Sensory information processing in mouse barrel cortex

Processazione dell'informazione sensoriale nella "barrel cortex" di top

Encoding strategies of primary somatosensory cortex for touch and pain in physiological and pathological conditions

학위논문(박사)--서울대학교 대학원 :의과대학 의과학과,2019. 8. 김상정.일차 체성감각 피질은 촉각과 통증을 지각하고 구별하는 데에 있어서 매우 중요한 역할을 한다. 전통적으로, 일차 체성감각피질을 포함한 체성감각계의 신경세포는 브러쉬, 핀치와 같은 무해한 자극과 유해한 자극에 대한 그 세포의 전기생리학적 반응에 따라 저역치, 고역치 또는 광동적범위 신경세포로 분류되어 왔다. 브러쉬와 포셉을 이용한 이 자극은 유해성뿐만 아니라 브러쉬와 포셉의 촉감, 동적/정적인 역동성과 같은 다른 특성들도 포함하고 있다. 하지만 감각 자극의 이렇게 다양한 특성들을 일차 체성감각피질의 개별세포와 집단 수준에서 종합적으로 어떻게 부호화하고 있는 지에 대한 연구는 부족하다. 조직 및 신경 손상은 이질통, 통각 과민과 같은 과민증을 동반하는 염증성 또는 신경병성 통증을 초래한다. 하지만 통증 과민성일 때 무해하고 유해한 기계적 자극에 대한 일차 체성감각 피질 신경 세포의 반응 속성이 어떻게 달라지며, 이 변화가 통각 과민증과 어떻게 연관되어 있는 지에 대한 연구는 부족하다. 나는 일차 체성감각 피질 세포가 촉각과 통증에 대한 다양한 자극 특성을 동시다발적으로 어떻게 암호화 하고 있는 지 조사했다. 또한 촉각 및 통증 자극에 대한 그 세포들의 반응 속성이 통증 과민증 일 때 어떻게 달라지는 지 조사했다. 이 조사를 위해, 나는 이광자 칼슘 이미징을 통해 무해하고, 유해한 촉각 및 통증 자극을 생쥐의 발바닥에 가하면서 생쥐의 일차 체성감각 피질 신경세포의 칼슘 반응을 기록했다. 이 논문은 촉각 및 통증 자극에 대한 일차 체성감각 피질 세포의 반응 속성을 설명하는 두 가지 부분으로 구성되어 있다. 제 1장에서는, 일차 체성감각 피질 신경세포가 촉감이 다른 자극에 대해서 높은 선택적 반응을 보인 것을 확인했다. 하지만 역동성 또는 유해성 특성에 대해서는 낮은 선택성을 보였으며, 그 중 역동성에 약간 더 높은 선택성을 보인 것을 확인했다. 제 2장에서는, 통증 과민증 일 때, 유해-선호 신경세포가 무해한 촉각 자극에도 반응하는 것을 확인했다. 하지만, 촉각 및 통증 자극 모두에 반응한 세포 (광범위하게 조정된 세포)는 자극에 대한 튜닝 속성이 과민증일 때 유지됐고, 그 세포의 일부는 촉각 및 통증 자극에 대한 반응성이 증가했다. 이 논문은 일차 체성감각 피질 신경세포가 자극 특성-의존적 방식으로 특이성 부호화와 패턴 부호화의 혼합 된 전략을 사용하는 걸 제시했다. 또한, 통증 과민증 일 때, 일체 체성감각 피질은 자극에 대한 반응 속성이 바뀌고, 광범위하게 조정된 세포의 반응성이 전반적으로 증가하는 방식으로 과민증에 기여하고 있다는 것을 보여줬다. 본 논문은 생리 및 병리적 조건에서 촉각 및 통증 자극에 대한 일차 체성감각 피질의 암호화 전략과 반응 속성에 대해 이해하기 위한 중요한 정보를 제공한다.Primary somatosensory cortex (S1) plays an important role in the perception and discrimination of touch and pain. Conventionally, neurons in the somatosensory system including S1 cortex have been classified by noxiousness feature with innocuous brush and noxious pinch stimuli. Besides this noxiousness feature, each stimulus also includes other stimulus features, such as different textures or dynamics. However, it remains unexplored how S1 neurons comprehensively encode such diverse features of cutaneous stimuli at single-cell and population levels. Tissue or nerve injury can lead to an inflammatory or neuropathic pain, in which hypersensitivity is accompanied. However, it is unclear how the response properties of S1 neurons towards mechanical stimuli are altered. It is also unknown how these S1 response changes are involved in pain hypersensitivity. I investigated how S1 neurons comprehensively encode multiple stimulus features for touch and pain in physiological conditions and how the response properties of S1 neurons are changed in pain hypersensitivity. To explore this, using in vivo two-photon Ca2+ imaging, I recorded neural activities of S1 neurons in mice while applying innocuous and noxious mechanical stimuli into hind paw. This thesis is composed of two research parts on response properties of S1 neurons to touch and pain. In chapter 1, it is shown that S1 neurons exhibited highly selective response to the difference in texture (specificity coding), but low selectivity to the difference in dynamics or noxiousness with slightly more specificity to dynamics (pattern coding). In chapter 2, I found some of the noxious-preferred neurons, which responded to noxious pinch stimuli at normal states, responded to innocuous touch stimuli in CFA-induced hypersensitivity. The majority of broadly tuned neurons, however, maintained their normal tuning properties during hypersensitivity, but some of those showed increased responses to both innocuous and noxious mechanical stimuli in CFA-induced hypersensitivity. This thesis demonstrates that S1 neurons use a mixed strategy of specificity coding and pattern coding for multiple stimulus features in a feature-dependent manner. In addition, it is also revealed how S1 cortex contributes to CFA-induced hypersensitivity in a way that tuning properties are changed and activities of broadly tuned neurons are generally increased in CFA-induced hypersensitivity. These findings would be important to understand the encoding rules and response properties of S1 to touch and pain in physiological and pathological conditions.Abstract 1 Chapter 1. Differential selectivity of S1 neurons to multiple stimulus features of touch and pain Introduction 5 Results 9 Discussion 29 Chapter 2. Alterations in response properties of S1 neurons to innocuous and noxious stimuli in CFA-induced hypersensitivity Introduction 41 Results 43 Discussion 51 Materials and Methods 58 References 66 Abstract in Korean 71Docto

