Mechanotransduction is fundamental to many sensory processes, including balance, hearing and motor co-ordination. However, for such an essential feature, the mechanism(s) that underlie it are poorly understood. The mechanotransducing stretch receptors that relay information on the tonicity and length of skeletal muscles have been well-defined, particularly at the gross anatomical level, in a wide variety of species, encompassing both vertebrates and invertebrates. To date, there exists a wealth of data describing them, anatomically, as well as good electrophysiological data from stretch receptors of some larger organisms. However, comparatively few studies have succeeded in identifying putative mechanotransducing molecules in such systems. Nonetheless, this class of sensory mechanotransducers perhaps offer the best means of identifying molecules that permit the stretch-sensitivity of such endings, revealing new information about the underlying mechanisms of stretch receptors, and mechanoreceptors more generally. However, a different approach is clearly needed; a theoretical approach, utilising mathematical modelling, offers a powerful means of pooling the current wealth of knowledge on the reported electrophysiological behaviour of muscle stretch receptors. This study, therefore, develops an extended theoretical model of a stretch receptor system in order to reproduce, in silico, the reported behaviour of both vertebrate and invertebrate stretch receptors, within the same modelling environment, thus enabling the first quantitative framework for comparing these data, and moreover, making predictions of the likely roles of specific molecular entities within a stretch receptor system. Subsequently, this study utilises a model in vivo system to test these theoretical predictions. The genetic toolbox of D. melanogaster offers a wide range of tools that are extremely suitable for identifying mechanotransducing molecules in stretch receptors. However, very little is currently known about such endings in this organism. This study, therefore, firstly characterises a putative stretch receptor organ in larval Drosophila, the dbd neuron, via a novel experimental approach. It is shown that this neuron exhibits known properties of stretch receptors, as previously observed in other, similar organs. Furthermore, these observations bear out the predictions of the mathematical model. Having defined the dbd neuron as a muscle stretch receptor, pharmacological and genetic assays in this system, combined with predictions from the mathematical model, identify a key role for the recently-discovered DmPiezo protein as an amiloride-sensitive, mechanically-gated sodium channel (MNaC) in dbd neurons, with TRPA1 also acting in this system in a supporting role. These data confirm the essential role of an MNaC in mechanosensory systems, but also supply important evidence that, whilst the electrophysiological mechanisms in stretch receptors are remarkably similar across taxa, different species likely employ various molecular mechanisms to achieve this.

Somatosensory neurons are essential for detecting diverse environmental stimuli, thus critical for survival of mammals. In order to achieve sensory modality specificity, many somatosensory subtypes emerge with various receptor and ion channel expression, as well as terminal morphologies. How the somatosensory system achieves such a high variety of neuronal subtypes is unknown. In this thesis, I used a newly discovered subtype, VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs), as a model to try to answer this question. C-LTMRs have been proposed to play a role in pleasant touch in humans or pain in mice. Previously, our lab has identified the Runt domain transcriptional factor Runx1 to be pivotal for the development of a cohort of sensory neurons such as pain related nociceptors, thermal receptors, as well as itch related pruriceptors. Here I found that Runx1 is also required to establish all known features associated with C-LTMRs. In search of the mechanism of how Runx1 controls C-LTMR development, I found that the zinc finger protein Zfp521 is predominantly expressed in C-LTMRs and its expression is Runx1 dependent. By generating and analyzing Zfp521 conditional knock out animals, I found Zfp521 is required for part of C-LTMR molecular identities and nerve terminal morphologies. Our studies suggest that Runx1 acts through Zfp521-dependent and Zfp521-independent pathways to specify C-LTMR identities. To study C-LTMR functions, we performed a series of behavioral analysis and found the loss of VGLUT3 and mechanosensitivities in C-LTMRs does not markedly affect acute or chronic mechanical pain measured from the hind paws, which argues against the proposed role of VGLUT3 in C-LTMRs in mediating mechanical pain in mice. In the future, we will continue to use our mutant mice to study physiological functions of C-LTMRs.