Atomic force microscopic studies of inner ear structure and mechanics

The transduction processes occurring in the inner ear, from sound induced vibration or head movements to nerve impulses sent to the brain, are to a large part dependent on the mechanical properties of the different components of this organ, especially the sensory hair cells. The function of the hearing organ is for example dependent on the unique mechanoelectric transduction properties of the cochlear outer hair cells (OHCs), which are able to respond to electrophysiological and mechanical stimulations by changing their shape and their mechanical properties. The study of these cells requires the development of nanomechanics tools that are enough sensitive to probe the cells with high resolution. The aim of this thesis was to develop and apply atomic force microscopy (AFM) for the characterisation of structural and mechanical properties of different structures from the inner ear. The work was articulated in five different projects: (1) In a first study, a novel AFM technique was applied to non-biological materials. The interphase of composite materials was characterized with the modified Scanning Local Acceleration Microscopy (SLAM). Using this technique the "contact stiffness" between the tip and surface, related to the elastic modulus of the sample, was detected. (2) The morphology of otoconia crystals from the vestibular system was investigated with AFM in air. The nanostructure of crystals from both the saccula and utricle of the guinea pig were imaged and compared. The surface of single otoconia exhibited a dense packing of round units whose characteristic dimensions were analyzed. (3) In order to explore the role of the protein prestin in outer hair cell electromotility, the AFM was used in combination with the Patch Clamp technique. It was reported to augment voltage-dependent movement when expressed in HEK-293 cells. We measured the differences between control and transfected cells. The study showed that prestin transfected cells exhibited an electromotile response of similar magnitude but opposite polarity to control cells. This phase change was removed when intracellular C1- was substituted with F-. (4) By scanning cells over the surface and acquiring force curves as a function of lateral position (the so-called force volume mapping), we evaluated local elastic properties at different positions on the OHC membrane. (5) AFM was used to investigate the mechanical responses of isolated OHCs to indentation by the AFM tip. Indentation curves showed a break at the contact point, a feature characteristic of an indentation of a stiff membrane surrounding a softer elastic medium (core-shell organization of the cell). Further, we showed that the responses of the OHC lateral wall are highly nonhysteretic at deformation rates of more than 50 µm/s. This suggests OHCs are highly elastic structures with little viscous dissipation compared to other cell types. In summary, atomic force microscopy provides unique possibilities to investigate the morphology and biomechanics of structures from the inner ear, especially the sensory outer hair cells. This technique allows one to image the sample at nanometer resolution, both on dry preparations and in fluid (which is most important for the study of living cells). The findings on the OHCs highlight the importance of the mechanical properties of these cells for hearing, and have consequences for the very fast motility that these cells are believed to undergo in vivo

Activation of class I metabotropic glutamate receptors limits dendritic growth of Purkinje cells in organotypic slice cultures

The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. We showed previously that the blockade of glutamatergic excitatory neurotransmission has little effect on Purkinje cell dendritic development. We have now studied the effects of glutamate receptor agonists on the development of Purkinje cell dendrites in mouse organotypic slice cultures. The activation of N-methyl-D-aspartate receptors had no major effect on Purkinje cell dendrites and the activation of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptors was strongly excitotoxic so that no analysis of its effects on dendritic development was possible. The activation of metabotropic glutamate receptors led to a very strong inhibition of dendritic growth, resulting in Purkinje cells with very small stubby dendrites. This effect was specific for the activation of class I metabotropic glutamate receptors and could not be reduced by blocking synaptic transmission in the cultures, indicating that it was mediated by receptors present on Purkinje cells. Pharmacological experiments suggest that the signaling pathway involved does not require activation of phospholipase C or protein kinase C. The inhibition of dendritic growth by activation of class I metabotropic glutamate receptor could be a useful negative feedback mechanism for limiting the size of the dendritic tree of Purkinje cells after the establishment of a sufficient number of parallel fiber contacts. This developmental mechanism could protect Purkinje cells from excitotoxic death through excessive release of glutamate from an overload of parallel fiber contacts

Evidence for a Highly Elastic Shell-Core Organization of Cochlear Outer Hair Cells by Local Membrane Indentation

Cochlear outer hair cells (OHCs) are thought to play an essential role in the high sensitivity and sharp frequency selectivity of the hearing organ by generating forces that amplify the vibrations of this organ at frequencies up to several tens of kHz. This tuning process depends on the mechanical properties of the cochlear partition, which OHC activity has been proposed to modulate on a cycle-by-cycle basis. OHCs have a specialized shell-core ultrastructure believed to be important for the mechanics of these cells and for their unique electromotility properties. Here we use atomic force microscopy to investigate the mechanical properties of isolated living OHCs and to show that indentation mechanics of their membrane is consistent with a shell-core organization. Indentations of OHCs are also found to be highly nonhysteretic at deformation rates of more than 40 μm/s, which suggests the OHC lateral wall is a highly elastic structure, with little viscous dissipation, as would appear to be required in view of the very rapid changes in shape and mechanics OHCs are believed to undergo in vivo

The role of filaggrin mutations leading to a decrease in the amount of protein in the development of atopic dermatitis and bronchial asthma in children

Atopic diseases remain one of the most common childhood diseases. At the beginning of life, atopic dermatitis (AD) occurs, and only then bronchial asthma (BA). This staged development of sensitization and transformation of clinical manifestations is called the atopic march. Are the genetic factors of predisposition to AD the same for BA? There is still no definite answer to this question. Mutations in the filaggrin gene (FLG) are known to impair skin barrier function. Filaggrin is expressed not only in the skin, but also in the respiratory organs of the nasal mucosa, lungs, and bronchi. Filaggrin defects lead not only to disruption of the skin barrier, but also to an increase in the Th2 response and increased production of IgE, typical of bronchial asthma. Therefore, mutations in the FLG gene can be a risk factor for the development of not only AD, but also BA. The aim of this study was to compare the values of the association of mutations in the FLG gene with AD and BA in the Russian sample. Material and methods. Case-control study design. We used 265 blood samples from children. 4 mutations in the filaggrin gene were identified by real-time PCR. The association of mutations with disease was assessed by odds ratio. Results. We showed a strongly pronounced association of the deletion of 4 nucleotides (2282del4) with AD, but not with BA, although for patients with atopic BA the indicator of the association of this mutation with the disease was higher than for the group with symptoms of bronchial asthma identified by the ISAAC questionnaire. These results lead to the conclusion that the role of the filaggrin gene for BA is much less significant than for AD