Sex and Age Differences in Ontogeny of Alloparenting: A Relation to Forebrain DRD1, DRD2, and HTR2A mRNA Expression?

Alloparenting refers to the practice of caring for the young by individuals other than their biological parents. The relationship between the dynamic changes in psychological functions underlying alloparenting and the development of specific neuroreceptors remains unclear. Using a classic 10-day pup sensitization procedure, together with a pup preference and pup retrieval test on the EPM (elevated plus maze), we showed that both male and female adolescent rats (24 days old) had significantly shorter latency than adult rats (65 days old) to be alloparental, and their motivation levels for pups and objects were also significantly higher. In contrast, adult rats retrieved more pups than adolescent rats even though they appeared to be more anxious on the EPM. Analysis of mRNA expression using real-time-PCR revealed a higher dopamine D2 receptor (DRD2) receptor expression in adult hippocampus, amygdala, and ventral striatum, along with higher dopamine D1 receptor (DRD1) receptor expression in ventral striatum compared to adolescent rats. Adult rats also showed significantly higher levels of 5-hydroxytryptamine receptor 2A (HTR2A) receptor expression in the medial prefrontal cortex, amygdala, ventral striatum, and hypothalamus. These results suggest that the faster onset of alloparenting in adolescent rats compared to adult rats, along with the psychological functions involved, may be mediated by varying levels of dopamine DRD1, DRD2, and HTR2A in different forebrain regions.

Adult neurogenesis is necessary for functional regeneration of a forebrain neural circuit.

In adult songbirds, new neurons are born in large numbers in the proliferative ventricular zone in the telencephalon and migrate to the adjacent song control region HVC (acronym used as proper name) [A. Reiner et al., J. Comp. Neurol. 473, 377-414 (2004)]. Many of these new neurons send long axonal projections to the robust nucleus of the arcopallium (RA). The HVC-RA circuit is essential for producing stereotyped learned song. The function of adult neurogenesis in this circuit has not been clear. A previous study suggested that it is important for the production of well-structured songs [R. E. Cohen, M. Macedo-Lima, K. E. Miller, E. A. Brenowitz, J. Neurosci. 36, 8947-8956 (2016)]. We tested this hypothesis by infusing the neuroblast migration inhibitor cyclopamine into HVC of male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) to block seasonal regeneration of the HVC-RA circuit. Decreasing the number of new neurons in HVC prevented both the increase in spontaneous electrical activity of RA neurons and the improved structure of songs that would normally occur as sparrows enter breeding condition. These results show that the incorporation of new neurons into the adult HVC is necessary for the recovery of both electrical activity and song behavior in breeding birds and demonstrate the value of the bird song system as a model for investigating adult neurogenesis at the level of long projection neural circuits.

The Effect of Intranasal Administration of Gangliosides on the Viability of CA1 Hippocampal Neurons in Rat Two-Vessel Occlusion Model of Forebrain Ischemia/Reperfusion Injury.

Intranasal administration of total bovine brain gangliosides (6 mg/kg) to rats protected the CA1 hippocampal neurons from the death caused by two-vessel occlusion model (with hypotension) of forebrain ischemia/reperfusion injury. The immunohistochemical reaction of specific antibodies to marker proteins of activated microglia (Iba1) and astrocytes (GFAP) in hippocampal slices revealed the neuroprotective effect of exogenous gangliosides which can be mostly explained by their ability to suppress neuroinflammation and gliosis. The expression of neurotrophic factor BDNF in the CA1 region of hippocampus did not differ in sham-operated rats and animals exposed to ischemia/reperfusion. However, the administration of gangliosides increased the BDNF expression in both control and ischemic groups. The intranasal route of administration allows using lower concentrations of gangliosides preventing the death of hippocampal neurons.

Parental developmental experience affects vocal learning in offspring.

Cultural and genetic inheritance combine to enable rapid changes in trait expression, but their relative importance in determining trait expression across generations is not clear. Birdsong is a socially learned cognitive trait that is subject to both cultural and genetic inheritance, as well as being affected by early developmental conditions. We sought to test whether early-life conditions in one generation can affect song acquisition in the next generation. We exposed one generation (F1) of nestlings to elevated corticosterone (CORT) levels, allowed them to breed freely as adults, and quantified their son's (F2) ability to copy the song of their social father. We also quantified the neurogenetic response to song playback through immediate early gene (IEG) expression in the auditory forebrain. F2 males with only one corticosterone-treated parent copied their social father's song less accurately than males with two control parents. Expression of ARC in caudomedial nidopallium (NCM) correlated with father-son song similarity, and patterns of expression levels of several IEGs in caudomedial mesopallium (CMM) in response to father song playback differed between control F2 sons and those with a CORT-treated father only. This is the first study to demonstrate that developmental conditions can affect social learning and neurogenetic responses in a subsequent generation.