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24660号 / 農博第2543号 / 新制||農||1098(附属図書館) / 学位論文||R5||N5441(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 大村 和香子, 教授 藤井 義久, 教授 今井 友也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Reflex effects evoked by graded electrical stimulation of the posterior articular nerves (PAN) of the ipsi- and contralateral knee joints were investigated using both micro-electrode recordings from 7 - motoneurones and recordings from single muscle muscle spindle afferents. Spindle afferent responses were also recorded using natural stimulation of different types of receptors, to elucidate if the articular reflexes onto the y -motoneurones were potent enough to significantly alter the muscle spindle afferent activity. Stretches of the ipsilateral posterior (PCL) and anterior (ACL) cruciate ligaments, pressure on the ipsi- and contralateral knee and ankle joint capsules, and passive flexion/extension movements of the joints in the contralateral hind limb were performed. The occurrance of different sensory endings in the ACL and PCL was examined using gold chloride staining for neuronal elements. All experiments were performed on chloralose anaesthetized cats. More than 90% of the static and dynamic y -motoneurones were responsive to electrical stimulation of the PAN. Most 7-cells responded to low intensity electrical stimulation. Excitatoiy reflex effects predominated on both static and dynamic posterior biceps-semitendinosus (PBSt) 7 -cells, while excitatory and inhibitory effects occurred with an about equal frequency on triceps-plantaris (GS) 7-cells. The fastest segmental route for excitatory PAN effects on hind limb 7-motoneurones seems to be di- or trisynaptic, while the path for inhibitory effects seems to be at least one synaps longer. Physiological stimulations of ipsi- and contralateral joint capsules and of ipsilateral cruciate ligaments were all found to evoke frequent and potent changes in spindle afferent responses from the GS and PBSt muscles. It was shown that these effects were due to reflexes onto dynamic and static fusimotor neurones caused by physiological activation of articular sensory endings. Both ipsi- and contralateral joint receptor stimulation evoked excitatory as well as inhibitory fusimotor effects. The highest responsiveness was found during stimula­tion of the cruciate ligaments, i.e. 58% for GS and 47% for PBSt primary spindle afferents to PCL stimula­tion, and 73% for GS and 55% for PBSt primary spindle afferents to ACL stimulation. Significant altera­tions in spindle afferent activity was encountered at very low traction forces applied to the cruciate ligaments (5-10 N). The low thresholds, the tonic character of the stimuli, and the fact that different types of sensory endings were demonstrated in the cruciate ligaments (i.e. Ruffini endings, Pacinian corpuscles, Golgi ten­don organ like endings and free nerve endings), indicate that the fusimotor effects observed were caused by activation of slowly adapting mechanoreceptors, most likely Ruffini endings and/or Golgi tendon organ like endings. The potent reflex effects on the muscle spindle afferents elicited by increased tension in the cruciate ligaments indicate that these ligaments may play a more important sensory role that hitherto believed, and it is suggested that they may be important in the regulation of the stiffness of muscles around the knee joint, and thereby for the joint stability. The possible clinical relevance and the mechanisms by which joint receptor afferents, via adjustment of the muscle stiffness, may control joint stability are discussed. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1989, härtill 7 uppsatser.</p> / digitalisering@umu

Cat hair receptors were studied to determine whether they could be distinguished by the following receptive field characteristics: thickness of innervated guard hairs, distance between innervated follicles and receptive field size. Initially the receptors were classified as G1, GI, G2 or D on the basis of their velocity requirements for excitation, their degree of linear directionality, their vibrational sensitivity, and whether they were activated by movement of down hairs. It was found that the thickest guard hairs on the posterior aspect of a cat's hindleg were usually 4-5 times thicker than the thinnest guard hairs from the same area and that G1, GI and G2 neurons innervated the full range of guard hair thicknesses available. Although there was a tendency for thicker guard hairs to be more heavily innervated, none of the neurons studied innervated thick guard hairs exclusively. While movement of the down hair and most guard hairs within D-mechanoreceptive fields easily evoked activity, a few guard hairs were regularly found for which mechanical displacement did not elicit a discharge even though they were well within the receptive field. Receptive field sizes and nearest neighbor distances between innervated follicles were smaller for D than for G1, GI and G2 receptors and greater for G1 than GI and G2 receptors.

