Hereditary cerebellar degenerations are a heterogeneous group of diseases often having a detrimental impact on patients’ quality of life. Unfortunately, no sufficiently effective causal therapy is available for human patients at present. There are several therapies that have been shown to affect the pathogenetic process and thereby to delay the progress of the disease in mouse models of cerebellar ataxias. The second experimental therapeutic approach for hereditary cerebellar ataxias is neurotransplantation. Grafted cells might provide an effect via delivery of a scarce neurotransmitter, substitution of lost cells if functionally integrated and rescue or trophic support of degenerating cells. The results of cerebellar transplantation research over the past 30 years are reviewed here and potential benefits and limitations of neurotransplantation therapy are discussed. Hereditary cerebellar ataxias are neurodegenerative diseases usually having a detrimental impact on patients’ quality of life or even leading to premature death. Patients suffer from a cerebellar syndrome, also combined in most of the hereditary ataxias with extracerebellar neurological symptoms, and in some cases other organ systems are also affected (for review see –5).Unfortunately, no sufficiently effective causal therapy is available for human patients at present . TheHereditary ataxias are heterogeneous from the point of view of the genetics, pathogenesis and manifestations at both morphological and functional level. In many of them, the molecular mechanisms of pathogenesis are not known. Thus, development of causal therapy is difficult and there might not be any universal approach applicable for all of them. Several experimental studies in laboratory animals have already succeeded in targeting pathogenetic processes, and neurotransplantation research has also shown some promising results .Intervention into the pathogenic process seems to be an optimal strategy since it could theoretically prevent or delay development of degeneration and improve the functionality of remaining cells, if started in time. Several experimental studies targeting pathogenic processes have been done in transgenic mouse models carrying the human pathological allele causing cerebellar degeneration. These studies have shown not only a potential direction for the development of a specific therapeutic method but also confirmed the role of particular mechanisms in pathogenesis of the diseases –15.In some hereditary cerebellar degenerations, a noxious effect of accumulated abnormal protein which is more resistant to proteolytic degradation is expected. Thus, silencing of the mutated gene’s expression might be helpful. In a mouse model of spinocerebellar ataxia type 3 (SCA3), halting the expression of ataxin-3 cleared nuclear accumulation of the abnormal protein in SCA3 mice . SilenciAnother possibility is to enhance elimination of the pathological protein. Stimulation of proteasome activity by Rho-kinase, promoting mutant ataxin-3 degradation, alleviated the neurological phenotype in SCA3 mice . Treatme2+) release channel, and sensitize it to activation [Mutant ataxin-2 and ataxin-3 with expanded polyglutamine tracts have been shown to interact specifically with type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular calcium  neural tissue, embryonic  or adult neural stem cells, embryonic stem cells, adult stem cells, mesenchymal stem cells isolated from various tissues , and carcinoma stem cells that are considered as dangerous for the risk of tumorigenesis. All these cells have some advantages and disadvantages dealing with biological as well as ethical aspects, which would become of much higher importance in humans. These various grafts have been transplanted into various cerebellar mutant mice and thus we have many but not all pieces of a large and diverse mosaic.Purkinje cell degeneration (pcd) mice suffering from almost complete loss of Purkinje cells, slowly progressive granule cell degeneration, moderate reduction of cerebellar nuclei and some extracerebellar disorders [isorders –29 have isorders –36 and Lisorders –41. In tisorders . Graftedisorders , 35, 38.isorders  reportedisorders . Carlettisorders  also fouGrafted Purkinje cells synaptically integrated within the host cerebellar cortex, having synaptic contacts like normal Purkinje cells but lacking basket fiber-pinceau formations around the initial segment of the axon , 33, 35.Bilateral transplantation of a fetal cerebellar cell suspension into the deep cerebellar nuclei partially restored corticonuclear projections in the cerebellum and improved motor coordination and increased locomotor activity of pcd mice , 41.Intravenous injection of bone marrow-derived cells at the age of 20 days led to reduction of the degenerative process in the olfactory bulb and improvement of olfaction in pcd mice, but did not change the degeneration of cerebellar Purkinje cells .Lurcher mutant mice lose virtually all Purkinje cells and their granule cells, other cerebellar cortex interneurons and inferior olive neurons are substantially reduced as a secondary consequence, while cerebellar nuclei are less affected [Grid2Lc mutation in the δ2 glutamate receptor (GluRδ2) encoding gene [Grid2Lc mutation is a gain of function mutation, which changes the receptor into a leaky membrane channel that chronically depolarizes the cells inducing their degeneration [affected –51. Priming gene . GluRδ2 ing gene . The Grineration .In these mice, embryonic cerebellar grafts have been also studied several times. Tomey and Heckroth , who graA significant improvement in performance on motor tests was observed by Jones et al.  after trWeaver mice carry the missense GrikWv mutation of the gene encoding a G-protein coupled with inward rectifying potassium channel [ channel . They su channel , 63. In  channel –66. Graf channel –66.In Weaver mutant mice, grafted cerebellar-derived multipotent astrocytic stem cells and embryonic stem cell-derived neural precursors were not capable of adopting region-specific cell identities, but the milieu of the granuloprival host cerebellum provided signals slightly supporting neuronal differentiation of donor cells .Nervous mutant mice are characterized by mitochondrial abnormalities and degeneration of Purkinje cells [je cells . The degje cells , 70. Li je cells  have shoje cells . The resje cells . Thus, ije cells .Meander tail mice are recessive mutants showing ankylosis or fusion of tail vertebrae [ertebrae  and defeertebrae , 75. Rosertebrae  grafted ertebrae . In thisertebrae .Harlequin mutant mice are a model of oxidative stress-mediated cerebellar degeneration and ataxia [d ataxia , 78. Humd ataxia . Furtherd ataxia .spinocerebellar ataxias (SCAs). In SCA1 mice, a transgenic mouse model of human spinocerebellar ataxia type 1, transplantation of an embryonic cerebellar cell suspension at the time of ataxia onset led to improvement in motor function as compared with sham-operated controls [Several strains of transgenic mice serve as models of particular human autosomal dominant controls . The gracontrols . Neverthcontrols .Chintawar et al.  grafted Intrathecal injection of mesenchymal stem cells mitigated cerebellar disorganization, suppressed atrophy of Purkinje cell dendrites and normalized motor coordination deficits in SCA1 mice .SCA2 transgenic mice. After intravenous injection, grafted cells were detected in the cerebellar white matter, molecular layer, in the lumen of blood vessels in the white matter and in the cerebral cortex [Chang et al.  investigl cortex . In micel cortex . In the l cortex .SCA3 (Machado-Joseph disease) is a polyglutamine disorder in pathogenesis of which inflammatory processes play a role [y a role . In a moy a role . Graftedy a role . The trey a role . The tray a role . Furthery a role .Niemann-Pick disease is an autosomal recessive lysosomal storage disease with accumulation of sphingomyelin in many tissues. Niemann-Pick disease type C is a defect of sphingomyelin transport and typically affects the cerebellum. Niemann-Pick disease types A and B are caused by defects of sphingomyelinase enzyme. Unlike human patients, Niemann-Pick type A mice suffer from progressive degeneration of Purkinje cells in the cerebellum and develop ataxia [p ataxia .In a mouse model of Niemann-Pick disease type C, therapy with mesenchymal stem cells has been tested with promising results –89. BoneIn a mouse model of Niemann-Pick disease, bone marrow-derived cells were also used for ex vivo gene therapy . Normal Injection of neural stem cells into the cerebellum of Niemann-Pick disease type C1 mice lengthened their life span, but did not influence the decline of motor performance as measured by the rotarod test . Similardelivery of a scarce neurotransmitter. The most successful neurotransplantation therapy used in human patients is that for Parkinson’s disease. Rossi and Cattaneo [substantia nigra are grafted directly onto the striatum which is the target structure for the dopaminergic projection, and that paracrine release of dopamine is sufficient to restore function without the need to reestablish specific cell-to-cell projections. Furthermore, fetal substantia nigra directly provides the desired cell phenotype. Thus the nature of Parkinson’s disease represents an optimal situation to be solved by substitution of lost neurons since the task of the graft is just to deliver an adequate amount of a neurotransmitter, in this case dopamine.Experiments in models of neurological diseases including cerebellar degeneration have shown that grafts can employ their effects via various mechanisms. One relatively simple way is Cattaneo  pointed complete substitution of lost cells and precise reconnection of neural circuitry by grafted cells would be optimal, since it would completely restore normal structure and function. However, this is probably the most difficult endpoint to achieve since it requires sufficient numbers of surviving grafted cells, migration into appropriate positions, differentiation into appropriate cell types, fiber sprouting and establishment of precise cell-to-cell connections . The ceview see . A good view see , 80, 94.view see , 81, 86 view see . The difview see  showed tview see . On the view see  . The taThe most effective source of cells having determined specific cerebellar phenotypes is embryonic cerebellar tissue. These cells have to be harvested from embryos and they cannot be expanded in vitro to sufficient amounts. Thus, obtaining grafts for clinical use would be problematic from an ethical point of view as well as that of quality and quantity. Nevertheless, it has been shown that induction of differentiation of embryonic stem cells into cerebellar Purkinje cells and granule cell precursors is also possible . Since lBeyond the investigation of therapeutic approaches for neurological diseases, neurotransplantation also remains a tool for studying the mechanisms and factors playing a role in brain development, cell differentiation and migration, and neural plasticity and regeneration , 113.


