Studies of the defence capacity of ancient hominins against toxic substances may contribute importantly to the reconstruction of their niche, including their diets and use of fire. Fire usage implies frequent exposure to hazardous compounds from smoke and heated food, known to affect general health and fertility, probably resulting in genetic selection for improved detoxification. To investigate whether such genetic selection occurred, we investigated the alleles in Neanderthals, Denisovans and modern humans at gene polymorphisms well-known to be relevant from modern human epidemiological studies of habitual tobacco smoke exposure and mechanistic evidence. We compared these with the alleles in chimpanzees and gorillas. Neanderthal and Denisovan hominins predominantly possess gene variants conferring increased resistance to these toxic compounds. Surprisingly, we observed the same in chimpanzees and gorillas, implying that less efficient variants are derived and mainly evolved in modern humans. Less efficient variants are observable from the first early Upper Palaeolithic hunter-gatherers onwards. While not clarifying the deep history of fire use, our results highlight the long-term stability of the genes under consideration despite major changes in the hominin dietary niche. Specifically for detoxification gene variants characterised as deleterious by epidemiological studies, our results confirm the predominantly recent appearance reported for deleterious human gene variants, suggesting substantial impact of recent human population history, including pre-Holocene expansions. The capacity to neutralise the adverse health effects of toxic substances is an important asset which increases “Darwinian” fitness, especially through dietary flexibility, but also by improved resistance to environmental poisons. The latter has been particularly important for the human lineage during the tens, or perhaps hundreds of thousands of years in which it enjoyed the benefits of fire use. Cooking made it possible to utilize a wider range of food resources more effectively by improving digestibility and detoxification, and fire usage enabled survival in colder regions –4. WhileHomo erectus in Africa at 1.8 million years ago (mya) to significantly later introduction by H. sapiens at the end of the Pleistocene [Use of biomass-fuelled fires leads to exposure to smoke toxicants, such as polycyclic aromatic hydrocarbons , signifiistocene , 17, 18.istocene –24, wereistocene , but wheThe most important toxic compounds produced during combustion of biomass , and thrConsequently, exposure to smoke toxicants increases the risk of developing various types of cancer in humans  and laboGSTM1 gene [Numerous epidemiological and biochemical studies of toxic compounds from tobacco smoke or heated food items are available , 41–45, TM1 gene , but advTM1 gene , 48, 49.TM1 gene , 37. FocThe genes involved in neutralising toxic smoke components and food heating products mostly display a wide range of target compounds and are representative for the major detoxification capacities , 51 and We postulate that, during human evolution, selection pressure towards increased resistance against fire-related toxicants was generated once humans started to use fire on a routine basis. This adaptive evolutionary model also implies that these protective variants are expected to be new beneficial mutants that will be derived alleles as compared to non-exposed great apes carrying the less efficient ancestral variant. Once present, these new beneficial variants will be under positive selection resulting in adaptation towards improved defence. In this study we explore the available genomic information for evidence of such genetic adaptations and discuss the implications of the results for the evolutionary history of human detoxification capacity. Although our results turned out to be non-conclusive regarding the time depth of fire usage, they opened a new, surprising perspective on the evolution of the involved detoxification capacities, the implications of which are discussed.The genetic variants occurring in Neanderthal and Denisovan genomes were derived from two high-coverage genomes, one from a Denisovan , 23 and et al. (2012) [Variant comparisons were performed using the UCSC Genome Browser, or variants were retrieved from the NCBI Sequence Read Archive. The chimpanzee variants were taken from the chimpanzee reference genome , and fro. (2012) , except . (2012) .CYP1A1 and CYP1B1 genes in ancient hominins, their genomic sequence was extracted from the corresponding Variant Call Format (VCF) file and all positions in translated sequences that were different from the human reference genome were filtered out using Excel.To analyse the complete protein-coding sequence of the The distribution of the number of low-/high-risk alleles within a global population of 2504 present-day individuals of the 1000 Genome Project  was deteSee Nineteen genes relevant for detoxification of toxic smoke components and food heating products were identified on the basis of biochemical knowledge of their detoxification mechanism Tables –3, and bNAT1 homozygous ancestral low-risk; GSTP1 heterozygous ancestral high-risk/derived low risk in the Vi33.26 Neanderthal). Together, in 33 out of 36 cases at least one of the ancient hominin species studied, Neanderthal or Denisovan, displays the ancestral allele (complete overview in GSTP1) where one of the low-coverage genomes clearly displayed another allele than the Altai Neanderthal (in the heterozygous state) , the haplotype with the highest catalytic efficiency observed in modern humans and a 3.4 times higher catalytic efficiency than the modern human wild-type [For the majority of gene polymorphisms studied (29 out of 36) we found that both the Altai Neanderthal and Denisovan genomes were still homozygous for the ancestral variant observed in chimpanzees and gorilla Tables . In 23 oate) see . Therefoild-type . AltogetFor 7 out of 36 loci we observed a derived allele in Neanderthal and/or Denisovan. Tables . In 3 caAHR1 was not included here because its risk status was not confirmed in a human background), 25 carried the protective (low-risk) allele in both Neanderthal and Denisovan hominins, whereas the high-risk variant was found consistently in both species at only 6 of these loci , the key regulator of CYP1A1, CYP1B1, and other important phase 1 and 2 detoxification enzyme genes. The modern human AHR has a valine residue at position 381 in the ligand-binding domain, resulting in lower binding affinity for prototype AHR agonists [Ahr, which carries Ala at homologous position 375 [AHR1 gene with an Ala residue at position 381 , dated to approximately 45,000 years ago , is cleaIn present-day humans, however, the high-risk variants occur at all 35 loci investigated, attain global allele frequencies up to 81%, exceed the 5% limit to become a common variation at 29 of these 35 loci, and are the major allele at 11 loci  affecting sensitivity to the adverse effects of genotoxic combustion and food heating products we observed that Neanderthal and/or Denisovan hominins carried the protective (low-risk) allele. Since we analysed 35 loci in 19 functionally related genes in at least 2 ancient hominin genomes  coincidence because of the low number of ancient hominin genomes analysed can be excluded as the explanation of the significant genetic pattern observed. Four population-genetic principles provide support to this conclusion  due to cultural adaptations, such as clothing and housing. Alternatively, the increase in high-risk detoxification gene variants could be part of a larger phenomenon. One recent study found that a high proportion of single nucleotide variants are rare, and of those predicted to be deleterious 86% are estimated to have arisen during the last 10,000–5,000 years . This coet al. [At the other end of the chronological spectrum, our study raises the question of the evolutionary origins of the low-risk variants. Answering this question will require more extensive phylogenetic comparison, which is complicated by the greater difficulty of establishing similarity in the haplotype context of the alleles. Since chimpanzees and gorillas are not exposed to smoke and cooked foods, there must be another reason for the early predominance of the low-risk alleles. One possible factor is that the diet of extant great apes contains deleterious compounds that deploy biotransformation mechanisms similar to toxic combustion products. Many plant species contain substantial levels of flavonoids  and otheet al.  found a et al. , and mayWe studied the evolution of detoxification capacity by determining the genetic variants found in Neanderthal and Denisovan hominins for genes known to have an impact on the sensitivity to reproductive impairment, when exposed to genotoxic combustion and food heating products. We hypothesized that selection occurred in favour of lower risk genotypes in the human lineage after the most recent common ancestor of humans and great apes. We observed a predominance of protective alleles not only in Neanderthal and Denisovan hominins, but also in chimpanzees, showing that these protective variants are mainly ancestral. Therefore, our results turned out to be non-conclusive regarding the time depth of fire usage and our hypothesis did not find any support in our study. However, our results do show that high-risk detoxification gene alleles became more numerous in modern humans, an increase which may have already started in early Upper Palaeolithic hunter-gatherers, suggesting that it preceded the major behavioural and dietary changes of the Neolithic. This finding supports previous studies , 80, 99 S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor regulating various processes in embryonic development and tissue homeostasis. On a cellular level PTK7 affects the establishment of cell polarity, the regulation of cell movement and migration as well as cell invasion. The PTK7 receptor has been shown to interact with ligands, co-receptors, and intracellular transducers of Wnt signaling pathways, pointing to a function in the fine-tuning of the Wnt signaling network. Here we will review recent findings implicating PTK7 at the crossroads of Wnt signaling pathways in development and disease. PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor with a broad range of functions in tissue development and homeostasis. Originally identified as a gene upregulated in colon carcinoma cells and accordingly named colon carcinoma kinase 4 (CCK-4)  and Disheveled (Dsh) were identified. Other core PCP proteins include the four-pass transmembrane protein Van Gogh , the atypical cadherin Flamingo  and intracellular components like Prickle (Pk) and Diego (Dgo). These proteins confer intra- and intercellular signaling, thereby aligning PCP in neighboring cells. Complementary studies in vertebrates revealed that these core PCP proteins are also required for the polarization of vertebrate tissues, like the orientation of hair follicles in the epidermis or the sensory hair cells in the inner ear , and off-track2 (otk2). These two genes, which are the result of a tandem gene duplication, function redundantly in the tubular morphogenesis of the male ejaculatory duct, leading to male sterility in the otk, otk2 double mutant  mutant zebrafish exhibit vertebral abnormalities at larval stages, phenotypically resembling congenital scoliosis. Further, zygotic ptk7 (Zptk7) mutants show late onset spinal curvatures consistent with the idiopathic form of scoliosis  is apparent at birth and involves abnormal vertebrae development, while idiopathic scoliosis is diagnosed during adolescence and does not show vertebral malformations. During the last two decades our understanding of the function of PTK7 has significantly advanced. Diverse biological processes that are regulated by PTK7 have been identified and its role in the establishment of polarity and coordinated cell movements has been acknowledged. Recent publications shed light on a literary “complex” function of PTK7 in Wnt signaling. While its role in non-canonical PCP signaling has been confirmed in different animal model systems and biological contexts, its function with respect to canonical Wnt signaling remains controversial. Possibly, these contradictory findings can be explained by the cell type-specific formation and subcellular localization of distinct co-receptor complexes. Further characterization of the formation and dynamics of these ligand-receptor complexes may help us to understand how PTK7 affects development as well as disease-related processes.Drosophila and the cancer section and edited the manuscript; HB wrote the section on scoliosis and designed the two tables.AB conceptualized and wrote the main part of the paper. AW wrote the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


