ICCls), the primary source of input to the ICX. In the ICX, device rearing caused adaptive, frequency-dependent changes in ITD tuning, as well as changes in frequency tuning. These changes in tuning were similar to changes that occurred in the optic tectum in the same owls. In contrast, in the ICCls, tuning for ITD and frequency was unaffected by device rearing. The data indicate that plasticity at the level of the ICX is largely responsible for the adaptive adjustments in ITD tuning and frequency tuning that are observed in the optic tecta of owls raised with abnormal auditory experience.

Recent advances in the study of the developmental processes that direct afferent axons toward their final destinations are beginning to elucidate the cellular and molecular bases for the early development of retinotectal topography. The present review focuses on two guidance components (repulsive and adhesive) and their cellular localization as revealed by a newly established retinotectal co-culture system. These co-cultures enable general outgrowth patterns to be examined when retinal ganglion cell (RGC) axons attempt to traverse cells dissociated from their target nuclei. Additionally, the recording of RGC growth cone responses to single living target cells allows a cellular localization of guidance components that is not possible by other methods. This co-culture system, therefore, complements the repertoire of cell-culture techniques used to investigate the sequence of cellular events that underlie the establishment of retinotopic development. With this new approach, time-lapse micrographs were collected when RGC growth cones from temporal or nasal regions of embryonic chick retinae encountered individual cells dissociated from optic tecta. Temporal RGC growth cones collapsed and retracted with a high probability from neuronal cells dissociated from posterior tecta, indicating that repellent components were enriched on posterior target neurons. The response to non-neuronal cells revealed a separate effect on axonal outgrowth that was less dependent upon the particular region of retina or tectum from which the cells originated: most RGC axons adhered to the edge of non-neuronal cells, without retracting. Together, the localization of repellent and adhesive components suggests a sequence of events that occurs during the early stages of tectal innervation and that results in the rudiments of retinotopic projection, and furthermore, it raises a number of experimentally approachable questions concerning the functional expression of several guidance components.

The optic tectum of Xenopus offers a readily manipulated system for testing the hypothesis that polysialylation of the neural cell adhesion molecule is associated with axonal plasticity. Axons relaying input to the tectum from the ipsilateral eye employ visual input to establish a topographic map in register with the contralateral map, despite naturally-occurring or surgically-induced repositioning of the eyes. This capacity for activity-dependent refinement or re-organization of the ipsilateral map is normally confined to a period between about one and four months postmetamorphosis but can be restored in adults by local application of N-methyl-D aspartate to the tectum. In addition, dark-rearing prolongs plasticity indefinitely. We have used immunohistochemical staining with antibodies to polysialic acid to determine whether conditions of high plasticity are correlated with high levels of polysialylated neural cell adhesion molecule in the tectum. We find that the staining level is high in tecta from one to three month postmetamorphic frogs but is low both before and after this period. Thus, in normal Xenopus frogs, anti-polysialic acid staining is heavier in the period of high plasticity than in the preceding or following postmetamorphic periods. As a further test of this relationship, we examined brains of adults with experimentally-induced plasticity. Tecta of N-methyl-D-aspartate-treated adults and of dark-reared adults showed higher levels of staining than did the tecta of normally-reared adults. These results also support the hypothesis that the presence of high levels of polysialic acid on neural cell adhesion molecules is causally related to activity-related changes in axonal growth patterns.