Radix as model group and the Lake Donggi Cona drainage system, located in the north-eastern part of the plateau, as model site. First, we performed plateau-wide phylogenetic analyses using mtDNA data to assess the overall relationships of Radix populations inhabiting the Lake Donggi Cona system for revealing refugial lineages. We then conducted regional phylogeographical analyses applying a combination of mtDNA and nuclear AFLP markers to infer the local structure and demographic history of the most abundant endemic Radix clade for identifying location and type of (sub-)refugia within the drainage system. Our phylogenetic analysis showed a high diversity of Radix lineages in the Lake Donggi Cona system. Subsequent phylogeographical analyses of the most abundant endemic clade indicated a habitat-related clustering of genotypes and several Late Pleistocene spatial/demographic expansion events. The most parsimonious explanation for these patterns would be a scenario of an intra-plateau glacial refugium in the Lake Donggi Cona drainage system, which might have consisted of isolated sub-refugia. Though the underlying processes remain unknown, an initial separation of lake and watershed populations could have been triggered by lake-level fluctuations before and during the Last Glacial Maximum. This study inferred the first intra-plateau refugium for freshwater animals on the Tibetan Plateau. It thus sheds new light on the evolutionary history of its endemic taxa and provides important insights into the complex refugial history of a high-altitude ecosystem.Although only relatively few freshwater invertebrate families are reported from the Tibetan Plateau, the degree of endemism may be high. Many endemic lineages occur within permafrost areas, raising questions about the existence of isolated intra-plateau glacial refugia. Moreover, if such refugia existed, it might be instructive to learn whether they were associated with lakes or with more dynamic ecosystems such as ponds, wetlands, or springs. To study these hypotheses, we used pulmonate snails of the plateau-wide distributed genus  Until recently, the freshwater diversity of the Tibetan Plateau and adjacent areas remained poorly understood. However, a number of phylogenetic and biogeographical studies, conducted in the past years, provided exciting new insights into the evolutionary history of plateau freshwater biota , particuGammarus [Gyraulus [Radix [Studies on Tibetan Plateau invertebrates, for example, have shown that taxa with a high passive dispersal capacity, such as amphipods of the genus Gammarus  and bivaGammarus , appear Gyraulus  and Radis [Radix , the degs [Radix ,7, 25–15s [Radix .This biogeographical pattern–the occurrence of endemic lineages within permafrost areas–parallels patterns in other Palearctic regions that are discussed within the concept of ‘northern glacial refugia’ –17. AccoHowever, it remains unknown whether potential intra-plateau refugia were associated with lakes or peripheral, highly dynamic systems. It is even conceivable that a complex system of sub-refugia, i.e., ‘refugia within refugia’ , may havTesting these scenarios is non-trivial as it requires a model study area that fulfills several criteria: i) it should provide suitable freshwater habitats , ii) it should be limnologically diverse , and iii) it should be accessible for researchers allowing a reasonable sampling. One of the few freshwater systems on the Tibetan Plateau, satisfying all of these criteria, is the Lake Donggi Cona drainage system –23, locaRadix populations from different habitats and locations scattered throughout the Lake Donggi Cona drainage system in order to infer the refugial history of this highly diverse and widespread group of plateau gastropods. Our working hypothesis is that the actual lake has served as a freshwater refugium that allowed both the intralacustrine survival of gastropod populations during glaciation events and the re-colonization of the drainage system after the LGM.In the present study, we examined Based on a combination of mitochondrial DNA (mtDNA) data, suitable for addressing older phylogenetic events, as well as genome-wide nuclear DNA (nuDNA) fingerprinting data, reflecting younger phylogeographical and demographic events, we conducted a hierarchical set of analyses.Radix populations inhabiting the Lake Donggi Cona system in a plateau-wide context to identify potential refugial lineages.We first performed phylogenetic analyses based on mtDNA data to assess the overall relationships of Radix lineage in the area to infer location and type of (sub-)refugia within the Lake Donggi Cona system.We then conducted phylogeographical analyses  using a combination of mtDNA and nuDNA data to assess the local structure and demographic history of the most abundant endemic Our study might not only help biogeographers and evolutionary biologists to better understand the evolution and Pleistocene history of Tibetan Plateau freshwater invertebrates . It might also provide important insights into the stability and complex refugial structure of isolated high-altitude ecosystems.2 [2 [The Lake Donggi Cona drainage system .In October 2008 and 2011, specimens of the freshwater gastropod genus ters see . Seven sc oxidase subunit I (COI) gene fragment, the forward primer LCO1490 [Genomic DNA was extracted using the protocol described by Winnepenninckx et al. . For the LCO1490  and the  LCO1490  were useAFLP genotyping was performed for a subset of 91 specimens belonging to the most abundant endemic watershed lineage see , Fig 1, 2O, 100x BSA, 10x Ligase buffer, EcoRI and MseI adapter , MseI (4 u/μl) and EcoRI (20 u/μl), 400 u/μl T4 ligase, and template DNA (~50 ng).Digestion/ligation reaction; carried out in a total reaction volume of 10 μl containing ddH2O, 10x ThermoPol buffer, dNTPs (2.0 mM each), pre-selective primers , Taq polymerase (5 u/μl), fluorescent labeled EcoRI primers (1 μM each), selective MseI primers , with bands recognized as present (1) or absent (0). Only bands with a length ranging from 100–500 bp and occurring in at least two samples were used to reduce homoplasy and to increase reproducibility .After AFLP genotyping, we tested the reproducibility of our analyses. For this, ~10% of the samples were randomly selected and reanalyzed (steps 1 to 4), using fresh reagents as suggested by Meudt and Clarke .Radix populations in a plateau-wide context for identifying potential refugial lineages, we complemented our COI data set with data available from GenBank for the Tibetan Plateau, the adjacent Himalaya mountain range, and several locations in Africa and Eurasia. The final dataset comprised 308 Radix specimens (120 unique haplotypes) as well as two outgroup taxa: Planorbarius corneus (GenBank accession number AY282590) and Physa fontinalis (EU818796).To assess the overall relationships of Lake Donggi Cona Prior to subsequent analyses, the GTR + I + Γ model was inferred as best-fit substitution model based on the Akaike information criterion using jModelTest v2.1.4 . The COIThe phylogenetic reconstruction was done in MrBayes v3.2.2  using twFor subsequent phylogeographical analyses, only specimens were used that belonged to the most abundant endemic Lake Donggi Cona clade  would have to be rejected [Radix-specific substitution rate for the COI gene, we used the published trait-specific COI Protostomia molecular clock rate (μ) of 0.0122  substitutions per site and one million years for the Jukes-Cantor model [-1 . To accK) ranging from two to twelve. As previously suggested [K values (ΔK). The lowest K value with the highest likelihood was then selected and the respective STRUCTURE outputs were averaged with CLUMPP v1.1.1 [A NeighborNet analysis of the AFLP dataset was conducted with the software tool SplitsTree4 v4.13.1 . In ordeuggested , the onluggested  was usedP v1.1.1  using a P v1.1.1 .Radix specimens belonging to three distinct clades  including the Tibetan Plateau. Within this non-endemic clade, specimens from the Lake Donggi Cona system formed a highly supported sub-clade (BPP = 1.0) together with other plateau specimens. Clade 9 (BPP = 1.0) exclusively contained specimens form the plateau. Within this endemic clade, specimens from the Lake Donggi Cona system build a sub-clade supported by a BPP of 1.0. Finally, endemic clade 13 contained a single haplotype shared by three specimens from the Lake Donggi Cona system (BPP support for the split with the sister group is 0.96).The Bayesian inference analysis of 310 specimens revealed that the Lake Donggi Cona drainage system harbors Lake Donggi Cona snails belonging to the non-endemic clade 1 occurred in 7 out of 20 locations (35%), while the endemic clade 13 was only found in a single location (5%). Most specimens clustered within the endemic clade 9 and originated from a total of 17 locations (85%).The TCS network analysis of the COI dataset for clade 9 (123 specimens) resulted in a single network with 18 haplotypes separated from each other by a maximum of nine mutational steps see . All hapThe AMOVA revealed that 64% of the total molecular variation is distributed among and 21% within the two sub-clusters (lake vs. drainage system); about 15% of variation was found within populations see .p-values below 0.05 . The AFLP NeighborNet analysis of 91 specimens revealed some degree of structuring of lake vs. drainage system populations see . HoweverThe AMOVA showed that 8% of the total molecular variation was distributed among groups (lake vs. drainage system), 20% within groups, and 72% within populations see . The sofRadix populations from the Lake Donggi Cona drainage system in order to unravel the refugial history of plateau freshwater organisms. Our working hypothesis was that the actual lake served as a freshwater refugium, supporting the survival of gastropod populations during glacial phases and enabling the re-colonization of the drainage system after the LGM.In this study we examined Radix populations from the Lake Donggi Cona system with other plateau and non-plateau populations for identifying potential refugial lineages. Then, we performed phylogeographical analyses based on mtDNA and nuDNA data, using specimens of the most common refugial clade  to assess their population structure and demographic history. These data, in turn, might be informative for inferring location and type of potential sub-refugia within the drainage system.Specifically, we conducted phylogenetic analyses based on mtDNA data to assess the relationships of Radix spp. in the Lake Donggi Cona system is surprisingly high and that the respective specimens belong to three well-supported, unrelated clades . Moreover, the degrees of plateau endemicity and even narrow-range endemicity  are significant. Within clade 1, Lake Donggi Cona specimens form a highly supported endemic sub-clade together with other plateau specimens .Radix clades, on the one hand, and the considerable genetic