U tests, Principal Components Analysis and General Linear Models). Distance to the hydrological network was consistently the most relevant variable for both parks and taxonomic classes. Topographic variables  were the second most predictive variables, followed by climatic variables. Differences in variable contribution were observed between parks and taxonomic classes. Variables related to water availability had the larger contribution to the models in the Mediterranean park, while topography variables were decisive in the Atlantic park. Specific response curves to environmental variables were in accordance with the biogeographic affinity of species (Mediterranean and non-Mediterranean species) and taxonomy (amphibians and reptiles). Interestingly, these results were observed for species located in both parks, particularly those situated at their range limits. Our findings show that ecological niche models built at local scale reveal differences in habitat preferences within a wide environmental gradient. Therefore, modelling at local scales rather than assuming large-scale models could be preferable for the establishment of conservation strategies for herptile species in natural parks.Ecological Niche Models (ENMs) are widely used to describe how environmental factors influence species distribution. Modelling at a local scale, compared to a large scale within a high environmental gradient, can improve our understanding of ecological species niches. The main goal of this study is to assess and compare the contribution of environmental variables to amphibian and reptile ENMs in two Spanish national parks located in contrasting biogeographic regions, i.e., the Mediterranean and the Atlantic area. The ENMs were built with maximum entropy modelling using 11 environmental variables in each territory. The contributions of these variables to the models were analysed and classified using various statistical procedures (Mann–Whitney  Ecological niche models (ENMs) describe the realised niches of species, i.e., the subset of fundamental niches where species are restricted due to their interspecific interactions . ENMs caOne way to mitigate these large-scale difficulties could be modelling at lower scale . Low-scaIn contrast to other terrestrial vertebrates, amphibians and reptiles are small ectothermic animals that are very sensitive to environmental and habitat attributes and have low dispersal capabilities . These fWe have built ENMs of amphibian and reptile communities in two Spanish National Parks, Picos de Europa and Cabañeros, located in two contrasted biogeographic regions of the Iberian Peninsula, the Atlantic and Mediterranean provinces . The MedIn summary, our general objective is to build local-scale ENMs of amphibian and reptile species in two contrasted environmental sites and to understand what variables are more relevant for describing their ecological niches. Specifically, we have addressed the following issues: (1) how does dependency between species or communities and environmental variables vary between the two national parks; (2) how does dependency between species or communities and environmental variables vary between taxonomic classes; (3) what is the variability of species-specific responses to environmental variables; (4) are there differences in ecological niche models for the species present in both territories?2. This protected area is completely under a Mediterranean climate of continental influence, defined by a dry summer season with less than 50 mm of average precipitation in this season and high temperatures (34°C mean maximum temperature in July). Its topography is adjusted to the apalachense relief, which lacks significant slopes and altitudes. Most of the territory is located below 1,000 m and consists of vast plains filled with eroded material called “rañas.” The tallest point in the Northwest of the park has an altitude of 1,448 m and consists of Paleozoic mountains in east–west alignment. CNP is mainly composed of Mediterranean forest characterised by a community of sclerophyllous species, mainly holm oak (Quercus ilex L.) and cork oak (Quercus suber L.). A complex mosaic of vegetation  coexists with the Mediterranean forest due to long-term human activity in the area  and Picos de Europa National Park (hereafter referred to as PENP), which are situated in the Iberian-Western-Mediterranean and Atlantic-European provinces, respectively , Fig. 1Fig. 1. Cthe area .2. The dominant climate is Atlantic, with marginal Alpine and Mediterranean influences on mountain tops and in lowland areas, respectively. Most of the territory has an annual precipitation of more than 1,300 mm/year, particularly in the northern mountains. Its topography is formed by three mountainous massifs  in the middle of the Cantabrian Mountains. In contrast to Cabañeros, there is a marked variation in environmental conditions due to a wide altitudinal gradient from 48 m to 2,460 m. Consequently, PENP has a wide representation of habitats. Grassland occurs on lowland pastures (Cynosurus cristatus), medium altitude slopes and subalpine meadows on the highest parts of the mountains (Nardus stricta and Festuca burnatii). There is a large representation of scrublands . Deciduous forests on valley bottoms and mid-slopes are dominated by beech (Fagus sylvatica). Riparian forest (Alnus glutinosa) is confined to valley bottoms, and prickly juniper (Juniperus oxycedrus) and holm oak woodlands (Quercus rotundifolia) are restricted to the exposed slopes of limestone gorges. Finally, there are fragmented stands of conifer plantations.PENP is situated north of the Iberian Peninsula and occupies an extension of approximately 638 kmThe data set for building ENMs was composed of 529 and 662 exact locations of 12 amphibian and 18 reptile species, respectively, in CNP and 1,108 and 1,196 exact locations of 9 amphibian and 12 reptile species, respectively, in PENP . We consWe used a set of 48 ecogeographical variables (EGVs) to build ENMs grouped into the following categories: climate, topography, potential solar radiation, vegetation and hydrology . These ETo create climatic variables, we used a climatic series constructed by the research group Kraken (University of Extremadura) and applying the method of kriging on specific data of weather stations, transferred by the State Agency of Meteorology (Government of Spain) between 1971–2007. Altitude, slope and radiation variables were obtained from a Digital Terrain Model with a mesh size of 5 m; the data is available on the website of the National Centre for Geographic Information (Government of Spain). Radiation variables were calculated using algorithms which estimate both direct and diffuse radiation . Vegetatr > 0.75), using loosely correlated environmental layers  were used to check for differences of the contribution of each EGV between taxonomic groups and biogeographic regions at both study areas; (2) Principal Components Analysis (PCA) with all EGVs contribution was performed to reduce the variance of these variables into a set of main components. Additionally, the VARIMAX rotation was used to minimise the number of variables with high loadings on each factor; (3) the values of PCA axes 1 and 2 for each species were examined by General Linear Models using taxonomic group, national park and biogeographic affinity and their interactions as factors. Biogeographic affinity was established, according to the classification of each amphibian/reptile species into their main geoclimatic regions  Mann–Whitney  regions , as MediDistance to the hydrological network was consistently the most relevant variable for both parks and taxonomic classes , althougSalamandra salamandra, Natrix natrix and Lacerta schreiberi) in CNP are associated to the factors distance to the hydrological network, annual mean precipitation or both. Furthermore, the Mediterranean species models (Natrix maura and Timon lepidus) in PENP are strongly associated with the second component, in the direction of altitude and minimum mean temperature of the coldest month.PCA showed a clear discrimination between ENMs from CNP and PENP according to the first and second axis  and second PCA axis  axis. When we repeated these analyses with site and biogeographic affinity (Mediterranean and non-Mediterranean), the interaction between both became marginally significant for the first axis . Mediterranean species did not show different values for the first PCA axis between both study areas (Using taxonomy (amphibians and reptiles) and site (CNP and PENP) as factors, GLMs showed only significant differences for site, both for values of the first . Likewise, in PENP, amphibians and reptiles have opposing responses to minimum mean temperature of the coldest month, i.e., maximum probability of occurrence at low values for several reptiles and vice versa. The response plots of this variable were in accordance with those of thermopile species such as Malpolon monspessulanus and Hemorrhois hippocrepis. For vegetation structure, there were species-specific responses according to particular habitat types, for example agrosylvopastoral system on Chalcides striatus and wetland on Zootoca vivipara  for their most predictive variables in CNP  were more accentuated.Species that inhabit both study areas had opposing response curves for some variables: for example, In this study, the importance of climate variables for modelling is replaced by other factors, such as altitude, slope and, in particular, the presence of watercourses and ponds. While several studies have reported that climatic variables are the best predictors to explain the ecological niches of amphibians and reptiles at regional or higher geographic scales , our resWe found that all the EGV categories were relevant in predicting the potential distribution of species in the following descending order of importance: (1) Hydrology, (2) Topography, (3) Climate, (4) Vegetation and (5) Insolation. The distance to the hydrological network is clearly the variable that most contributes to the distribution models. On the one hand, amphibian breeding is closely linked to freshwater environments. Beyond reproduction, numerous reptiles and most amphibians spend much of their life spans near water . In termThe PCA results demonstrated a clear segregation of the contribution of environmental variables to species models between CNP and PENP. Mediterranean climate is characterised by a dry summer with high temperatures. Consequently, there is a pronounced water deficit for at least three months. Despite a moderate influence of continental climate, CNP is completely dominated by Mediterranean features; for this reason, the variables related to water availability  had, as we expected, the larger contribution to the models in this park. Moreover, human management in CNP has created a landscape mosaic , and thiIberolacerta monticola or Zootoca vivipera). Therefore, altitude plays a decisive role in predicting reptile distribution. Several reptile species showed a high contribution of spatial distribution of tree vegetation, indicating the sensibility of reptile species for canopy , whereas other species showed a preference for more uniform areas (Lacerta schreiberi in PENP). Fraction of canopy cover and vegetation structure in treeless areas were relevant for species such as Zootoca vivipara (Vipera seoanei). These results show that factors related to solar energy at ground level are the most relevant ones for reptiles . We noted in these cases that just one or a few variables had far higher values of useful information in the models, except for Lacerta schreiberi , whereas typical Mediterranean species prefer sunny spots . Thus, the response to insolation and tree cover is consistent with the different needs of each species for direct sunlight and optimal thermal environment  have the lowest contributions of distance to the hydrological network. Soil texture is another not available environmental variable that can influence microhabitat selection  and the distance between their ecological optimum and environmental conditions  may be useful to obtain baseline information for conservation programs . Unfortuelection , particucinereus  or Pelobultripes . Future 10.7717/peerj.2405/supp-1Figure S1The figure shows as logistic prediction changes as distance of hydrological network is varied, keeping all other environmental variables at their average sample value: (A) Response curve in Cabañeros national park; (B) Response curve in Picos de Europa national park.Click here for additional data file.10.7717/peerj.2405/supp-2Figure S2The figure shows as logistic prediction changes as vegetation structure is varied in Picos de Europa national park. This variable is reclassified into 14 categories (that reflect the land use): (1) Natural Forest; (2) Forest Plantation; (3) Agrosylvopastoral system; (4) Forest temporarily treeless; (5) Scrub; (6) Pasture and meadows; (7) Devoid of vegetation; (8) Banks; (9) Agricultural land and agricultural mosaic; (10) Artificial; (11) Wetland; (12) Water; (13) Out of range; (14) Area of human influence.Click here for additional data file.10.7717/peerj.2405/supp-3Figure S3Territory at the edge of specie distribution correspond to left graphics. Saturated columns represent gain with only the one variable and less saturated columns represent gain without that variable. Variable abbreviations are detailed in Click here for additional data file.10.7717/peerj.2405/supp-4Table S1Coefficients of the cross correlations between grids inputs of selected environmental variables , its biogeographic affinity (B. affinity) that can be Mediterranean (Med.) and non-Mediterranean (non-Med.), the area under the ROC curve of test data (AUC’) and the training data (AUC”) and contribution of the environmental variables , its biogeographic affinity (B. affinity) that can be Mediterranean (Med.) and non-Mediterranean (non-Med.), the area under the ROC curve of test data (AUC’) and the training data (AUC”) and contribution of the environmental variables (abbreviations are shown in Click here for additional data file.10.7717/peerj.2405/supp-7Data S1Click here for additional data file.


