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Natural history collections include rare and significant taxa that might otherwise be unavailable for comparative studies. However, curators must balance the needs of current and long-term research. Methods of data extraction that minimize the impact on specimens are therefore favored. Micro-CT has the potential to expose new character systems based on internal anatomy to taxonomic and phylogenetic analysis without dissection or thin sectioning for histology. However, commonly applied micro-CT protocols involve critical point drying, which permanently changes the specimen. Here, we apply a minimally destructive method of specimen preparation for micro-CT investigation of spider neuroanatomy suitable for application to legacy specimens in natural history collections. We used two groups of female spiders of the common species Araneus diadematus—freshly captured (n = 11) vs. legacy material between 70 and 90 years old (n = 10)—to qualitatively and quantitatively assess the viability of micro-CT scanning and the impact of aging on their neuroarchitecture. We statistically compared the volumes of the supraesophageal ganglion (syncerebrum) and used 2D geometric morphometrics to analyze variations in the gross shape of the brain. We found no significant differences in the brain shape or the brain volume relative to the cephalothorax size. Nonetheless, a significant difference was observed in the spider size. We considered such differences to be explained by environmental factors rather than preservation artifacts. Comparison between legacy and freshly collected specimens indicates that museum specimens do not degrade over time in a way that might bias the study results, as long as the basic preservation conditions are consistently maintained, and where lapses in preservation have occurred, these can be identified. This, together with the relatively low-impact nature of the micro-CT protocol applied here, could facilitate the use of old, rare, and valuable material from collections in studies of internal morphology.

Introduction Studying neuro‐structural markers of intellectual giftedness (IG) will inform scientific understanding of the processes helping children excel academically. Methods Structural and diffusion‐weighted MRI was used to compare regional brain shape and connectivity of 12 children with average to high average IQ and 18 IG children, defined as having IQ greater than 145. Results IG had larger subcortical structures and more robust white matter microstructural organization between those structures in regions associated with explicit memory. TD had more connected, larger subcortical structures in regions associated with implicit memory. Conclusions It was found that the memory systems within brains of children with exceptional intellectual abilities are differently sized and connected compared to the brains of typically developing children. These different neurodevelopmental trajectories suggest different learning strategies. A spectrum of intelligence types is envisioned, facilitated by different ratios of implicit and explicit system, which was validated using a large external dataset., A double dissociation was found between the size and connectomics of explicit and implicit memory systems within brains of children with exceptional intellectual abilities and the brains of typically developing children. These divergent neurodevelopmental trajectories appear to support different learning systems and therefore strategies. A heuristic for the spectrum of intelligence types was envisioned, facilitated by different ratios of neural implicit and explicit memory systems, validated using a large external dataset.

Standard templates are widely used in human neuroimaging processing pipelines to facilitate group-level analyses and comparisons across subjects/populations. MNI-ICBM152 template is the most commonly used standard template, representing an average of 152 healthy young adult brains. However, in patients with neurodegenerative diseases such as frontotemporal dementia (FTD), high atrophy levels lead to significant differences between individuals’ brain shapes and MNI-ICBM152 template. Such differences might inevitably lead to registration errors or subtle biases in downstream analyses and results. Disease-specific templates are therefore desirable to reflect the anatomical characteristics of the populations of interest and reduce potential registration errors. Here, we present MNI-FTD136, MNI-bvFTD70, MNI-svFTD36, and MNI-pnfaFTD30, four unbiased average templates of 136 FTD patients, 70 behavioural variant (bv), 36 semantic variant (sv), and 30 progressive nonfluent aphasia (pnfa) variant FTD patients and a corresponding age-matched template of 133 controls (MNI-CN133), along with probabilistic tissue maps for each template. Public availability of these templates will facilitate analyses of FTD cohorts and enable comparisons between different studies in an appropriate common standardized space., Measurement(s)average brain templateTechnology Type(s)T1-weighted magnetic resonance imagingFactor Type(s)sex • age • education • Clinical Dementia RatingSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14974848

Ecological or sensory system shifts associated with brain shape and size change may have contributed to unique avian survivorship., Birds today are the most diverse clade of terrestrial vertebrates, and understanding why extant birds (Aves) alone among dinosaurs survived the Cretaceous-Paleogene mass extinction is crucial to reconstructing the history of life. Hypotheses proposed to explain this pattern demand identification of traits unique to Aves. However, this identification is complicated by a lack of data from non-avian birds. Here, we interrogate survivorship hypotheses using data from a new, nearly complete skull of Late Cretaceous (~70 million years) bird Ichthyornis and reassess shifts in bird body size across the Cretaceous-Paleogene boundary. Ichthyornis exhibited a wulst and segmented palate, previously proposed to have arisen within extant birds. The origin of Aves is marked by larger, reshaped brains indicating selection for relatively large telencephala and eyes but not by uniquely small body size. Sensory system differences, potentially linked to these shifts, may help explain avian survivorship relative to other dinosaurs.