diversity within populations, on the other hand, found in the current study are not consistent with a scenario of post-glacial recolonization of the Lake Donggi Cona drainage system. Although these patterns, in theory, could also have resulted from multiple colonization events from different areas and/or during different times [Radix populations indicative of local adaptation (see Radix specimens originating from different areas outside the plateau (for more information on the timing of demographic events see the section “Location and type of (sub-)refugia within the Lake Donggi Cona system” below).Therefore, the high divergence and phylogenetic distinctness of local nt times –57, thistion see  and 3. TRana kukunoris [Unfortunately, it remains challenging to discuss these findings in a comparative context due to the low number of plateau-wide distributed freshwater species and the virtual absence of respective phylogeographical data. Therefore, our current understanding of the refugial history of the Tibetan Plateau is largely based on data from terrestrial species ,59. Mostukunoris  may haveComparing these findings with the results of the current study, we suggest that the Lake Donggi Cona drainage system might be the first intra-plateau refugium inferred for freshwater animals, in general, and for invertebrates, in particular. Moreover, the survival of the LGM in a region where glaciers might have descended to altitudes as low as 3,500 m a.s.l.  indicateRadix clade in order to test for location and type of sub-refugia (lake vs. drainage). Our working hypothesis was that the actual lake served as a freshwater refugium. Accordingly, we would have expected to observe i) a higher genetic diversity among lake populations, ii) no clear structuring of lake vs. drainage populations, and iii) possibly derived haplotypes in the drainage system.Phylogeographical analyses were performed for specimens of the most abundant endemic Lake Donggi Cona However, the phylogeographical patterns inferred are not consistent with these operational criteria. The mtDNA data resulted in a network in which lake and drainage populations form distinct sub-clusters. Though the number of mutational steps between the sub-clusters is low, there are no shared haplotypes among them . This seThese patterns of distinct phylogeographical histories of lake vs. drainage populations are, in principle, confirmed by our nuDNA data, although the AFLP-based structures appear to be less pronounced. In the NeighborNet , some drHowever, the differences between the patterns inferred from the mtDNA and nuDNA datasets are not surprising as the highly variable AFLP data (which target variable non-coding regions with a high substitution rate) typically reflect more recent evolutionary processes compared to mtDNA data . One parRadix populations during the last glaciations. Though the underlying processes remain unknown, an initial Pleistocene separation of lake and watershed populations could have been triggered by strong lake-level fluctuations  before and during the LGM, likely caused by a desiccation of major Lake Donggi Cona inflows [Although the mtDNA and nuDNA pattern are not fully in concordance, both suggest a relative distinctness of lake vs. drainage populations. Therefore, our working hypothesis that the actual lake served as (the sole) refugium has to be rejected. Instead, the patterns inferred might be better explained by a scenario in which the lake and the surrounding drainage system provided isolated sub-refugia  for Rad inflows . These eRadix populations from the Lake Donggi Cona drainage system may have continued after the LGM. It also suggests that an initial system of Pleistocene sub-refugia has resulted in complex patterns of biodiversity potentially driven by adaptation and isolation processes.On first sight, the AFLP data  may suggest a ‘leveling-out’ of an initial vicariance-driven structure of lake vs. drainage populations as seen in the mtDNA data. These changes over time could have been caused by, for example, increasing gene flow (see the relatively low among-group variation in the AMOVA of the AFLP data). However, a closer look at the NeighborNet, and particularly at the STRUCTURE outputs Figs  and 4, mRadix and thus belongs to the most comprehensive analyses of plateau-wide distributed animals. Nonetheless, we have to note three limitations of our approach. First, the reproducibility of the AFLP data is relatively low (see above). This might be related to the sub-optimal sampling and preservation conditions during expeditions to the plateau. In addition, our highly conserved test for reproducibility  may havreagents . OverallRadix spp. belong to the best-studied freshwater gastropods worldwide, little is known about their occurrences north and northeast of the plateau . In ourwork see  do not sRadix-specific substitution rates for the mismatch analyses. As selfing frequently occurs in Radix, it is not yet clear whether commonly accepted substitution rates for (dioeceous) animals [Radix specimens in our phylogenetic analysis refugia, in particular, for generating the exiting patterns of endemic biodiversity seen on the ‘Roof of the World’.Radix individuals belonging to three well-supported, unrelated clades . These clades either consist exclusively of Lake Donggi Cona narrow-range endemics or Lake Donggi Cona specimens form sub-clades of endemics/narrow-range endemics. The most parsimonious explanation for these patterns involves a scenario of an intra-plateau refugium in the Lake Donggi Cona drainage system. This assumption is confirmed by detailed phylogeographical and demographic studies of Radix specimens that belong to the most abundant endemic Radix clade. Within this clade, the mtDNA network analysis indicates distinct clusters of lake vs. drainage populations. These findings are also supported by the AMOVA and by the results of the mismatch distribution analyses, which suggested distinct spatial extension events in both group during the Late Pleistocene. These results are not consistent with the operational criteria for our working hypothesis. Therefore, the hypothesis assuming that Lake Donggi Cona served as freshwater refugium, has to be rejected. Instead, a complex system of sub-refugia, i.e., ‘refugia within refugia’, both within the lake and the surrounding watershed is assumed.Our comparative phylogenetic analysis showed that the Lake Donggi Cona system is inhabited by Radix populations form the Lake Donggi Cona drainage system may have continued after the LGM, resulting in the complex patterns of biodiversity that can be observed today.These findings are largely confirmed by our AFLP data, although the latter show a more complex picture. In the NeighborNet, some drainage populations clustered within the group of lake populations and the variance between lake and drainage population in the AMOVA, although significant, was relatively low. Moreover, the STRUCTURE analysis did not suggest two (lake vs. drainage populations) but eight genetic clusters with a considerable degree of fine-scale structure, particularly among drainage populations. A possible explanation would be that a Pleistocene vicariance event, potentially driven by strong lake-level fluctuations, caused an initial separation of lake and watershed populations. However, isolation processes in This study inferred the first intra-plateau refugium for freshwater animals on the Tibetan Plateau and thus sheds new light on the evolutionary history of its endemic taxa. We are confident that future research will likely unravel glacial refugia for other plateau freshwater animals and in other isolated intra-plateau areas. This will subsequently help to obtain a more comprehensive picture about evolutionary processes on the ‘Roof of the World’.S1 FigRadix spp. from the Lake Donggi Cona drainage system; an asterisk indicates Tibetan Plateau endemic clades. Bayesian posterior probabilities (BPP) are given next to the respective node when BPP were higher than 0.5. The scale bar represents substitutions per side according to the applied model of sequence evolution.Specimens inhabiting the Lake Donggi Cona drainage system are highlighted in bold and are labeled with haplotype codes (for details see (TIF)Click here for additional data file.S1 TableIt includes haplotype number, system , geographical coordinates, haplotype code, clade number according to the phylogenetic analysis, DNA voucher number, and GenBank accession numbers. Specimens used for the AFLP analyses are marked with √.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Differential diagnosis of severe progressive dementia includes a wide spectrum of inflammatory and neurodegenerative diseases. Particularly challenging is the differentiation of potentially treatable autoimmune encephalitis and Creutzfeldt-Jakob disease. Such a coincidence may indeed complicate the correct diagnosis and influence subsequent treatment.A 75-year-old woman was admitted due to rapid progressive cognitive impairment. Her husband observed a temporal disorientation and confusion. The initial neurological examination and an extensive neuropsychological evaluation showed significant impairments in almost all tested cognitive domains. All other neurological functions including motor, sensory and coordinative function were intact. Initial diagnostics included EEG, MRI and lumbar puncture with unspecific results. Complementary blood testing revealed a positive result for antineural antibodies to Contactin-associated protein 2 (CASPR2) and the patient received treatment for CASPR2 autoimmune encephalitis. Further symptoms and results, including 14-3-3 proteins, led to suspected Creutzfeldt-Jakob disease. The postmortem examination supported the diagnosis of a definitive Creutzfeldt-Jakob disease.One could argue that global screening for antineural antibodies may lead to a false diagnosis triggering intense and potentially dangerous procedures. We believe, however, that potentially treatable causes of dementia should aggressively sought out and subsequently treated in an attempt to curtail the course of disease and ultimately reduce the rate of mortality. Differential diagnosis of severe progressive dementia includes a wide spectrum of inflammatory and neurodegenerative diseases. Particularly challenging is the differentiation of potentially treatable autoimmune encephalitis and Creutzfeldt-Jakob disease (CJD). Such a coincidence may indeed complicate the correct diagnosis and influence subsequent treatment. The diseases can mimic one another to such an extent that one is led to believe that the patient is suffering from CJD when he or she has in fact a treatable autoimmune encephalitis -3 or vicContactin-associated protein 2-Antibody (CASPR2) production during ongoing CJD, discuss the potential biological causes and the clinical consequences.We here report a recent case of A 75-year-old woman was admitted to our hospital