Current main topics of our research:
The neural basis of sound localisation
[Supported by the CRC/TRR 31 "The active auditory system"]
Localisation of stimuli is an essential task of sensory systems but the localisation of sound is less straightforward than for most other senses. Unlike, e.g., the retina of the eye, the auditory receptor organ, the cochlea, contains no map-like representation of stimulus location. Sound location needs to be computed and this task is carried out by the central auditory pathways in the brain.
Animals and humans use the minute differences in the timing and intensity of the acoustic input to both ears: When sound comes from one side of the body, it reaches one ear before the other and it is louder in that ear. These so-called interaural differences are determined by the brain by comparing the inputs from both ears and are used to synthesise a neural map of auditory space.
Many steps are involved in this and the individual stages of neural processing are quite well known for the derivation of interaural time differences. Research on barn owls has contributed much to our understanding of temporal processing in the auditory system. Data from small mammals, however, have suggested an alternative mechanism for coding interaural time differences.
We are recording from the relevant brainstem centre (Nucleus laminaris) in chickens and barn owls to learn more about the neural coding of interaural time differences across frequencies and across species.
The black structure in this microscope image is a single dye-labelled nerve cell in the chicken's brainstem. This particular neurone responded best when sound in the left ear was leading the same sound in the right ear by 60 µs. For the chicken, this means a sound originating from about 30° to its left. Neighbouring neurones responded best to other time differences, together they form a map of horizontal auditory space.
But what does the chicken hear exactly?
During electrophysiological recordings, stimuli are played through headphones which allows testing of arbitrarily small or large interaural time difference. Another aspect of our ongoing study of sound localization in birds is thus to clarify exactly what interaural time differences are experienced by chickens naturally - not as easy as it sounds! Unlike mammals, birds have their middle ears connected by open Eustachian tubes and airspaces in the skull bones. This has the interesting consequence that sound waves can reach their eardrums from both outside and inside. This in turn may change the exact timing of sound input to each ear and thus the perceived interaural time difference. We are measuring cochlear potentials and eardrum vibration (in collaboration with colleagues in Denmark) to determine exactly what the chicken hears.
Schematic illustration of sound paths to a bird's eardrums. Instead of just travelling around the head (blue arrows), there are additional paths entering through the contralateral eardrum and passing through the head (green arrows) - literally in one ear and out the other! The net eardrum vibration (red waveforms) is the sum of the sound pressure impinging on the outside (blue curves) and the inside (green curves) of the eardrum. Note the slight but significant difference this makes to the timing.
Mechanisms of ultrafast temporal processing in the inner ear
Hearing is our fastest sense. In particular, the resolution of temporal processing used in measuring interaural time differences for localising sound sources in space is amazing. The barn owl, a nocturnal bird of prey, is a well-known example for the extreme performance of a basic mechanism that is used by many animals, including humans. Via neural phase locking, the afferent nerve fibres leaving the owl's cochlea for the brainstem encode the temporal occurrence of a stimulus with a precision of about 30µs.
Temporal dispersion (or jitter) of phase coding in the auditory nerve as a function of stimulus frequency. Inset shows schematically how spike timing is measured. The coloured curves show median values for large samples of single-unit data in different species of birds and mammals. Note the scaling in microseconds - this degree of temporal precision is an amazing feat of the auditory system! The barn owl (red curve) holds the record, with temporal dispersions below 30µs.
How are ribbon synapses involved in that?
Although well documented, it is still unknown how that kind of temporal precision is achieved. Specialisations both at the level of the hair cells and the afferent neurones are probably necessary. The specialised ribbon synapses between a hair cell and its afferent fibres are commonly assumed to play a crucial role. We are currently comparing numbers of ribbons and expression levels of salient proteins between hair cells in the chicken, an average bird, and the barn owl, an auditory specialist.
Cross-section of a chicken cochlea at high magnification under the light microscope, focussing through successive layers in depth. Several sensory hair cells are labelled blue, their cell nuclei in a pale red-green shade. Ribbon synapses at the bases of the hair cells are labelled by antibodies against synaptic proteins and appear as little, bright red and green dots.
Development of the inner ear
Vertebrate inner ears consist of several sensory organs (e.g. 5-6 vestibular organs in mammals and 7 in birds, respectively, in addition to the auditory organ). All develop from specified regions of the common embryonic otocyst (see diagram). The dorsal ear comprises the vestibular apparatus. Ventrally, the cochlear duct of birds and mammals houses the auditory organ and the vestibular lagena macula (although most mammals lack the latter).
