Converging Prefronto-Insula-Amygdala Pathways in Negative Emotion Regulation in Marmoset Monkeys

BACKGROUND: Impaired regulation of emotional responses to potential threat is a core feature of affective disorders. However, while the subcortical circuitry responsible for processing and expression of fear has been well characterized, the top-down control of this circuitry is less well understood. Our recent studies demonstrated that heightened emotionality, as measured both physiologically and behaviorally, during conditioned fear and innate/ social threat was induced, independently, by excitotoxic lesions of either the anterior orbitofrontal cortex (antOFC) or ventrolateral prefrontal cortex (vlPFC). An important outstanding question is whether the antOFC and vlPFC act on common or distinct downstream targets to regulate negative emotion. METHODS: The question was addressed by combining localized excitotoxic lesions in the PFC of a nonhuman primate and functional neuroimaging ([$^{18}$F]fluorodeoxyglucose positron emission tomography) with a fear-regulating extinction paradigm. Marmoset monkeys with unilateral lesions of either the antOFC or vlPFC were scanned immediately following exposure to a fearful or safe context, and differences in [$^{18}$F]fluorodeoxyglucose uptake were evaluated. RESULTS: [$^{18}$F]fluorodeoxyglucose uptake in the insula and amygdala of the intact hemisphere was significantly increased in response to the fearful context compared with the safe context. Such discrimination between the two contexts was not reflected in the activity of the insula-amygdala of the antOFC or vlPFC-lesioned hemisphere. Instead, uptake was at an intermediate level in both contexts. CONCLUSIONS: These findings demonstrate that the distinct control functions of the antOFC and vlPFC converge on the same downstream targets to promote emotion regulation, taking us closer to a mechanistic understanding of different forms of anxiety.This research was supported by a Medical Research Programme Grant (No. MR/M023990/1) from the Medical Research Council (to ACR) and was carried out within the Behavioural and Clinical Neuroscience Institute supported by a consortium award from the Wellcome Trust and the Medical Research Council

Effects of hyperoxia on 18F-fluoro-misonidazole brain uptake and tissue oxygen tension following middle cerebral artery occlusion in rodents: Pilot studies.

PURPOSE: Mapping brain hypoxia is a major goal for stroke diagnosis, pathophysiology and treatment monitoring. 18F-fluoro-misonidazole (FMISO) positron emission tomography (PET) is the gold standard hypoxia imaging method. Normobaric hyperoxia (NBO) is a promising therapy in acute stroke. In this pilot study, we tested the straightforward hypothesis that NBO would markedly reduce FMISO uptake in ischemic brain in Wistar and spontaneously hypertensive rats (SHRs), two rat strains with distinct vulnerability to brain ischemia, mimicking clinical heterogeneity. METHODS: Thirteen adult male rats were randomized to distal middle cerebral artery occlusion under either 30% O2 or 100% O2. FMISO was administered intravenously and PET data acquired dynamically for 3hrs, after which magnetic resonance imaging (MRI) and tetrazolium chloride (TTC) staining were carried out to map the ischemic lesion. Both FMISO tissue uptake at 2-3hrs and FMISO kinetic rate constants, determined based on previously published kinetic modelling, were obtained for the hypoxic area. In a separate group (n = 9), tissue oxygen partial pressure (PtO2) was measured in the ischemic tissue during both control and NBO conditions. RESULTS: As expected, the FMISO PET, MRI and TTC lesion volumes were much larger in SHRs than Wistar rats in both the control and NBO conditions. NBO did not appear to substantially reduce FMISO lesion size, nor affect the FMISO kinetic rate constants in either strain. Likewise, MRI and TTC lesion volumes were unaffected. The parallel study showed the expected increases in ischemic cortex PtO2 under NBO, although these were small in some SHRs with very low baseline PtO2. CONCLUSIONS: Despite small samples, the apparent lack of marked effects of NBO on FMISO uptake suggests that in permanent ischemia the cellular mechanisms underlying FMISO trapping in hypoxic cells may be disjointed from PtO2. Better understanding of FMISO trapping processes will be important for future applications of FMISO imaging

Brain hypoxia mapping in acute stroke: Back-to-back T2′ MR versus 18F-fluoromisonidazole PET in rodents