due to rapid progressive cognitive impairment. During the previous year, the patient had shown mild cognitive impairment due to moderate leukoariosis, thought to be associated with arterial hypertension and hypercholesterolemia. There was no positive family history of dementia or dementia-like symptoms in the anamnesis. Ten days before admission, her husband observed a temporal disorientation and confusion, e.g. the patient could not recall the present date and put a saltshaker into the refrigerator. When admitted to our care the patient was conscious but disorientated to place, time and person. The initial neurological examination and an extensive neuropsychological evaluation showed significant impairments in almost all tested cognitive domains including attention, concentration, memory, executive function and visual-constructional ability. In addition, there was evidence of a right-sided visual neglect, aphasia in terms of language comprehension disorder and pronounced apraxic impairments corresponding to dysfunctions of her left-sided parietal circuits. All other neurological functions including motor, sensory and coordinative function were intact. An initial electroencephalography showed unspecific encephalopathy patterns. The MRI showed multiple microangiopathic lesions: left-sided lesions in the thalamus, parietooccipital, temporo mesial, thalamic, frontal and parietal cortices, as well as right-sided lesions in the basal ganglia. The brain-SPECT showed hypometabolism in the frontoparietal and parietooccipital cortices, more obvious on the left side, with normal nuclide accumulation in motor and occipital cortex. The primary investigation of cerebrospinal fluid revealed a pleocytosis of 7 Leukocytes/μl [<5 Leukocytes/μl] with a Total Protein of 701 mg/l [<450 mg/l] and 2,31 mmol/l Lactate . Simultaneously, thyroperoxidase antibodies (serum titre 1606 IU/ml [<60 IU/ml]) were detected. It was initially concluded based on these results that the patient was suffering from autoimmune encephalitis, believed to be caused by autoimmune thyroiditis. High dosage intravenous methylprednisolone therapy was initiated . The patA rapidly progressive dementia with symptoms such as disorientation, apraxia, aphasia, extrapyramidal dysfunction, and psychiatric symptoms caused by CJD may mimic treatable types of autoimmune encephalitis. As shown in the present case, thyroperoxidase- and CASPR2-antibodies known to be associated with cognitive and neuropsychological symptoms , can accOur findings expand upon prior reports in which VGKC complex antibodies were shown to be associated with CJD. In previous studies, the patient had not been tested for CASPR2 or LG1  and the Another case series reported about a 68-year-old patient with sporadic CJD tested positive for serum antibodies to VGKC and GlyR antibodies but negative for CASPR2 and LG1 antibodies .The biological cause of this molecular mimicry is currently unknown and deserves further investigation. One may hypothesize that prion-induced neurodegeneration may have triggered an immunological reaction in peripheral lymph nodes causing antibody production against certain neuronal structures such as elements of voltage gated potassium channels.With the diagnosis still unconfirmed, we chose to initiate therapy for the potentially treatable differential diagnosis. One could argue that global screening for antineural antibodies led us to a false diagnosis triggering intense and potentially dangerous procedures. We believe, however, that potentially treatable causes of dementia should be aggressively sought out and subsequently treated to avoid poor outcome. In fact, encephalitic syndromes are treatable conditions that may mimic CJD ,3.The development of a CJD case into a progressive and untreatable encephalopathy, as presented here, is certainly very rare. Nevertheless, such unique cases are important as they present new opportunities to study the complex role of the immune system in the CNS.Written informed consent was obtained from the legal guardian of the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Individuals with high-trait anxiety frequently report decreased perceived control. However, it is unclear how these processes are instantiated at a neural level. Prior research suggests that individuals prone to anxiety may have exaggerated activity in the anterior insula and altered activity in the cingulate cortex during anticipation of aversive events. Thus, we hypothesized that anxiety proneness influences anterior insula activation during anticipation of unpredictable threat through decreased perceived control. Forty physically healthy adults underwent neuroimaging while they explored computer-simulated contexts associated either with or without the threat of an unpredictable shock. Skin conductance, anxiety ratings and blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging were used to assess responses to threat versus no threat. Perceived control was measured using the Anxiety Control Questionnaire-Revised. Mediation analysis examined how anxiety proneness influenced BOLD activity. Anticipation of unpredictable threat resulted in increased skin conductance responses, anxiety ratings and enhanced activation in bilateral insula, anterior midcingulate cortex (aMCC) and bed nucleus of the stria terminalis. Individuals with greater anxiety proneness and less perceived control showed greater activity in dorsal anterior insula (dAI). Perceived control mediated the relationship between anxiety proneness and dAI activity. Increased dAI activity was associated with increased activity in aMCC, which correlated with increased exploratory behavior. Results provide evidence that exaggerated insula activation during the threat of unpredictable shock is directly related to low perceived control in anxiety-prone individuals. Perceived control thus may constitute an important treatment target to modulate insula activity during anxious anticipation in anxiety-disordered individuals. Perceived control over aversive events is an important psychological construct that links a specific cognitive schema to an experienced emotion,2 and may be a possible mediator between high-trait anxiety and exaggerated neural processing of emotionally significant events. Theories of psychopathology posit that a lack of perceived control is central to the etiology and maintenance of negative affect and emotional disorders.4 Supporting this view, the results of a recent meta-analysis suggested that reduced perceptions of control are associated with greater trait anxiety in both nonclinical and clinical samples, particularly in healthy adults and in individuals with generalized anxiety disorder.5 In a related study, treatment-seeking individuals with generalized anxiety disorder, panic disorder, social phobia and obsessive-compulsive disorder who initiated cognitive-behavioral therapy, subsequently reported large increases in perceived control and greater reductions in anxiety symptoms compared with untreated individuals, suggesting that perceived control might predict recovery from some anxiety disorders following cognitive-behavioral therapy.6 Elucidating whether anxiety proneness affects neural systems indirectly through perceived control conceivably may facilitate the development of more effective interventions capable of enhancing perceptions of control and reducing the detrimental effects of diminished perceived control.Trait anxiety, a predisposition to respond anxiously, has been linked to dysregulation of mood and anxiety,8 loud sounds,9 monetary loss,10 thermal stimuli,11 electric stimuli12 and aversive unconditioned stimuli predicted by either cue or context conditioned stimuli.14 Although these prior reports suggest that the insula is broadly involved in anxious anticipation, studies that have compared anticipation of temporally predictable and unpredictable aversive events indicate that activity in anterior insula is heightened during anticipation of unpredictable aversive events compared with predictable aversive events.16 In addition, studies that differentiated sustained from transient anticipatory processing found that activity in anterior insula is particularly enhanced during sustained versus transient anticipation of aversive events.19 These findings strongly suggest that anterior insula has an important role in the anticipation of aversive events, especially sustained anticipation of unpredictable aversive events.Functional neuroimaging data obtained in nonclinical subjects have suggested that insular cortex may be critically involved in the anticipation of aversive events, including negatively valenced pictures,24 For example, individuals with major depressive disorder show enhanced activity in right anterior insula during the anticipation of painful temperature stimuli compared with healthy controls.26 Compared with non-traumatized controls, women with a history of intimate partner violence and consequent posttraumatic stress disorder show greater hemodynamic activity in right anterior insula during the anticipation of negative images.27 Similarly, compared with combat-exposed controls, combat veterans with posttraumatic stress disorder show increased activation in right anterior insula during the anticipation of negative combat-related images presented unpredictably.28 The magnitude of right anterior insula activity in response to unpredictably presented versus predictably presented images was associated with posttraumatic stress disorder severity, such that right anterior insula activity is heightened in combat veterans with more severe posttraumatic stress disorder. Moreover, in the combat-exposed healthy controls from this study, activity in right anterior insula was associated with greater perceived threat. Several studies also report that nonclinical subjects with high trait anxiety compared with those having low-trait anxiety show enhanced activation in anterior insula during emotional processing and the anticipation of aversive images.31 These results indicate that individuals with dysregulated mood or anxiety manifest altered activity in anterior insula during the anticipation of aversive events.Notably, evidence of altered insula activation is also emerging as a key finding in individuals with dysregulated mood or anxiety.32 or have examined anxious anticipation of threat, that is, aversive events,18 frequently report concomitant activation in the anterior insula and the supracallosal portion of the anterior cingulate gyrus referred to as the midcingulate cortex (MCC).34 While much remains to be clarified about the functional relationships between these regions, their joint activation during the anticipation of threat is supported by the substantial neuroanatomical connections extant between anterior insula and the anterior MCC (aMCC).36 These regions are emerging as key nodes in the ‘salience network', a brain system thought to have a central role