Feedforward and feedback pathways of nociceptive and tactile processing in human somatosensory system: A study of dynamic causal modeling of fMRI data

Nociceptive and tactile information is processed in the somatosensory system via reciprocal (i.e., feedforward and feedback) projections between the thalamus, the primary (S1) and secondary (S2) somatosensory cortices. The exact hierarchy of nociceptive and tactile information processing within this ‘thalamus-S1-S2’ network and whether the processing hierarchy differs between the two somatosensory submodalities remains unclear. In particular, two questions related to the ascending and descending pathways have not been addressed. For the ascending pathways, whether tactile or nociceptive information is processed in parallel (i.e., 'thalamus-S1′ and 'thalamus-S2′) or in serial (i.e., 'thalamus-S1-S2′) remains controversial. For the descending pathways, how corticothalamic feedback regulates nociceptive and tactile processing also remains elusive. Here, we aimed to investigate the hierarchical organization for the processing of nociceptive and tactile information in the ‘thalamus-S1-S2’ network using dynamic causal modeling (DCM) combined with high-temporal-resolution fMRI. We found that, for both nociceptive and tactile information processing, both S1 and S2 received inputs from thalamus, indicating a parallel structure of ascending pathways for nociceptive and tactile information processing. Furthermore, we observed distinct corticothalamic feedback regulations from S1 and S2, showing that S1 generally exerts inhibitory feedback regulation independent of external stimulation whereas S2 provides additional inhibition to the thalamic activity during nociceptive and tactile information processing in humans. These findings revealed that nociceptive and tactile information processing have similar hierarchical organization within the somatosensory system in the human brain

Modulation of vestibular input by short-term head-down bed rest affects somatosensory perception: implications for space missions

IntroductionOn Earth, self-produced somatosensory stimuli are typically perceived as less intense than externally generated stimuli of the same intensity, a phenomenon referred to as somatosensory attenuation (SA). Although this phenomenon arises from the integration of multisensory signals, the specific contribution of the vestibular system and the sense of gravity to somatosensory cognition underlying distinction between self-generated and externally generated sensations remains largely unknown. Here, we investigated whether temporary modulation of the gravitational input by head-down tilt bed rest (HDBR)–a well-known Earth-based analog of microgravity—might significantly affect somatosensory perception of self- and externally generated stimuli.MethodsIn this study, 40 healthy participants were tested using short-term HDBR. Participants received a total of 40 non-painful self- and others generated electrical stimuli (20 self- and 20 other-generated stimuli) in an upright and HDBR position while blindfolded. After each stimulus, they were asked to rate the perceived intensity of the stimulation on a Likert scale.ResultsSomatosensory stimulations were perceived as significantly less intense during HDBR compared to upright position, regardless of the agent administering the stimulus. In addition, the magnitude of SA in upright position was negatively correlated with the participants’ somatosensory threshold. Based on the direction of SA in the upright position, participants were divided in two subgroups. In the subgroup experiencing SA, the intensity rating of stimulations generated by others decreased significantly during HDBR, leading to the disappearance of the phenomenon of SA. In the second subgroup, on the other hand, reversed SA was not affected by HDBR.ConclusionModulation of the gravitational input by HDBR produced underestimation of somatosensory stimuli. Furthermore, in participants experiencing SA, the reduction of vestibular inputs by HDBR led to the disappearance of the SA phenomenon. These findings provide new insights into the role of the gravitational input in somatosensory perception and have important implications for astronauts who are exposed to weightlessness during space missions

Spatial Patterns of Brain Activity Preferentially Reflecting Transient Pain and Stimulus Intensity

How pain emerges in the human brain remains an unresolved question. Neuroimaging studies have suggested that several brain areas subserve pain perception because their activation correlates with perceived pain intensity. However, painful stimuli are often intense and highly salient; therefore, using both intensity- and saliency-matched control stimuli is crucial to isolate pain-selective brain responses. Here, we used these intensity/saliency-matched painful and non-painful stimuli to test whether pain-selective information can be isolated in the functional magnetic resonance imaging responses elicited by painful stimuli. Using two independent datasets, multivariate pattern analysis was able to isolate features distinguishing the responses triggered by (1) intensity/saliency-matched painful versus non-painful stimuli, and (2) high versus low-intensity/saliency stimuli regardless of whether they elicit pain. This indicates that neural activity in the so-called "pain matrix" is functionally heterogeneous, and part of it carries information related to both painfulness and intensity/saliency. The response features distinguishing these aspects are spatially distributed and cannot be ascribed to specific brain structures