A proportion of cases with mood disorders have elevated inflammatory markers in the blood that conceivably may result from stress, infection and/or autoimmunity. However, it is not yet clear whether depression is a neuroinflammatory disease. Multiple histopathological and molecular abnormalities have been found postmortem but the etiology of these abnormalities is unknown. Here, we take an immunological perspective of this literature. Increases in activated microglia or perivascular macrophages in suicide victims have been reported in the parenchyma. In contrast, astrocytic markers generally are downregulated in mood disorders. Impairment of astrocytic function likely compromises the reuptake of glutamate potentially leading to excitotoxicity. Inflammatory cytokines and microglia/macrophage-derived quinolinic acid (QA) downregulate the excitatory amino acid transporters responsible for this reuptake, while QA has the additional effect of inhibiting astroglial glutamine synthetase, which converts glutamate to glutamine. Given that oligodendroglia are particularly vulnerable to inflammation, it is noteworthy that reductions in numbers or density of oligodendrocyte cells are one of the most prominent findings in depression. Structural and/or functional changes to GABAergic interneurons also are salient in postmortem brain samples, and may conceivably be related to early inflammatory insults. Although the postmortem data are consistent with a neuroimmune etiology in a subgroup of depressed individuals, we do not argue that all depression-associated abnormalities are reflective of a neuroinflammatory process or even that all immunological activity in the brain is deleterious. Rather, we highlight the pervasive role of immune signaling pathways in brain function and provide an alternative perspective on the current postmortem literature. The most salient of these abnormalities are reductions in the size and/or density of GABAergic neurons and reductions in glial cell densities that are concurrent with altered gene expression. These data have been reviewed in detail elsewhere1 and are not comprehensively covered here. Instead we focus on one potential cause of the histopathological changes associated with mood disorders—inflammation. That is, we highlight those postmortem cellular and molecular abnormalities that may be directly or indirectly related to immune dysregulation. This is not intended to be a systematic review of the literature. Nor do we argue that all changes observed postmortem in mood disorders are related to inflammation. Rather we provide an alternative perspective on a substantial and diverse body of work.The question of whether mood disorders are neuropathological conditions has been the subject of considerable debate.8 Concentrations of pro-inflammatory cytokines are also reportedly elevated during mania or hypomania.10 Although most studies have focused on cytokine proteins, additional support for the role of inflammation in mood disorders is derived from gene expression studies of peripheral blood mononuclear cells, which have demonstrated the existence of increased messenger RNA (mRNA) expression of pro-inflammatory mediators in patients with mood disorders.14 Notably, Padmos et al.11 performed whole-genome expression profiling on microarrays using purified cluster of differentiation 14 (CD14+) monocytes and reported elevated mRNAs of inflammatory , trafficking, survival and mitogen-activated protein kinase pathway genes in BD subjects in various illness phases, and in the affected offspring of other BD subjects. Similarly, the expression of several genes previously implicated in neurological and inflammatory disorders, including TNF, were found to be upregulated in the peripheral blood mononuclear cells of a combined sample of depressed MDD and BD participants.14 Further, Pandey et al.13 found that the mRNA expression of IL-1, IL-6 and TNF, as well as their receptors, IL-1R1, IL-1RA and TNFR1 was significantly greater in the lymphocytes from BD patients than those of healthy controls. These data demonstrate the potentially important pathological role of membrane-bound cytokine receptors, which mediate the functional and biological effects of cytokines. In a follow-up study by the same group, similar increases in the mRNA expression of cytokines and their membrane-bound receptors were found in hospitalized MDD patients.15 Soluble cytokine receptors may also be relevant to the pathophysiology of depression. Elevations in the protein concentrations of soluble IL-1R (sIL-1R), sTNFR1, sTNFR2 and sIL-6R also have been reported in patients with mood disorders.18A large number of studies have reported increased levels of inflammatory proteins such as tumor necrosis factor (TNF), interleukin 6 (IL-6) and C-reactive protein (CRP) in the serum or plasma of depressed individuals with MDD and BD, and these results have been confirmed in meta-analyses.19 as has been demonstrated several times for CRP or IL-6 in the case of MDD and psychosis.22 Second, it has been well established that treatment of hepatitis C or melanoma patients with interferon alpha (IFN-α) or interleukin 2 (IL-2) induces a depressive episode in about 40% of patients.25 Importantly, there is a temporal disjunction between the ‘psychological' and ‘physical' manifestations of this immune-stimulating treatment, with the neurovegetative symptoms appearing within 1 week, whereas the mood and cognitive symptoms peak 8–12 weeks post initiation of treatment.27 Moreover, it is the mood and cognitive symptoms rather than the neurovegetative symptoms that are responsive to antidepressant treatment.27 Similarly, the typhoid vaccine and low-dose endotoxin, which has been shown to activate microglia in vivo,28 induce transient, mild depressive symptoms in healthy controls.31 These data prompted investigations into the efficacy of anti-inflammatory medications for the treatment of bipolar depression. Results have been mixed32 although low-dose aspirin, minocycline, infliximab and n-acetyl cysteine show some therapeutic promise.35 Finally, positron emission tomography studies have found evidence for microglial activation in the PFC and anterior cingulate cortex (ACC) of patients with MDD as well as the hippocampus in patients with BD as indexed by an increase in the distribution volume of the ligand for the translocator protein ligand, TPSO, which is expressed by microglia.37It is not yet clear whether the association between mood disorders and inflammation is causal. Regarding prospective studies, elevated CRP levels have been shown to predict the subsequent development of BD39 as well as hypoactivity of a ‘reward network' centered on the ventral striatum, thus providing a clear link with the behavioral trait of anhedonia.41 Nevertheless, postmortem studies have generally pointed to abnormalities in glutamatergic and/or GABAergic circuits in BD, and thus immune-mediated modulation of the balance between excitatory and inhibitory neurotransmission will be the primary focus here.Inflammatory mediators likely affect neuronal function and neurotransmission by an array of different mechanisms. For instance, pro-inflammatory cytokines such as interferon gamma (IFN-γ), IL-1 and TNF can reduce the availability of monoamines  by upregulating synaptic reuptake transporters, as well as reducing monoamine synthesis by decreasing the availability of tetrahydrobiopterin (BH4), a co-factor for the enzyme tyrosine hydroxylase  receptor.51 In contrast, QA is produced by microglia and is neurotoxic at elevated concentrations partly via its agonistic effect on the NMDA receptor.52 Although QA is usually found in low nanomolar concentrations in the human brain and cerebrospinal fluid, a significant increase in QA levels to micromolar concentrations is observed in patients with inflammatory neurological diseases.53 In addition to its effect on the NMDA receptor, QA has been shown to increase the production of reactive oxygen and nitrogen species,54 inhibit the reuptake of glutamate into synaptic vesicles,55 promote the formation of hyperphosphorylated tau proteins56 and upregulate transcription of pro-inflammatory chemokines and cytokines in astrocytes.57As will be discussed below, activated microglia release glutamate pathway . Specifi58 and we previously reported a reduction in the ratio of KynA to 3HK and KynA to QA in the serum of patients with both MDD and BD.60 In addition, the ratio of 3HK to KynA and/or QA to KynA in the serum was inversely correlated with hippocampal volume in both MDD and BD participants, as well as depressed football players with concussion, raising the possibility that neuroactive kynurenine metabolites may affect brain structure in the context of depression.61Decreases in KynA and increases in QA in the cerebrospinal fluid of predominantly depressed subjects have been found up to 2 years after a suicide attempt,Although the evidence for immune dysregulation in a subgroup of individuals with mood disorders is robust, the upstream cause of this immune dysregulation is less clear. Below, we discuss three potential, non-mutually exclusive causal factors, stress, pathogens and autoimmunity.62 In complementary studies, Cole and colleagues have demonstrated that social isolation and loneliness is associated with a consistent pattern of gene expression in peripheral blood leukocytes, that is, an increase in inflammatory markers involved in wound healing and bacterial immunity together with a downregulation of transcripts involved in Type I interferon antiviral responses and IgG antibody production.65 In the brain, this immunological dysregulation may lead to increased activity of the amygdala, deficient hippocampal neuroplasticity, and more generally, morphological and/or atrophic changes to pyramidal neurons.66Stress hormones  and ‘hard-wired' sympathetic nervous system innervation of lymphoid organs exert pleiotrophic effects on the immune system via their receptors on immune cells. This stress response causes  ‘low-grade' inflammation characterized by increases in pro-inflammatory mediators such as TNF, IL-6 and IFN-γ, as well as impairment of the adaptive immune system leading to increased vulnerability to viral disease, deficient response to vaccination and poorer prognosis for diseases such as cancer.67 whereas more than half of the US population is seropositive for cytomegalovirus.68 Generally, these viruses remain latent after initial infection but they may undergo reactivation during periods of both physical and psychological stress without producing significant clinical disease. Reactivation is usually accompanied by a noticeable increase in specific antibody titer to the virus, even in the absence of detectable virus. For instance, IgG antibody titers to cytomegalovirus, Epstein–Barr virus and herpes simplex 1 virus were found to be significantly elevated in medical students during exams compared with when they returned after summer vacation.69 Several studies have reported higher IgG cytomegalovirus antibody titers in