in the detection of behaviorally relevant stimuli and the coordination of adaptive responses.38Studies that have compared individuals with high-trait and low-trait anxiety during decision making,42 consistent with anatomical studies in monkeys.43 Converging evidence from these studies suggest that anterior insula consists of a dorsal anterior subregion that is relatively more involved in cognitive processes and a ventral anterior subregion more involved in affective processes. On the basis of this research and the presumption that perceived control is primarily a cognitive process, we hypothesized that any influence of perceived control on insula activity would specifically involve dorsal anterior insula. We therefore examined whether anxiety proneness affects the anterior insula through diminished perceived control during anxious anticipation. We used blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI), electrodermal recording and anxiety ratings to explore the relationships between perceived control and the brain responses, skin conductance and subjective anxiety elicited during the anticipation of unpredictable threat (AUT). Using mediation analysis, we tested the hypothesis that anxiety proneness specifically influences BOLD activity in the dorsal anterior insula indirectly through perceived control. Correlation analyses were applied to examine the relationship between brain activation and behavioral measures, and to explore the functional relationship between dorsal anterior insula and MCC. Finally, because the bed nucleus of the stria terminalis (BNST) has been implicated in modulating sustained anxiety responses analogous to those elicited herein to a context in which an aversive event may occur that otherwise is unpredicted by any sensory cue,44 and in preclinical and clinical studies of the functional anatomy of sustained apprehensive states,45 we also examined activity in the BNST. The main results suggest that the link between anxiety proneness and activity in the dorsal anterior insula is mediated by reduced perceptions of control.Recent meta-analyses examining the functional organization of the insula indicate that anterior insula consists of important functional subdivisions in humans2 also were excluded from participation to minimize difficulty obtaining electromyographic recordings during individual threshold testing before scanning. All the subjects received monetary compensation for their participation.Forty physically healthy volunteers  between the ages of 18 and 50 years were recruited  to participate in fMRI while performing an ‘anticipation of unpredictable threat' task. All the subjects provided informed consent according to procedures approved by the Western Institutional Review Board, and underwent a Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I) performed by a Master's-level clinician with experience and training in the administration of the SCID-I interview. Exclusion criteria included past or current psychiatric disorders as per SCID-I, major neurological or medical disorders, substance abuse, current pregnancy and exposure to psychotropic medications or other drugs likely to influence cerebral function or blood flow within 3 weeks. Subjects with a body mass index >35 kg /m46 Participants also completed the ACQ-R (Anxiety Control Questionnaire-Revised), a 15-item self-report measure of perceived control over aversive events,4 the Quick Inventory of Depressive Symptomatology-C47 and the Inventory of Depression and Anxiety Symptoms-I.48 The Pain Anxiety Symptoms Scale-20, a 20-item self-report measure of fear and anxiety of pain49 and the Pain Catastrophizing Scale, a 13-item measure of the tendency to catastrophize during painful situations50 also were included. Handedness was assessed using the Edinburgh Handedness Inventory.51Participants completed the STAI (State-Trait Anxiety Inventory), Form Y, a 40-item self-report measure of both trait anxiety (STAI-T) assessing individual differences in anxiety proneness, and state anxiety (STAI-S) assessing feelings of apprehension in response to current circumstances.The stimuli for the scanner task consisted of two computer-simulated rooms generated by a customized software application  during scanning. Following threshold testing, participants practiced using the response pad to navigate the task contexts for 2 min in a full-scale MRI simulator (mock scanner) with no threat of receiving stimulation. Because the present report focuses on SCRs and ratings collected only during task scans, we only briefly describe the overall scanning procedure here. During scanning, participants wore a respiration belt over the abdomen and a nasal cannula at the tip of the nose to monitor breathing, and a finger pulse oximeter to measure heart rate. To minimize unwanted movement in response to electric stimulations, participants wore velcro straps over the hips and left ankle that could be easily fastened and unfastened as needed. Once participants were positioned in the MRI scanner with stabilizing head cushions in place, scanning began with structural and resting-state fMRI scans. Participants then underwent a 4-min practice scan during which they explored the task contexts according to task instructions including receiving one electric stimulation in the threat context. Immediately following the practice scan, participants completed a contingency awareness test in which they confirmed accurate understanding of which context was associated with threat and which was not. The AUT task then commenced.Before scanning, participants were informed that during the task they were to virtually explore two computer-simulated rooms projected onto a screen in the scanner, and that later they would be asked what they learned about each room. Half the participants were told that whenever they were in the ‘purple room' they could receive a stimulation on the ankle at any time, and that whenever they were in the ‘peach room' they would never receive a stimulation; the other half were given the opposite instructions. During each of the four fMRI scans, five threat and five safe contexts were each semi-randomly presented for a duration of 18 s followed by an interstimulus interval of 14–18 s. Order of scan presentation  was counterbalanced across participants. An unpredictable  electrical stimulation served as an unconditioned stimulus (US) and was delivered during one to two threat contexts each fMRI scan for a total of five unconditioned stimuli . No US was administered during the safe context. During the interstimulus interval, participants performed a low-level vigilance task in which they fixated on a central plus sign and pressed a button on the response pad anytime it changed color . Following each threat context in which an electric stimulus was administered, participants rated the intensity of the stimulus received on a scale from 0 to 100 presented during the interstimulus interval. Starting from the left, the scale was labeled 0 (no sensation), 25 (uncomfortable), 50 (painful), 75 (very painful) and 100 (maximum tolerable). Participants were allowed 7 s to register an intensity rating.13 the AUT task involves explicit verbal instruction to participants from the outset about which context is associated with threat and which is not. Therefore, it was expected that throughout the task, participants would engage in sustained AUT during the threat context compared with the safe context.At the conclusion of each fMRI scan, which lasted 350 s, participants retrospectively rated how fearful they were in the threat and safe contexts using a 0 to 100 scale. Starting from the left, the scale was labeled 0 (no fear), 25 (mild fear), 50 (moderate fear), 75 (strong fear) and 100 (extreme fear). Participants were under no time limit when registering anxiety ratings. Although the AUT task shares similarities with unsignaled contextual fear conditioning, which involves subjects gradually learning context-US associations through direct experience,www.scralyze.sourceforge.net).53 For details on threshold testing and the assessment of SCRs, see The US during the AUT task was delivered with a Digitimer DS7A  constant current stimulator triggered by a presentation computer and waveform generator . Stimulus intensity was determined before scanning during individual threshold testing. The maximum intensity of stimulation was set at 40 mA. The electric stimulus was delivered via surface electrodes attached to the skin over the left ankle to minimize the amount of head movement during scanning. During each fMRI scan, SCRs were obtained using a Biopac Systems electrodermal activity module. Offline data analysis of SCRs was performed using a model-based general linear model with SCRalyze software .Imaging experiments were conducted on a Discovery MR750 3 Tesla MRI scanner . A receive-only 32-element head array coil  optimized for parallel imaging was used for MRI signal reception. The size and location of the BNST, like other small structures,http://afni.nimh.nih.gov/afni) and spatial alignment of functional images was performed using Advanced Normalization Tools (http://stnava.github.io/ANTs/) to optimize spatial alignment of functional data to the TT_N27 T1-weighted template  and the time span of the BOLD response following context offset. For the contrast of threat context versus safe context, the voxel-wise analysis included only regressors for the 10 time points (0–18 s) spanning each context. The first time point (at 0 s) was assumed to have zero magnitude to account for the expected delay in the BOLD response to context onset. The time points following context offset were treated as regressors of non-interest.Functional image preprocessing and analysis was performed using AFNI , thresholded per voxel at P<0.0001. The group sample size was chosen to ensure adequate power to detect effects using these relatively conservative threshold levels.56The behavioral and demographic analyses were carried out using SPSS . Paired-samples The STAI-T and ACQ-R were used to conduct correlation analyses examining the relationships among anxiety proneness, perceived control and anterior insula activation during the AUT. For these analyses, the Pearson correlation coefficient was computed using total scores and insula activity was extracted from dorsal (dAI) and ventral (vAI) subregions of anterior insula. More specifically, the average beta values for threat context versus safe context were extracted per individual from dAI and vAI subregions defined by 3-mm-radius spheres placed at the voxel coordinates showing peak activity in the group-level statistical map. The volume of these spheres was selected to balance between preserving the precision of anatomical localization enabled by our high spatial resolution fMRI methods, while also accommodating a modest amount of anatomical variability across subjects. Data from the left and right hemispheres of each subregion