How do large and unique brains evolve? Historically, comparative neuroanatomical studies have attributed the evolutionary genesis of highly encephalized brains to deviations along, as well as from, conserved scaling relationships among brain regions. However, the relative contributions of these concerted (integrated) and mosaic (modular) processes as drivers of brain evolution remain unclear, especially in non-mammalian groups. While proportional brain sizes have been the predominant metric used to characterize brain morphology to date, we perform a high-density geometric morphometric analysis on the encephalized brains of crown birds (Neornithes or Aves) compared to their stem taxa—the non-avialan coelurosaurian dinosaurs and Archaeopteryx. When analyzed together with developmental neuroanatomical data of model archosaurs (Gallus, Alligator), crown birds exhibit a distinct allometric relationship that dictates their brain evolution and development. Furthermore, analyses by neuroanatomical regions reveal that the acquisition of this derived shape-to-size scaling relationship occurred in a mosaic pattern, where the avian-grade optic lobe and cerebellum evolved first among non-avialan dinosaurs, followed by major changes to the evolutionary and developmental dynamics of cerebrum shape after the origin of Avialae. Notably, the brain of crown birds is a more integrated structure than non-avialan archosaurs, implying that diversification of brain morphologies within Neornithes proceeded in a more coordinated manner, perhaps due to spatial constraints and abbreviated growth period. Collectively, these patterns demonstrate a plurality in evolutionary processes that generate encephalized brains in archosaurs and across vertebrates.

The taxonomy, phylogeny, and biology of theParanthropusspecies have been the center of debates since the earliest discoveryof the TM 1517 cranium (now attributed toParanthropus robustus)from the Plio-Pleistocene site of Kromdraai (South Africa) in 1938by R. Broom (Broom, 1938). In particular, whetherParanthropusboiseiandP. robustusrepresent two distinct taxa, and whether thetwo species emerged from a common ancestor (i.e., the hypothesisof a monophyletic group) remain largely unresolved (reviewed inConstantino and Wood, 2004,2007;Wood and Schroer, 2017).Besides the taxonomic and phylogenetic aspects, the presence oftwo contemporaneous‘robust’species in the eastern and southernAfrican hominin fossil records raises critical questions regardingpotential occurrences of homoplasies in the hominin clade, withsubstantial implications for our understanding of early hominin paleobiology (Wood and Schroer, 2017). Moreover, the absence ofassociated cranial and postcranial remains attributed toPara-nthropuscomplicate the reconstruction of the behavior of thisenigmatic‘megadont’hominin genus (reviewed in Constantino and Wood, 2007).

Background: Idiopathic intracranial hypertension (IIH) and normal pressure hydrocephalus (NPH) represent a cluster of typical clinical and imaging findings, with no evident etiological cause noted. In this study, we have proposed a model for IIH and NPH called Monroe–Kellie 3.0 (MK 3.0). IIH and NPH may be entities which represent opposite sides of the same coin with venous system and cerebrospinal fluid (CSF) as core drivers for both these entities. Materials and Methods: IIH and NPH volume data were collected, voxel-based morphometry analysis was performed without normalization, and the distribution of the individual volumes of gray matter, white matter, and CSF was statistically analyzed. Visual morphometry analyses of segmented data were performed, and the findings in routine magnetic resonance imaging (MRI) were noted to build a model for IIH and NPH. Results: In IIH and NPH when the volumes were compared with controls, the distribution was similar. Furthermore, the morphometric changes noted in the MRI and segmented volume data were analyzed and the results were suggestive of changes in elastic property of brain causing a remodeling of brain shape and resulting in minor brain shift in the skull vault, and the resulting passive displacement of CSF which has been termed as MK 3.0. Conclusion: This model helps to put the clinical and imaging findings and complications of treatment in single perspective.