Morphogenesis from the otocyst through to the 3-dimensional labyrinth of semicircular canals and the cochlear duct that houses the auditory sensory organ.
Inner ear development involves multiple processes from sensory fate specification to cell differentiation, axon guidance, synaptogenesis, and specialization of mechanosensory cells with individual physiological and morphological properties. These processes are all under tight genetic control.
In chicken embryos, a cartilaginous otic capsule (Alcian blue staining) surrounds the developing cochlear duct, which houses two sensory epithelia (brown), the auditory basilar papilla and the vestibular lagenar macula. Sensory hair cells labeled with anti-HCA antibody (brown); embryonic day 10, transverse section.
Although information has recently accumulated rapidly, our picture of the molecular network controlling these processes is broadly incomplete and in many cases we don't know the role of interacting signaling pathways or genes. Thus, investigation of gene function is at the heart of our interest.
Gene expression - Mapping of gene expression patterns at different stages of inner ear development
Exploration of the genes that are active during inner ear development is our first approach (method: RT-PCR), followed by detailed studies of their spatial and temporal distribution.
We perform systematic mapping of the expression patterns of a large catalogue of genes (such as members of the Wnt gene family) and document their expression on serial sections at different embryonic stages (method: in-situ hybridization).
Such studies allow us to pinpoint putative key players in particular developmental events and select these genes for further investigation and functional analyses.
Wnts are an ancient class of signaling molecules that play many varied roles in development and disease. These secreted cysteine-rich glycoproteins have at least three major intracellular signaling pathways (beta-catenin pathway, calcium pathway, planar cell polarity pathway). A plethora of Wnt-related genes has been shown to accompany many steps of the formation of the inner ear in chickens (Sienknecht and Fekete, 2008, 2009). Wnt pathways are hypothesized to play a role in numerous distinct aspects of inner ear development, such as:
Specifying sensory organ fates
Controlling growth and morphogenesis
Cell proliferation, differentiation and programmed cell death
Axon guidance, and synaptogenesis
Polarity of sensory hair cell stereovillar bundles, the mechanoreceptive apparatus
Complementary expression of two Wnt receptors (Frizzled, Fz) in the developing auditory epithelium. The micrographs show co-labeling of mRNA (blue) by in situ hybridization (probed for Fz9 and Fz10, respectively) and neurofilament- associated antigen (brown); chicken, embryonic day 10, transversal sections.
Gene function - Planar cell polarity (PCP) formation during sensory organ development
One of the major developmental processes during the embryonic formation of mechanosensory epithelia is the development of planar cell polarity (PCP), i.e., the systematic orientation of cells within an epithelial sheet.
Our investigation of gene function is exemplified here by examining this developmental phenomenon in the bird's auditory epithelium.
Deflection of the sensory hair cell's stereovillar bundle along its mechanosensitive axis is the functional element for stimulus transduction, thus a precise orientation angle of the hair-cell bundle is crucial.
Developmental processes move and direct the differentiating hair-cell bundle to the periphery of the cell's surface and then lead to an adjustment of the proper orientation angle (see diagram).
Schematic diagram of the developmental steps that first define a rough polarity at the apical surface of young sensory cells (left) and, secondly, control the refinement of cell orientation in the auditory sheet that displays a characteristic pattern of cell polarity. This finally involves regional re-orientation of groups of cells that rotate their stereovillar bundles accordingly, the mechanoreceptive apparatus of sensory hair cells (right).
We are interested in the molecular mechanisms that control this kind of PCP achievement and seek to know which genes are involved and what their function is in this machinery.
Example of the critical involvement of the gene Vangl2 for proper sensory cell orientation in the auditory epithelium of the chicken (Sienknecht et al. 2011). Confocal microscopy images of whole-mount preparations of the sensory epithelium show hair cell stereovillar bundles labeled for f-actin with phalloidin (red). A) control, normal hair-cell bundle orientation. B) disturbed hair-cell bundle orientation after gene (Vangl2) misexpression.
In addition to the bird's inner ear, we follow up on comparative aspects as well. For example we investigate PCP development in lizards such as geckos. Lizard inner ears display an interesting hair-cell orientation pattern in their auditory epithelium, with facing groups of cells of mirrored orientation. Using comparative studies we aim to gain insights into common or derived mechanistic aspects of developmental genetic control systems.