depressed subjects relative to controls72 and more recently, the Detroit Neighborhood Health Study showed that individuals with cytomegalovirus antibodies in the top quartile were four times more likely to be depressed than those individuals in the bottom three quartiles of the population.73The impaired adaptive immunity associated with chronic stress may lead to increased susceptibility to the negative sequelae of pathogens, especially viruses. Herpesvirus infections are a salient example. The majority of the population of the United States (US) is seropositive for Epstein–Barr virus and herpes simplex 1 virus,Toxoplasma gondii.74 A meta-analysis of 23 studies found that serological evidence of infection with T. gondii was associated with a 2.73-fold increased risk of schizophrenia75 and several recent studies have reported associations between T. gondii seropositivity and depression, mania and suicidal behavior more generally.79 Like the herpesviruses, T. gondii is neurotrophic and additionally encodes proteins with homology to tyrosine hydroxylase and the D2 receptor, raising the possibility that it may modulate dopaminergic neurotransmission.80 Further, T. gondii-infected mice display increased production of neuroactive kynurenine pathway metabolites in the brain, suggesting another possible mechanistic connection between T. gondii and the pathophysiology of mood disorders.81Another pathogen that may have a role in the pathophysiology of mood disorders is the protozoan, 82 There are two predominant sources of evidence for autoimmune illness in depression and psychosis. First, there is a greater prevalence of various autoimmune disorders in patients with mood disorders than that of the general population.87 Further, there is persuasive epidemiological evidence to suggest that autoimmune disease is a risk factor for the development of de novo mood disorders. For instance, using the Danish Psychiatric Central Register, which included data on 90 000 inpatient admissions for depression, Benros et al.88 found that a history of any prior autoimmune disease increased the risk of a subsequent diagnosis with a mood disorder by 45%, a history of hospitalization for infection increased the risk of later mood disorders by 62% and the two factors interacted in synergy to increase the risk of subsequent mood disorders by 235%.Certain microbial infections may increase the risk of developing autoimmune disease via molecular mimicry and/or bystander activation and stimulation of pattern recognition receptors.90 a finding that may be related to a shared genetic vulnerability to BD and autoimmune thyroiditis.84 In another one of many examples, Ching et al.91 reported that 2 out of 20 patients with a diagnosis of MDD had raised levels of autoantibodies to glutamic acid decarboxylase 65 (GAD65) and Ro52 in both the serum and cerebrospinal fluid, with one of the patients meeting criteria for stiff-person syndrome. Whether these antibodies have a causal role in the development of the illness or are a consequence of an independent pathological process is still unknown. However, at least some cases of psychosis and/or depression appear to be caused by antibodies that have functional effects on neurotransmission. Dalmau and colleagues first reported the existence of autoantibodies, which cause cross-linking and internalization of the NMDA receptor in limbic encephalitis patients with a variety of psychiatric symptoms including auditory and visual hallucinations, delusions, depression and mania.93 Further, ~4% of the cases in the Kayser et al.93 series presented with isolated psychiatric symptoms. Subsequently, anti-NMDA receptor antibodies also have been reported to be present at an increased frequency in patients initially diagnosed with MDD (3%)94 and post-partum psychosis (2%).95 On the basis of these and other studies, it has become increasingly recognized that a small minority of patients diagnosed with psychosis or depression may in fact have a variant of autoimmune encephalitis. In a recent editorial, Lennox et al.96 wrote that ‘Antibody screening in young people presenting with psychosis, seizures and cognitive disturbance is now part of routine clinical practice in neurological and intensive care settings [in the United Kingdom]'.Second, there is evidence to suggest that a subset of patients with mood disorders have elevated levels of circulating autoantibodies. For instance, thyroperoxidase antibodies are significantly more common in patients with MDD and BD even after accounting for lithium exposure,98 Furthermore, the same team found that the diversity of these cells is more important in the human neocortex, with astrocytic subtypes displaying projections spanning several cortical layers or columns. The great diversity of human astrocytes was subsequently confirmed and extended to other brain regions, with the discovery of novel subtypes in the hippocampus and, more recently, in subcortical regions such as the thalamus and caudate nucleus.100 The notion that human astrocytes may also display different functional properties than in animal models further highlights the importance of clinical and postmortem studies to better understand the biological roots of mental illnesses.Among the different approaches used to investigate psychiatric illnesses, postmortem brain research offers a unique window through which underlying cellular and molecular alterations can be observed and described with the highest resolution and sensitivity. Moreover, postmortem studies can provide invaluable information on cerebral cells and networks that are only found in humans. This is particularly crucial when it comes to mental illnesses unique to humans such as BD. For instance, our view of human astrocytes has changed considerably in the past few years since Nedergaard and colleagues elegantly highlighted that both the size and complexity of cortical astrocytes are disproportionately greater in humans than in rodents.Despite these advantages, postmortem studies also present some limitations, and the results should always be interpreted with consideration for factors such as: (i) the quality of sample preservation, which is generally assessed by measuring tissue pH and RNA quality (RNA Integrity Number); (ii) the necessity of adequately matched controls ; and (iii) the amount of information  available for each donor, which can be considerably increased by psychological autopsies. Even when generated in optimal conditions, postmortem data showing significant differences between cases and controls should be viewed as a ‘snapshot' of cellular and molecular events unfolding in brain tissues before death, without precluding the possibility that the observations may reflect cumulative changes having occurred during the course of the illness. Consideration should also be given to possible postmortem changes to brain tissues between death and their preservation by freezing or fixation, although a careful matching of samples should eliminate this concern.et al.101 reported a significant increase in the expression of IL-4 in women and of IL-13 in men. As highlighted by the authors, this study had important limitations, such as a lack of diagnosis for the majority of suicides, the absence of toxicological data, and the fact that groups were not matched by age.101 The roles of IL-4 and IL-13 in the brain, similar cytokines released by T-helper 2 cells in allergic inflammation, remain mostly to be determined  inflammatory molecules and mediators in patients with mood disorders, little is currently known about the expression of inflammatory markers in the brain of such individuals. The few postmortem investigations that have addressed this question have all examined cortical areas and are, unfortunately, largely inconsistent. Using real-time polymerase chain reaction to compare the expression of TNF, IL-1, IL-4, IL-5, IL-6 and IL-13 in the orbitofrontal cortex of suicides vs non-psychiatric controls, Tonelli et al.104 found that both mRNA and protein levels of pro-inflammatory cytokines TNF, IL-1 and IL-6 were significantly increased in prefrontal cortex  relative to matched controls. These findings are consistent with the results of a later study by this group on the expression of the Toll-like receptors (TLR) found on macrophages and microglia.105 They showed that the expression (mRNA and protein) of TLR3 and TLR4 was significantly upregulated in the dorsolateral PFC of depressed suicides vs non-depressed suicides. TLR3 and TLR4 mRNA, but not protein, was similarly increased in depressed non-suicides, suggesting that suicide, independent of diagnosis, is associated with a stronger dysregulation in the expression of these receptors.105In their investigation of teenage suicides (various diagnoses), Pandey et al.106 also detected a highly significant depression-associated increase in protein levels of transmembrane—but not soluble—TNF that was restricted to BA46. A more extensive follow-up study by these authors showed a greater than three-fold increase in transmembrane TNF expression in ACC (BA24) samples from BD patients, as well as a 51 and 67% decrease in TNFR2 expression in BA46 samples from MDD and BD subjects, respectively.107 These results suggest illness-specific regional disruptions in TNF signaling. In contrast, Rao et al.108 did not detect any difference in TNF expression in the frontal cortical samples from BD patients relative to controls. They did however, report significantly increased mRNA and protein levels for IL-1 and IL-1R, as well as of astrocytic  and microglial (CD11b) markers in the same tissues.108 Using microarrays, a more high-throughput approach, some investigators have reported a widespread upregulation of both pro- and anti-inflammatory cytokines in the dorsolateral PFC of MDD patients,109 whereas others have been unable to implicate cerebral inflammation in any of brain regions examined in individuals with BD.110Focusing on TNF protein expression in prefrontal cortical samples (BA24 and BA46) from 10 adult MDD patients (mostly suicides) and matched controls, Dean 111 Fillman et al.111 performed postmortem gene expression analyses with Stanley Array Cohort frontal/prefrontal cortex samples . Using eight markers of inflammation , the authors were able to cluster the samples into a high inflammation subgroup consisting of 16 samples with schizophrenia (46%), 10 samples with BD (29%) and 9 controls (26%), and a low inflammation subgroup consisting of 19 samples with BD, 20 samples with schizophrenia and 25 controls. The samples also were clustered into high and low stress groups on the basis of the expression of several glucocorticoid signaling-related markers. The stress and inflammation markers were further combined to yield a high stress/inflammation subgroup and a low stress/inflammation subgroup with the former consisting of 46% of the schizophrenia group, 32% of the BD group and 18% of the controls. In addition, a microarray analysis demonstrated that 57 genes were differentially expressed between the high