Subsystem macroarchitecture of the intrinsic midbrain neural network and its tectal and tegmental subnetworks.

The midbrain is the smallest of three primary vertebrate brain divisions. Here we use network science tools to reveal the global organizing principles of intramidbrain axonal circuitry before adding extrinsic connections with the remaining nervous system. Curating the experimental neuroanatomical literature yielded 17,248 connection reports for 8,742 possible connections between the 94 gray matter regions forming the right and left midbrain. Evidence for the existence of 1,676 connections suggests a 19.2% connection density for this network, similar to that for the intraforebrain network [L. W. Swanson et al., Proc. Natl. Acad. Sci. U.S.A. 117, 31470-31481 (2020)]. Multiresolution consensus cluster analysis parceled this network into a hierarchy with 6 top-level and 30 bottom-level subsystems. A structure-function model of the hierarchy identifies midbrain subsystems that play specific functional roles in sensory-motor mechanisms, motivation and reward, regulating complex reproductive and agonistic behaviors, and behavioral state control. The intramidbrain network also contains four bilateral region pairs designated putative hubs. One pair contains the superior colliculi of the tectum, well known for participation in visual sensory-motor mechanisms, and the other three pairs form spatially compact right and left units (the ventral tegmental area, retrorubral area, and midbrain reticular nucleus) in the tegmentum that are implicated in motivation and reward mechanisms. Based on the core hypothesis that subsystems form functionally cohesive units, the results provide a theoretical framework for hypothesis-driven experimental analysis of neural circuit mechanisms underlying behavioral responses mediated in part by the midbrain.

Nucleus Isthmi Is Required to Sustain Target Pursuit during Visually Guided Prey-Catching.

Animals must frequently perform a sequence of behaviors to achieve a specific goal. However, the neural mechanisms that promote the continuation and completion of such action sequences are not well understood. Here, we characterize the anatomy, physiology, and function of the nucleus isthmi (NI), a cholinergic nucleus thought to modulate tectal-dependent, goal-directed behaviors. We find that the larval zebrafish NI establishes reciprocal connectivity with the optic tectum and identify two distinct types of isthmic projection neuron that either connect ipsilaterally to retinorecipient laminae of the tectum and pretectum or bilaterally to both tectal hemispheres. Laser ablation of NI caused highly specific deficits in tectally mediated loom-avoidance and prey-catching behavior. In the context of hunting, NI ablation did not affect prey detection or hunting initiation but resulted in larvae failing to sustain prey-tracking sequences and aborting their hunting routines. Moreover, calcium imaging revealed elevated neural activity in NI following onset of hunting behavior. We propose a model in which NI provides state-dependent feedback facilitation to the optic tectum and pretectum to potentiate neural activity and increase the probability of consecutive prey-tracking maneuvers during hunting sequences.

Aging is associated with larger brain mass and volume in homing pigeons (Columba livia).

In mammals, the brain decreases in mass and volume as a function of age. The current study is, to the best of our knowledge, the first to investigate age-related changes in brain mass and volume in birds. Following perfusion, brains from young and old homing pigeons were weighed on a balance and orthogonal measurements of the telencephalon, cerebellum, and tecta were obtained with a digital caliper. It was found that older pigeons had heavier brains than younger pigeons, a difference that remained after controlling for body mass. Additionally, older pigeons had on average greater estimated telencephalon volumes than younger pigeons, again also after controlling for body mass. Cerebellum and right tectum volumes also differed between age groups after controlling for body mass, with older pigeons having a larger cerebellum and right tectum than younger pigeons. In sum, brains are on average heavier and larger in old pigeons, which display age-related cognitive decline, compared to young adult pigeons. The larger brain in older homing pigeons also lies in stark contrast with aging of the mammalian brain.

The nucleus pretectalis principalis: A pretectal structure hidden in the mammalian thalamus.