Studies on Cellular Mechanisms and Single-Cell Transcriptional Profiling

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 뇌인지과학과, 2020. 8. 오석배이인아.Pain sensations experienced in the body, as well as in the orofacial region, convey to the central nervous system (CNS) by peripheral sensory neurons such as the dorsal root ganglion (DRG) and the trigeminal ganglion (TG). Nociceptive pain, called nociception, begins at peripheral nerve endings of specialized peripheral sensory neurons known as nociceptors, which exclusively respond to noxious stimuli. Up to date, it has been well studied how the distinct nociceptors respond to various noxious stimuli such as heat, cold, chemical, or mechanical. However, it remains relatively little understood on mechanisms for how innocuous or non-painful stimuli, which would never be considered noxious, can cause pain. In this study, I have explored these polymodalities of nociception that may be elicited by innocuous stimuli or endogenous mediators of inflammation in several pathophysiological conditions as below. In the first chapter, I examined the contribution of peripheral γ–aminobutyric acid type A receptor (GABAAR) that may be activated by endogenous GABA produced in the inflamed tissue to inflammatory pain. Using complete Freunds adjuvant (CFA)-induced persistent inflammatory pain mouse model, I demonstrated that CFA-induced spontaneous nociceptive behavior and mechanical hypersensitivity were inhibited by intraplantar (i.pl.) injection of GABAAR antagonists. Moreover, local blockage of endogenous GABA with injection (i.pl.) of anti-GABA antibody attenuated CFA-induced mechanical hypersensitivity, whereas, i.pl. injection of a positive allosteric modulator of GABAAR facilitated mechanical allodynia in naïve mice. These findings suggest that peripheral GABAAR signaling contributes to CFA-induced hypersensitivity, and its modulation can potentially be a therapeutic target for inflammatory pain alleviation. In the second chapter, I investigated cellular mechanisms by which sweet substances excite dental primary afferent (DPA) neurons, thereby leading dentin hypersensitivity. Sweet substances contain high sugar concentrations so that they cause hyperosmolar conditions at the teeth. I thus demonstrated that the transient receptor potential cation channel, subfamily M, member 8 (TRPM8), a well-known cold- and menthol-receptor, also served as a hyperosmosensor in DPA neurons. By applying a hyperosmolar sucrose solution to the mouse exposed tooth dentin, I investigated whether TRPM8 in DPA neurons mediates upregulation of c-fos expression as a marker of hyperosmolarity-induced nociception in the CNS level. I showed that hyperosmolarity-induced dental nociception was significantly attenuated by a selective TRPM8 antagonist, implying innocuous stimuli such as sweet substances can be sufficient to result in dental nociception via TRPM8 channels. In recent years, an increasing number of studies have successfully applied single-cell transcriptomics to characterize a population of cells and to identify rare subtypes or novel therapeutic targets in heterogeneous sensory system. In the third chapter, I thus employed transcriptional profiling using single-cell RNA sequencing (scRNA-seq) and specific gene-expression validation with in situ hybridization in order to identify unique molecular signatures in the adult mouse DPA neurons. The single-cell transcriptome analysis detected six distinct clusters of DPA neurons. Interestingly, a