Sleep Disruption Precedes Forebrain Synaptic Tau Burden and Contributes to Cognitive Decline in a Sex-Dependent Manner in the P301S Tau Transgenic Mouse Model.

Sleep disruption and impaired synaptic processes are common features in neurodegenerative diseases, including Alzheimer's disease (AD). Hyperphosphorylated Tau is known to accumulate at neuronal synapses in AD, contributing to synapse dysfunction. However, it remains unclear how sleep disruption and synapse pathology interact to contribute to cognitive decline. Here, we examined sex-specific onset and consequences of sleep loss in AD/tauopathy model PS19 mice. Using a piezoelectric home-cage monitoring system, we showed PS19 mice exhibited early-onset and progressive hyperarousal, a selective dark-phase sleep disruption, apparent at 3 months in females and 6 months in males. Using the Morris water maze test, we report that chronic sleep disruption (CSD) accelerated the onset of decline of hippocampal spatial memory in PS19 males only. Hyperarousal occurs well in advance of robust forebrain synaptic Tau burden that becomes apparent at 6-9 months. To determine whether a causal link exists between sleep disruption and synaptic Tau hyperphosphorylation, we examined the correlation between sleep behavior and synaptic Tau, or exposed mice to acute or chronic sleep disruption at 6 months. While we confirm that sleep disruption is a driver of Tau hyperphosphorylation in neurons of the locus ceruleus, we were unable to show any causal link between sleep loss and Tau burden in forebrain synapses. Despite the finding that hyperarousal appears earlier in females, female cognition was resilient to the effects of sleep disruption. We conclude sleep disruption interacts with the synaptic Tau burden to accelerate the onset of cognitive decline with greater vulnerability in males.

Altered nucleocytoplasmic export of adenosine-rich circRNAs by PABPC1 contributes to neuronal function.

Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.

Forebrain commissure formation in zebrafish embryo requires the binding of KLC1 to CRMP2.

Formation of the corpus callosum (CC), anterior commissure (AC), and postoptic commissure (POC), connecting the left and right cerebral hemispheres, is crucial for cerebral functioning. Collapsin response mediator protein 2 (CRMP2) has been suggested to be associated with the mechanisms governing this formation, based on knockout studies in mice and knockdown/knockout studies in zebrafish. Previously, we reported two cases of non-synonymous CRMP2 variants with S14R and R565C substitutions. Among the, the R565C substitution (p.R565C) was caused by the novel CRMP2 mutation c.1693C > T, and the patient presented with intellectual disability accompanied by CC hypoplasia. In this study, we demonstrate that crmp2 mRNA could rescue AC and POC formation in crmp2-knockdown zebrafish, whereas the mRNA with the R566C mutation could not. Zebrafish CRMP2 R566C corresponds to human CRMP2 R565C. Further experiments with transfected cultured cells indicated that CRMP2 with the R566C mutation could not bind to kinesin light chain 1 (KLC1). Knockdown of klc1a in zebrafish resulted in defective AC and POC formation, revealing a genetic interaction with crmp2. These findings suggest that the CRMP2 R566C mutant fails to bind to KLC1, preventing axonal elongation and leading to defective AC and POC formation in zebrafish and CC formation defects in humans. Our study highlights the importance of the interaction between CRMP2 and KLC1 in the formation of the forebrain commissures, revealing a novel mechanism associated with CRMP2 mutations underlying human neurodevelopmental abnormalities.

An atlas and database of neuropeptide gene expression in the adult zebrafish forebrain.

Zebrafish is a useful model organism in neuroscience; however, its gene expression atlas in the adult brain is not well developed. In the present study, we examined the expression of 38 neuropeptides, comparing with GABAergic and glutamatergic neuron marker genes in the adult zebrafish brain by comprehensive in situ hybridization. The results are summarized as an expression atlas in 19 coronal planes of the forebrain. Furthermore, the scanned data of all brain sections were made publicly available in the Adult Zebrafish Brain Gene Expression Database (https://ssbd.riken.jp/azebex/). Based on these data, we performed detailed comparative neuroanatomical analyses of the hypothalamus and found that several regions previously described as one nucleus in the reference zebrafish brain atlas contain two or more subregions with significantly different neuropeptide/neurotransmitter expression profiles. Subsequently, we compared the expression data in zebrafish telencephalon and hypothalamus obtained in this study with those in mice, by performing a cluster analysis. As a result, several nuclei in zebrafish and mice were clustered in close vicinity. The present expression atlas, database, and anatomical findings will contribute to future neuroscience research using zebrafish.

