Final Report Summary - TOUCH IN SITU (Mechanotransduction in situ)
The main research objective of this research project was to clarify the molecular mechanisms of mechanotransduction in sensory neurons by direct recording of mechano-activated ion channel activity from dorsal root ganglion (DRG) neurons innervating the skin. To overcome the technical constrains due to the distance between site of the transduction (skin) and the cell body of the sensory neuron (in DRG), we proposed to set up a novel electrophysiological preparation in which DRG neurons dissected from one mouse are confronted directly with the skin of a second mouse in vitro.
Therefore, the fellow with the collaboration of the Doctor of Philosophy (PhD) student Jan Walcher started to set up the best conditions to obtain a reproducible growth of the DRG neuron transplanted in the host mouse. We dissected the DRG neurons from adult mice and they are dissociated by enzymatic digestion with collagenase and trypsin and in order to visualise them after transplantation. Initial experiments were carried out to evaluate the ability of isolated sensory neurons to survive in mouse skin acutely in vitro. This approach turned out not to be fruitful as it was not possible to retrieve labelled neurons from the mouse skin even after a few hours of co-incubation.
Therefore, we decided to try to set up a more simple preparation. Since a subclass of skin mechanoreceptor innervated the hair follicles we established co-culture of mouse hair follicles with DRG neurons. These co-cultures proved to be viable as the many sensory neurons in vitro grow out to the hair folliocle and innervate the follicle which in theory allowed us to study the neuronal response after mechanical stimulation of the hair. Immunocytochemistry experiments demonstrated the ability of the DRG neurons to established physical contacts with hair follicles in cultures resembling the native anatomical structure occurring in the skin. Patch clamp recordings in whole configuration were performed on these co-cultured DRG neurons comparing the response upon mechanical stimulation of the hair innervated by the recorded neuron with that obtained by direct stimulation of the soma or neurites of the neuron. We did not observe significant differences in the properties of the mechano-gated current evoked by the two types of stimulation indicating that the transduction channel function does not depend on the interaction between the nerve ending and the hair follicles. Indeed we also carried out experiment on plasma-cleaned hair follicles (that are nbot living) and found that the same innervation by DRG neurons was achieved. This data indicated that a critical cellular factor was probably missing in such experiments and we now believe this may be the terminal Schwann cell.
Moreover the fellow started to address other aspects of the research proposal using cultured dissociated DRG neurons directed stimulated by a glass probe guided by a piezoelectric-controlled nanomotor. In particular he investigated the role of myosin VIIa in modulation of mechanosensitive channel in skin mechanoreceptors contributing to show that Ca2+ dependent activation of myosin VIIa is necessary to maintain the mechanosensitivity upon repetitive stimulation. Moreover, he started to clarify the molecular mechanisms by which STOML-3 controls the mechanosensitivity of DRG mechanorecepors. In particular he tested the effect of different small compounds selected by large screening as modulator of oligomerisation of STOML-3. He found that molecules inhibiting the self-association of STOML-3 are able to reduce the number of mechanosensitive DRG neurons and to alter the kinetics of mechano-gated current.
Project management
The management of the project is handled by administrative staff of the host Institute in connection the fellow and the scientist in charge. All experiments, including mice were carried out with the appropriate permission from local authorities.
The main research objective of this research project was to clarify the molecular mechanisms of mechanotransduction in sensory neurons by direct recording of mechano-activated ion channel activity from DRG neurons innervating the skin. To overcome the technical constrains due to the distance between site of the transduction (skin) and the cell body of the sensory neuron (in DRG), we proposed to set up a novel electrophysiological preparation in which DRG neurons dissected from a mouse are confronted with the skin of another mouse in vitro.
Many different experimental approaches were taken here but we had to conclude finally that the original technical approach was not actually feasible. We then adopted another approach in which a much simpler preparation was used. Since a subclass of skin mechanoreceptor innervated the hair follicles we established a co-culture of hair follicles with DRG neurons allowing us to study the neuronal response after mechanical stimulation of the hairs. Immunocytochemistry experiments have been demonstrated the ability of the DRG neurons to established physical contacts with hair follicles in cultures resembling the native anatomical structure occurring in the skin. Patch clamp recordings in whole configuration were performed on these co-cultured DRG neurons comparing the response upon mechanical stimulation of the hair innervated by the recorded neuron with that obtained by direct stimulation of the soma or neurites of the neuron. We did not observe significant differences in the properties of the mechano-gated current evoked by the two types of stimulation indicating that the transduction channel function does not depend on the interaction between the nerve ending and the hair follicles.
Therefore other aspects of the research proposal were addressed by using cultured dissociated DRG neurons directed stimulated by a glass probe guided by a piezoelectric-controlled nanomotor. In particular it has been investigated the role of myosin VIIa in modulation of mechanosensitive channel in skin mechanoreceptors contributing to show that Ca2+ dependent activation of myosin VIIa is necessary to maintain the mechanosensitivity upon repetitive stimulation. Moreover, experiments had been performed to clarify the molecular mechanisms by which STOML-3 controls the mechanosensitivity of DRG mechanorecepors. In particular, the effect of several compounds selected by large screening as modulator of oligomerisation of STOML-3 had been tested showing that molecules inhibiting the self-association of STOML-3 are able to reduce the number of mechanosensitive DRG neurons and to alter the kinectis of mechanogated current.
The data produced during this project increases knowledge of the basic physiological mechanisms of mechanotransduction and they will useful to further development of the field. Moreover, since pain is very often associated with mechanical stimuli, and touch-evoked pain is a hallmark of naturopathic pain, i.e. touch evoked pain that emerges after nerve damage, these data will be useful in the future design / testing of new treatments for pain treatment.