inflammation/stress subgroup of psychiatric samples and the low inflammation/stress subgroup of controls. A pathway analysis of these 57 genes identified a network of differentially expressed genes involving immune, growth factors, inhibitory signaling and cell death factors.111 Thus, a robust immune activation in the brain does seem to be occurring in subgroups of patients with BD and schizophrenia. More recently, Clark et al.112 compared the postmortem mRNA expression of various cytokines in ventrolateral prefrontal cortical samples from patients with depression not otherwise specified to matched controls. Unexpectedly, this study showed that the expression of TNF, IFN-γ, IL-13, IL-33, IL-12 and of Chemokine (C-C Motif) Ligand 2 (CCL-2), was significantly decreased in the samples from depressed patients. Furthermore, these findings were correlated with evidence of a compromised kynurenine pathway in the same samples. Taken together, these studies suggest that the important variations in expression levels of inflammation-related molecules and pathways measured in postmortem brain samples potentially reflect the clinical heterogeneity of samples analyzed, both between and within diagnoses.The inconsistencies in the literature presented above could be attributable to differences in experimental approaches or to the diversity of postmortem brain samples. As mentioned above, however, this diversity could be further enhanced by the clinical heterogeneity of patients suffering from mental illness. Molecular differences associated with such heterogeneity were recently highlighted in a postmortem study aimed at examining genes involved in stress and inflammation.113 Upon immune activation, microglia release pro-inflammatory cytokines as well as other factors, such as glutamate, chemokines and growth factors, and can undergo graded morphological changes leading up to a highly motile ameboid phenotype.115 Other types of macrophages can be found in the brain, such as infiltrated monocytes and perivascular macrophages, both of which are highly responsive to alterations of the blood–brain barrier integrity in pathological conditions. The best way to distinguish these different cell populations in postmortem brain samples remains the observation of their fine morphological properties and spatial distribution in immunostained tissues.116 With this approach, it is particularly easy to distinguish microglia from perivascular macrophages, as the former occupy the parenchyma in non-overlapping domains, whereas the latter are clearly associated with blood vessels.115 Although many macrophage-specific markers have been identified, it is currently impossible to differentiate macrophage cell populations based on the expression of these markers alone. The scavenger receptor CD163 may represent an exception, as it is more highly expressed by perivascular macrophages than microglia.117Microglia are the resident immune cells of the brain . In theiet al.118 provided the first evidence suggesting increased microglial activation in psychiatric illnesses. These authors examined the immunohistochemical distribution of human leukocyte antigen-DR, a major histocompatibility complex (MHC class) II cell surface receptor specifically expressed by macrophages, in samples of dorsolateral prefrontal cortex, ACC and mediodorsal thalamus from individuals having died with MDD, BD and schizophrenia as well as matched non-psychiatric controls.118 They observed increased densities of human leukocyte antigen-DR-immunoreactive (-IR) microglial cells in all the three brain regions. Interestingly, this observation concerned samples from individuals having died from suicide, irrespective of diagnosis.118 The same group subsequently published another postmortem study showing increased microglial quinolinic acid-IR within ACC subregions of severely depressed individuals compared with matched controls.119 More recently, support to the notion of depression-associated microglial/immune activation in the brain was provided by independent postmortem investigations focused on other microglial markers. On the basis of previously established morphometric criteria,116 the first of these studies assessed through stereology the relative abundance of microglial phenotypes immunostained for the macrophage-specific calcium-binding protein IBA1 in the dorsal ACC white matter of depressed suicides and matched sudden-death controls.120 Although this comparison suggested a relative increase of primed microglia in depressed suicides, the most striking observation was that samples from depressed suicides displayed significant more blood vessels surrounded by a high density of IBA-IR macrophages than matched controls.120 This may reflect increased recruitment of circulating bone marrow-derived monocytes in depressed suicides, a phenomenon that has been associated in mice with anxiety-like behavior in response to repeated social defeat stress.121 The distinction between resident microglia cells and monocyte-derived macrophages is important because the latter have been more closely linked with neurotoxicity, for instance producing 32 times more QA than resident microglia.122Few studies have examined microglia/macrophages in postmortem samples from individuals having suffered from mood disorders. Steiner 123 In this study, the group of cases was composed of individuals who had suffered from affective disorders or from schizophrenia, and the results were significant only in the subgroup of patients who died by suicide. It remains to be explored whether this phenomenon occurs solely in the white matter or whether the blood vessels in neocortical gray matter are similarly affected. Moreover, it will be important to determine whether the perivascular space is affected in this manner only in regions associated with mood disorders and suicide, such as the ACC and PFC, or whether this is a more global phenomenon in the brain.The postmortem study conducted concomitantly by Schnieder and colleagues suggested that the accumulation of macrophages in the perivascular space is not specific to the ACC. Indeed, their stereological investigation of IBA1 and of CD68-IR cells in the prefrontal white matter also showed an increase in perivascular macrophages in suicides relative to controls.124 Hashimoto et al.125 reported increased levels of glutamate in the frontal cortex of BD patients and several studies identified increases in the glutamine+glutamate (GLX) signal in multiple brain regions in BD patients.127 The GLX signal is constituted predominantly by intracellular glutamate and glutamine and is thought to reflect the total glutamatergic pool available for neurotransmission in the form of glutamate or glutamine.126 The smaller number of studies that distinguished between glutamate and glutamine, generally suggested elevations in glutamate in patients with BD.127 Thus it is unclear how best to interpret the increase in GLX, but the data are at least consistent with glutamate-induced neuronal hyperactivation.128Glutamate released by activated microglia may induce excitotoxicity and contribute to neuronal damage and/or dysfunction. A recent proton magnetic resonance spectroscopy study reported elevations in glutamate in the basal ganglia and dorsal ACC in patients receiving treatment with IFN-α, and higher levels of glutamate correlated with increases in depressive symptoms during the course of treatment.129 Consistent with the changes described above concerning macrophages in ACC white matter, morphometric data generated from the analysis of reconstructed Golgi-stained cells indicated that fibrous astrocytes in this cortical compartment displayed a hypertrophic phenotype in depressed suicides (MDD and BD) relative to controls.130 This observation may be indicative of mild astrogliosis in response to local low-grade neuroinflammation. In this regard, it is puzzling that postmortem studies examining the expression (gene and protein) of GFAP, an astrocyte-specific intermediate filament known to be upregulated with inflammation, have consistently found it to be significantly decreased in depressed cases vs matched controls.132 Initially described in prefrontal cortical areas, this downregulation was also recently reported in subcortical regions implicated in depression, but not in other unrelated cortical areas , suggesting that specific networks of astrocytes are affected in mood disorders, and that downregulation of GFAP is not a brain-wide phenomenon.99Astrocytes  have att133 and connexins 43 and 30  isoform 30 refs , 135 are137 However, this balance may be disrupted by oxidative stress or inflammation leading to necrosis and/or apoptosis through excessive stimulation of glutamate receptors.49 Several researchers47 have shown that activated microglial cells release glutamate via the system xc− cystine/glutamate transporter (Xc−) that exchanges cysteine for glutamate, and that this process may be accentuated by inflammation-induced downregulation of the astrocytic EAAT1 and/or reversed function  of EAAT2, potentially leading to excitotoxicity.49Glutamate reuptake is critical for regulating glutamate concentrations in the synaptic cleft and maintaining normal synaptic activity, and thus an impairment in glutamate transport may result in excessive or dysregulated glutamate receptor signaling. Under physiological conditions, astrocytes prevent excitotoxicity by maintaining extracellular glutamate in the micromolar range via the high-affinity glutamate transporters, excitatory amino acid transporter (EAAT) 1 and EAAT2.138 These results were subsequently extended to the locus coeruleus139 and hippocampus.140 It is not immediately clear from these studies whether the decrease in glutamate transporter expression was driven by a reduction in glial cell number. However, at least in the case of the locus coeruleus, the depression-associated decrease in EAAT1 and EAAT2 expression was found to be present when expression was normalized to the number of astrocyte cells (but not oligodendrocytes), suggesting an astrocyte-specific dysfunction.141A microarray analysis of samples of the ACC (BA24) and the dorsolateral PFC (BA9 and BA46) from people with MDD showed reduced expression of the genes coding for the high-affinity glutamate transporters glial excitatory amino acid EAAT1 and EAAT2 along with decreased expression of glutamine synthetase, the enzyme that converts glutamate to glutamine.142 As ∼90% of glutamate is removed from the synapse by EAAT2, Rao et al. hypothesize that their results remain largely consistent with the premise that BD is characterized by hyperglutamatergic signaling.142 Of note, riluzole, which increases the expression of EAAT2, has shown some efficacy in treatment-resistant depression and as adjunctive therapy for bipolar depression in mostly open-label studies.143Regarding BD, the picture appears to be more complicated. Both mRNA and protein levels of EAAT1 were reported to be increased, whereas EAAT2 mRNA and protein levels were decreased relative to controls in samples