In the visual pathway of frogs it is possible to apply low levels of NMDA chronically to the optic tectum and study the mechanisms underlying the stabilization of synapses developing within the CNS. Earlier studies (Cline and Constantine-Paton, 1990) found that chronic NMDA treatment of tecta innervated by two retinas results in a reduction of branching within the terminal arbors of retinal ganglion cells (RGCs). We now report that this same chronic NMDA treatment produces fine-structural changes in synaptic morphology as well as local synaptic rearrangements within the retinotectal neuropil. Chronic NMDA treatment of doubly innervated tecta was associated with a thickening or darkening of both pre- and postsynaptic densities. These changes in synapse morphology were restricted to the superficial neuropil of tecta in regions where reductions in branches of RGC axonal arbors were observed at the light microscopic level. The fine- structural effects were absent from similarly treated tecta innervated by only one eye, where RGC axonal arbor pruning was not observed. Stereological analyses indicated that the incidence of two or more presynaptic profiles converging on the same postsynaptic process was significantly increased in the NMDA-treated, doubly innervated tecta. This observed increase in synaptic clustering was not associated with a larger synaptic active zone, or with an increase in the number of synapses per unit volume. These data are discussed in the context of the hypothesis that chronic NMDA treatment raises the threshold for synapse stabilization in tectal neurons, causing the selective loss of poorly correlated synapses of both retinal and non-retinal origin from tectal neuropil that is innervated by two retinas: increased pre- and postsynaptic thickening could reflect greater efficiency in the remaining synaptic contacts and their closer spatial proximity on the same postsynaptic process is consistent with greater cooperativity and less competition.

The optic tectum of the dogfish Scyliorhinus canicula L. was studied by using the methods of Nissl, reduced silver nitrate, Golgi-aldehyde, and Golgi-Cox. Six layers and eight types of neurons were recognized. These cell types are not restricted to one layer; in fact some are found in all six tectal layers. The types of cells found are A) monopolar, B) triangular, C) radial bipolar, D) horizontal fusiform, E) large tectal, F) small tectal, G) pyriform, and H) stellate cells. In at least six of the cell types a series of dendritic specializations can be observed, such as spines in the form of "drumsticks" and thin varicose appendages, similar to those reported previously in the optic tecta of amphibians and teleosts. The optic tectum of the dogfish shows a degree of complexity comparable to that of amphibians and teleosts.

Optic axons in the goldfish retina generally run radially from their origins to the optic nerve. However, horseradish peroxidase (HRP), introduced into small groups of axons through lesions in the tecta of normal fish, consistently filled some which ran parallel to the retinal margin for long distances before turning centrally. Since all the HRP-filled ganglion cells lay close together, their axons evidently reached adjacent tectal sites by these widely divergent routes.

The temporal, nasal or dorsal half (40–60%) of the eye primordium was excised in Xenopus frog embryos stage 25/26 (optic vesicle), 31/32 (optic cup) or 38 ± 1 (early larval eye). The residual nasal (NF) or temporal (TF) or ventral (VF) fragment gradually rounded-up to form a grossly normal eye of normal or reduced size. Electrophysiologic analysis of the visuotectal projection after metamorphosis showed that (independent of stage of surgery and final eye size) most eyes of each type generated normal retinotectal maps (orderly and continuous retinotopic projection across the entire tectum). A minority of NF eyes generated double-nasal compound maps, in which nasal and temporal halves of the adult retina projected as mirror images (of the nasal pattern) over the whole tectum. A minority of TF eyes generated double-temporal compound maps, while a minority of VF eyes generated double-ventral compound maps. Respectively, these compound maps simulated those generated by NN, TT or VV “compound-eyes” prepared by fusing two nasal half eyes, or two temporal half eyes, or two ventral half eyes. The incidence of double-maps was lowest in TF eyes at all stages, and after stage 25/26 surgery in all eye types. Lure tests and post-synaptic recording confirmed that these eyes formed functional retinotectal synapses, and that the (presynaptic) map accurately mirrored the connectivity pattern. The results are considered in terms of (1) the patterning of locus specificities in the retinal ganglion cell population, (2) embryonic “regulation” and “reduplication” in non-neural systems, and (3) alternative mechanisms by which partial extirpation might modify the deployment or expression of ganglion cell locus specificities to produce the retinotectal connectivity patterns we observed.

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ipsilaterally into the remaining tectal lobe. Initially fibers from the two eyes overlap in the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsilateral eye and tectum at this time. At longer time periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a significant number of synapses from the ipsilateral eye are present. These findings have important implications for how eye-specific termination bands are formed in doubly innervated tecta.