were combined to create dAI and vAI subregions, respectively. Therefore, activity in dAI and vAI subregions, respectively, reflect bilateral activation during the AUT.Additional correlation analyses were performed to explore the relationship between activity in dAI and aMCC and posterior MCC (pMCC) to assess whether aMCC specifically influenced time spent exploring the virtual room in the threat context. The average beta values were derived in the same manner as the data for anterior insula, with aMCC and pMCC activity extracted from subregions defined by 3-mm-radius spheres placed at the voxel coordinates showing peak activity in the group-level statistical map. The average time spent exploring the threat context was measured using the period before any electrical stimulation, and was indexed using the metric total time spent pressing the forward and backward buttons minus total time spent pressing any button (including left and right buttons). Because participants seldom moved in a backward direction, and pressing left and right buttons during navigation would enable participants to orient toward the left and right but not advance forward, it was hypothesized that this metric would best index exploratory or approach behavior during the threat context during AUT. To assess the average time spent exploring overall, in both the threat and safe contexts, the metric used was total time spent pressing the forward and backward buttons minus total time spent pressing any button irrespective of the timing of any electric stimulus in reinforced threat trials.post hoc tests aimed at assessing the specificity of the results in the dAI. These analyses were conducted using ordinary least squares path analysis in the SPSS version of PROCESS57 using the identical data used in the correlational analyses. Post hoc analyses were performed to examine the relationships between SCR or subjective anxiety with anterior insula and BNST activation. The data for the correlational analyses involving activity in the BNST also were extracted from a 3-mm-radius sphere placed at the peak voxel coordinates in the group-level statistical map.To examine whether anxiety proneness influenced dAI activation through perceived control, a simple mediation analysis was conducted for dAI. Additional mediation analyses were conducted for vAI, aMCC and the BNST as M=49, s.d.=15). SCRs and subjective anxiety were greater during threat than safe contexts (SCR: t(39)=6.24, P<0.001; anxiety: t(39)=7.95, P<0.001). No significant difference was observed between female and male participants in anxiety proneness or perceived control, the factors of primary interest (t(38)=0.12, P=0.91; anxiety: t(38)=−1.72, P=0.09; US: t(38)=−0.27, P=0.78). Thus, the remaining results are based on all participants irrespective of sex.On average, participants rated the US intensity as moderately painful  and the BNST . The hem). The hemodynamic response function indicated that the BOLD signal elevation in these regions was sustained over the entire epoch of the unpredictable threat condition . In addiSupplementary Table S2).r=0.32, P<0.05, r=−0.40, P<0.05, r=−0.15, P=0.36; ACQ-R: r=0.20, P=0.21). Moreover, the levels of anxiety proneness correlated inversely with perceived control , but was not significantly correlated with that of the pMCC . In addition, greater aMCC activation was associated with increased time spent exploring the threat context during AUT . Note that no significant difference was observed between threat and safe contexts in time spent exploring overall (t(39)=0.14, P=0.89, The magnitude of the BOLD signal elevation in dAI correlated positively with the extent of anxiety proneness (STAI-T: a=−1.183), whereas those with lower perceptions of control tended to respond to anticipation of threat with greater activation in dAI (b=−0.051). A bias-corrected 95% bootstrap confidence interval (CI) for the indirect effect (ab=0.060) based on 10,000 bootstrap samples was entirely above zero (0.003 to 0.121). There was no evidence that anxiety proneness influenced dAI activation independent of its effect on perceived control (P=0.787). In contrast, the corresponding analysis with BOLD activity in vAI, aMCC and BNST, respectively, yielded no support for an indirect effect of anxiety proneness on activity in these regions through perceived control .Mediation analyses were conducted to test whether diminished perceived control may be a mechanism through which anxiety proneness exerts influence specifically on dAI activation. As conveyed in Post hoc correlations suggested multiple relationships between levels of autonomic arousal, subjective anxiety and activation in insula and BNST. Autonomic arousal assessed via SCR was associated with increased subjective anxiety as measured by anxiety ratings . The change in BOLD activity in the dAI correlated positively with both SCR  and subjective anxiety . In contrast, the change in BOLD signal in vAI correlated significantly only with SCR . Similarly, the change in BOLD activity in the BNST correlated positively with SCR , but the relationship between BNST activation and subjective anxiety was not significant .17 consistent with translational evidence from studies in experimental animals.45 The magnitude of autonomic arousal correlated positively with greater activity in the BNST, as well as in the vAI and dAI. Taken together, these results supported the a priori hypothesis and demonstrated an extended functional anatomy of contextual anxiety, which includes the aMCC and the BNST as well as the anterior insula.We tested the hypothesis that high-trait anxiety may influence the hemodynamic activity of the anterior insula during AUT indirectly through perceived control. The study yielded six major results. First, individuals who showed greater activation in dAI during anticipation of threat relative to no threat, also showed increased levels of trait anxiety and decreased levels of perceived control. Second, individuals with greater levels of trait anxiety also reported lower levels of perceived control, consistent with the extant literature. Third, the magnitude of activation in dAI during anticipation of threat correlated moderately with activation in aMCC. Fourth, greater activation in aMCC during anticipation of threat was related to increased time spent exploring the threat context. Fifth, perceived control mediated the relationship between trait anxiety and activation in dAI during anticipation of threat; moreover, the magnitude of dAI activity correlated positively with the levels of both autonomic arousal and subjective anxiety during the anticipation of unpredictable shock. Sixth, we replicated and extended our previous finding that, in humans, the BNST is activated by sustained anxiety in response to a context associated with unpredictable aversive stimulation,19 and that adult anxiety-prone individuals from a nonclinical sample may have exaggerated anterior insula activity31 and altered activity in anterior cingulate during emotional processing.32 It has been also demonstrated that there is a large, negative association between trait anxiety and perceived control, such that higher levels of anxiety proneness are related to greater deficits in perceived control.5 However, until now, the functional role of hyperactivity in anterior insula in the relationship between elevated trait anxiety and perceived control had not been empirically examined. Our data suggest that the link between anxiety proneness and exaggerated anterior insula activity is mediated by reduced perceived control.Other studies have demonstrated that healthy adults have enhanced activity in insular cortex during the anticipation of aversive events,60 Over time, according to etiological theories of emotional disorders,61 perceived control becomes trait-like and influences anxiety by modulating related brain function. We propose that contexts that signal temporally unpredictable aversive events induce uncertainty about the occurrence of future threat. When individuals face such uncertainty, the anterior insula generates a signal predicting an expected aversive body state that results in increased arousal and anticipatory anxiety, the latter of which appears to involve primarily the dorsal region of anterior insula, potentially because of its greater putative involvement in cognitive evaluative processes. Because dAI is engaged by various forms of cognitive evaluation63 and has strong functional connectivity to aMCC,40 which is thought to modulate action in response to negative affective information,33 we suggest that dAI uses perceptions of control in performing calculations of the likelihood and consequences of anticipated threat. This information is used then to amplify or dampen subjective anxiety, and to influence behavior possibly by relaying threat assessment information to aMCC.Perceived control is generally characterized by an individual's beliefs about how much control they have and has been implicated in a wide range of life domains.45 and suggests that anticipatory responses to unpredictable threat involve multiple processes and their neural substrates.64 On the basis of prior studies that have implicated ventromedial prefrontal cortex in responding to safety or pleasure,68 and data indicating that ventromedial prefrontal cortex and anterior hippocampus  may regulate emotional responses69 including the contextual retrieval of conditioned stimuli-no US memory following fear extinction,71 we interpret greater activity in these regions in the safe versus threat contexts as contributing to safety processing. However, because anxiety ratings were near zero and there were no independent safety ratings acquired, behavioral correlations to support this interpretation were not obtained.The association between increased BNST activation and autonomic arousal supports the hypothesis that the BNST has a role in modulating sustained anxiety responses,This study has some limitations. First, the data are cross-sectional, thus, prospective data will be required to definitively separate trait anxiety from current levels of perceived control. Second, although ecologically valid, the measure used to examine behavior under threat reflects relatively small differences in exploratory behavior. Third, although we detected a significant relationship between BNST activation and autonomic arousal, greater statistical power or the use of an online anxiety measure may be needed to detect a similar relationship with reported anxiety.In conclusion, individuals prone to anxiety show hyperactivity in dAI during anticipation of unpredictable aversive events. Anxiety proneness appears to affect dAI activity indirectly through perceived control, suggesting that the perception of control might contribute to the formation and maintenance of dysregulated anxiety, and may serve as an important treatment target for attenuating insula hyperactivity in anxious individuals.