particular cluster of DPA neurons was characterized by high expression of a low-threshold mechanosensitive Piezo2 ion channel and a pain-related neuropeptide Calca encoding CGRP (calcitonin gene-related peptide). These findings provide an insight into one of the previously proposed mechanisms underlying dentin hypersensitivity (i.e., hydrodynamic theory; Brännström and Astroem 1964), which is a common occurrence by innocuous mechanical irritations such as brush or air puffs. I further discussed mechanosensitive ion channels that may play critical roles in generating pain within the tooth pulp and their clinical implications.신체와 구강악안면 영역에서 경험되는 통증 감각은 후근신경절(dorsal root ganglion)과 삼차신경절(trigeminal ganglion)과 같은 말초 감각 신경을 통해 중추 신경계로 전달된다. 일반적인 통증 감각은 유해감수기(nociceptor)로 불리는 특수한 말초 감각 신경의 말단에서 시작하고 이러한 유해감수기는 유해 자극에 주로 반응한다. 지금까지 열, 저온, 화학 물질 또는 기계적 자극과 같은 다양한 유해 자극에 반응하는 유해감수기에 대해서는 잘 연구되었지만 특수한 병태생리학적 상황에서는 무해한 자극도 통증을 일으킬 수 있는데, 이에 대한 기전은 아직 완전하게 규명되지 않았다. 따라서 본 학위 논문은 여러 유형의 병태생리 상태에서 무해 자극 또는 염증으로 인한 내생적 매개체(endogenous mediator)에 의해서 활성화될 수 있는 통증 감각의 기전을 세포, 분자 생물학 및 동물행동학적 수준에서 다음과 같이 탐구하고자 했다. 첫 번째 장에서는 염증 상태에서 내생적 감마아미노 낙산(GABA)에 의해 활성화 될 수 있는 감마아미노 낙산 유형 A 수용체(GABAAR)의 역할을 조사했다. CFA로 유도된 염증성 통증 마우스 모델을 사용하여, 후근신경절의 말단에 발현하고 있는 감마아미노 낙산 유형 A 수용체의 활성을 줄이기 위한 선택적 억제제의 사용 또는 항-감마아미노 낙산 항체를 염증 부위에 주사하여 내생적 감마아미노 낙산을 직접적으로 차단함으로써 조절했을 때, CFA로 유도된 자발적 통증 행동(spontaneous nociceptive behavior)과 기계적 자극에 대한 통각과민(mechanical hypersensitivity)이 감소했다. 이와는 반대로 감마아미노 낙산 유형 A 수용체의 활성을 양성 입체다른자리 조절자로 증가시켰을 때는 정상 마우스에서도 기계적 자극에 대한 이질통(mechanical allodynia)이 증가했다. 이를 통해 말초 감각 신경의 감마아미노 낙산 유형 A 수용체를 매개로 하는 신호 전달이 염증성 통증 조절을 위한 새로운 치료 표적이 될 수 있음을 입증했다. 두 번째 장에서는 단 물질이 어떻게 치수유래 일차 구심성 신경(dental primary afferent neuron)을 자극하여 상아질 과민증(dentin hypersensitivity)을 유발하는지에 대한 세포 기전을 탐색했다. 설탕 농도가 높은 단 물질은 삼투압이 높기 때문에 우리가 단 음식을 섭취할 때 치아는 고삼투압 상태가 되며, 단 음식은 치통을 빈번하게 유발한다. 따라서, TRPM8 이온 채널-저온과 멘톨 수용체로 잘 알려져 있지만 특별히 각막 신경에서는 각막의 건조와 같은 고삼투압 상태에서 눈의 깜빡임을 조절하는 것으로 보고됨-이 치수유래 일차 구심성 신경에서 고삼투압 자극에 활성화되어 통증 감각을 전달할 수 있음을 확인하고자 했다. 마우스의 치수유래 일차 구심성 신경 세포에는 TRPM8이 기능적으로 발현하고 있었으며, 살아있는 마우스의 치아 상아질을 노출시키고 고삼투압성의 설탕 용액을 적용함으로써 유발된 통증 감각이 TRPM8의 선택적 억제제에 의해서 현저하게 감소되는 것을 중추신경계 수준에서 확인했다. 따라서, 단 물질과 같은 무해한 자극이 TRPM8 채널을 통해 치통을 유발할 수 있음을 증명했다. 최근에 점점 더 많은 연구가 단일 세포 수준의 전사유전체학(single-cell transcriptomics)을 사용하여 혼합된 세포 집단 또는 복잡한 감각 신경 시스템에서 세포의 이종성, 희귀한 세포 유형, 새로운 치료 표적을 성공적으로 발견하고 있다. 세 번째 장에서는 마우스의 치수유래 일차 구심성 신경 세포에서 보이는 독특한 유전자 발현을 탐색하기 위해 단일 세포 수준의 RNA 시퀀싱(single-cell RNA sequencing) 기술과 현장혼성화(in situ hybridization) 조직 염색 기법을 도입했다. 단일 세포 수준의 전사체 프로파일링을 통해 치아 신경에서 여섯 개의 구별된 세포 집단(cluster)들이 존재하는 것을 발견했다. 흥미롭게도 가장 큰 집단은 약한 기계적 자극에 활성화 되는 Piezo2 이온 채널과 CGRP(칼시토닌 연관 단백질)를 부호화(encoding)하는 통증 관련 신경전달물질인 Calca의 높은 발현을 특징으로 했다. 이러한 발견은 치아의 신경세포가 피부 영역에 분포 되어 있는 다른 감각 신경과는 달리, 칫솔질 또는 공기 분사와 같은 약한 기계적 자극이 빈번하게 상아질 과민증을 발생시키는 기전을 설명하며, 이는 기존에 중요하게 제안된 유체역학 이론(hydrodynamic theory)을 입증했다. 추가적으로 나는 치통에 중요하게 관여할 수 있는 기계적 감각 이온 채널들과 본 연구의 임상적 적용에 대해서 논의했다.CHAPTER 1: Functional Roles of Peripheral GABAA Receptors in Persistent Inflammatory Hypersensitivity 29 Abstract 30 Introduction 32 Materials and methods 34 Results 39 Discussion 56 CHAPTER 2: Cellular Mechanisms of TRPM8 Channels Contributing to Dentin Hypersensitivity 62 Abstract 63 Introduction 64 Materials and methods 66 Results 74 Discussion 87 CHAPTER 3: Gene-Expression Signatures of the Adult Mouse Dental Sensory System 93 Abstract 94 Introduction 95 Materials and methods 98 Results 104 Discussion 117 Conclusion 123 Bibliography 125 국문초록 142Docto