Dissemination measures
The results of this project will be the subject of publications that are in preparation for high impact scientific journals that will give visibility to the Marie Curie Action. Moreover some results of the project were presented at the 'Force transduction and emerging ion channel' meeting held in Berlin in May 2012.
Therefore, the fellow with the collaboration of the Doctor of Philosophy (PhD) student Jan Walcher started to set up the best conditions to obtain a reproducible growth of the DRG neuron transplanted in the host mouse. We dissected the DRG neurons from adult mice and they are dissociated by enzymatic digestion with collagenase and trypsin and in order to visualise them after transplantation. Initial experiments were carried out to evaluate the ability of isolated sensory neurons to survive in mouse skin acutely in vitro. This approach turned out not to be fruitful as it was not possible to retrieve labelled neurons from the mouse skin even after a few hours of co-incubation.
Therefore, we decided to try to set up a more simple preparation. Since a subclass of skin mechanoreceptor innervated the hair follicles we established co-culture of mouse hair follicles with DRG neurons. These co-cultures proved to be viable as the many sensory neurons in vitro grow out to the hair folliocle and innervate the follicle which in theory allowed us to study the neuronal response after mechanical stimulation of the hair. Immunocytochemistry experiments demonstrated the ability of the DRG neurons to established physical contacts with hair follicles in cultures resembling the native anatomical structure occurring in the skin. Patch clamp recordings in whole configuration were performed on these co-cultured DRG neurons comparing the response upon mechanical stimulation of the hair innervated by the recorded neuron with that obtained by direct stimulation of the soma or neurites of the neuron. We did not observe significant differences in the properties of the mechano-gated current evoked by the two types of stimulation indicating that the transduction channel function does not depend on the interaction between the nerve ending and the hair follicles. Indeed we also carried out experiment on plasma-cleaned hair follicles (that are nbot living) and found that the same innervation by DRG neurons was achieved. This data indicated that a critical cellular factor was probably missing in such experiments and we now believe this may be the terminal Schwann cell.
Moreover the fellow started to address other aspects of the research proposal using cultured dissociated DRG neurons directed stimulated by a glass probe guided by a piezoelectric-controlled nanomotor. In particular he investigated the role of myosin VIIa in modulation of mechanosensitive channel in skin mechanoreceptors contributing to show that Ca2+ dependent activation of myosin VIIa is necessary to maintain the mechanosensitivity upon repetitive stimulation. Moreover, he started to clarify the molecular mechanisms by which STOML-3 controls the mechanosensitivity of DRG mechanorecepors. In particular he tested the effect of different small compounds selected by large screening as modulator of oligomerisation of STOML-3. He found that molecules inhibiting the self-association of STOML-3 are able to reduce the number of mechanosensitive DRG neurons and to alter the kinetics of mechano-gated current.
Project management
The management of the project is handled by administrative staff of the host Institute in connection the fellow and the scientist in charge. All experiments, including mice were carried out with the appropriate permission from local authorities.
The main research objective of this research project was to clarify the molecular mechanisms of mechanotransduction in sensory neurons by direct recording of mechano-activated ion channel activity from DRG neurons innervating the skin. To overcome the technical constrains due to the distance between site of the transduction (skin) and the cell body of the sensory neuron (in DRG), we proposed to set up a novel electrophysiological preparation in which DRG neurons dissected from a mouse are confronted with the skin of another mouse in vitro.
Many different experimental approaches were taken here but we had to conclude finally that the original technical approach was not actually feasible. We then adopted another approach in which a much simpler preparation was used. Since a subclass of skin mechanoreceptor innervated the hair follicles we established a co-culture of hair follicles with DRG neurons allowing us to study the neuronal response after mechanical stimulation of the hairs. Immunocytochemistry experiments have been demonstrated the ability of the DRG neurons to established physical contacts with hair follicles in cultures resembling the native anatomical structure occurring in the skin. Patch clamp recordings in whole configuration were performed on these co-cultured DRG neurons comparing the response upon mechanical stimulation of the hair innervated by the recorded neuron with that obtained by direct stimulation of the soma or neurites of the neuron. We did not observe significant differences in the properties of the mechano-gated current evoked by the two types of stimulation indicating that the transduction channel function does not depend on the interaction between the nerve ending and the hair follicles.
Therefore other aspects of the research proposal were addressed by using cultured dissociated DRG neurons directed stimulated by a glass probe guided by a piezoelectric-controlled nanomotor. In particular it has been investigated the role of myosin VIIa in modulation of mechanosensitive channel in skin mechanoreceptors contributing to show that Ca2+ dependent activation of myosin VIIa is necessary to maintain the mechanosensitivity upon repetitive stimulation. Moreover, experiments had been performed to clarify the molecular mechanisms by which STOML-3 controls the mechanosensitivity of DRG mechanorecepors. In particular, the effect of several compounds selected by large screening as modulator of oligomerisation of STOML-3 had been tested showing that molecules inhibiting the self-association of STOML-3 are able to reduce the number of mechanosensitive DRG neurons and to alter the kinectis of mechanogated current.
The data produced during this project increases knowledge of the basic physiological mechanisms of mechanotransduction and they will useful to further development of the field. Moreover, since pain is very often associated with mechanical stimuli, and touch-evoked pain is a hallmark of naturopathic pain, i.e. touch evoked pain that emerges after nerve damage, these data will be useful in the future design / testing of new treatments for pain treatment.
Dissemination measures
The results of this project will be the subject of publications that are in preparation for high impact scientific journals that will give visibility to the Marie Curie Action. Moreover some results of the project were presented at the 'Force transduction and emerging ion channel' meeting held in Berlin in May 2012.