In the cerebellar cortex, the granular layer is at the first stage of processing information from other brain regions delivered via mossy fibers. The major components of this neural network are numerous and tiny granule cells (GrC), which are the only excitatory neurons, and Golgi cells (GoC) that provide the only inhibitory inputs to GrCs. Despite such structural simplicity, many questions about their functions remain unanswered.5 GrC and ~2000 GoC models with their physiological synaptic and electrical connectivity. With background and constant mossy fiber inputs, the model shows network-wide oscillations driven by the synchronized GoC firing, as in previous simulation and experimental studies [Here we investigate the signal transformation property of the granular layer neural network with our three-dimensional large-scale and detailed computer model, composed of the biophysically detailed ~8×10 studies . Oscilla studies , while t


There are no definitive treatment guidelines for caval-filter thrombosis in the postoperative setting. Clinical management for partial or complete postoperative inferior vena cava (IVC) occlusion relies solely on expert opinion, anecdotal evidence, and small clinical trials. As such, the primary objective of the present report is to offer a complex case of extensive IVC filter occlusion in a neurosurgical patient with past medical history significant for protein C deficiency. The presentation, unique radiological findings, management, and outcome will be discussed. No similar cases of massive IVC-occlusive disease in a thrombophilic patient early in the postoperative course following neurosurgical intervention are documented in the medical literature.  Over recent years, the use of inferior vena cava (IVC) filters for primary thromboprophylaxis in surgical patients has substantially increased . CurrentDespite dramatically increased use of IVC filters in recent years, the indications, safety, and effectiveness of the filter  remain understudied and controversial , 9-12. RA 57-year-old male with past medical history significant for protein C deficiency presented to the authors' practice with severe trigeminal neuralgia refractory to medical management. After seven years of conservative therapy, the patient complained of worsening neuralgic pain in a left V3 distribution complicated by neurocognitive side effects secondary to his high-dose antineuralgic medications. With progressing symptoms and a side effect profile that limited any further escalation in medical management, the patient opted for operative intervention. Preoperative prophylactic IVC filter placement was arranged since the patient would have to stop his anticoagulation in the perioperative period. A hematology consultation was obtained preoperatively for postoperative co-management. Informed patient consent was obtained for this study.An IVC filter was placed on the day prior to left-sided microvascular decompression surgery. Venous access was obtained under sonographic guidance via the right internal jugular vein. A retrievable Celect IVC  filter was deployed below the renal veins. Spot images of filter placement were acquired to achieve accurate positioning. An inferior venacavogram was obtained during the procedure and confirmed normal caliber IVC with no evidence of stenosis or pre-existing thrombus Figure .With the IVC filter in place, the patient was then taken to the operating room to undergo microvascular decompression. A lumbar drain was placed and a left retromastoid craniotomy was performed. The cerebellum was gently retracted inferolaterally to allow visualization of the trigeminal nerve. A large, tortuous loop of the superior cerebellar artery was identified extending from the basilar artery, coursing beneath the fifth cranial nerve, and looping back up superiorly to the entry zone of the trigeminal nerve root. Teflon pledgets were inserted between the nerve and vessel to achieve adequate decompression. The surgery itself was without complication.Postoperatively, the patient reported complete resolution of the trigeminal neuralgia. After an uncomplicated two-day hospital course, he was discharged home on an enoxaparin bridge to warfarin with hematology follow-up scheduled for continued management. Thirteen days postoperatively, the patient noticed acute-onset lumbar back pain with associated radicular pain. He endorsed painful episodes that emanated from a point of origin in his lumbar spine, traveled bilaterally down the posterior aspect of his legs, and terminated at mid-calf. These symptoms progressed for several days until postoperative day 16, when he presented to the Emergency Department with incapacitating pain. The patient was unable to stand or ambulate independently. He had 2+ pitting edema of both ankles and calves. An ultrasound revealed occlusive thrombosis in the right greater saphenous vein, right common femoral, and right femoral vein. A nonocclusive thrombus was found in the right popliteal vein. The patient’s INR upon arrival to the ED was subtherapeutic at 1.3.Magnetic resonance (MR) imaging of the lumbar spine was ordered to rule out hematoma or abscess formation as the underlying cause of the patient's myelopathic symptoms. The MR imaging revealed enhancing tissue within the ventral epidural space, extending from the level of L3 through the sacrum. Additionally, prominent flow voids could be seen within the lateral recesses on T2-weighted sequences, consistent with engorged epidural venous plexuses. The engorged collateral vessels were compressing the thecal sac at the L4 vertebral level , balloon push thrombectomy, and bilateral pelvic vein and infrarenal IVC angioplasty. Although extensive residual thrombosis remained following endovascular intervention, a slight degree of circulatory restoration was achieved and the patient reported mild improvement of his lower extremity pain and weakness. He was then admitted for observation and started on full dose anticoagulation therapy . Over the course of the following two weeks, the patient was successfully transitioned to therapeutic anticoagulation on warfarin. Complete resolution of his lower extremity edema and continued improvement in his lower extremity pain was achieved within one week at therapeutic levels. He was discharged home in stable condition. At his three-month follow-up visit, the warfarin anticoagulation was within the therapeutic range, the lower extremity edema had resolved, and the resolution of his trigeminal neuralgia pain continued.The following noteworthy aspects of this case will be discussed and supported by comprehensive review of the medical literature: (1) the timing of IVC thrombosis within two weeks postoperatively, 2) the degree of occlusion extending caudally from 2 cm above the infrarenal IVC filter into the iliac veins bilaterally, (3) the myelopathy-mimicking symptomatology arising secondary to epidural and paravertebral venous engorgement, (4) the highly unique diagnostic MR imaging that revealed engorgement of Batson’s plexus with prominent flow voids indicating collateral circulation around the thrombosed filter (Figure  the degrThere are no consensus guidelines for the optimal screening frequency to monitor IVC patency following perioperative filter placement. As major general surgery is a significant risk factor for VTE, close monitoring for IVC occlusive disease is required to minimize morbidity in patients who undergo perioperative IVC filter placement. This case highlights the importance of early diagnostic CT or MR-imaging in surgical patients with an IVC filter who present with myelopathic signs.The diagnostic imaging included as part of this case is unique. The lumbar MR imaging shows enhancing tissue within the ventral epidural space, prominent flow voids, and tortuous collateral vessels compressing the thecal sac Figure . The ensIndeed, the majority of IVC thromboses culminate in only partial IVC occlusion with minimal or no clinical manifestations . Ahmad eThe optimal management for extensive IVC thrombosis remains to be established . AnticoaFor this case, the patient’s uniquely extensive thrombosis encouraged immediate invasive management while still within the two-week postoperative window for which venous thrombolysis remained contraindicated. Endovascular intervention with Angiojet thrombolysis, suction thrombectomy, balloon push thrombectomy, and bilateral iliac angioplasty were all attempted with minimal technical success. In this rare context of repeatedly achieving minimal mechanical circulatory restoration in a patient with extensive occlusive disease, the authors suggest admission on a case-by-case basis for observation and initiation of full-dose anticoagulation. Within one week of initiating this treatment, there was a complete resolution of symptoms with the patient recovering to baseline functional capacity.IVC thrombosis is an under-recognized and understudied complication that is diagnostically challenging in a postoperative setting. The authors encourage careful postoperative surveillance following perioperative IVC filter placement and early CT abdominal imaging when presented with myelopathic symptoms, edema, dilation of superficial abdominal wall veins, pyrexia, or oliguria. In the rare case of a postoperative patient with incomplete mechanical clot retrieval, initiation of therapeutic anticoagulation is recommended.