A defining feature of the amniote tecto-fugal visual pathway is a massive bilateral projection to the thalamus originating from a distinct neuronal population, tectal ganglion cells (TGCs), of the optic tectum/superior colliculus (TeO/SC). In sauropsids, the thalamic target of the tecto-fugal pathway is the nucleus rotundus thalami (Rt). TGCs axons collateralize en route to Rt to target the nucleus pretectalis principalis (PT), which in turn gives rise to bilateral projection to the TeO. In rodents, the thalamic target of these TGCs afferents is the caudal division of the pulvinar complex (PulC). No pretectal structures in receipt of TGC collaterals have been described in this group. However, Baldwin et al. (Journal of Comparative Neurology, 2011;519(6):1071-1094) reported in the squirrel a feedback projection from the PulC to the SC. Pulvino-tectal (Pul-T) cells lie at the caudal pole of the PulC, intermingled with the axonal terminals of TGCs. Here, by performing a combination of neuronal tracing, immunohistochemistry, immunofluorescence, and in situ hybridization, we characterized the pattern of projections, neurochemical profile, and genoarchitecture of Pul-T cells in the diurnal Chilean rodent Octodon degus. We found that Pul-T neurons exhibit pretectal, but not thalamic, genoarchitectonical markers, as well as hodological and neurochemical properties that match specifically those of the avian nucleus PT. Thus, we propose that Pul-T cells constitute a pretectal cell population hidden within the dorsal thalamus of mammals. Our results solve the oddity entailed by the apparent existence of a noncanonic descending sensory thalamic projection and further stress the conservative character of the tectofugal pathway.

The dorsal tectal longitudinal column (TLCd): a second longitudinal column in the paramedian region of the midbrain tectum.

The tectal longitudinal column (TLC) is a longitudinally oriented, long and narrow nucleus that spans the paramedian region of the midbrain tectum of a large variety of mammals (Saldaña et al. in J Neurosci 27:13108-13116, 2007). Recent analysis of the organization of this region revealed another novel nucleus located immediately dorsal, and parallel, to the TLC. Because the name "tectal longitudinal column" also seems appropriate for this novel nucleus, we suggest the TLC described in 2007 be renamed the "ventral tectal longitudinal column (TLCv)", and the newly discovered nucleus termed the "dorsal tectal longitudinal column (TLCd)". This work represents the first characterization of the rat TLCd. A constellation of anatomical techniques was used to demonstrate that the TLCd differs from its surrounding structures (TLCv and superior colliculus) cytoarchitecturally, myeloarchitecturally, neurochemically and hodologically. The distinct expression of vesicular amino acid transporters suggests that TLCd neurons are GABAergic. The TLCd receives major projections from various areas of the cerebral cortex (secondary visual mediomedial area, and granular and dysgranular retrosplenial cortices) and from the medial pretectal nucleus. It densely innervates the ipsilateral lateral posterior and laterodorsal nuclei of the thalamus. Thus, the TLCd is connected with vision-related neural centers. The TLCd may be unique as it constitutes the only known nucleus made of GABAergic neurons dedicated to providing massive inhibition to higher order thalamic nuclei of a specific sensory modality.

Magnetic resonance imaging mesencephalic tectum dimensions according to age and gender.

OBJECTIVE: To analyze and classify normal MRI tectum length and colliculus dimensions according to age and gender. METHODS: Tectum length and colliculus diameters were measured on the T1 midsagittal and axial cranial MR images in the radiology archive of 532 (344 women, 188 men) patients aged 37.36+/-21.49 (range: 4-91) years old on average, and with no disorders affecting the mesencephalic tectum. All 532 patients underwent clinical MR imaging of the cranium at the MRI Unit of Sivas Numune Hospital and Sivas Cumhuriyet University Hospital, Sivas, Turkey between February and December 2011. RESULTS: Although there was a positive linear correlation between tectum length and age, there was a negative correlation between the anteroposterior diameter of the colliculus superior and colliculus inferior and age (p<0.01). While tectum length (M3) increases with age, the anteroposterior diameter of the colliculus superior and inferior (M1 and M2) decreased (p<0.01). The colliculi were larger, and the tectum was longer in men. Although there was no difference in size between right and left superior colliculi, the left colliculus inferior was larger than the right one. CONCLUSION: In addition to the fact that normal mesencephalic tectum dimensions provide information on the brain development of individuals, they may also be beneficial for the detection and treatment of related pathologies.

Bilateral efferents from nucleus isthmi to the optic tectum in goldfish (Carassius auratus) are spatially restricted.