Slow-wave activity in the S1HL cortex is contributed by different layer-specific field potential sources during development

Spontaneous correlated activity in cortical columns is criticalfor postnatal circuit refinement.We used spatial discriminationtechniques to explore the late maturation of synaptic pathways through the laminar distribution of the field potential (FP) generators underlying spontaneous and evoked activities ofthe S1HL cortex in juvenile (P14 –P16) and adult anesthetized rats. Juveniles exhibit an intermittent FP pattern resembling Up/Down states in adults, but with much reduced power and different laminar distribution. Whereas FPs in active periods are dominated by a layer VI generator in juveniles, in adults a developing multipart generatortakes over, displaying current sinks in middle layers (III–V). The blockade of excitatory transmission in upper and middle layers of adults recovered the juvenile-like FP profiles. In additiontothe layer VI generator, a gamma-specific generator in supragranular layers wasthe same in both age groups.While searching for dynamical coupling among generators in juveniles we found significant cross-correlation in one-half of the tested pairs, whereas excessive coherence hindered their efficient separation in adults. Also, potentials evoked by tactile and electrical stimuli showed different short-latency dipoles between the two age groups, and the juveniles lacked the characteristic long latency UP state currents in middle layers. In addition, the mean firing rate of neurons was lower in juveniles. Thus, cortical FPs originate from different intracolumnar segments as they become active postnatally. We suggest that although some cortical segments are active early postnatally, a functional sensory-motor control relies on a delayed maturation and network integration of synaptic connections in middle layers