Segregated zones of termination between converging inputs that arise from different presynaptic populations are a common property of topographically organized zones within the vertebrate central nervous system. Increasing evidence suggests that such segregation is at least in part established on the basis of competitive interactions that depend upon the activity patterns within each afferent population. However, the cellular mechanisms of these interactions are poorly understood. We have used a preparation in which a stereotyped interdigitating pattern of retina-specific termination stripes are produced in frog tecta innervated by two retinas as a result of embryonic implantation of a third eye primordia. In these animals it has been possible to examine the relationship between the number of retinal ganglion cells in each of the retinas innervating a striped tectum, the volumetric changes in the tectum as a result of this double innervation, and the pattern of eye-specific segregation that is produced. Counts of retinal ganglion cells in the retinas of the three-eyed frogs with one completely striped tectal lobe revealed no significant differences between cell numbers in the doubly innervating retinas and the normal retinas of the same animals. The average increase in retinal ganglion cell innervation to the striped tecta of these animals was 100%. However the tecta only increased in total volume by 26%. This later increase consisted of a 25% increase in the volume of the deep lying and predominantly cellular tectal laminae and a 37% increase in the superficial retinotectal synaptic zone. In many of these same animals HRP and 3H-proline were used to differentially label the set of stripes from each retina and measurements of the extent of each projection were performed. We found that the volume of tectal neuropil occupied by a striped projection is relatively unrelated to the number of ganglion cells making up that projection. Observations of the striping pattern after HRP processing to visualize stripes in whole unsectioned tecta indicate that the periodicities and rostrocaudal orientation of stripes are robust over a wide range of relative innervation densities. When one projection is much smaller than the other, stripes appear to break down into a series of "puffs" or islands of retina-specific termination zones. Nevertheless, these puffs still have a rostrocaudal alignment and the spacing of fully formed stripes. These observations suggest that the formation of exclusive termination zones may be a threshold phenomenon: so after a certain innervation density is reached one input can take over a unit of target neuropil in an all-or-none manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Tectal anatomy and physiology of the blind cave characin, Astyanax hubbsi , have been compared with that of its sighted ancestor Astyanax mexicanus (the river fish) and with goldfish. Normal and experimental neuroanatomic methods have revealed that, with the exception of a greatly reduced retinotectal projection, connectivity and structure of cave fish tecta are similar to those described in sighted species. It appears that the rudimentary retinotectal input is nonfunctional, since no tectal evoked responses could be elicited with electrical or visual stimulation of the optic cyst, and all attempts to visually condition cave fish were unsuccessful. Attempts have also been made to record somatosensory, auditory and lateral line activity in the tecta of the blind and sighted fish. A sparse somatic representation was found in the deeper portion of the sighted fish tecta which contrasts with a dense, well-organized one in the cave fish. No tectal responses were found to auditory or lateral line stimuli. CNS plasticity is discussed in relation to studies of fish, amphibians, reptiles, birds and mammals, in which a reduction of sensory input by any one of a number of means has resulted in alterations of structure and function.

Retinal pieces taken from known positions of 6-day chick embryos were vitally labeled with the fluorescent dye Rhodamine-B-isothiocyanate (RITC). They were then transferred onto the surfaces of optic tecta following early bilateral removal of the embryo's optic vesicles. One to 5 days after transplantation the tecta were fixed and transplants that issued fibers were examined on tectal wholemounts or were sectioned and viewed with a fluorescence microscope. Retinal fibers growing out from transplants on day E6 tecta showed a capacity for changing their initial outgrowth directions and for reorienting themselves towards their specific retinotopic projection area. Frequently, changes in growth direction appeared in a right-angled pattern. The capacity for turning was strongest for fibers of nasal retinal origin, less strong for fibers of temporal origin, and occurred rarely but unquantifiably in the case of fibers of ventral retinal origin. Fibers of all investigated retinal quadrants were found to reach their corresponding projection areas and to arborize there, that is, fibers of nasal retinal transplants in the posterior tectum, of temporal transplants in the anterior tectum, and of ventral transplants in the dorsal tectum. Furthermore, once in their target region, the fibers left the outer layer of the tectum and turned, again in right angles, to invade deeper layers. Capacity of fibers to turn towards their projection area was not observed for fibers issued from transplants placed on the tectum later than day E8. We suggest that there is a specific guidance of retinal axons on the tectum.