The projection from nucleus isthmi (NI) to the optic tectum (OT) was investigated in the goldfish, Carassius auratus, by retrograde transport of biocytin applied at various sites in a tectal lobe. In previous studies, this projection is described as predominantly from the ipsilateral NI and maps topographically along the approximate rostrocaudal axis of both brain areas. However, the rostromedial tectal lobe, the tectal region representing the binocular visual field, receives afferents from both the ipsilateral and the contralateral NI. The contralateral isthmic neurons are found at the most caudal position in NI and are not topographic with the tectum. The bilateral projection from NI to the tectum may play a role in functions requiring the coordination of both eyes.

Retrograde tracing with fluorescent microspheres reveals bifurcating projections from central retina to tectum and thalamus in chicks.

The goal of this study is to demonstrate the dual-projection pattern of retinal ganglion cells (RGCs) projecting to the tectum and visual thalamus in chick using retrograde fluorescent tracers and also to define the morphological properties of these RGCs with dual projections by intracellular injection of Lucifer Yellow (LY) combined with immunohistochemistry. Thirty-two chicks received double injections of green and red fluorescent microspheres into their thalamus and tectum in the same side. In the central retina, most of the labelled RGCs were tec-RGCs (RGCs projecting to the tectum), a quarter was tha-RGCs (RGCs projecting to the thalamus), and almost all of the tha-RGCs were double-labelled RGCs. An intracellular injection of LY into the double-labelled RGCs showed all six groups of RGCs without specific populations in each group (J. Comp. Neurol., 2004, 469: 360). These dendritic patterns were mostly mono- and bistrata, which extended horizontally in the deeper part of the inner plexiform layer.

Organization of the cholinergic systems in the brain of two lungfishes, Protopterus dolloi and Neoceratodus forsteri.

Lungfishes (dipnoans) are currently considered the closest living relatives of tetrapods. The organization of the cholinergic systems in the brain has been carefully analyzed in most vertebrate groups, and major shared characteristics have been described, although traits particular to each vertebrate class have also been found. In the present study, we provide the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two representative species of lungfishes, the African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry against the enzyme choline acetyltransferase (ChAT). Distinct groups of ChAT immunoreactive (ChAT-ir) cells were observed in the basal telencephalon, habenula, isthmic nucleus, laterodorsal tegmental nucleus, cranial nerve motor nuclei, and the motor column of the spinal cord, and these groups seem to be highly conserved among vertebrates. In lungfishes, the presence of a cholinergic cell group in the thalamus and the absence of ChAT-ir cells in the tectum are variable traits, unique to this group and appearing several times during evolution. Other characters were observed exclusively in Neoceratodus, such as the presence of cholinergic cells in the suprachiasmatic nucleus, the pretectal region and the superior raphe nucleus. Cholinergic fibers were found in the medial pallium, basal telencephalon, thalamus and prethalamus, optic tectum and interpeduncular nucleus. Comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that the cholinergic systems in lungfishes largely resemble those of amphibians and other tetrapods.

Anatomic study of the quadrigeminal cistern in patients with 3-dimensional magnetic resonance cisternography.

OBJECT: The aim of this study was to demonstrate the anatomy of the quadrigeminal cistern, define the anatomic landmarks, and measure the extension of the cistern in the living by using magnetic resonance (MR) cisternography with 3-dimensional reconstruction. METHODS: The quadrigeminal cistern was examined in 38 patients. We focused on measurements of the superior, posterior, and lateral limits; the anterior and posterior maximal rostrocaudal diameter; the distance between the right and left superior colliculus and the right and left inferior colliculus; and the angle between the quadrigeminal plate and pineal gland. RESULTS: The highest variability was observed for the posterior rostrocaudal diameter with a standard deviation of 3.1 and a range from 8 to 21.1 mm followed by the anterior-posterior diameter with a standard deviation of 2.8 and a range from 6.4 to 16.5 mm. In all cases the distance between the right and left superior colliculus (13.3 +/- 1.8 mm; mean +/- SD) was longer than the distance between the right and left inferior colliculus (11.4 +/- 1.3 mm; mean +/- SD). We classified 2 types of cisterns: closed cisterns with angles between the quadrigeminal plate and the pineal gland ranging from 39 degrees to 63 degrees and open cisterns with angles ranging from 63 degrees to 76 degrees . The analysis of variability by age and sex showed no significant differences. CONCLUSIONS: The MR cisternography with 3-dimensional reconstruction was a simple and noninvasive tool providing detailed anatomic information in the living. It allowed measurement of the high variability of morphology of the quadrigeminal cistern. We defined the lateral landmarks and identified the lateral limit of the cistern. We classified the different shapes of the quadrigeminal cistern as open or closed cisterns. This can be helpful in the choice of the surgical approach to the lesions arising in this area.

