Mechanisms of trigeminal perception [Elektronische Ressource] : characterization of the cellular properties of sensory neurons of the trigeminal system in rats and mice / submitted by Markus Rothermel

Mechanisms of trigeminal perception –
Characterization of the cellular properties of sensory
neurons of the trigeminal system in rats and mice


Dissertation to obtain the degree
Doctor Rerum Naturalium (Dr. rer. nat.)
at the Faculty of Biology and Biotechnology
Ruhr-University Bochum


International Graduate School of Biosciences
Ruhr-University Bochum
Department of Cellphysiology


submitted by
Markus Rothermel


from
Recklinghausen, Germany



Bochum
April 2009  
Mechanismen trigeminaler Wahrnehmung–
Charakterisierung der zellulären Eigenschaften
sensorischer Neurone im trigeminalen System von Ratte
und Maus


Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
der Fakultät für Biologie und Biotechnologie
an der Internationalen Graduiertenschule Biowissenschaften
der Ruhr-Universität Bochum


angefertigt im
Lehrstuhl für Zellphysiologie


vorgelegt von
Markus Rothermel


aus
Recklinghausen, Deutschland


Bochum
April 2009
  





















Dedicated to
Brunhilde Grenz

  

Erklärung


Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen
Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel
verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um
fünf in Wort und Bild völlig übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und
in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.


Bochum, den



_________________________

(Unterschrift)



  
Content


Abstract ............................................................................................................................8 
1.  Introduction............................................................................................................10 
1.1.  The Mammalian Trigeminal System..........................12 
1.1.1.  Functional Anatomy of the Mammalian Trigeminal System................12 
1.1.2.  Nasal Trigeminal Chemosensation........................................................................................16 
1.1.3.  Trigeminal Somatosensation.................................21 
1.2.  The Skin – Structural Anatomy and Function ..........................................28 
1.2.1.  The Epidermis .......................................................................................28 
1.2.2.  Keratinocytes.........................................................29 
1.3.  Tracing the Trigeminal System with Pseudorabies Viruses....................32 
1.3.1.  Retrospect: Tracing History ..................................................................................................32 
1.3.2.  Advantages of Viral Tracers..33 
1.3.3.  Functional Neuronal Network Exploration using Viral Tracing Tools.35 
1.3.4.  The Pseudorabies Virus.........................................................................................................36 
1.3.5.  Viral Influence on Neuronal Physiology...............39 
2.  Objectives................................................................................................................43 
3.  Material and Methods...........................45 
3.1.  Animals..........................................................................................................................................45 
3.2.  Establishment of the Trigeminal Ganglion in vivo Preparation..............45 
3.2.1.  In vivo voltage-sensitive dye loading of the Trigeminal Ganglia..........46 
3.2.2.  Optical Imaging and Electrical Recordings...........................................................................46 
3.2.3.  Stimulus Delivery..................................................47 
3.2.4.  Odor Concentration...............48 
3.2.5.  In vivo Trigeminal Ganglion Drug Application ....................................................................48 
3.2.6.  Nasal Drug Application.........................................48 
3.2.7.  Data Analysis........................................................48 
3.3.  Cell Cultures .................................................................................................50 
3.3.1.  Primary Cell Culture of Trigeminal Ganglion and Trigeminal Brainstem Neurons .............50 
3.3.2.  Primary Human Keratinocyte Cell Culture / Coculture Approach.......51 
3.3.3.  Cell Lines: HEK293, PK15 and MDBK ...............................................................................51 
3.3.4.  Transient Transfection of HEK293 Cells..............52 
3.3.5.  Growth Media and Buffers for Cell Cultures........53 
3.4.  The Pseudorabies Virus...............................................................................................................55 
3.4.1.  PrV Strains............................55 
3.4.2.  Production of Viral Stocks....56 
3.4.3.  Plaque Assay and Determination of Viral Titre ....................................................................57 
5 
 3.4.4.  Inoculation Procedure............................................................................................................58 
3.5.  Histology........................................58 
3.5.1.  Whole-Mount Preparation.....................................................................................................58 
3.5.2.  Cryosections ..........................................................58 
3.5.3.  Fixatives59 
3.6.  Epifluorescence, Confocal and Multiphoton Laser-Scanning Microscopy ............................59 
3.7.  Imaging of Intracellular Calcium Levels ...................................................................................60 
3.8.  Patch Clamp Recordings .............................................62 
3.9.  Statistical Analysis.......................................................................................62 
3.10.  Human Psychophysical Experiments.........................63 
3.10.1.  Experiment 1: Threshold Tests for CO and Citral Combination .........................................63 2
3.10.2.  Experiment 2: Suprathreshold CO Exposure Followed by Suprathreshold Citral...............64 2
4.  Results .....................................................................................................................66 
4.1.  Anterograde Transsynaptic Tracing in the Murine Somatosensory System using PrV .......66 
4.2.  PrV mediated functional Expression of Fluorescent Calcium Indicator Proteins.................73 
4.3.  In vitro Investigation of Chemosensory Properties of Trigeminal Ganglion Neurons ..........77 
4.4.  Spatiotemporal Dynamics of Odor Representation in the Trigeminal Ganglion in vivo
Visualized by voltage-sensitive dye Imaging...........................................................................................83 
4.4.1.  In vivo voltage-sensitive dye Recording of the Trigeminal Ganglion...................................83 
4.4.2.  In vivo voltage-sensitive dye Imaging of Odor Evoked Activity Patterns in the Trigeminal
Ganglion ...............................................................................................................84 
4.4.3.  Cellular Localization of the voltage-sensitive dye Signal.....................................................87 
4.4.4.  The voltage-sensitive dye Signal Correlates with Odor Evoked Spikes ...............................88 
4.4.5.  Suppression in the voltage-sensitive dye Signal Correlates with Trigeminal Ganglion
Spontaneous Activity Suppression..........................................................................................................90 
4.4.6.  Modulation of the voltage-sensitive dye Signal....92 
4.5.  Human Psychophysical Experiments .........................................................................................95 
4.5.1.  Experiment 1: Threshold Tests for CO and Citral Combination .........................................95 2
4.5.2.  Experiment 2: Suprathreshold CO Exposure Followed by Suprathreshold Citral...............96 2
5.  Discussion................................................................................................................97