Optic fibre regeneration in young Xenopus was studied by [ 3 H]proline autoradiography and by mapping the visuotectal projection electrophysiologically from 5 to 210 days after sectioning the right optic nerve. Regenerated optic fibers were demonstrated morphologically both in the contra and ipsilateral tecta. In contrast, electrophysiological studies showed that the visuotectal projection was detectable only in the contralateral tectum. The apparent disparity between morphological and electrophysiological results is discussed.

Right double nasal (NN) and left double temporal (TT) compound eyes were formed in the same animal by the fusion of two similar halves at early embryonic stages in Xenopus. After metamorphosis either the right or left optic nerve was sectioned to induce optic fiber regeneration from one of the two eyes to both tecta. Retinal projections from each eye to the monocularly and dually innervated tecta were later investigated by visuotectal mapping and by [3H]proline autoradiography. Visuotectal and autoradiographic maps from NN and TT eyes showed the restoration of typical compound eye projections where each of the hemiretinal projections extended across the entire contralateral (monocularly innervated) tectum. In contrast, the tectum ipsilateral to the optic nerve section was dually innervated by fibers from the TT and NN eyes. The rostral and caudal poles of the tectum were innervated by fibers from the TT and NN eyes respectively, whereas the middle third received partially overlapping projections from both eyes. Thus the full expansion of the hemiretinal projection across the dually innervated tectum was prevented by fiber-fiber interactions between optic fibers from the TT and NN eyes. It is concluded that tectal polarity cues determine the proper orientation, and fiber-fiber interaction the extent and orderliness of the retinotectal map. The formation of the latter two features of the retinotectal map does not require the existence of independent tectal positional markers.

An assay is described that can measure the rates of adhesion of isotopically labeled cell bodies from either the dorsal or ventral regions of the chick embryo retina to dorsal and ventral tectal halves. Immediately after dissociation of the retina, both ventral and dorsal retinal cells adhere preferentially to ventral tectal halves. With increasing time after dissociation, however, the preference of the ventral retinal cells shifts to dorsal tectal halves. The dorsal retinal cells continue to show a specificity for ventral tectal halves regardless of the length of time after their dissociation. When the developmental age of the tectal halves is varied from 8–14 days, there is no change in the specificities shown by retinal cells toward these tecta. When retinal age is varied, ventral retinal cells do not adhere preferentially to dorsal tecta until day 6 and older. Dorsal retinas show their specificity toward ventral tecta as early as day three. Control experiments include the use of nonretinal tissues, noninnervated tectal halves and pigmented retinal cells.

Rapid significant changes in tectal receptor populations occur one day after enucleation. A predominant increase of serotonin receptor binding in the denervated tecta was observed when compared to control tecta. This change seemed to be due to an increase exposure receptor sites rather than to any alterations of the binding affinity.

Right compound eyes were formed in Xenopus embryos at stages 32–33 by the fusion of two nasal (NN), two ventral (VV) or two temporal (TT) halves. Shortly after metamorphosis the optic nerve from the compound eye was sectioned and the left intact eye removed. The retinotectal projections from the compound eye to the contralateral and ipsilateral tecta were studied by [3H]proline autoradiography and electrophysiological mapping between 6 weeks and 5 months after the postmetamorphic surgery. The results showed that NN and VV eyes projected to the entire extent of both tecta. In contrast, optic fibre projection from TT eyes, although more extensive than the normal temporal hemiretinal projection, failed to cover the caudomedial portion of the tecta. The visuotectal projections in all three combinations corresponded to typical reduplicated maps to be expected from such compound eyes, where each of the hemiretinae projected across the contralateral and ipsilateral tecta in an overlapping fashion. The rapid expansion of the hemiretinal projections of the compound eyes in the ipsilateral tectum following the removal of the resident optic fibre projection suggests that tectal markers may be carried and deployed by the incoming optic fibres themselves.