Rigor and reproducibility in human brain organoid research: Where we are and where we need to go.

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

DLX genes and proteins in mammalian forebrain development.

The vertebrate Dlx gene family encode homeobox transcription factors that are related to the Drosophila Distal-less (Dll) gene and are crucial for development. Over the last ∼35 years detailed information has accrued about the redundant and unique expression and function of the six mammalian Dlx family genes. DLX proteins interact with general transcriptional regulators, and co-bind with other transcription factors to enhancer elements with highly specific activity in the developing forebrain. Integration of the genetic and biochemical data has yielded a foundation for a gene regulatory network governing the differentiation of forebrain GABAergic neurons. In this Primer, we describe the discovery of vertebrate Dlx genes and their crucial roles in embryonic development. We largely focus on the role of Dlx family genes in mammalian forebrain development revealed through studies in mice. Finally, we highlight questions that remain unanswered regarding vertebrate Dlx genes despite over 30 years of research.

Variations in touch representation in the hummingbird and zebra finch forebrain.

Somatosensation is essential for animals to perceive the external world through touch, allowing them to detect physical contact, temperature, pain, and body position. Studies on rodent vibrissae have highlighted the organization and processing in mammalian somatosensory pathways.1,2 Comparative research across vertebrates is vital for understanding evolutionary influences and ecological specialization on somatosensory systems. Birds, with their diverse morphologies, sensory abilities, and behaviors, serve as ideal models for investigating the evolution of somatosensation. Prior studies have uncovered tactile-responsive areas within the avian telencephalon, particularly in pigeons,3,4,5,6 parrots,7 and finches,8 but variations in somatosensory maps and responses across avian species are not fully understood. This study aims to explore somatotopic organization and neural coding in the telencephalon of Anna's hummingbirds (Calypte anna) and zebra finches (Taeniopygia guttata) by using in vivo extracellular electrophysiology to record activity in response to controlled tactile stimuli on various body regions. These findings reveal unique representations of body regions across distinct forebrain somatosensory nuclei, indicating significant differences in the extent of areas dedicated to certain body surfaces, which may correlate with their behavioral importance.

Spatially resolved cell atlas of the teleost telencephalon and deep homology of the vertebrate forebrain.

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking variations in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely "everted" telencephalon, which has confounded comparisons of their brain regions to other vertebrates. Here we combine spatial transcriptomics and single nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the Mchenga conophorus cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell-types in the fish telencephalon and subpallial, hippocampal, and cortical cell-types in tetrapods, and find support for partial eversion of the teleost telencephalon. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

A histological and diceCT-derived 3D reconstruction of the avian visual thalamofugal pathway.

Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.

Adult Neurogenesis of Teleost Fish Determines High Neuronal Plasticity and Regeneration.

Studying the properties of neural stem progenitor cells (NSPCs) in a fish model will provide new information about the organization of neurogenic niches containing embryonic and adult neural stem cells, reflecting their development, origin cell lines and proliferative dynamics. Currently, the molecular signatures of these populations in homeostasis and repair in the vertebrate forebrain are being intensively studied. Outside the telencephalon, the regenerative plasticity of NSPCs and their biological significance have not yet been practically studied. The impressive capacity of juvenile salmon to regenerate brain suggests that most NSPCs are likely multipotent, as they are capable of replacing virtually all cell lineages lost during injury, including neuroepithelial cells, radial glia, oligodendrocytes, and neurons. However, the unique regenerative profile of individual cell phenotypes in the diverse niches of brain stem cells remains unclear. Various types of neuronal precursors, as previously shown, are contained in sufficient numbers in different parts of the brain in juvenile Pacific salmon. This review article aims to provide an update on NSPCs in the brain of common models of zebrafish and other fish species, including Pacific salmon, and the involvement of these cells in homeostatic brain growth as well as reparative processes during the postraumatic period. Additionally, new data are presented on the participation of astrocytic glia in the functioning of neural circuits and animal behavior. Thus, from a molecular aspect, zebrafish radial glia cells are seen to be similar to mammalian astrocytes, and can therefore also be referred to as astroglia. However, a question exists as to if zebrafish astroglia cells interact functionally with neurons, in a similar way to their mammalian counterparts. Future studies of this fish will complement those on rodents and provide important information about the cellular and physiological processes underlying astroglial function that modulate neural activity and behavior in animals.