Background Mapping the hypoxic brain in acute ischemic stroke has considerable potential for both diagnosis and treatment monitoring. PET using (18)F-fluoro-misonidazole (FMISO) is the reference method; however, it lacks clinical accessibility and involves radiation exposure. MR-based T2' mapping may identify tissue hypoxia and holds clinical potential. However, its validation against FMISO imaging is lacking. Here we implemented back-to-back FMISO-PET and T2' MR in rodents subjected to acute middle cerebral artery occlusion. For direct clinical relevance, regions of interest delineating reduced T2' signal areas were manually drawn. Methods Wistar rats were subjected to filament middle cerebral artery occlusion, immediately followed by intravenous FMISO injection. Multi-echo T2 and T2* sequences were acquired twice during FMISO brain uptake, interleaved with diffusion-weighted imaging. Perfusion-weighted MR was also acquired whenever feasible. Immediately following MR, PET data reflecting the history of FMISO brain uptake during MR acquisition were acquired. T2' maps were generated voxel-wise from T2 and T2*. Two raters independently drew T2' lesion regions of interest. FMISO uptake and perfusion data were obtained within T2' consensus regions of interest, and their overlap with the automatically generated FMISO lesion and apparent diffusion coefficient lesion regions of interest was computed. Results As predicted, consensus T2' lesion regions of interest exhibited high FMISO uptake as well as substantial overlap with the FMISO lesion and significant hypoperfusion, but only small overlap with the apparent diffusion coefficient lesion. Overlap of the T2' lesion regions of interest between the two raters was ∼50%. Conclusions This study provides formal validation of T2' to map non-core hypoxic tissue in acute stroke. T2' lesion delineation reproducibility was suboptimal, reflecting unclear lesion borders.This study was funded by an EU Grant (EUSTROKE Health-F2-2008-2022131). DJW was funded by an MRC collaborative grant (G0600986), RM by the NIHR Cambridge Biomedical Research Centre, and UJK by a fellowship from the Deutsche Forschungsgemeinschaft (Je 598/1-1)

From impulses to maladaptive actions: the insula is a neurobiological gate for the development of compulsive behavior.

Impulsivity is an endophenotype of vulnerability for compulsive behaviors. However, the neural mechanisms whereby impulsivity facilitates the development of compulsive disorders, such as addiction or obsessive compulsive disorder, remain unknown. We first investigated, in rats, anatomical and functional correlates of impulsivity in the anterior insular (AI) cortex by measuring both the thickness of, and cellular plasticity markers in, the AI with magnetic resonance imaging and in situ hybridization of the immediate early gene zif268, respectively. We then investigated the influence of bilateral AI cortex lesions on the high impulsivity trait, as measured in the five-choice serial reaction time task (5-CSRTT), and the associated propensity to develop compulsivity as measured by high drinking levels in a schedule-induced polydipsia procedure (SIP). We demonstrate that the AI cortex causally contributes to individual vulnerability to impulsive-compulsive behavior in rats. Motor impulsivity, as measured by premature responses in the 5-CSRTT, was shown to correlate with the thinness of the anterior region of the insular cortex, in which highly impulsive (HI) rats expressed lower zif268 mRNA levels. Lesions of AI reduced impulsive behavior in HI rats, which were also highly susceptible to develop compulsive behavior as measured in a SIP procedure. AI lesions also attenuated both the development and the expression of SIP. This study thus identifies the AI as a novel neural substrate of maladaptive impulse control mechanisms that may facilitate the development of compulsive disorders.This research was carried-out within the Department of Psychology and the Department of Pharmacology of the University of Cambridge as well as the INSERM AVENIR team Psychobiology of Compulsive Disorders of the University of Poitiers. It was supported by an INSERM AVENIR grant and a FYSSEN foundation grant to DB. MLD was supported by a PhD fellowship from the Fondation pour la Recherche Médicale (FRM) and ABR was supported by a post-doctoral fellowship from the INSERM. BJE was supported by the United Kingdom Medical Research Council (MRC) Grant 9536855.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/mp.2015.14

Molecular neuropathology of the synapse in sheep with CLN5 Batten disease

© 2015 Published by Wiley Periodicals, Inc. Aims: Synapses represent a major pathological target across a broad range of neurodegenerative conditions. Recent studies addressing molecular mechanisms regulating synaptic vulnerability and degeneration have relied heavily on invertebrate and mouse models. Whether similar molecular neuropathological changes underpin synaptic breakdown in large animal models and in human patients with neurodegenerative disease remains unclear. We therefore investigated whether molecular regulators of synaptic pathophysiology, previously identified in Drosophila and mouse models, are similarly present and modified in the brain of sheep with CLN5 Batten disease. Methods: Gross neuropathological analysis of CLN5 Batten disease sheep and controls was used alongside postmortem MRI imaging to identify affected brain regions. Synaptosome preparations were then generated and quantitative fluorescent Western blotting used to determine and compare levels of synaptic proteins. Results: The cortex was particularly affected by regional neurodegeneration and synaptic loss in CLN5 sheep, whilst the cerebellum was relatively spared. Quantitative assessment of the protein content of synaptosome preparations revealed significant changes in levels of seven out of eight synaptic neurodegeneration proteins investigated in the motor cortex, but not cerebellum, of CLN5 sheep (α-synuclein, CSP-α, neurofascin, ROCK2, calretinin, SIRT2, and UBR4). Conclusions: Synaptic pathology is a robust correlate of region-specific neurodegeneration in the brain of CLN5 sheep, driven by molecular pathways similar to those reported in Drosophila and rodent models. Thus, large animal models, such as sheep, represent ideal translational systems to develop and test therapeutics aimed at delaying or halting synaptic pathology for a range of human neurodegenerative conditions

MRI-guided histology of TDP-43 knock-in mice implicates parvalbumin interneuron loss, impaired neurogenesis and aberrant neurodevelopment in amyotrophic lateral sclerosis-frontotemporal dementia.