Multimodal assessment of neonatal pain

Pain assessment is critical to prevent suffering and harm in infants admitted to the neonatal care unit. As pain is a subjective experience, its assessment in nonverbal infants relies on surrogate measures. Current infant pain assessment tools that are based on behaviour and autonomic nervous system measurements lack face validity — they are unlikely to reflect pain in all its dimensions. In recent years, EEG-derived measures of pain have been developed in late preterm and term infants. Multimodal tools which include these cerebral measurements are conceptually more appropriate to measure pain. Yet, their use is still limited to specific research applications. This thesis focuses on outstanding questions that need to be addressed in order to advance the development of multimodal pain assessment tools that incorporate cerebral measurements. In the first part of this thesis, I focus on the characterisation of preterm infants’ noxious-evoked responses and their development. Across several modalities, premature infants have dampened or altered responsiveness compared to term infants, and it is uncertain if these responses can be reliably discriminated from tactile-evoked responses. In particular, a discriminative pattern of noxious-evoked EEG activity that is present in term infants, is unlikely to be present in preterm infants. In addition, it is unclear how noxious-evoked responses, especially brainderived responses, change with age. In this thesis, I use a classification model to show that infants aged 28–40 weeks postmenstrual age display discriminable multimodal responses to a noxious clinical procedure and a tactile control procedure, and I provide examples of how a such a model could be used in clinical trials of analgesics. I show that noxious-evoked responses change magnitude and morphology across this age range, and that discriminative brain activity emerges in early prematurity. In the second part of this thesis, I focus on improving the neuroscientific validity of a noxious-evoked EEG response measured at the cot-side, as the spatial neural correlates of these responses are still poorly understood. I present an EEG-fMRI pilot study to investigate the spatial neural correlates of inter-individual differences in noxious-evoked EEG responses and provide recommendations for a larger follow-up study. Overall, this thesis provides a characterisation of infants’ noxious-evoked responses and their development across multiple modalities, a crucial next step in improving multimodal neonatal pain assessment

Genetic approaches to understanding pain mechanisms: Zfhx2 and peripheral sensory neuron ablation mouse transgenic models

Latest cutting-edge sequencing has allowed researchers to obtain a full array of differentially expressed neuronal genes within the peripheral nervous system. Understanding this heterogeneity and functional implication could unveil new therapeutic targets towards a more precise medicine. Combining a novel reporter mouse with Cre recombinase strategies, I examined the spatial and functional organization of transcriptomically different subpopulations of neurons in the mouse DRG in pathological and nonpathological states. Results herein include: confirmation of Cre activity and specificity in all lines studied by RNA scope when compared to previous reports and transcriptomic analysis; significant upregulation of DRG gal expression after Complete Freund's Adjuvant (CFA) induced inflamation; normal weight and exploratory behaviour for all lines tested; motor activity assessed by Rotarod not significant, but further motor coordination tests on animals missing Th DRG neurons showed significant impairment; noxious mechanosensation reduced in animals lacking SCN10aCre and Tmem45b DRG; confirmation of CGRP-positive neurons role in heat and cold perception as well as in the formalin inflammatory model; Von Frey hypersensitivity on animals lacking CGRP; and lastly TrkBpositive neurons responsible for significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model whilst no effect in cancer induced bone pain model. Parallelly, by reverse genetics approach, I explore the contribution of the Zfhx2 gene, whose mutation has been identified as responsible for the Marsili pain insensitivity syndrome, in two different animal models of nociception. Behavioural characterisation of bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine mutation, as well as Zfhx2 null mutant mice, shows significant deficits in pain sensitivity in thermal and mechanical tests respectively. In summary, as well as validating several new useful transgenic mouse lines, this thesis provides insights into genes and neuronal subpopulations important in pain pathways and provides potential platforms for translational studies of pain syndromes