Le traitement et la gestion de la douleur sont depuis toujours une priorité pour le corps médical. Malgré leur importance pour la qualité de vie, les analgésiques couramment utilisés possèdent un ratio bénéfice/risque faible. La recherche de nouveaux concepts thérapeutiques pour lutter contre la douleur est donc une priorité. Afin de répondre à ce besoin, il faut d'abord comprendre les mécanismes de la perception de la douleur ainsi que, plus globalement, ceux permettant de percevoir son environnement. Dans ce contexte, de nombreuses études ont mis en évidence l'implication du canal calcique à bas seuil Cav3.2 dans les voies de la transmission de l'information douloureuse. Il représente donc une cible de choix pour le traitement de la douleur mais l'identité des neurones exprimant ces canaux ainsi que la fonction de Cav3.2 dans la physiologie des neurones sensoriels étaient jusqu'à présent inconnues. Au cours de cette thèse nous avons dans un premier temps décrit un nouvel inhibiteur des canaux calciques à bas seuil : le TTA-A2. Nous avons ainsi démontré que le TTA-A2 est un inhibiteur spécifique des canaux Cav3.1, Cav3.2, et Cav3.3. Il permet de diminuer l'excitabilité des neurones sensoriels exprimant Cav3.2, ce qui provoque une analgésie sur des animaux sains et pathologiques. Dans un deuxième temps nous nous sommes servis de ce nouvel outil en parallèle d'un nouveau modèle murin possédant une étiquette fluorescente (Knock in GFP) sur le canal Cav3.2 pour explorer la localisation et la fonction de Cav3.2 dans les neurones sensoriels. Nous avons ainsi découvert que Cav3.2 est exprimé dans des mécanorécepteurs à bas seuil impliqués dans la perception des stimuli mécaniques et thermiques nocifs ou non-nocifs. Le canal en lui-même se trouve aux endroits clés de la genèse et de la propagation du message nerveux périphérique, et module le seuil et la vitesse de conduction des potentiels d'action. Replacé dans le contexte de la bibliographie, l'ensemble de nos résultats montre que Cav3.2 permet de donner la modalité à bas seuil aux neurones l'exprimant. / Pain management and treatment have always been a priority for life quality. Despite this fact, analgesics commonly used present a bad benefice/risk ratio. Discovery of new therapeutic concepts to fight pain is highly required. To complete this task, we first need to better understand pain perception mechanisms, and more globally, mechanisms involved in the perception of our environment. In this context, numerous studies have shown that low threshold calcium channels Cav3.2 are involved in pain information transmission. Thus, it represents a good target for the treatment of pain. However, neuronal identity of Cav3.2-expressing sensory neurons and Cav3.2 functions in neuronal physiology are unknown. During this PhD we first described a new low voltage activated channel antagonist named TTA-A2. We demonstrated that TTA-A2 is a powerful nanomolar specific agonist of Cav3.1, Cav3.2 and Cav3.3. This molecule is able to reduce excitability in sensory neurons expressing Cav3.2, and is able to generate a strong analgesic effect on naive and pathologic animals. In the other part of this PhD, we used this new tool combined to a new transgenic mouse that expressed Cav3.2 tagged with a fluorescent protein (Knock-in GFP). With these new tools we discovered that Cav3.2 is expressed in low threshold mechanoreceptors involved in detection of painful and non painful mechanical and thermal stimuli. Cav3.2 itself is expressed at key localisations that allow action potential generation and propagation, and modulate threshold and speed conduction of action potential. Taken together, these results show that Cav3.2 gives the “low threshold” modality to neurons.