How do large and unique brains evolve? Historically, comparative neuroanatomical studies have attributed the evolutionary genesis of highly encephalized brains to deviations along, as well as from, conserved scaling relationships among brain regions. However, the relative contributions of these concerted (integrated) and mosaic (modular) processes as drivers of brain evolution remain unclear, especially in non-mammalian groups. While proportional brain sizes have been the predominant metric used to characterize brain morphology to date, we perform a high-density geometric morphometric analysis on the encephalized brains of crown birds (Neornithes or Aves) compared to their stem taxa—the non-avialan coelurosaurian dinosaurs. When analyzed together with developmental neuroanatomical data of model archosaurs (Gallus, Alligator), crown birds exhibit a distinct allometric relationship that dictates their brain evolution and development. Furthermore, analyses by neuroanatomical regions reveal that the acquisition of this derived shape-to-size scaling relationship occurred in a mosaic pattern, where the ‘avian’-grade optic lobe and cerebellum evolved first among non-avialan dinosaurs, followed by major changes to the evolutionary and developmental dynamics of cerebrum shape after the origin of Avialae.Notably, the brain of crown birds is a more integrated structure than non-avialan archosaurs, implying that diversification of brain morphologies within Neornithes proceeded in a more coordinated manner, perhaps due to spatial constraints and abbreviated growth period. Collectively, these patterns demonstrate a plurality in evolutionary processes that generate encephalized brains in archosaurs and across vertebrates.

Gray matter volume for cortical, subcortical, and ventricles all vary with age. However, these volumetric changes do not happen on their own, there are also age-related changes in cortical folding and other measures of brain shape. Fractal dimensionality has emerged as a more sensitive measure of brain structure, capturing both volumetric and shape-related differences. For subcortical structures it is readily apparent that segmented structures do not differ in volume in isolation—adjacent regions must also vary in shape. Fractal dimensionality here also appears to be more sensitive to these age-related differences than volume. Given these differences in structure are quite prominent in structure, caution should be used when examining comparisons across age in brain function measures, as standard normalisation methods are not robust enough to adjust for these inter-individual differences in cortical structure.

Image decomposition plays an important role in Alzheimer’s disease classification. Existing image decomposition methods can extract features but their performance is insufficiently accurate due to loss of major characteristics of brain shape in image decomposition. In this paper, a variational feature extraction of two-dimensional variational mode decomposition is proposed for classifying Alzheimer’s disease from brain MRI images. This method extracts and selects the variational features of the major characteristics of the brain, and then eliminates some features which lose the brain information. For 60 brain images from the OASIS database, the proposed method yields 94.44%, higher accuracy than the state-of-the-art methods. Our method is able to extract the variational features with the major characteristics of the brain, thereby improving the performance of Alzheimer’s disease classification.

Endocasts (i.e., replicas of the inner surface of the bony braincase) constitute a critical proxy for qualifying and quantifying variations in brain shape and organization in extinct taxa. In the absence of brain tissues preserved in the fossil record, endocasts provide the only direct evidence of brain evolution. However, debates on whether or not information inferred from the study of endocasts reflects brain shape and organization have polarized discussions in paleoneurology since the earliest descriptions of cerebral imprints in fossil hominin crania. By means of imaging techniques (i.e., MRIs and CT scans) and 3D modelling methods (i.e., surface‐based comparisons), we collected consistent morphological (i.e., shape) and structural (i.e., sulci) information on the variation patterns between the brain and the endocast based on a sample of extant human individuals (N = 5) from the 3D clinical image database of the Steve Biko Academic Hospital in Pretoria (South Africa) and the Hôpitaux Universitaires Pitié Salpêtrière in Paris (France). Surfaces of the brain and endocast of the same individual were segmented from the 3D MRIs and CT images, respectively. Sulcal imprints were automatically detected. We performed a deformation‐based shape analysis to compare both the shape and the sulcal pattern of the brain and the endocast. We demonstrated that there is close correspondence in terms of morphology and organization between the brain and the corresponding endocast with the exception of the superior region. By comparatively quantifying the shape and organization of the brain and endocast, this work represents an important reference for paleoneurological studies., Is the endocast a reliable proxy for reconstructing the brain of fossil specimens? By quantitatively comparing extant human brains and endocasts using MRIs and CT‐scans, here we address this fundamental question and support the significance of fossil endocasts in paleoneurological studies.

