The prefrontal cortices, in particular lateral prefrontal cortex (LPFC) and anterior cingulate cortex (ACC), have been implicated in top-down control of attention switching and behavioral flexibility. These cortices and their networks are disrupted in autism, a condition in which diverse behaviors such as social communication and attention control are dysregulated. However, little is known about the typical development of these cortical areas or the ways in which this process is altered in neurodevelopmental disorders. In order to identify changes that could affect the local processing of signals transmitted by the short-range pathways connecting the ACC and LPFC I assessed developmental changes in the distinct cortical layers, which send and receive different pathways and have unique inhibitory microenvironments that dictate excitatory-inhibitory balance. Normative developmental trends were compared with those seen in individuals with autism to identify changes that may contribute to symptoms of attention dysfunction. Unbiased quantitative methods were used to study overall neuron density, the density of inhibitory neurons labeled by the calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV), and the density, size, and trajectory of myelinated axons in the individual cortical layers in children and adults with and without a diagnosis of autism. There was a reduction in neuron density and an increase in the density of myelinated axons in both areas during neurotypical development. Axons in layers 1-3 of LPFC were disorganized in autism, with increased variability in the trajectory of axons in children and a decrease in the proportion of thin axons in adults. These findings were most significant in layer 1, the ultimate feedback-receiving layer in the cortex. While there were no differences in neuron populations between cohorts in children, in adults with autism there was a significant reduction in the density of CR-expressing neurons in LPFC layers 2-6 and a significant increase in the density of PV-expressing neurons in ACC layers 5-6. In autism, these findings suggest that dysregulation of the normal development of axonal networks, seen in children, may induce compensatory developmental changes in cell and axon populations in adults that could be connected to attention dysregulation. / 2021-10-07T00:00:00Z

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The anterior cingulate has been implicated in a number of cognitive processes that are at risk following traumatic brain injury (TBI), such as executive function and emotional processing. While the cingulate is believed to play a role in the above-mentioned cognitive processes, the relative roles of gray and white matter in functional outcomes post-TBI are not fully understood. The current study investigated various quantifiable brain properties (e.g., cortical thickness and volume, volume of underlying white matter, and white matter integrity) of the caudal anterior cingulate (CAC) gyrus and their relationships with behavioral measures of cognitive control following pediatric TBI. Parent ratings at three months post-injury indicated that TBI children demonstrated greater difficulty inhibiting inappropriate behavior and effectively transitioning between tasks. Reductions of CAC white matter integrity were observed in TBI participants, in the absence of significant morphometric group differences in this region. Neither CAC morphometrics nor fractional anisotropy (FA) were associated with experimental measures of cognitive control. The current findings indicate that DTI metrics may be more sensitive to brain changes in the region of the CAC following TBI. While strong relationships were not observed between CAC properties and measures of cognitive control, it is possible that study limitations may have obscured potential findings.