遺伝学的、光遺伝学的手法を用いた、ラットにおける脳機能局在性の研究

学位の種別:課程博士University of Tokyo(東京大学

Cholinergic modulation of cortical sensory-evoked responses

Cholinergic inputs to the cortex from the basal forebrain are essential for performing complex behaviours that require precise integration of sensory and motor information. However, the role of the basal forebrain cholinergic system in cortical sensory processing in awake behaving animals is unknown. Acetylcholine modulates the cortical microcircuitry in complex ways, with opposing effects on different neuronal types leading to both inhibitory and disinhibitory actions for excitatory pyramidal neuron circuitry. Therefore, it is crucial to understand how cholinergic modulation influences anatomically registered cell types. I focused on Layer 2/3 pyramidal neurons since they are the primary source of cortico-cortical connections responsible for broadcasting information across cortical areas, areal connections, presumably to integrate sensory and motor behaviours. To assess the role of cholinergic modulation of this system, I examined how a chronic depletion of basal forebrain cholinergic fibres alters Layer 2/3 network activity across cortical areas in response to brief tactile stimulations. First, I determined how recruitment of cortical areal connections occurs in mice with an intact cholinergic system. To do this, I used mice expressing the voltage indicator VSFP Butterfly 1.2 specifically in cortical Layer 2/3 pyramidal neurons and widefield fluorescence imaging through a thinned skull window in head-fixed awake mice as they received single or double brief stimulation to the forepaw or air puff to the whiskers. I also generated real-time maps of sensory-evoked changes in areal Layer 2/3 activity. Following these experiments, I standardised a chronic but selective cholinergic lesion in the cortex using the neurotoxin mu p75SAP both at the anatomical and behavioural levels. Mice with this lesion only had difficulty performing complex behavioural tasks. Finally, I obtained and analysed real-time maps of sensory, paw-evoked changes in areal Layer 2/3 activity in mice with a chronic cholinergic lesion. The areal Layer 2/3 activity maps showed fast depolarisation of initiated within distinct areas of the sensory cortex in response to whisker and paw stimulation. Sensory evoked responses after paw and whisker stimulation responses exhibited a similar fast depolarisation, but their time scale and broadcast patterns differed markedly. Notably, we observed different patterns of slower hyperpolarisation (presumed inhibition). Double stimulation to the forepaw revealed a second smaller depolarisation consistent with recruitment of inhibition and also with the well-known adaptation of sensory-evoked responses. In mice with the unilateral lesion, the sensory-evoked responses in the forelimb area exhibited an increased initial depolarisation and significantly prolonged and widespread hyperpolarisation that disrupted the areal Layer 2/3 pyramidal neuron activity maps and adaptation after paired stimulation. The results indicate that cholinergic inputs from the basal forebrain are essential for cortical sensory processing and sensory adaptation by the Layer 2/3 cortical microcircuitry. Disruption of this sensory processing may underlie the degraded performance of complex behaviours in disorders where cholinergic transmission is altered, as in Alzheimer’s disease

Discovery and Characterization of Novel Selective NKCC1 Inhibitors for Down Syndrome and Brain Disorders with Depolarizing GABAergic Transmission

Proper GABAergic transmission through Cl-permeable GABAA receptors is fundamental for physiological brain development and function. Indeed, defective GABAergic signaling -due to a high ratio of expression of the Cl importer NKCC1 and Cl exporter KCC2- has been implicated in several neurodevelopmental disorders (e.g., Down syndrome, DS). Interestingly, NKCC1 inhibition by the FDAapproved diuretic bumetanide reverts cognitive deficits in the TS65Dn mouse models of DS and core symptoms in a number of models of other brain disorders. However, the required chronic treatment with bumetanide is burdened by its diuretic side effects caused by the antagonism of the kidney Cl- importer NKCC2, which leads to hypokalemia and jeopardizes drug compliance. Crucially, these issues would be solved by selective NKCC1 inhibitors, thus devoid of the diuretic effect. Starting from bumetanide\u2019s structure, we applied a computational ligand-based approach to design new molecular entities that we tested in vitro for their capacity to selectively block NKCC1. Among the 3 newly-identified and highly promising NKCC1 inhibitors, one showed excellent solubility and metabolic stability in vitro. Moreover, analysis of WT and Ts65Dn mice systemically treated with this NKCC1 inhibitor revealed no diuretic effect. Finally, chronic treatment with our novel, selective NKCC1 inhibitor was able to rescue cognitive deficits in Ts65Dn mice in four different memory tasks, with no major signs of toxicity. Thus, our selective NKCC1 inhibitor devoid of the diuretic effect could represent a suitable and solid therapeutic strategy for the treatment of Down syndrome and all the brain disorders with depolarizing GABAergic transmission

The endocannabinoid system in experimental models of Alzheimer's disease

415 p.Durante la progresión de la enfermedad de Alzheimer (EA) se produce una degeneración del sistema de neurotransmisión colinérgico del prosencéfalo basal, que conduce a una pérdida progresiva e irreversible de funciones cognitivas, así como alteraciones del sistema endocannabinoide (eCB) y cambios en la composición lipídica cerebral. Cada vez más estudios apuntan al posible papel neuroprotector del sistema eCB en procesos neurodegenerativos y ya han sido demostrados los efectos beneficiosos de agonistas cannabinoides para el tratamiento de alteraciones conductuales asociadas a la EA. El sistema eCB modula entre otros al sistema colinérgico a través del receptor CB1 y, la síntesis de sus ligandos está ligada a la señalización muscarínica colinérgica en áreas que controlan procesos cognitivos. En este trabajo se ha estudiado el papel del sistema eCB en diferentes modelos de degeneración colinérgica y de EA. La inmunotoxina 192IgG-saporina elimina específicamente neuronas colinérgicas del prosencéfalo basal y se ha utilizado en cultivos organotípicos y ratas adultas como modelos ex vivo e in vivo de EA. Además se han administrado cannabinoides en el modelo 3xTg-AD para la EA y evaluado aprendizaje y memoria. El tratamiento con cannabinoides muestra protección sobre la muerte celular asociada a la lesión colinérgica en el modelo ex vivo. Los estudios in vivo, tras la administración de la inmunotoxina, muestran un deterioro cognitivo, una gran pérdida de inervación colinérgica cortical, un proceso adaptativo del sistema eCB a través del receptor CB1 y una profunda alteración del perfil fosfolipídico, todo ello asociado a la lesión colinérgica. El modelo 3xTg-AD presenta alteraciones conductuales asociadas a una desregulación del sistema eCB, que son parcialmente revertidas tras la activación subcrónica del receptor CB1. Proponemos al sistema eCB como modulador de la neurotransmisión colinérgica en neurodegeneración y potencial diana de intervención terapéutica en la EA