Our analysis of endocranial imprints of apes and humans sheds new light on human brain asymmetry., Brain lateralization is commonly interpreted as crucial for human brain function and cognition. However, as comparative studies among primates are rare, it is not known which aspects of lateralization are really uniquely human. Here, we quantify both pattern and magnitude of brain shape asymmetry based on endocranial imprints of the braincase in humans, chimpanzees, gorillas, and orangutans. Like previous studies, we found that humans were more asymmetric than chimpanzees, however so were gorillas and orangutans, highlighting the need to broaden the comparative framework for interpretation. We found that the average spatial asymmetry pattern, previously considered to be uniquely human, was shared among humans and apes. In humans, however, it was less directed, and different local asymmetries were less correlated. We, thus, found human asymmetry to be much more variable compared with that of apes. These findings likely reflect increased functional and developmental modularization of the human brain.

Large brains are a defining feature of primates, as is a clear allometric trend between body mass and brain size. However, important questions on the macroevolution of brain shape in primates remain unanswered. Here we address two: (i), does the relationship between the brain size and its shape follow allometric trends and (ii), is this relationship consistent over evolutionary time? We employ three-dimensional geometric morphometrics and phylogenetic comparative methods to answer these questions, based on a large sample representing 151 species and most primate families. We found two distinct trends regarding the relationship between brain shape and brain size. Hominoidea and Cercopithecinae showed significant evolutionary allometry, whereas no allometric trends were discernible for Strepsirrhini, Colobinae or Platyrrhini. Furthermore, we found that in the taxa characterized by significant allometry, brain shape evolution accelerated, whereas for taxa in which such allometry was absent, the evolution of brain shape decelerated. We conclude that although primates in general are typically described as large-brained, strong allometric effects on brain shape are largely confined to the order's representatives that display more complex behavioural repertoires.

Shifts in brain regions with brain size Brain size among normal humans varies as much as twofold. Reardon et al. surveyed the cortical and subcortical structure of more than 3000 human brains by noninvasive imaging (see the Perspective by Van Essen). They found that the scaling of different regions across the range of brain sizes is not consistent: Some brain regions are metabolically costly and are favored in larger brains. This shifts the balance between associative and sensorimotor brain systems in a brain size–dependent way. Science , this issue p. 1222 ; see also p. 1184

Human populations that migrated out of Africa interbred with Neandertals. A new study assesses the effects of Neandertal gene variants on brain shape in modern humans, providing insights into the genomic basis of the uniquely globular human brain.

The TD6-2 level of the Gran Dolina cave site (Sierra de Atapuerca, Burgos, Spain) has yielded an assemblage of about 170 human fossil remains dated to > 800 ka (probably MIS 21) and assigned to the species Homo antecessor. In this study, we describe for the first time a large portion of a parietal bone (ATD6-100/168). The morphology of the fractures on the bones is compatible with a peri-mortem trauma. The superior parietal areas are flat. There is a large parietal foramen and one smaller accessory parietal foramen. Middle meningeal vessels are not particularly developed, but they are distributed in both anterior and posterior districts, with the parietal vasculature originating from the posterior branch. The meningeal vessels show multiple minor connections with the pericranial and diploic vascular systems. The diploe is not particularly developed, and large diploic channels are not detected. The bone is thin when compared with adult fossil humans, and equivalent to juvenile values. All these characters suggest that the parietal ATD6-100/168 probably belonged to a juvenile individual, with plesiomorphic endocranial traits similar to those described for H. ergaster/erectus. The derived temporal, maxillary and dental traits in Homo antecessor and the primitive parietal morphology further point to distinct (mosaic) patterns of morphological evolution of face and braincase.

When compared to the brains of our closest living relatives, chimpanzees and bonobos, the brains of modern humans are larger and differently shaped. This chapter reviews what we know about the evolutionary history of these differences. We can make an educated guess about the size and shape of the brains of the hypothetical common ancestor of modern humans and chimpanzees/bonobos, but between ca. 8 million years ago and the present day evidence about the size and shape of the brain comes from either natural endocasts, which are literally brain-shaped rocks, or from individuals for which enough of the brain case is preserved to provide estimates of endocranial volume and/or the relative proportions of the different regions of the cerebral hemispheres and the cerebellum. The tempo and mode of brain size increase in the hominin clade has been the subject of spirited debate, but we suggest that some of this controversy is the combination of an overreliance on frequentist statistical tests and researchers addressing these issues at different taxonomic scales. The existence and significance of shape changes are also controversial topics, made more so by the dearth of reliable evidence.