Neural and Psychological Mechanisms of Oral Sensation

This thesis set out to explore oral sensory processing. Oral sensory processing extends beyond taste perception, the nerves that innervate the mouth and carry taste information to the brain also carry chemosensations, thermal sensations and somatosensations. While a great deal is understood about oral chemo and thermal perception, this thesis focuses on the not fully recognised oral somatosensory processes. A substantial amount of movement occurs within the mouth, from movement while speaking to chewing food. As food moves around the mouth, different oral receptors are activated and the consumption experience changes. Taste perception varies between individuals in a way that has led to the identification of the taster status genetic polymorphism of taster status where three taster groups (hyper-taster, taster, tolerant taster) with differing sensitivity to bitter tastes were identified. This sensitivity is further represented in anatomical differences with differing densities of fungiform papillae on the tongue. Using psychophysical methods and the taster status phenotype, this thesis examined if different regions of the tongue and mouth experienced different chemostimulant intensity and if dynamic touch changed the intensity perception of chemostimulants in chapter 4. This identified that different regions of the oral cavity experience chemostimulant intensity differently with the tip of the tongue being the most sensitive and the vermillion of the lower lip the least sensitive to sensation. Furthermore, whilst there was no main effect of touch on sensation intensity an interaction between touch type, taster status and oral locations was found when using 10-ppm capsaicin and Sichuan pepper. A dynamic touch on the lip with mint oil was also considered more intense than a static touch. Chapter 5 investigated the possibility that C tactile (CT) afferents were present in the lower lip, the structure of the lip skin widely suggests that CTs are not present but their regular use in the affective behaviour of lip-to-lip contact between individuals suggests otherwise. By applying the standardised psychophysical stroking approach to the lip, cheek and mucosa the classic psychophysical inverted U associated with CT like behavioural responses to touch was found on the cheek where CTs are known to be present as well as on the lower lip. This CT like response on the lip warrants further detailed investigation. Serotonin (5-HT) is widely associated with hedonic experiences and reduced 5-HT levels are a linked with depression and anhedonia. 5-HT is also a candidate neurotransmitter associated with taste transduction. Chapter 6 describes an acute tryptophan depletion (ATD) study that examined the peripheral and central effect of reduced 5-HT levels on taste perception. The primary findings highlight that tryptophan levels do not effect sweet, sour, salt and bitter taste detection ability. A significant difference in bitter taste intensity and pleasantness was identified with tryptophan depletion increasing the taste intensity and decreasing bitter pleasantness at suprathreshold concentration. An effect of taster status was identified in bitter intensity ratings with tolerant-tasters reporting a greater intensity of sensation in the tryptophan depletion session than in the control. During the course of the experimental phase of this thesis, it became clear that describing oral sensations was a difficult task. When asked to describe how sensations felt within their mouth in chapter 4, participants were unable to find words to describe sensations. Therefore, the final study in chapter 7 describes the development of a candidate oral lexicon to aid in describing mouth feel and oral sensations highlighting that the approach to lexicon development previously used to develop the McGill Pain Questionnaire and the Touch Perception Task can successfully be applied to the development of an Oral Lexicon

Analgesia mediated by the TRPM8 cold receptor in neuropathic pain

Chronic pain, particularly neuropathic pain, is a major clinical problem which currently represents a largely unmet therapeutic need.To identify novel analgesic strategies for chronic pain, we investigated the phenomenon of analgesia produced by cutaneous cooling. The recent identification of specific cold sensory receptors has allowed, for the first time, investigation of the molecular mechanism underlying cooling-induced analgesia.We have shown that the cold-and-menthol receptor, TRPM8, is critically involved in cooling-induced analgesia. Activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, and inhibits the characteristic sensitisation of dorsal horn neurons that occurs ipsilateral to nerve injury. This analgesia is abolished following antisense knockdown of the TRPM8 receptor.In contrast, activation of the related putative cold-receptor TRPA1 produces hyperalgesia in naive and neuropathic rats.TRPM8 expression was observed in small diameter sensory neurons in dorsal root ganglia and on afferent terminals in the spinal cord, with increases in specific subsets of sensory neurons following nerve injury.We further found that the central mechanism of TRPM8-mediated analgesia is mediated through inhibitory Group II/III metabotropic glutamate receptors, and is opioid-independent.These results identify TRPM8 as an essential molecular mediator of coolinginduced analgesia. We propose a novel analgesic axis in which activation of TRPM8- expressing afferents by innocuous cooling or chemical ligands leads to activation of inhibitory Group II/III metabotropic glutamate receptors in the spinal cord, which then exert inhibition over nociceptive inputs. These findings suggest that both TRPM8 and the inhibitory metabotropic glutamate receptors are promising targets for the development of novel analgesics for the treatment of neuropathic pain states