Neurodevelopmental effects of chronic exposure to elevated levels of pro-inflammatory cytokines in a developing visual system.

BACKGROUND: Imbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with a number of severe and prevalent neurodevelopmental disorders, including autism spectrum disorder, schizophrenia and Down syndrome. Although several studies have shown that cytokines have potent effects on neural function, their role in neural development is still poorly understood. In this study, we investigated the link between abnormal cytokine levels and neural development using the Xenopus laevis tadpole visual system, a model frequently used to examine the anatomical and functional development of neural circuits. RESULTS: Using a test for a visually guided behavior that requires normal visual system development, we examined the long-term effects of prolonged developmental exposure to three pro-inflammatory cytokines with known neural functions: interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. We found that all cytokines affected the development of normal visually guided behavior. Neuroanatomical imaging of the visual projection showed that none of the cytokines caused any gross abnormalities in the anatomical organization of this projection, suggesting that they may be acting at the level of neuronal microcircuits. We further tested the effects of TNF-alpha on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-alpha resulted in enhanced spontaneous excitatory synaptic transmission in tectal neurons, increased AMPA/NMDA ratios of retinotectal synapses, and a decrease in the number of immature synapses containing only NMDA receptors, consistent with premature maturation and stabilization of these synapses. Local interconnectivity within the tectum also appeared to remain widespread, as shown by increased recurrent polysynaptic activity, and was similar to what is seen in more immature, less refined tectal circuits. TNF-alpha treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-alpha-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures. CONCLUSIONS: Taken together our data are consistent with a model in which TNF-alpha causes premature stabilization of developing synapses within the tectum, therefore preventing normal refinement and synapse elimination that occurs during development, leading to increased local connectivity and epilepsy. This experimental model also provides an integrative approach to understanding the effects of cytokines on the development of neural circuits and may provide novel insights into the etiology underlying some neurodevelopmental disorders.

Projections from the inferior colliculus to the tectal longitudinal column in the rat.

We have used the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) to study with albino rats the projections from the inferior colliculus (IC) to the tectal longitudinal column (TLC), a newly discovered nucleus that spans the midbrain tectum longitudinally, on each side of the midbrain, immediately above the periaqueductal gray matter. We studied the projections of the medial IC, which includes the classical central nucleus (CNIC) and the dorsal cortex (DCIC), and those of the lateral IC, equivalent to the classical external cortex (ECIC). Following unilateral injections of PHA-L into the medial IC, numerous terminal fibers are labeled bilaterally in the TLC. The ipsilateral projection is denser and targets the entire nucleus, whereas the contralateral projection targets significantly only the caudal half or two-thirds of the TLC. Fibers from the medial IC reach the TLC by two routes: as collaterals of axons that travel in the commissure of the IC and as collaterals of thick ipsilateral colliculogeniculate axons; the latter travel through the deep superior colliculus on their way to the TLC. Within the TLC, individual IC fibers tend to run longitudinally. The injection of PHA-L into the lateral IC indicates that this subdivision sends a weak, bilateral projection to the TLC whose trajectory, morphology and distribution are similar to those of the projection from the medial IC. These results demonstrate that all subdivisions of the IC send projections to the TLC, suggesting that the IC may be one of the main sources of auditory input to this tectal nucleus.

Selective projection patterns from subtypes of retinal ganglion cells to tectum and pretectum: distribution and relation to behavior.