from the frontal cortex (BA10).144 The decrease in EAAT3 and EAAT4 expression may diminish the capacity of the synapse to clear glutamate, resulting in increased levels of synaptic glutamate in mood disorders.144 Supporting the potential relevance of EAAT3 function to depression and psychosis, a deletion of the solute carrier family 1 member 1 (SLC1A1) gene that codes for EAAT3 was found to co-segregate with psychosis (BD or schizophrenia) in a large five-generation pedigree, increasing disease risk by 18-fold.145 Similarly, both rare and more common variants of the genes coding for EAAT1 and EAAT2 have been reported to be associated with BD.146Another study found decreased expression of the neuronal transporters, EAAT3 and/or EAAT4 but not EAAT1 or EAAT2 in the striatum of MDD and BD (as well as schizophrenia) samples relative to controls.147 and a case of treatment-resistant depression.148 EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex and the binding of AQP4 IgG has been shown to cause the rapid internalization of both AQP4 and EAAT2.149Interestingly, autoantibodies to the water channel, aquaporin-4 (AQP4) which are usually associated with neuromyelitis optica, have also been reported in one case of schizophrenia150 perhaps explaining previous reports of decreased glutamine synthetase expression in the amygdala and PFC of suicide completers.151After synaptic uptake into astrocytes by EAATs, glutamate is converted into glutamine by glutamine synthetase and glutamine is, in turn, delivered to neurons where it is re-converted into glutamate. Thus, the expression of glutamine synthetase may affect glutamate cycling and availability to neurons. QA not only inhibits the uptake of glutamate by astrocytes but inhibits astroglial glutamine synthetase153 Lipopolysaccharide is toxic to oligodendrocytes when they are co-cultured with microglia, and in rodents, administration of endotoxin preferentially damages oligodendrocytes; an effect that may be mediated by TLR4 as transgenic mice lacking TLR4, through which endotoxin primarily acts, are less vulnerable to oligodendrocyte damage.155 Oligodendroglia in turn are capable of modulating immune function by, for instance, producing CCL-2 (previously known as MCP-1) and IL-1 post injury to open up the blood–brain barrier and recruit peripheral immune cells into the brain parenchyma as well to redirect themselves to areas of demyelination.157 Interestingly, inflammation-associated white matter damage is associated with pre-term births, and the risk for BD putatively increases monotonically with (shorter) length of gestation.158 Nevertheless, pre-term birth is more generally associated with a variety psychiatric and neurodevelopmental disorders, and thus the correlation between pre-term birth and risk for mood disorders is not necessary indicative of an underlying inflammatory etiology.Relative to other cell types, oligodendroglia  are part159 A reduction in the number of glial cells together with an increase in neuronal density initially was found in the subgenual ACC in BD and MDD samples, although the identity of the abnormal glia subtype could not be determined with the nonspecific staining methods applied.160 These nonspecific glia cell abnormalities were extended to the supragenual ACC, BA9 of the PFC, the amygdala and the entorhinal cortex. Cotter et al. reported a 22% decrease in the glial cell density of layer VI of the supragenual ACC and 30% decrease in the glial cell density in layer V of BA9 in MDD, but not BD, samples.162 On the other hand, after a stereological analysis of Nissl-stained tissues, Hercher et al.163 found no difference in glial densities nor in neuronal densities and average neuronal soma size between supracallosal ACC (BA24a) samples from MDD individuals vs matched controls. However, those samples from MDD subjects who had also been alcohol dependent had significantly higher densities of glial cells in this region compared with the samples from controls or from MDD subjects who were not alcohol dependent.163 Further, in the BA9 samples derived from BD subjects, Rajkowska et al.164 did find evidence for a decrease in the density (16–22%) of glial cells in layer III in conjunction with glial cell enlargement. These discrepancies in the literature may be related to various confounders, including treatment with lithium and valproic acid. Indeed, Price and colleagues reported reductions in glial cell density in the amygdala  of MDD samples but not in the BD samples.165 However, further analysis showed that the reduction in glial cell density was in fact present in the untreated BD cases.165Reductions in numbers or density of glia cells—most likely oligodendroglia cells are one of the most prominent findings in postmortem mood disorder samples.167 Similarly, a 19% reduction in oligodendrocyte density in the BD samples that reached trend-level significance was found in the amygdala,168 while signs of necrosis or apoptosis-related damage to oligodendrocyte cells have been observed in the BD samples from the caudate.169 Further, immunoreactivity of myelin basic protein, a surrogate marker of myelination that is expressed by oligodendroglia, was decreased in layer I of the hippocampus in female, but not male subjects with BD.170 In the case of MDD, the staining of deep white matter in the dorsolateral PFC was reported to be significantly less intense in MDD subjects compared with controls.171 Subsequent studies using specific staining methods or flow activated cell sorting methods, reported reduced numbers of oligodendroglia cells in the amygdala168 and frontopolar cortex.172Follow-up studies using methods that allowed for the differentiation of oligodendrocytes from other glial cell types, specifically reported reduced numbers of oligodendroglia in layers III and VI of BA9 in BD.173 Specifically, the expression of proteolipid protein 1, myelin associated glycoprotein, oligodendrocyte specific protein, myelin oligodendrocyte glycoprotein, and transferrin (TF) was reduced by 2- to 4-fold in the BD patients relative to psychiatrically healthy controls, while the transcription factors, OLIG2 and SOX10, which are involved in oligodendrocyte differentiation and maturation, were downregulated by 2- to 3-fold in BD.173 Although this decrease in oligodendrocyte-related gene expression conceivably could result from cell loss, not all of the oligodendrocyte-related genes were downregulated, suggesting that these abnormalities are more likely to reflect cellular dysfunction than cell death.173These morphological studies receive support from studies that have measured oligodendrocyte cell-related gene and protein expression changes in mood disorders. For instance, Bahn and colleagues performed a quantitative PCR analysis of BA 9 tissue, finding a significant reduction in mRNA expression of key protein markers of myelination and oligodendrocyte function.174 and BD.177 Interestingly, in patients with BD, a recent study reported a significant positive association between several pro-inflammatory cytokines and radial diffusivity, and an inverse association between these cytokines and fractional anisotropy, leading Benedetti et al.178 to hypothesize that inflammation is associated with demyelination or dysmyelination in BD. Oligodendrocytes are also involved in the turnover of N-acetylaspartate, a marker of neuronal integrity. Males with BD were reported to display a reduced concentration of (N-acetylaspartate), in the dorsolateral PFC -expressing oligodendrocytes or a chronic social stress-induced decrease in their density, leads to an impairment of astrocytic glutamate reuptake and depressive-like behaviors which can be rescued by repopulation with endogenous oligodendrocyte progenitor cells.184 Interestingly, MDD co-morbidity is high in MS, with a lifetime prevalence rate of about 50%, that is, 3- to 5-fold that of the general population.185 Similarly, BD in MS patients is twice as frequent as in the general population.185 Furthermore, MS patients can also present pseudobulbar affect , with prevalence estimates varying widely in the literature (6.5–95%).186 Pseudobulbar affect, which is often misdiagnosed as MDD or BD, is characterized by inappropriate and uncontrolled crying and/or laughing, and is thought to result from a dishinibition within the corticopontine-cerebellar circuits.187 Given these epidemiological and clinical observations, it can be speculated that the altered connectivity arising from neuroinflammation-related oligodendroglial loss and myelination deficits may represent a common path leading to disorders of mood and affect.Given the evidence discussed above for mood disorders, it is tempting to draw parallels with the psychopathological features observed in multiple sclerosis (MS). Indeed, MS is a chronic neuroinflammatory disorder that is characterized by a compromised blood–brain barrier, infiltration of immune cells, and loss of myelin and oligodendrocytes.2+ channel that is assembled from four or five subunits with an obligatory NR1 subunit associated with different combinations of NR2A–D subunits. The NR1 gene is alternatively spliced, producing eight different isoforms of the NR1 subunit, which combine with different NR2 and NR3 subunits to produce NMDA receptors with distinct pharmacological properties. Activation of the receptor requires multiple signals, that is, glutamate binding, AMPA receptor-mediated depolarization of the postsynaptic membrane, and binding of glycine or d-serine to the NR1 subunit.188 The NMDA receptors are found both in the synapse and in extrasynaptic locations on neurons but are predominantly expressed in the postsynaptic membrane where they interact with an intracellular protein complex termed the postsynaptic density (PSD).The NMDA receptor is membrane-bound ligand-gated Ca189 For instance, IL-1 can enhance NMDA signaling by inducing NR1  at postnatal day 21 together with neuronal excitability and long-term potentiation in the hippocampus.194Pro-inflammatory cytokines such as IL-1, IL-6 and TNF can enhance the release of glutamate from presynaptic neurons and increase NMDA receptor currents in postsynaptic neurons potentially leading to excitotoxicity.NR1 ref.  and NR2BNR1 ref.  subunit 195 In contrast, elevations in NR2A and NR2C and PSD-95 were reported by the same group to be elevated in the locus coeruleus and lateral amygdala, respectively.197 A more recent study using quantitative PCR in a large sample of 53 MDD subjects and 32 controls reported increased expression of all the NMDA receptor subunit genes in BA9/BA46 of depressed females, an effect that was accentuated in the individuals who had committed suicide.198Regarding postmortem studies, a western blot analysis showed approximately 50% reductions in NR2A, NR2B (but not NR1) as well as the anchoring protein, PSD-95, in the anterior region of the PFC of MDD samples.in situ hybridization studies generally have reported decreased expression of NR1. Specifically, decreased expression of NR1 was observed in the Cornu Ammonis 3 (CA3) and hippocampal subiculum of BD samples with a history of psychosis,199 while decreased expression of synapse- associated protein 102 (SAP102), NR1 and NR2A  was independently reported in hippocampal samples from individuals with BD (but not schizophrenia) relative to controls.200 Partially consistent with these data, NR1 (but no other NMDA subunit) expression was decreased in the oriens layer of CA1 but not CA2/3.201 Regarding the frontal cortex, NR1 expression was reported to be decreased in the dorsolateral PFC of MDD, BD and schizophrenia samples vs controls.202 Similarly, decreased mRNA and protein levels of NR1 and NR3A (but not NR2A or NR2B) together with increased concentrations of inflammatory and excitotoxicity markers such as IL-1, nuclear factor-κB and inducible nitric oxide synthase were reported in BD samples relative to controls.108In BD, In aggregate, this literature is suggestive of abnormal glutamatergic signaling in depression. Although the changes in expression of the NMDA receptor subunits could conceivably result from the direct actions of pro-inflammatory cytokines, there may be many other factors driving these changes such as compensatory up- or downregulation of receptors as a result of astrocyte dysfunction-mediated decreases or increases in glutamate levels.203 is an endogenous inhibitor of vesicular glutamate transporters (VGLUT).204 The vesicular glutamate transporters are responsible for loading glutamate into synaptic vesicles at excitatory neurons thereby regulating presynaptic glutamate release.205 VGLUT1 primarily is expressed in the cerebral cortex, hippocampus and cerebellar cortex, whereas VGLUT2 transcripts are expressed subcortically (including in dopamine neurons) and in layer V of the cerebral cortex.207 VGLUT3 is expressed in the subsets of serotonergic, cholinergic and GABAergic interneurons.206The kynurenine metabolite, xanthurenic acid, which is produced by the transamination of 3HK  and is u208 Consistent with these data, Gray et al.198 reported increased VGLUT1 expression in the dorsolateral PFC of female MDD samples using quantitative PCR. This abnormality may be region-specific since decreased expression of VGLUT1 also has been reported in mood disorders. Using in situ hybridization, Uezato et al.206 reported decreased VGLUT1 mRNA expression in both MDD and BD in the entorhinal cortex as well as decreased VGLUT2 expression in MDD, (trending for BD) in the middle temporal gyrus. Consistent with these data, decreased expression of VGLUT1 was reported in layer V of both MDD and BD samples from the dorsolateral PFC (BA9).209 A reduction in VGLUT1/2 mRNA may be due to structural loss of presynaptic terminals or alternatively a functional decrease in glutamate release while conversely, increased VGLUT expression may be indicative of greater numbers of glutamate neurons and/or greater presynaptic innervation and glutamate neurotransmission.206Eastwood and Harrison reported increased VGLUT1 expression in the supragenual ACC (BA24) in BD, a result they hypothesize is indicative of abnormally increased glutamate neurotransmission, rather than, or in addition to, alterations in glutamate metabolism or cycling.1 For instance, in the rostral ACC, Woo et al.210 found a reduction in the density of NR2A-expressing GABAergic interneurons in subjects with BD (and schizophrenia) relative to controls and in a separate study, the density of GABAergic neurons expressing the GluR5 subunit of the kainate receptor was found to be decreased by 40% in layer II of this region.211 Similarly, the density of calbindin (CB)-expressing GABAergic neurons in layer II of the supracallosal gyrus was reduced by 33% in BD212 and in the dorsolateral cortex, the density of glutamic acid decarboxylase 67 (GAD67) mRNA-containing (GABAergic) neurons in layers II through V of BA9 was decreased by approximately 25–33%.213 These data are supported by additional studies reporting decreased expression of GABAergic neuron-associated proteins such as GAD67 in both cortical and subcortical regions,217 as well as a meta-analysis showing a reduction in the density of CB-expressing neurons in layer VlI of BA9 in BD.218 Similarly, MDD appears to be characterized by reduced density of GABAergic interneurons labeled with calretinin and/or CB in the dorsolateral PFC,219 occipital cortex220 and auditory cortex.221Structural and/or functional changes to GABAergic interneurons are one of the most robust postmortem findings in BD.223 which are implicated in normal cortical function . Expression of mRNA for neuronal activity-regulated pentraxin, a protein that is secreted at presynaptic glutamate synapses that terminate on parvalbumin-containing interneurons, is reduced in the dorsolateral PFC of subjects with BD,225 suggesting that excitatory drive onto this interneuron subclass is disrupted which, in turn, could lead to a disruption of gamma oscillations and associated cortical function.These data suggest that GABAergic neurotransmission in at least a subset of local neuronal circuits is attenuated; however, the functional implications are not well understood. In addition to disinhibition of pyramidal neurons, other studies demonstrate that parvalbumin-containing interneurons are critical for the generation of gamma oscillations,67-staining neurons in the hippocampus -exposed mothers who were subjected to unpredictable subchronic stress during peripubertal development displayed significant reductions of parvalbumin-expressing interneurons in the dentate gyrus.229The mechanisms through which inflammation specifically affects the structure and/or function of GABAergic interneurons is not yet clear. However, there are preclinical data showing that inflammation impacts GABAergic circuits. Multiple rodent studies have reported that prenatal exposure to lipopolysaccharide or polyinosinic: polycytidylic acid (poly I:C) is associated with a reduction in the density of parvalbumin and/or GADT. gondii infection was shown to shift the distribution of GAD67 expression such that GAD67 becomes diffusely located throughout the neuropil rather than clustering on presynaptic termini where it catalyzes GABA synthesis in the brain.230In a different vein, 231 However, without the rapid clearance of myelin debris or toxins from the brain, microglial cells may instead mount a detrimental pro-inflammatory response, resulting in demyelination, synaptic dysfunction and ultimately neurodegeneration.233 Thus a delicate equilibrium between under- and overactivation of microglial cells may determine whether the conditions conducive to neuronal repair or neuronal damage, predominate. There are likely to be many mechanisms beyond removal of debris and oligodendroglia-genesis underlying these neuroprotective effects. For instance, activated macrophages and microglial cells express high-affinity glutamate transporters and glutamine synthetase during both acute and chronic central nervous system inflammation, potentially partially compensating for astrocytic dysfunction.234 Mice given intraperitoneal injections of 1.0 mg kg−1 of endotoxin for four consecutive days to globally activate central nervous system microglia, are protected from a subsequent traumatic brain injury challenge235 by microglia-mediated stripping of inhibitory GABAergic synapses, which facilitates NMDA receptor signaling allowing for greater expression of neurotrophic and anti-apoptotic molecules such as brain-derived neurotrophic factor and B-cell lymphoma 2 (Bcl-2).236 Thus conceivably, the increased expression of glutamate-related genes together with the decreased expression of GABA observed in postmortem tissue in BD may be an adaptive response to injury rather than a cause of neuronal pathology.Although we have emphasized the potentially detrimental effects of neuroinflammation on the brain, microglial cells also exert neuroprotective effects. Toll-like receptor signaling caused by cellular damage or infectious agents triggers a rapid activation of microglial cells, clearing away debris, promoting angiogenesis, neurogenesis, increasing the recruitment of oligodendrocyte progenitor cells, favoring remyelination and conferring neuroprotection.237 Rather than (or at least in addition to) defending against microbial infection, CA4 in the brain appears to have a key role in synaptic pruning during neurodevelopment.237 Thus postmortem alterations in inflammation-related proteins do not necessarily reflect an immune-related etiology—at least in the classical sense of the word.The assumption that molecules that are involved in the peripheral immune response automatically play the same functional role in the brain is potentially faulty. A case in point is the recent discovery that a variant in the gene coding for a component of the complement (CA4) pathway constitutes a risk factor for schizophrenia.The most prominent morphological abnormalities observed in postmortem samples from individuals with BD include decreases in the density and/or number of GABAergic neurons and glial cells. The literature describing microglial activation in mood disorders is smaller but appears robust. These data are complemented by gene expression studies, which are consistent with functional deficits in myelination, astrocytic glutamate recycling and in the regulation of inhibitory/excitatory neurotransmission. Immune processes could conceivably account for these abnormalities, but with the possible exception of the microglia data it is not possible to draw a direct link between neuroinflammation and histopathology. It is likely that there is a great deal of heterogeneity within the MDD and BD syndromes with respect to pathophysiology and etiology such that postmortem samples encompass patients with a range of conditions that appear clinically related but are neurobiologically distinct. This lack of a precise and biologically verifiable definition of illness, together with contrasting experimental approaches presumably contribute to the inconsistencies observed within the literature. Future studies should give greater consideration to these factors to better clarify the possible contribution of neuroinflammation to the etiology of mood disorders.It will be particularly importance for future postmortem studies to be carried out using thoroughly characterized samples, that is, case and control tissues for which substantial clinical information is available and complemented retrospectively with psychosocial data. Moreover, in addition to common tissue-based immunohistochemical and molecular approaches, the field should increasingly take advantage of techniques allowing for single cell analyses, such as laser capture microdissection and fluorescence-activated cell sorting. These approaches offer the opportunity to explore and compare, within a given brain region or circuit, the epigenetic and molecular profiles of different cellular subtypes . Such characterizations should lead to a more detailed understanding of the causes and consequences of cerebral neuroinflammation in mood disorders.