Att utvärdera samband mellan subjektivt skattad smärta och transmittorsubstanser med magnetresonansspektroskopi Bakgrund: Smärta är en komplex upplevelse, som involverar olika delar av hjärnan. Regionen anterior cingulate cortex (ACC) är kopplad till upplevelsen av smärta och delas in i ett flertal mindre regioner, till exempel den pregenuala regionen (pgACC) och dorsala regionen (dACC). För att studera olika metaboliter och transmittorsubstanser kan magnetresonansspektroskopi (MRS) användas. MRS och sekvensen MEGA-PRESS kan mäta specifika transmittorsubstanser såsom Gamma-AminoButyric Acid (GABA) och glutamin-glutamat (Glx).  Motiv: Det finns kunskapsluckor kring hur individens subjektiva smärtupplevelse i relation till transmittorsubstanser objektivt kan mätas och utvärderas.  Syfte: Att med MRS och MEGA-PRESS undersöka GABA+ och Glx-nivåer i hjärnområdena pgACC och dACC samt undersöka samband mellan smärtkänslighet och GABA+ och Glx i pgACC och dACC.  Metod: En kvantitativ, experimentell pilotstudie genomfördes med tio friska deltagare. Initialt skannades deltagarna i MRT och smärtstimulerades, sedan skattade de den upplevda smärtan med hjälp av Numeric Rating Scale. MRS och tekniken MEGA-PRESS användes för att mäta transmittorsubstansnivåerna.   Resultat: Studien visade att det fanns en statistiskt signifikant negativ korrelation mellan skattad smärtintensitet och uppmätta nivåer av GABA+ i pgACC (Spearman´s rho = -0,67; p = 0,04). Det fanns även ett statistiskt signifikant positivt samband mellan skattad smärtintensitet och uppmätta nivåer av Glx i dACC (Spearman´s rho =0,73; p=0,02). Vidare fanns signifikant skillnad i Glx mellan pgACC och dACC och en icke signifikant skillnad i GABA+.  Konklusion: Sammanfattningsvis visar resultatet att MRS och MEGA-PRESS kan kvantifiera transmittorsubstanser vid utvärdering av smärtkänslighet och att det finns en positiv korrelation mellan Glx och skattad smärtintensitet, samt en negativ korrelation mellan GABA+ och skattad smärtintensitet. Detta kan ge fördjupad insikt i individens smärtupplevelse och kan främja den individuella behandlingen. Genom att ta hänsyn till sambandet mellan smärta och transmittorsubstanser kan det bidra till ökad förståelse kring individens smärtupplevelse. / To evaluate the relation between subjectively estimated pain and neurotransmitters using magnetic resonance spectroscopy  Background: Pain is a complex experience that involves different parts of the brain. The region anterior cingulate cortex (ACC) is connected to the experience of pain and can be divided into several smaller areas, such as the pregenual region (pgACC) and the dorsal region (dACC). To study different metabolites and neurotransmitters, magnetic resonance spectroscopy (MRS) can be used. MRS and the sequence (MEGA-PRESS) can measure specific neurotransmitters such as Gamma-AminoButyric Acid (GABA) and glutamin-glutamate (Glx).  Motive: There are knowledge gaps about how the individual's subjective pain experience in relation to neurotransmitters can be objectively measured and evaluated.  Aim: Using MRS and MEGA-PRESS to examine levels of GABA+ and Glx in the brain regions pgACC and dACC and to examine the relationship between pain sensitivity and GABA+ and Glx in pgACC and dACC.  Methods: A quantitative, experimental pilot study was conducted which included ten healthy participants. The participants were initially scanned in the MRI and subjected to pain-stimulation, thereafter the participants rated the perceived pain using Numeric Rating Scale. MRS and the sequence MEGA-PRESS were used to quantify the neurotransmitters of interest.  Result: There was a significant, negative correlation between rated pain intensity and measured GABA+ levels in pgACC (Spearman´s rho = -0,67; p = 0,04). There was also a significant, positive correlation between rated pain intensity and measured levels of Glx in dACC (Spearman´s rho =0,73; p=0,02). Furthermore, there was a significant difference in Glx between pgACC and dACC as well as a non-significant difference in GABA+ between regions.  Conclusion: In summary, the result shows that MRS and MEGA-PRESS can quantify neurotransmitters when evaluating pain sensitivity and that there is a positive correlation between Glx and estimated pain intensity, and also a negative correlation between GABA+ and estimated pain intensity. This can provide a deeper insight into the individual’s pain experience and promote individual treatment. Further research regarding the meaning of the different brain regions when measuring neurotransmitters is recommended.

The lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC) are two key regions of the frontal executive control network. Ascending cholinergic pathways differentially innervate these two functionally distinct cortices to modulate arousal and motivational signaling for higher-order functions. The action of acetylcholine (ACh) in sensory cortices is constrained by layer, anatomical cell type, and subcellular localization of distinct receptors, but little is known about the nature and organization of frontal-cholinergic circuitry in primates. In this dissertation, we characterized the anatomical localization of muscarinic acetylcholine receptors (mAChRs), m1 and m2–the predominant subtypes in the cortex–and their expression profiles on distinct cell types and pathways in ACC and LPFC of the rhesus monkey, using immunohistochemistry, anatomical tract-tracing, whole cell patch-clamp recordings, and single nucleus RNA sequencing. In the first series of studies (Chapter 2), we used immunohistochemistry and high-resolution confocal microscopy to reveal regional differences in m1 and m2 receptor localization on excitatory pyramidal and inhibitory neuron subpopulations and subcellular compartments in ACC (A24) versus LPFC (A46) of adult rhesus monkeys (Macaca mulatta; aged 7-11 yrs; 4 males and 2 females). The ACC exhibited a greater proportion of m2+ inhibitory neurons and a greater density of presynaptic m2+ receptors localized on inhibitory (VGAT+) terminations on pyramidal neurons compared to the LPFC. This result suggests a greater cholinergic suppression of GABAergic neurotransmission in ACC. In a second set of experiments (Chapter 3), we examined the heterogeneity of m1 and m2 laminar expression in functionally distinct ACC areas A24, A25, and A32. These differ in their connections with higher order cortical areas and limbic structures, such as the amygdala (AMY). The density of m1+ and/or m2 expressing (m1+/m2+) pyramidal neurons was significantly greater in A24 compared to A25 and to A32, while A25 exhibited a significantly greater density of m2+VGAT+ terminals. In addition, we examined the substrates for cholinergic modulation of long-range cortico-limbic processing using bidirectional neural tracers to label one specific subtype, the AMY-targeting projection neurons in these ACC areas. Compared to A24 and A32, the limbic ventral A25 had a greater density of m1+/m2+ AMY-targeting pyramidal neurons across upper layers 2-3 and deep layers 5-6, suggesting stronger cholinergic modulation of amygdalar outputs. Lastly (Chapter 4), we assessed the functional effects of cholinergic modulation on excitatory and inhibitory synaptic activity as well as the molecular signatures related to m1 and m2 receptor expression. In experiments using in vitro whole-cell patch-clamp recordings of layer 3 pyramidal neurons in ACC and LPFC, we found that application of the cholinergic agonist carbachol (CCh) significantly decreased the frequency of excitatory postsynaptic currents (EPSCs) to a greater extent in ACC A24 than in LPFC A46. Using single nucleus RNA sequencing, we found that enriched m1 and m2 transcriptional profiles in distinct cell-types and frontal areas (ACC A24 and LPFC A46) had differentially expressed genes associated with down-stream signaling cascades related to synaptic signaling and plasticity. Together, these data reveal the anatomical, functional, and transcriptomic neural substrates of diverse cholinergic modulation of local excitatory and inhibitory circuits and long-range cortico-limbic pathways in functionally-distinct ACC and LPFC frontal areas that are important for cognitive-emotional integration.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24088号 / 医博第4864号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 渡邉 大, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pain is the primary reason why patients seek medical care and there is a great unmet need for the development of pain relieving medications. The treatments that are currently available either have limited efficacy or are accompanied by a multitude of unwanted side effects. However, discovering novel therapeutics for the treatment of pain has been challenging. Part of the reason for this may be that that the ways in which pain is assessed in the preclinical setting are different from the way that it is evaluated clinically in human trials. The most common method for evaluating pain in preclinical models is to measure responses to evoked stimuli. However, a change in the threshold of response to evoked pain likely does not measure whether the unpleasant component of pain has actually been reduced. The most clinically relevant question for pain is whether the treatment actually makes the patients "feel better". Here, we demonstrate that the aversiveness of pain can be captured using motivated behavior to seek pain relief. We used conditioned place preference (CPP) to establish that animals with ongoing pain will seek a context that has been paired with effective pain relief, likely as a result of negative reinforcement. These studies allowed for mechanistic investigation. Our results show that: 1) effective pain relief can be achieved by either blocking noxious peripheral input or by directly attenuating pain related unpleasantness in the brain, and 2) pain relief is rewarding and activates the reward circuitry. These studies provide a basis for development of a future platform for drug discovery for pain.