An important issue to understand is how visual information can influence the motor system and affect behavior. Using the lamprey (Petromyzon marinus) as an experimental model we examined the morphological subtypes of retinal ganglion cells and their projection pattern to the tectum, which controls eye, head, and body movements, and to the pretectum, which mediates both visual escape responses and the dorsal light response. We identified six distinct morphological types of retinal ganglion cell. Four of these distribute their dendrites in the inner plexiform layer (image forming layer) and project in a retinotopic manner to all areas of the tectum. The posterior part of the retina has the highest density of ganglion cells and projects to the rostral part of the tectum, in which the visual field in front of the lamprey will be represented. From this area both orienting and evasive behaviors can be elicited. In contrast, pretectum receives input from two ganglion cells types that send their dendrites only to the outer plexiform layer or the outer limiting membrane and therefore may directly contact photoreceptors, and transmit information without additional delay to pretectum, which may be particularly important for visual escape responses. One of these two types, the bipolar ganglion cell, is only found in a small patch of retina just ventral of the optic nerve. Due to its distribution, morphology, and projections we suggest that this cell may control the dorsal light response.

Eph/ephrin gradients in the retinotectal system of Rana pipiens: developmental and adult expression patterns.

Eph/ephrin-receptor/ligand A and B families play a variety of roles during CNS development, including patterning the retinotectal projection. However, the alignment of their expression gradients with developing retinotectal maps and gradients of cellular development is not well understood in species whose midbrain tecta undergo a protracted anterior to posterior development. By using anatomical tracing methods and (3)H-thymidine neuronography, we have mapped the retinotectal projection and the spatiotemporal progression of tectal cellular development onto Eph/ephrin expression patterns in the tectum of larval Rana pipiens, as studied by means of in situ affinity analysis with fusion proteins. EphA expression is maximal in anterior tectum (and temporal retina); ephrin-A expression is maximal at the posterior pole (and nasal retina). EphB expression is graded in the early larva, where it is maximal in the posterior tectum just anterior to the posterior pole (and in the ventral retina). Tectal EphB expression becomes uniform at later stages and remains so in the adult, although its retinal expression remains maximal ventrally. In the early larva, EphA, EphB, and ephrin-A protein gradients are parallel to each other and align with the temporonasal axis of the retinal projection. The early EphB expression maximum overlaps the boundary between the mantle layer of newly postmitotic cells and the posterior, epithelial region of cell proliferation, suggesting that the expression maximum is associated with the initial migrations of the postmitotic cells. Ephrin-B expression was detected in the olfactory bulb and dorsal retina at all ages, but not in the tectum.

Two parallel ascending pathways from the dorsal octavolateral nucleus to the midbrain in the paddlefish Polyodon spathula.

The paddlefish is a passive electrosensory ray-finned fish with a special rostral appendage that is covered with thousands of electroreceptors, which makes the fish extremely sensitive to electric fields produced by its primary prey, small water fleas. We reexamined the electrosensory pathways from the periphery to the midbrain by injecting the neuronal tracer BDA into different branches of the lateral line nerve and into different parts of the dorsal octavolateral nucleus (DON) and the tectum. Primary afferents from the anterior to posterior body axis terminate in different areas in the mediolateral axis of the DON, the first electrosensory processing station. Previous studies showed that DON neurons project to the tectum and two different areas in the tegmentum. Now, we have found differences between the anterior and the posterior DON. Fibers from the anterior DON project unilaterally to the contralateral tectum while its posterior neurons project bilaterally to two nuclei in the tegmentum, the torus semicircularis and the lateral mesencephalic nucleus. This study is the first to show that two different populations of ascending neurons project to two different targets in the midbrain. These two pathways are likely to have different functions and further investigations may reveal the functional significance of these two parallel ascending systems.

Posterior parietal cortex as part of a neural network for directed attention in rats.

A rodent model of directed attention has been developed based upon behavioral analysis of contralateral neglect, pharmacological manipulations, and anatomical analysis of neural circuitry. In each of these three domains the rodent model exhibits striking similarities to humans. We hypothesize that there is a specific thalamo-cortical-basal ganglia network that subserves spatial attentional functions. Key components of this network are medial agranular and posterior parietal cortex, dorsocentral striatum, and the lateral posterior thalamic nucleus. Several issues need to be addressed before we can hope to realistically understand or model the functions of this network. Among these are the roles of medial versus lateral posterior parietal cortex; cholinergic mechanisms in attention; interhemispheric interactions; the role of synchronous firing at the cortical, striatal, and thalamic levels; interactions between cortical and thalamic projections to the striatum; interactions between cortical and nigral inputs to the thalamus; the role of collicular inputs to the lateral posterior thalamic nucleus; the role of cerebral cortex versus superior colliculus in driving the motor output expressed as orienting behavior during directed attention; the extent to which the circuitry we describe for directed attention also plays a role in other forms of attention.