Amyotrophic lateral sclerosis and frontotemporal dementia are overlapping diseases in which MRI reveals brain structural changes in advance of symptom onset. Recapitulating these changes in preclinical models would help to improve our understanding of the molecular causes underlying regionally selective brain atrophy in early disease. We therefore investigated the translational potential of the TDP-43Q331K knock-in mouse model of amyotrophic lateral sclerosis-frontotemporal dementia using MRI. We performed in vivo MRI of TDP-43Q331K knock-in mice. Regions of significant volume change were chosen for post-mortem brain tissue analyses. Ex vivo computed tomography was performed to investigate skull shape. Parvalbumin neuron density was quantified in post-mortem amyotrophic lateral sclerosis frontal cortex. Adult mutants demonstrated parenchymal volume reductions affecting the frontal lobe and entorhinal cortex in a manner reminiscent of amyotrophic lateral sclerosis-frontotemporal dementia. Subcortical, cerebellar and brain stem regions were also affected in line with observations in pre-symptomatic carriers of mutations in C9orf72, the commonest genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Volume loss was also observed in the dentate gyrus of the hippocampus, along with ventricular enlargement. Immunohistochemistry revealed reduced parvalbumin interneurons as a potential cellular correlate of MRI changes in mutant mice. By contrast, microglia was in a disease activated state even in the absence of brain volume loss. A reduction in immature neurons was found in the dentate gyrus, indicative of impaired adult neurogenesis, while a paucity of parvalbumin interneurons in P14 mutant mice suggests that TDP-43Q331K disrupts neurodevelopment. Computerized tomography imaging showed altered skull morphology in mutants, further suggesting a role for TDP-43Q331K in development. Finally, analysis of human post-mortem brains confirmed a paucity of parvalbumin interneurons in the prefrontal cortex in sporadic amyotrophic lateral sclerosis and amyotrophic lateral sclerosis linked to C9orf72 mutations. Regional brain MRI changes seen in human amyotrophic lateral sclerosis-frontotemporal dementia are recapitulated in TDP-43Q331K knock-in mice. By marrying in vivo imaging with targeted histology, we can unravel cellular and molecular processes underlying selective brain vulnerability in human disease. As well as helping to understand the earliest causes of disease, our MRI and histological markers will be valuable in assessing the efficacy of putative therapeutics in TDP-43Q331K knock-in mice

Serotonergic, brain volume and attentional correlates of trait anxiety in primates.

Trait anxiety is a risk factor for the development and maintenance of affective disorders, and insights into the underlying brain mechanisms are vital for improving treatment and prevention strategies. Translational studies in non-human primates, where targeted neurochemical and genetic manipulations can be made, are critical in view of their close neuroanatomical similarity to humans in brain regions implicated in trait anxiety. Thus, we characterised the serotonergic and regional brain volume correlates of trait-like anxiety in the marmoset monkey. Low- and high-anxious animals were identified by behavioral responses to a human intruder (HI) that are known to be sensitive to anxiolytic drug treatment. Extracellular serotonin levels within the amygdala were measured with in vivo microdialysis, at baseline and in response to challenge with the selective serotonin reuptake inhibitor, citalopram. Regional brain volume was assessed by structural magnetic resonance imaging. Anxious individuals showed persistent, long-term fearful responses to both a HI and a model snake, alongside sustained attention (vigilance) to novel cues in a context associated with unpredictable threat. Neurally, high-anxious marmosets showed reduced amygdala serotonin levels, and smaller volumes in a closely connected prefrontal region, the dorsal anterior cingulate cortex. These findings highlight behavioral and neural similarities between trait-like anxiety in marmosets and humans, and set the stage for further investigation of the processes contributing to vulnerability and resilience to affective disorders.This research was supported by a Medical Research Programme Grant (G0901884) from the Medical Research Council UK (MRC) to Angela Roberts, and a PhD studentship from MRC and final-term funding from Trinity College, Cambridge, UK to Yevheniia Mikheenko.This is the author accepted manuscript. The final version is available from NPG at http://www.nature.com/npp/journal/v40/n6/full/npp2014324a.htm

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids $\textit{in vivo}$ and demonstrate that ECOs self-organize into bile duct–like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded $\textit{in vitro}$.This work was funded by ERC starting grant Relieve IMDs (281335; L.V., N.R.F.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre (L.V., N.R.F.H., S. Sinha., F.S.), the Evelyn Trust (N.H.) and the EU FP7 grant TissuGEN (M.C.D.B.) and was supported in part by the Intramural Research Program of the NIH/NIAID (R.L.G., C.A.R.). F.S. has been supported by an Addenbrooke's Charitable Trust Clinical Research Training Fellowship and a joint MRC–Sparks Clinical Research Training Fellowship. (MR/L016761/1) A.W.J. and A.E.M. acknowledge support from EPSRC (EP/L504920/1) and an Engineering for Clinical Practice Grant from the Department of Engineering, University of Cambridge. J.B. was supported by a BHF Studentship (Grant FS/13/65/30441)