The ecological origin of snakes remains amongst the most controversial topics in evolution, with three competing hypotheses: fossorial; marine; or terrestrial. Here we use a geometric morphometric approach integrating ecological, phylogenetic, paleontological, and developmental data for building models of skull shape and size evolution and developmental rate changes in squamates. Our large-scale data reveal that whereas the most recent common ancestor of crown snakes had a small skull with a shape undeniably adapted for fossoriality, all snakes plus their sister group derive from a surface-terrestrial form with non-fossorial behavior, thus redirecting the debate toward an underexplored evolutionary scenario. Our comprehensive heterochrony analyses further indicate that snakes later evolved novel craniofacial specializations through global acceleration of skull development. These results highlight the importance of the interplay between natural selection and developmental processes in snake origin and diversification, leading first to invasion of a new habitat and then to subsequent ecological radiations. Three alternatives have been proposed for the ecological state of the ancestral snake: fossorial (burrowing), aquatic, or terrestrial. Here, the authors use an integrative geometric morphometric approach that suggests evolution from terrestrial to fossorial in the most recent common ancestor of extant snakes. In addition, the evolution and development of the limb and axial skeleton have been recently assessed in snakes, suggesting a correlation between limb loss and body elongation8. However, studies assessing the early ecological and evolutionary origins of snakes have so far largely focused on discrete morphological differences, and the adaptive role of skull shape development in the origin and diversification of snakes remains to be tested at large scale. The interplay between development and natural selection in driving morphological skull disparities associated with the lizard-to-snake transition remains poorly understood. Therefore, in this study, we performed a large-scale and integrative geometric morphometric analysis of skull bones across squamates to help clarify the ecological and evolutionary origins of snakes.A century of anatomical and phylogenetic studies have established that snakes evolved from lizards11, terrestrial14, fossorial18, or even multiple habitats19, have been proposed based on cladistic analysis of discrete traits. Central to this debate are the paucity of intact well-preserved snake fossils19, the difficulty of deciphering squamate phylogenetics24, sampling variability and incompleteness26, and incompatible morphological character coding when convergence is expected26. In addition, ancestral ecologies have been typically hypothesized based on sister clades of snakes only, without formal ancestral character state estimates26. Recently, modern phylogenetic comparative methods have been developed to estimate the ecological state of snake ancestors23, but with a limited taxon sampling that hampered the phylogenetic signal. Similarly, investigations of the evolution of discrete ossification sequences in skull development could not discern between different ancestral ecological scenarios27.Conflicting ecological hypotheses for early snakes, including aquatic28. In support of that, the comparison of the inner-ear shape in a limited number of snake species has already been used in the context of snake origins30, and recent geometric morphometric studies integrating ecological and/or developmental data revealed new insights into skull evolutionary specializations in several lizard and snake radiations34. The diversity in cranial structure of squamates is remarkable and appears tightly linked to functional and constructional demands within specific clades of lizards or snakes39, suggesting that large-scale comparisons of skull shape and size across the whole of Squamata could offer a holistic framework to address the ecological origin of snakes. Equally, cranial shape associated with ontogeny and heterochronic processes—changes in the timing and/or rate of developmental events—have been implicated in cranial evolution at different taxonomic levels in squamates42 and other major vertebrate lineages such as mammals and archosaurs44. Thus far, studies tackling the evolutionary origin of snakes have largely ignored morphometric and ontogenetic information, as well as the importance of developmental mechanisms for understanding the ecological origins of snakes.The analysis of morphological data using complementary geometric morphometric approaches has the potential to shed light on these issues45, we quantified ontogenetic skull shape variation by analyzing multivariate ontogenetic trajectories in a unique data set of squamate embryos. Strikingly, our data reveal that while the most recent common ancestor (MRCA) of crown snakes had a small skull with a shape fully adapted to a fossorial lifestyle, all snakes plus their sister group evolved from a terrestrial form with non-fossorial or non-leaf-litter behaviors, thus indicating a surface-terrestrial-to-fossorial scenario at the origin of snakes. In addition, we demonstrate that the unique skull features of modern snakes later evolved by peramorphosis through global acceleration in the rate of craniofacial development during embryogenesis. This set of results demonstrates the importance of the relationships between skull form, function, and development in the major ecological radiations of snakes to different habitats, and provides a new framework to understand the origin and evolutionary history of snakes.Here we performed a large-scale and integrative characterization of skull shape evolution in squamates, by covering all major groups of lizards and snakes, using a geometric morphometric approach integrating developmental, embryological, ecological, phylogenetic, and paleontological data. We hypothesized that skull shape and size are linked to particular habitat features; hence, ancestral snake ecologies could be inferred from skull shape parameters. In complement, and to formally test the recently revived hypothesis that snake skulls evolved by heterochronyOur principal component analysis (PCA) performed on Procrustes coordinates of skulls from a large data set of adult squamate species  and three-dimensional (3D) data, respectively; Fig. 46 in a phylogenetic context, using a main topological hypothesis integrating fossil data and compiled from the most recent and inclusive molecular as well as combined molecular and morphological phylogenetic studies on squamate evolution 15, terrestrial (Yurlunggur and Wonambi)14, or terrestrial/burrowing (Dinilysia)29 species were found near these estimated ancestral skull shapes . However, only a small fraction of total shape variation could be accounted for by allometry , in contrast to scolecophidians . This argues for an independent origin of fossoriality between amphisbaenians and snakes, as also supported by recent phylogenetic studies50.To both assess size diversification within squamates and estimate the ancestral size of early snake skulls, we estimated centroid size evolution as a proxy for total skull size using squared-change parsimony algorithms in a similar way as for shape analysis. As shown in Fig. 23, but is in agreement with the range of relatively small skull fragments recovered for Cretaceous fossorial snakes such as Coniophis17, Najash16, and the enigmatic Tetrapodophis18. Unfortunately, the incomplete or damaged skull available for these latter fossils hampers their inclusion into our ecological analyses.Altogether, these results indicate that allometry was presumably critical in the early evolution of snakes toward a fossorial behavior, and suggest that allometry played a different role in the evolution of fossoriality among squamates. In addition, our predicted size for the MRCA of crown snakes strongly rejects the hypothesis of a large, terrestrial pro-boid snake ancestor44. To assess both cranial ontogeny and the impact of heterochrony on squamate skull evolution, we used a unique embryonic data set covering 50% of all lizard and snake families , an earlier onset (predisplacement), or an increased rate of development (acceleration) of descendent lineages compared to ancestors. The regression of shape changes onto centroid size as a proxy for ontogenetic time, a widely used method to detect heterochronic trends52, clearly demonstrates steeper regression slopes and angles for snakes in comparison to lizards  and temporal regulation of morphogenesis (heterochrony).http://www.reptile-database.org). Specimens were primarily sampled from the published literature, the Digital Morphology (DigiMorph) library, reptile colonies at the University of Helsinki (Finland) and Tropicario (Finland), as well as from collections at the Finnish Museum of Natural History (Finland), Museum für Naturkunde in Berlin (Germany), Museum of Comparative Zoology of Harvard University (USA), and American Museum of Natural History (USA). Staging of new unpublished embryos was performed based on the ‘standard event system’ using external characters61, and with the help of complete staging tables available for several snake and lizard species62.3D computed tomography (CT) scans and/or 2D high-quality photographs of skulls were obtained from 326 squamate species (408 skulls), including 279 extant adult or juvenile (298 skulls), 55 embryonic , and 23 well-preserved adult fossil (26 skulls) species covering all lineages and most families of squamates  for the same species when available. Bones absent in different squamate lineages such as the temporal bar and supratemporal bones were not included in the analysis. All data were scaled by voxel or pixel size in the respective 3D and 2D package. Landmark coordinates were aligned using Generalized Procrustes Analysis and projected onto tangent space in the package MorphoJ v1.0663. The evolutionary patterns of cranial shape disparity were visualized using a PCA as implemented in MorphoJ v1.06. The influence of allometry was tested using a multivariate regression analysis of independent contrasts of shape (Procrustes coordinates) on independent contrasts of size (centroid size), and statistical significance was assessed using a permutation test (10 000 permutations) against the null hypothesis of total independence. Residual scores were also used to correct allometry and verify its relevance to the patterns of shape distribution. TPS technique was used for data interpolation, allowing estimation and visualization of species-specific shape changes through deformation grids with depicted vectors reflecting both the direction and magnitude of changes.Skull shape was extracted by digitizing 61 for 3D data) and 20 (for 2D) landmarks using Amira 5.5.0  and tpsDig v2.17 software package, respectively , and phylogenetic relatedness was corrected in statistical analyses that require independent observations. To visualize the skull morphologies of reconstructed MRCAs, the closest species in multidimensional shape space  were warped toward these ancestral nodes using TPS method66. Ancestral skull sizes were also reconstructed using squared-change parsimony algorithms and visualized by mapping centroid size changes onto the phylogeny. All ancestral reconstruction calculations were performed in MorphoJ v1.06 using several alternative composite hypotheses but also different recent combined molecular and morphological phylogenies24, including or not fossil data, mosasauroids, and branch length information, as well as with Dinilysia and mosasauroids at different positions to address the phylogenetic uncertainty of these fossils24.To include a large data set of squamate specimens, including extant, fossil, and embryonic taxa, a main composite phylogenetic hypothesis was used by assembling the most recent and inclusive molecularion Fig. . For ass49 were computed using the R-package convevol v1.1. Significance was assessed in the same package using 1000 evolutionary simulations along the phylogeny according to a Brownian-motion model49. The influence of habitat modes on skull shape  was initially tested with multivariate analysis of variance (MANOVA) and phylogenetic MANOVA using simulations under a Brownian-motion model using the R-package geiger v2.0.667. The significant separation of different ecologies was then tested using univariate analyses of variance (ANOVAs) followed by post hoc pairwise comparisons as well as DFA, before predicting ancestral ecologies from both allometry-uncorrected and -corrected shape parameters using linear discriminant analysis in the R-package MASS v7.3-4768.Ecological habitat modes for squamate species were first gathered from the published literature, reptile databases, and/or personal field observations  between two ontogenetic points (stage 10 embryos and adults) were quantified in snake and lizard species, using the trajectory.analysis function in geomorph v3.0.565. Statistical significance was determined by a random permutation procedure of 1000 iterations. As both the angle and direction of trajectories did not differ in our data set, different heterochronic hypotheses were tested using multivariate regression of shape (Procrustes coordinates) onto log-centroid size as a proxy for developmental time51; the slope, length, and angle between descendant trajectories (snakes) in relation to ancestor trajectories (lizards) were compared to predict global heterochronic processes such as modification of the onset/offset, growth rate, or length of development52. Variation in the total duration of embryonic development was evaluated in 126 squamate species (only one representative species per genus) with documented incubation or gestation periods, using ANOVA. Finally, the degree of ossification of both parietal and frontal bones was used as a proxy to compare the offset of skull development in 35 different snake and lizard species at fixed pre-hatchling embryonic stage (stage 1061).The different ontogenetic trajectories for lizards and snakes were generated as vectors of shape changes in both PCA morphospace and regression analyseswww.digimorph.org (Supplementary Information). The remnant data are available through the corresponding author, upon reasonable request. Landmarks are available in the Supplementary Information.2D photographs and 3D CT data are in part publicly available in the published literature and Supplementary InformationPeer Review File