Evolutionary changes in the complexity of the tectum of nontetrapods: a cladistic approach.

BACKGROUND: The tectum is a structure localized in the roof of the midbrain in vertebrates, and is taken to be highly conserved in evolution. The present article assessed three hypotheses concerning the evolution of lamination and citoarchitecture of the tectum of nontetrapod animals: 1) There is a significant degree of phylogenetic inertia in both traits studied (number of cellular layers and number of cell classes in tectum); 2) Both traits are positively correlated accross evolution after correction for phylogeny; and 3) Different developmental pathways should generate different patterns of lamination and cytoarchitecture. METHODOLOGY/PRINCIPAL FINDINGS: The hypotheses were tested using analytical-computational tools for phylogenetic hypothesis testing. Both traits presented a considerably large phylogenetic signal and were positively associated. However, no difference was found between two clades classified as per the general developmental pathways of their brains. CONCLUSIONS/SIGNIFICANCE: The evidence amassed points to more variation in the tectum than would be expected by phylogeny in three species from the taxa analysed; this variation is not better explained by differences in the main course of development, as would be predicted by the developmental clade hypothesis. Those findings shed new light on the evolution of an functionally important structure in nontetrapods, the most basal radiations of vertebrates.

Developmental origins of species differences in telencephalon and tectum size: morphometric comparisons between a parakeet (Melopsittacus undulatus) and a quail (Colinus virgianus).

Parrots, including parakeets, evolved significantly larger brains than other birds, relative to their body size, and they possess a proportionately larger telencephalon. For example, the telencephalon occupies approximately 68% of the brain in parakeets but only 52% in bobwhite quail. The present study was designed to determine when and how this difference in brain region proportions arises during development. To that end, we present volumetric data on the major brain regions in parakeets and bobwhite quail at several stages of embryogenesis, at hatching and, for the parakeets, 1 week after hatching. We also report on the proportional sizes of each region's proliferative and postproliferative zones. One major finding is that parakeets develop a proportionately larger telencephalon relatively late in development and that this late increase correlates with a delay in telencephalic neurogenesis. The most prominent aspect of this delayed telencephalic development is a tremendous expansion of the proliferative subventricular zone in the telencephalon of late embryonic and posthatching parakeets. The second major finding is that the tectum is much smaller in parakeets than in quail at all developmental stages examined, suggesting that the tectum's reduced size is due to an evolutionary change in how much tissue was allocated to become tectum at the time of brain regionalization. Collectively these findings indicate that evolutionary changes in brain region proportions are caused not by a single type of change but by several distinct developmental mechanisms, including changes in brain regionalization and neurogenesis timing.

The pretectal nuclei in two monotremes: the short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus).

We have examined the organization of the pretectal area in two monotremes (the short beaked echidna-Tachyglossus aculeatus, and the platypus-Ornithorhynchus anatinus) and compared it to that in the Wistar strain rat, using Nissl staining in conjunction with enzyme histochemistry (acetylcholinesterase and NADPH diaphorase) and immunohistochemistry for parvalbumin, calbindin, calretinin and non-phosphorylated neurofilament protein (SMI-32 antibody). We were able to identify distinct anterior, medial, posterior (now called tectal gray) and olivary pretectal nuclei as well as a nucleus of the optic tract, all with largely similar topographical and chemoarchitectonic features to the homologous regions in therian mammals. The positions of these pretectal nuclei correspond to the distributions of retinofugal terminals identified by other authors. The overall size of the pretectum in both monotremes was found to be at least comparable in size, if not larger than, the pretectum of representative therian mammals of similar brain and body size. Our findings suggest that the pretectum of these two monotreme species is comparable in both size and organization to that of eutherian mammals, and is more than just an undifferentiated area pretectalis. The presence of a differentiated pretectum with similar chemoarchitecture to therians in both living monotremes lends support to the idea that the stem mammal for both prototherian and therian lineages also had a differentiated pretectum. This in turn indicates that a differentiated pretectum appeared at least 125 million years ago in the mammalian lineage and that the stem mammal for proto- and eutherian lineages probably had similar pretectal nuclei to those identified in its descendants.