The heterogeneity intrinsic to the ventromedial prefrontal cortex (vmPFC) is evidenced in both its anatomy and implicated function: vmPFC subregions have roles in positive affect, negative affect and autonomic/endocrine regulation. Whether different subregions serve fundamentally different functions, or whether they perform similar computations on different inputs, remains unclear. Nevertheless, the role of the vmPFC in psychopathology is widely appreciated - in mood and anxiety disorders, over-activity within constituent regions of the vmPFC is consistently implicated in symptomatology, together with its normalisation following successful treatment. However, the precise locus of change varies between studies. The work presented in this thesis investigates the causal contributions of over-activity within two key subregions of the vmPFC - the subgenual anterior cingulate cortex (sgACC, area 25) and perigenual anterior cingulate cortex (pgACC, area 32) - in discrete dimensions of behaviour and physiology affected in psychiatric disorders. Specifically, the impact of over-activity is assessed on (i) baseline physiological function; (ii) the regulation of anticipatory, motivational and consummatory aspects of reward-related behaviour; and (iii) negative affect including fear learning, stress recovery and the intolerance of uncertainty. To provide further insight into the mechanism of action of antidepressants, the efficacy of selected treatments is tested on changes induced by over-activity of these regions. Beyond the direct relevance of the results presented here to psychiatric disorders and their treatment, the thesis aims to emphasise the importance of broader themes associated with the measurement and quantification of emotion in preclinical animal studies. First, a multi-faceted approach is utilised enabling quantification of both the autonomic and behavioural aspects of emotion. In so doing, the experiments maintain relevance to studies which assess these correlates in isolation, both in humans (which typically measure subjective responses and physiology) and in rodents (which frequently assess behaviour in isolation). The assessment of more than one dimension of emotion confers these studies with improved power to detect maladaptive changes. Second, the experiments described were conducted in the marmoset, a new-world primate. The extensive anatomical homology between marmoset and human prefrontal cortex facilitates the forward-translation of functional results. In combination with the appropriate assays, this renders marmosets as an invaluable species to study the causal contributions of vmPFC subregions to symptoms of psychiatric disorders. I believe that the results of these experiments provide important insights into the causal role primate vmPFC has in relation to the behavioural and physiological aspects of psychiatric symptomatology. Most importantly, I hope that they serve as the foundation for future work to further elucidate the neuropathological processes underlying mental disorders.

The studies in this thesis explored how pain and its relief are represented in the human brain. Pain and relief are important survival signals that motivate escape from danger and search for safety, however, they are often evaluated by subjective descriptions only. Studying how humans learn and adapt to pain and relief allows objective investigation of the information processing and neural circuitry underlying these internal experiences. My research set out to use computational learning models to provide mechanistic explanations for the behavioural and functional neuroimaging data collected in pain/relief learning experiments with independent groups of healthy human participants. With a Pavlovian acute pain conditioning task in Experiment 1, I found that 'associability' (a form of uncertainty signal) had a crucial role in controlling the learning rates of different conditioned responses, and can be used to anatomically dissociate underlying neural systems. Experiment 2 focused on relief learning of terminating a tonic pain stimulus, in which the priority for relief-seeking is in conflict with the general suppression of cognition and attention. I showed that associability during active learning not only controls the relief learning rate, but also correlates with endogenously modulated (reduced) ongoing pain. This finding was confirmed in Experiment 3 using an independent active relief learning paradigm in a complex dynamic environment. Critically, both experiments showed that associability was correlated with responses in the pregenual anterior cingulate cortex (pgACC), a brain region previously implicated in aspects of endogenous pain control related to attention and controllability. This provided a potential computational account of an information-sensitive endogenous analgesic mechanism. In Experiment 4, I explored the implications of endogenous controllability for technology-based pain therapeutics. I designed an adaptive closed-loop system that learned to control pain stimulation using decoded real-time pain representations from the brain. Subjects were shown to actively enhance the discriminability of pain only in the pgACC, and uncertainty during learning again correlated with endogenously modulated pain and were associated with pgACC responses. Together, these studies (i) show the importance of uncertainty in controlling learning during both acute and tonic pain, (ii) describe how uncertainty also flexibly modulates pain to maximise the impact of learning, (iii) illustrate a central role for the pgACC in this process, and (iv) reveal the implications for future technology-based therapeutic systems.