Brain histamine improves colonic hyperpermeability through the basal forebrain cholinergic neurons, adenosine A2B receptors and vagus nerve in rats.

Intestinal barrier dysfunction, leaky gut, is implicated in various diseases, including irritable bowel syndrome (IBS) and neurodegenerative conditions like Alzheimer's disease. Our recent investigation revealed that basal forebrain cholinergic neurons (BFCNs), critical for cognitive function, receive signals from butyrate and orexin, playing a role in regulating intestinal barrier function through adenosine A2B signaling and the vagus. This study explores the involvement and function of brain histamine, linked to BFCNs, in the regulation of intestinal barrier function. Colonic permeability, assessed by quantifying absorbed Evans blue in rat colonic tissue, showed that histamine did not affect increased colonic permeability induced by LPS when administered subcutaneously. However, intracisternal histamine administration improved colonic hyperpermeability. Elevating endogenous histamine levels in the brain with SKF91488, a histamine N-methyltransferase inhibitor, also improved colonic hyperpermeability. This effect was abolished by intracisternal chlorpheniramine, an histamine H1 receptor antagonist, not ranitidine, an H2 receptor antagonist. The SKF91488-induced improvement in colonic hyperpermeability was blocked by vagotomy, intracisternal pirenzepine (suppressing BFCNs activity), or alloxazine (an adenosine A2B receptor antagonist). Additionally, intracisternal chlorpheniramine injection eliminated butyrate-induced improvement in colonic hyperpermeability. These findings suggest that brain histamine, acting via the histamine H1 receptor, regulates intestinal barrier function involving BFCNs, adenosine A2B signaling, and the vagus. Brain histamine appears to centrally regulate intestinal barrier function influenced by butyrate, differentiating its actions from peripheral histamine in conditions like IBS, where mast cell-derived histamine induces leaky gut. Brain histamine emerges as a potential pharmacological target for diseases associated with leaky gut, such as dementia and IBS.

Generation of rat forebrain tissues in mice.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

Prenatal identification of a pathogenic maternal FGFR1 variant in two consecutive pregnancies with fetal forebrain malformations.

OBJECTIVE: Holoprosencephaly (HPE) is the most common aberration of forebrain development, and it leads to a wide spectrum of developmental and craniofacial anomalies. HPE etiology is highly heterogeneous and includes both chromosomal abnormalities and single-gene defects. METHODS: Here, we report an FGFR1 heterozygous variant detected by prenatal exome sequencing and inherited from the asymptomatic mother, in association with recurrent neurological abnormalities in the HPE spectrum in two consecutive pregnancies. RESULTS: Individuals with germline pathogenic variants in FGFR1 (MIM: 136350) show extensive phenotypic variability, which ranges from asymptomatic carriers to hypogonadotropic hypogonadism, arhinencephaly, Kallmann's syndrome with associated features such as cleft lip and palate, skeletal anomalies, isolated HPE, and Hartsfield syndrome. CONCLUSION: The presented case supports the role of exome sequencing in prenatal diagnosis when fetal midline structural anomalies are suggestive of a genetic etiology, as early as the first trimester of gestation. The profound heterogeneity of FGFR1 allelic disorders needs to be considered when planning prenatal screening even in asymptomatic carriers.

Comparative MRI analysis of the forebrain of three sauropsida models.

The study of the brain by magnetic resonance imaging (MRI) allows to obtain detailed anatomical images, useful to describe specific encephalic structures and to analyze possible variabilities. It is widely used in clinical practice and is becoming increasingly used in veterinary medicine, even in exotic animals; however, despite its potential, its use in comparative neuroanatomy studies is still incipient. It is a technology that in recent years has significantly improved anatomical resolution, together with the fact that it is non-invasive and allows for systematic comparative analysis. All this makes it particularly interesting and useful in evolutionary neuroscience studies, since it allows for the analysis and comparison of brains of rare or otherwise inaccessible species. In the present study, we have analyzed the prosencephalon of three representative sauropsid species, the turtle Trachemys scripta (order Testudine), the lizard Pogona vitticeps (order Squamata) and the snake Python regius (order Squamata) by MRI. In addition, we used MRI sections to analyze the total brain volume and ventricular system of these species, employing volumetric and chemometric analyses together. The raw MRI data of the sauropsida models analyzed in the present study are available for viewing and downloading and have allowed us to produce an atlas of the forebrain of each of the species analyzed, with the main brain regions. In addition, our volumetric data showed that the three groups presented clear differences in terms of total and ventricular brain volumes, particularly the turtles, which in all cases presented distinctive characteristics compared to the lizards and snakes.