The aim of the present study was to compare metabolic connectivity, inferred from inter-regional [18F]FDG PET uptake correlations, in right-sided  and left-sided TLE  with healthy controls  using graph theory based network analysis. Comparing LTLE and RTLE and patient groups separately to HC, we observed higher lobar connectivity weights in RTLE compared to LTLE for connections of the temporal and the parietal lobe of the contralateral hemisphere (CH). Moreover, especially in RTLE compared to LTLE higher local efficiency were found in the temporal cortices and other brain regions of the CH. The results of this investigation implicate altered metabolic networks in patients with TLE specific to the lateralization of seizure focus, and describe compensatory mechanisms especially in the CH of patients with RTLE. We propose that graph theoretical analysis of metabolic connectivity using [18F]FDG-PET offers an important additional modality to explore brain networks.The human brain exhibits marked hemispheric differences, though it is not fully understood to what extent lateralization of the epileptic focus is relevant. Preoperative [ Temporal lobe epilepsy (TLE) is a common neurological disorder, which is defined by characteristic clinical features and a typical seizure semiology. Associated morphological brain alterations may be caused by recurrent seizure propagation and deafferation of brain regions connected to the hippocampus135Network analyses in TLE have been performed by determination of structural or functional connectivity using interregional correlation analysis. Cortical thickness analysis has been used to model structural connectivity, investigating connectivity patterns and correlations between brain areas, respectively in healthy control subjects and in different neuropsychiatrical disorders589718F] fluorodeoxyglucose (FDG) ([18F]FDG PET). [18F]FDG PET is one of the most accurate imaging methods to evaluate the functional deficit zone1415161819202122232418F]FDG PET, are metabolically connected.However, only a few studies utilizing nuclear medicine methodology to explore connectivity on a network level have been published. Studies applying positron emission tomography (PET) focus mainly on metabolic connectivity using fluorine-18 FDG PET neuroimaging studies investigate brain activity through observation of glucose metabolism at the synaptic level, which is the functional neuronal component of highest glucose turnover18F]FDG PET is used in epilepsy centers to define seizure focus in temporal lobe epilepsy, and beyond that, to display dysfunctional metabolism throughout the brain in epilepsy. Over the last three decades neuroimaging studies have demonstrated metabolic connectivity based on interregional correlation analyses in healthy control subjects and in various neuropsychiatric disorders2024FDG PET study demonstrated the ability to discriminate lateralization of seizure focus and to predict seizure prognosis via analysis of regional and global brain metabolism in patients with TLEA [18F]FDG PET for the analyses of metabolic connectivity using graph theory. A limitation of this study is an unknown effect of ongoing antiepileptic therapy or of subclinical seizure activity on network parameters. Present therapy was not evaluated separately, but should be equalized statistically for both patient groups. Further, seizures during PET investigations were excluded by observation, but there was no online EEG during PET acquisition. Consequently sleep or drowsiness during PET could not be excluded as well. Finally, flipping of the hemisphere in one patient group was required for analysis reasons, i.e., to have the affected hemisphere on the same side for all patients. Hence, one limitation is that we cannot rule out that our results are still influenced by inherent structural and/or functional differences between left and right hemispheres.Our results confirm the feasibility of [18F]FDG PET and graph theory. The results of this graph theoretical approach revealed a distinct neuronal pattern, dependent on lateralization of epileptogenic focus. Differences in hemispheric and lobar molecular connectivity have been observed between patients and HC and between RTLE and LTLE in the affected and contralateral hemisphere, confined to the temporal, parietal, frontal and occipital lobe. Higher contralateral molecular connectivity is found more pronounced in patients with RTLE, implicating more compensatory mechanisms opposing to the damaged neuronal tissue subsequent to seizures in patients with RTLE compared to LTLE. Hence, the results of this neuroimaging study underline the feasibility of [18F]FDG PET data for measurement of connectivity across brain regions and for further investigation of the pathophysiological mechanisms in TLE.The results of this investigation suggest significant altered metabolic networks in patients with TLE, which supports MRI investigations and captures similar information through utilizing [18F]FDG PET for pre-surgical assessment between 1999 and 2009 were recruited from the Epilepsy Monitoring Unit of the Department of Neurology, Medical University of Vienna. The diagnosis was based on clinical history, physical and neurological exams, results of prolonged video-EEG monitoring including clinical seizure semiology and interictal and ictal EEG results, high resolution MRI and inter-ictal [18F]FDG PET. 30 patients had RTLE  and 32 patients had LTLE . Except for TLE, patients had no history of any other neurological disorder. Subsequently to the comprehensive diagnostic procedure, all patients underwent selective amygdalo-hippocampectomy, except for two patients with RTLE, who underwent anteriormedial temporal resection, and hippocampal sclerosis, indicated in MRI, was confirmed histologically in all patients. Demographic patient data was assessed from patients’ clinical work-up  according to Hamacher et al.68Ge rod sources. The emission measurement was done in static 3D mode, which started 30 min after injection and lasted 10 minutes. Imaging procedures were carried out under resting and awake conditions and carefully controlled for ictal events. To minimize head movements, patient’s head was placed in a polyurethane head mould and straps were placed around the chin and the forehead.[http://www.fil.ion.ucl.ac.uk/spm/). Images were normalized to standard uptake values by dividing each voxel’s activity by the injected dose per kg body weight. For network analyses, images were further normalized to the average brain uptake5354PET images were spatially normalized to a tracer-specific template in MNI-space using SPM8 . First, clinical and epidemiological data were compared with unpaired two-tailed t-tests and Mann-Whitney U Tests. Differences in metabolism between affected and contralateral hemisphere were assessed by paired t-tests in the pooled patient group using SPM8. Graph metrics characterizing metabolic connectivity networks were evaluated using N = 5000 random permutations586060How to cite this article: Vanicek, T. et al. Insights into Intrinsic Brain Networks based on Graph Theory and PET in right- compared to left-sided Temporal Lobe Epilepsy. Sci. Rep.6, 28513; doi: 10.1038/srep28513 (2016).


Multiple intestinal atresias are rare and its treatment is challenging. Here, we present a case of multiple gastro-intestinal atresia associated with choledochal cyst posing us a surgical challenge.  A full-term, normally vaginal delivered, 10-hour-old male baby, born out of consanguineous marriage, presented with bilious vomiting and mild upper abdominal fullness. Antenatal USG had shown gross polyhydramnios and dilated bowel loops. A 3-year-old sibling is known case of classical galactosemia. Abdominal examination revealed epigastric fullness. Abdominal roentgenograph showed single dilated gastric bubble with no distal gas and few calcifications in lower abdomen . Exploratory laparotomy revealed multiple atresias involving duodenum, jejunum, ileum and colon. There were intra-luminal calcifications between the atretic segments. The stomach and duodenum were grossly dilated. Most of the jejunum and ileum were atretic with no lumen and inter atretic segment filled with calcified putty-like material. The large gut from caecum to transverse colon was filled with calcified material. There were further atresia present in the descending and sigmoid colon. Additionally, there was a large choledochal cyst measuring 5x4 cms . We initially planned for multiple resection and anastomoses, but in view of the long atretic segments with no lumen, calcified intra-luminal contents and intra-operative multiple bronchospasms, only resection of proximal jejunum was done. End duodenostomy and jejunostomy were done. Rest of the jejunum, ileum and large bowel till transverse colon were left as such and descending colostomy was done. Tube-drainage of choledochal cyst was done . A central line for total parenteral nutrition was also placed in internal jugular vein. Post-operatively, the baby was kept on total parenteral nutrition (TPN) for three weeks. The baby had persistent gastric distention and repeat x-ray showed a single bubble (missed pyloric atresia). On day 24, the baby expired due to TPN induced nosocomial sepsis, with blood culture positive for acinetobacter. Multiple intestinal atresia (MIA) is different from duodenal and jejunal atresia because of the extent of involvement from stomach to anus; and present a unique constellation of clinical, radiologic and pathologic findings secondary to malformative process occurring during early intrauterine life.[1] It has been seen in children with common ancestry.[2] Severe combined immunodeficiency is also associated.[3] Transmission pattern in several reported families with MIA had been known to be consistent with autosomal recessive inheritance. TTC7A was the likely causal gene for MIA.[4] Associated anomalies include malrotation, cystic dilatation of bile duct, omphalocele, cardiac anomalies, and congenital cystic adenomatoid malformation.[5] The index case had associated choledochal cyst. We hypothesize that in view of both proximal and distal obstruction of duodenum, the back-pressure led to cystic dilation of common bile duct and thus formation of choledochal cyst.MIA poses a serious surgical challenge. Surgical treatment has included tapering enteroplasty and resection and primary anastomoses.[6] Standard surgical therapy may not allow maximal preservation of residual bowel because of trimming process required for a safe and secure anastomosis. In addition, multiple anastomoses may require excessive operating time, jeopardizing the newborn’s clinical status. Hatch and Schaller described a “shish-kebab” technique in 1986, to perforate multiple membranous obstructions as an alternative to multiple resections.[7] Use of intraluminal Silastic stents to support multiple hand-sewn anastomoses is also described in literature.[8] However, none of them are feasible in these neonates in which whole of intestine is atretic with no intervening segment of normal intestine. Major cause of morbidity and mortality is short gut syndrome. There are no survivals reported in literature for the neonates with MIA. [5]Source of Support: NilConflict of Interest: The author is editor of the journal but the manuscript is handled independently by other editors and he is not involved in decision making of the manuscript.