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Orienting behaviours and attentional processes in the mouse and macaque : neuroanatomy, electrophysiology and optogenetics

PhD ThesisThe neuronal basis of orienting and attentional behaviours has been widely researched in higher animals such as non-human primates (NHPs). However the organisation of these behaviours and processes in rodent models has been less well characterised. This thesis is motivated to delineate the key neuroanatomical pathways and neuronal mechanisms that account for orienting behaviours in the mouse model and compare them, in part, to those seen in the macaque. A better understanding of the processes and networks involved with attention and orienting is necessary in order to relate findings in the mouse model to those seen in humans and NHPs. Further to this, the availability of highly targeted manipulations in the mouse, such as optogenetics, requires a more detailed picture of the neurophysiology underpinning those behaviours to effectively interpret findings and design experiments to exploit these techniques and animal models for maximum benefit. In this thesis, study one focuses on the neuroanatomical pathways that terminate in subregions of the midbrain superior colliculus (SC) in the mouse (mus musculus) using iontophoretic injection of the retrograde tracer fluorogold. This region has been implicated in various forms of orienting behaviours in both macaques and mice (Albano et al., 1982, Dean et al., 1988b, Felsen and Mainen, 2008). Furthermore study one examines the prefrontal connectivity that links to the SC subsections and which may govern approach and avoidance behaviours (motor cortex area 2 (M2) and cingulate area (Cg)) in the mouse via pressure injection of the anterograde tracer biotinylated dextran amine into these regions. It was found that the medial and lateral SC receive differential prefrontal input from the Cg and M2 respectively. And that these areas project to brain networks related to avoidance or approach. This section furthers our understanding of the partially segregated networks which exist in the prefrontal cortex and midbrain of the mouse, which are important in mediation of different orienting behaviours Study two focuses on the effects of one type of orienting, namely bottom-up attention (BU) in visual areas. This exogenous (automatic) form of visual attention has been studied extensively in human psychophysics (Posner, 1980, Nakayama and Mackeben, 1989) and the areas involved in the human brain have been delineated using brain imaging (Corbetta and Shulman, 2002, Liu et al., 2005). To understand the neurophysiology involved, some electrophysiological invasive studies have been performed in the macaque monkeys, II ( Luck et al., 1997, Buschman and Miller, 2007), but our understanding of the mechanisms involved is relatively sparse when compared to top-down (endogenous) attentional processing. To understand the similarities in this mechanism between macaques and mice it is therefore important to study both model systems using similar approaches. The research of this chapter aims to make direct comparisons between these two model species via electrophysiological recordings in a bottom-up attentional paradigm. It was found that in the macaque BU cues increased responses to visual stimuli in both V1 and V4, but no obvious pattern was seen in the mouse V1 and SC. This study goes some way in describing the similarities and differences in neural responses in visual areas of different species which are utilised for attention based paradigms Finally study three focuses on linking the previous two studies. In study two we investigated bottom-up attentional processes, which are thought to involve early, fast visuomotor pathways. Whereas in study one we found that SC and V1, areas known for their involvement in and ability to coordinate rapid visuomotor responses, respectively, also receive clear and structured input from higher-level prefrontal areas. Therefore we hypothesized that stimulating these prefrontal areas could modulate bottom-up attention. This is achieved by using optogenetic stimulation of prefrontal control regions, such as Cg, identified in this research whilst preforming electrophysiological recordings in a bottom-up attentional paradigm. In V1 is was found that optogenetic stimulation had no effect on neuronal activation. However in SC optogenetic activation increased the sustained stimulus response, regardless of cuing condition. Taken together, this research further investigates some brain regions involved in orienting and attention in both mice and macaques and partially bridges the gap in understanding between these two animal models