In Limnodynastes dorsalis neurogenesis of the optic tecta and the pattern of cellular lamination was determined by [3H]thymidine autoradiography. There was a rostral to caudal gradient of cell proliferation with peak neurogenesis in mid-larval life and by metamorphosis generation was complete. The cellular layers formed as each portion of tectum was generated. As evidenced by comparison of animals killed within 24 h of [3H]thymidine injection and those injected as larvae and killed at more mature stages, cells were generated in the ependymal and peri-ependymal layers and during development they migrated to form the peripheral layers. During late larval life, the soma diameter of cells in the most superficial layers, and the width of these layers increased. Tectal innervation during development was assessed by tracing axonal trajectories with horseradish peroxidase. Innervation recapitulated tectal growth with more caudal areas being progressively encompassed by primary optic fibres. By metamorphosis, the entire tectum was innervated. These results indicate that tectal generation, cellular lamination and innervation by optic fibres have a rostral to caudal gradient of development which is complete by metamorphic climax. Therefore to accommodate postmetamorphic retinal growth the developing retinotectal projection must remain labile beyond metamorphosis.

The projection of the binocular visual field on the optic tecta of Rana temporaria has been mapped by recording action potentials evoked in the tectum by a spot of light in the visual field. The binocular visual field extends for about 100° on the horizontal meridian. Only the central region of the binocular field is represented through both eyes on both ipsilateral and contralateral tecta. The binocular field to the left and right of this central region is represented through both eyes on one tectum only: the left side of the field projects through both eyes to the right optic tectum, while the right side of the field projects through both eyes to the left tectum. Corresponding retinal points project to the same tectal locus.

Xenopus with one double-nasal (NN), double-temporal (TT) or double-ventral (VV) eye were induced to regenerate optic fibres from the compound eye to both tecta after metamorphosis. The extent of the projection from the compound eye was then estimated autoradiographically at various intervals after nerve section. The regenerated projection covered the whole of the contralateral tectum but covered only a restricted part of the ipsilateral tectum; thus NN eyes innervated caudomedial tectum only, TT eyes innervated rostrolateral tectum only and VV eyes innervated medial tectum only. Since the tectum ipsilateral to the compound eye also received the projection from the normal eye these results are taken to indicate that NN, TT and VV eyes are unregulated systems in terms of the retinal positional markers that they carry; each such eye bears only the appropriate half-set of such markers.

Regenerating optic axons of goldfish were loaded with [3H]RNA by injecting [3H]uridine into the eye and allowing time for the radioactivity to be delivered to the optic tectum. The axons were subsequently removed from the tecta by cutting the optic nerve and allowing the optic axons in the tectum to degenerate. Analysis of tectal [3H]RNA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a selective loss of tritiated 4S RNA and not ribosomal RNA from the denervated tecta. These results support the hypothesis that regenerating optic axons of goldfish grow back into the tectum carrying 4C but not ribosomal RNA.

The retinotectal connections formed in adult goldfish after removal of the rostro-caudal strip of dorsal tectum were mapped electrophysiologically. Initial maps recorded 1 month after surgery revealed visual scotoma corresponding to the ablated tectum. Subsequent maps recorded 4-5 months after the operation showed that the entire visual field redistributed appropriately over the remaining tectum. It is suggested that, in spite of an abnormal optic tectum, the reformed projection demonstrates a tendency to retain its completeness.

Monosialogangliosides were extracted from the retinas and optic tecta of chick embryos at 6–12 days of incubation, and analyzed by high-performance liquid chromatography. The dominant monosialoganglioside was found to be N-acetyl-galactosaminyl (N-acetylneuraminyl) galactosylglucosylceramide (GM2). The specific concentration of GM2 in both the retina and tectum rose to a peak at 8–9 days of incubation, then declined. At day 10, GM2 was significantly lower in the dorsal than in the ventral or temporal quadrants of the retina. The same trend to a lesser degree was seen in the tectum. These results demonstrate striking variations in the monosialoganglioside content of the retina and tectum during formation of the retinotectal projection, and suggest that topological gradients may be a consequence of these developmental and spatial variations.