Understanding brain mechanics is crucial in the study of pathologies involving brain deformations such as tumor, strokes, or in traumatic brain injury. Apart from the intrinsic mechanical properties of the brain tissue, the topology and geometry of the mammalian brains are particularly important for its mechanical response. We use computational methods in combination with geometric models to understand the role of these features. We find that the geometric quantifiers such as the gyrification index play a fundamental role in the overall mechanical response of the brain. We further demonstrate that topological diversity in brain models is more important than differences in mechanical properties: Topological differences modify not only the stresses and strains in the brain but also its spatial distribution. Therefore, computational brain models should always include detailed geometric information to generate accurate mechanical predictions. These results suggest that mammalian brain gyrification acts as a damping system to reduce mechanical damage in large-mass brain mammals. Our results are relevant in several areas of science and engineering related to brain mechanics, including the study of tumor growth, the understanding of brain folding, and the analysis of traumatic brain injuries. P.S has been supported by the Generalitat de Catalunya under grants 2017-SGR-1278. N.A acknowledge funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013)/ERC grant agreement no. [615170]. This work was supported by a Engineering and Physical Sciences Research Council grant to Alain Goriely (EP/R020205/1).

Encephalization is a core concept in comparative neurobiology, aiming to quantify the neurological capacity of organisms. For measuring encephalization, many studies have employed relative brain sizes corrected for expected allometric scaling to body size. Here we highlight the utility of a multivariate geometric morphometric (GM) approach for visualizing and analyzing neuroanatomical shape variation associated with encephalization. GM readily allows the statistical evaluation of covariates, such as size, and many software tools exist for visualizing their effects on shape. Thus far, however, studies using GM have not attempted to translate the meaning of encephalization to shape data. As such, we tested the statistical relationship between size and encephalization quotients (EQs) to brain shape utilizing a broad interspecific sample of avian endocranial data. Although statistically significant, the analyses indicate that allometry accounts for

Computational neuroanatomical techniques that are used to evaluate the structural correlates of disorders in the brain typically measure regional differences in gray matter or white matter, or measure regional differences in the deformation fields required to warp individual datasets to a standard space. Our aim in this study was to combine measurements of regional tissue composition and of deformations in order to characterize a particular brain disorder (here, major depressive disorder). We use structural Magnetic Resonance Imaging (MRI) data from young adults in a first episode of depression, and from an age- and sex-matched group of non-depressed individuals, and create population gray matter (GM) and white matter (WM) tissue average templates using DARTEL groupwise registration. We obtained GM and WM tissue maps in the template space, along with the deformation fields required to co-register the DARTEL template and the GM and WM maps in the population. These three features, reflecting tissue composition and shape of the brain, were used within a joint independent-components analysis (jICA) to extract spatially independent joint sources and their corresponding modulation profiles. Coefficients of the modulation profiles were used to capture differences between depressed and non-depressed groups. The combination of hippocampal shape deformations and local composition of tissue (but neither shape nor local composition of tissue alone) was shown to discriminate reliably between individuals in a first episode of depression and healthy controls, suggesting that brain structural differences between depressed and non-depressed individuals do not simply reflect chronicity of the disorder but are there from the very outset., Highlights • We combine measurements of regional tissue composition and of deformations to characterize major depressive disorder. • We use structural MRI data from young adults in a first episode of depression. • The combination of hippocampal shape deformations and tissue composition was shown to discriminate between individuals.

Multi-atlas label fusion is an accurate but time-consuming method of labeling the human brain. Using an intermediate image as a registration target can allow researchers to reduce time constraints by storing the deformations required of the atlas images. In this paper, we investigate the effect of registration through an intermediate template image on multi-atlas label fusion and propose a novel registration technique to counteract the negative effects of through-template registration. We show that overall computation time can be decreased dramatically with minimal impact on final label accuracy and time can be exchanged for improved results in a predictable manner. We see almost complete recovery of Dice similarity over a simple through-template registration using the corrected method and still maintain a 3–4 times speed increase. Further, we evaluate the effectiveness of this method on brains of patients with normal-pressure hydrocephalus, where abnormal brain shape presents labeling difficulties, specifically the ventricular labels. Our correction method creates substantially better ventricular labeling than traditional methods and maintains the speed increase seen in healthy subjects.

Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us. In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion. In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion. In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli. The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning. Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.