Macro- and microscopic brain anatomy of the amazon lava lizard (Tropidurus torquatus) (WIED, 1820)

Abstract Reptiles have a key role in understanding amniotes’ reproductive independence of water. Many adaptations arose, including in locomotor patterns and behaviours, and the nervous system adapted to those new habits. We have described the macroscopic anatomy and cytoarchitecture of the Amazon Lava Lizard brain (Tropidurus torquatus), an abundant lizard in South America. Fifteen specimens were captured, euthanized and their brains were dissected, eight of these were processed and stained in haematoxylin-eosin. Their main areas of the brain are the telencephalon and diencephalon, in the forebrain, tectum and tegmentum, in the midbrain and medulla oblongata and cerebellum, in the hindbrain. The main and accessory olfactory bulbs are the most rostral structure of the brain and are composed of six layers. Brain hemispheres compose the telencephalon and are divided in pallium and subpallium. Medial, dorsomedial, lateral and dorsal cortices are part of the pallium. Striatum, pallidum and septum compose the subpallium. The diencephalon is composed of thalamus, epithalamus and hypothalamus. The midbrain has a ventral tegmentum, composed of torus semicircularis and a dorsal 14 layered optic tectum. Most part of the hindbrain is composed of the medulla oblongata, and the cerebellum arises from it, forming a three-layered plate like structure. In general, the brain of Tropidurus torquatus resembles those of other lizards, with its own adaptations.


Introduction
Reptiles' embryos present the amniotic membrane, an adaptation that arose with water independence in reproduction.But this feature is not the only one associated with the transition from water to land that happened in tetrapods.Adaptations to the head to accommodate the differences in feeding, hearing and other behaviours and specialized limbs to support the body off the ground were some of the changes that happened, with equivalent changes in the nervous system (1) .
Survival and reproduction of organisms in the environment in which they live is important to a species' success and the nervous system coordinate activities towards these goals.Mammalian and reptile brain share ancestry and a number of functional attributes and since the reptile brain is simpler, it may provide invaluable help in deciphering modern neuroscience questions (2) .
Lizards have been identified as model organisms for various types of studies due to their easy observation, capture and handling.One of these species, the amazon lava lizard (Tropidurus torquatus) (Wied, 1820) has been explored in several studies, including temperature (3) , diet (4) , reproduction (5) and embryonic development studies (6) .Its specimens are extremely abundant, being distributed from Brazil to Argentina, with a seasonal reproductive cycle in the rainy season.The species is diurnal and preferentially inhabits open environments, feeding on invertebrates, flowers and fruits (3,5) .We aimed to describe the macroscopic anatomic and cytoarchitecture of the brain of T. torquatus, highlighting its main regions and structures.

.Macroscopic analysis
This research is part of supported by the Biodiversity Authorization and Information System of Brazil (Sistema de Autorização e Informação em Biodiversidade -SISBIO), protocol number -SISBIO 61909-1, and by ethics committee of the Federal University of Goiás (Universidade Federal de Goiás -UFG / Regional Jataí), protocol number -CEUA 013/18, both of which permitted the collection, transportation and care of the animals.
Fifteen juvenile and adult specimens of T. torquatus were used.Animals of both sexes were collected with a noose at the Universidade Federal de Goiás -Regional Jataí.They were euthanized with an intraperitoneal lethal dose of bupivacaine hydrochloride (100 mg/kg) and dissected with the help of tweezers, scissors and a dissecting microscope (7) .The skin of the head was removed, followed by removal of eyes and muscles around the brain.Next the bones protecting the brain and the dura mater were extracted, exposing the structure.Finally, the brain was carefully removed from the remaining brain case and severed at the spinal cord and fixed in 10% formalin for 2 weeks.The terminology for the areas and structures was used according with Wright et al. (8) and Naumann et al. (2) for the reptile brain, and the Nomina Anatomica Veterinarian (9) .

Histologic analysis
Eight brains were stained with haematoxylineosin (HE).The material was dehydrated in a series of alcohol 100% (5 baths, 50 min each), followed by submersion in xylol (2 baths / 50 min each) and paraffin inclusion (3 baths / 50 min each).The brains were included in three sectioning planes (sagittal, frontal and transversal) and then sectioned with microtome at 5 μm.For the staining protocol the paraffin was melted in an incubator (1 hour) and the remaining paraffin was removed with xylene (2 baths, 20 min each).Then it was passed through a series of alcohol solutions (100, 90, 70 and 50%, 5 min each) and bathed in distilled water (10 min) before the haematoxylin staining (5 min).Next it was submitted to running tap water (10 min) and counter-stained with eosin (4 min).It was dehydrated through alcohol 70% (5 quick immersions), alcohol 80% (1 min), alcohol 90% (2 min), alcohol 100% (5 min) and finally submersed in xylene (2 baths, 5 min each) and mounted with entellan.

Image capturing and processing
The macroscopic external morphology and topography of the brain were described and documented with a dissecting microscope (Leica ICC50 HD ® ).Histological images were analysed and photographed using an optic microscope (LEICA DM750 ® ) with an embedded camera (LEICA ICC50 HD ® ), with objective lens 4x (0.10), 10x (0.22), 20x (0.40) and 40x (0.65).After capture, macro-and microscopic images were processed using the software Affinity Photo ® v1.5.2.69 to merge continuous pictures of the same region, Adobe Photoshop CS6 ® v13.0 for background processing and adjusting tone and light and CorelDRAW X7 ® v17.1.0.572 to assemble images and point structures.

3.Results and discussion
The brain of T. torquatus was smooth and it extended from the medulla oblongata to the olfactory bulbs.It was limited caudally from the spinal cord by the foramen magnum and rostrally by olfactory capsules (Fig. 1A).The olfactory bulbs were located rostrally to the eyes, being connected to the brain by the olfactory peduncles.It was composed of forebrain (telencephalon and diencephalon), midbrain (tectum and tegmentum) and hindbrain (medulla oblongata and cerebellum) (Fig. 1B, 2, 5-7).These divisions are found in reptiles in general, though each species presents their own adaptations, as will be presented below (2,10,11) .
A system of ventricles was associated with every region of the brain, and its ependyma formed an innermost layer in all regions containing ventricles (Fig. 3,  4).The choroid plexus was located inside the ventricles (Fig. 4D).Only two meninges were present covering the brain, pia and dura mater, and they were closely associated with the ventricular system.
In T. torquatus, the olfactory peduncles became thicker as they reached the brain hemispheres (Fig. 2), which was also observed in G. gecko, I. iguana iguana and T. teguixin (13,15,18) and this structure appeared to be significantly thinner in A. garmani (12) .Olfactory ventricles were present and connected to lateral ventricles in T. torquatus, as reported in G gecko and I. iguana iguana (13,18) .While there is no information for most species, Shanklin (17) noted the lack of an olfactory ventricle in C. Vulgaris.
Microscopically in T. torquatus, both olfactory bulbs presented six layers: olfactory nerve fibers, glomerular, external plexiform, mitral, internal plexiform and granular layers.In the main bulb the layers were located concentrically around the ventricle, while in the accessory one it was mainly on the medial wall because the ventricle was located laterally (Fig. 3).This is similar to A. garmani, A. grahami, A. lineatopus and P. hispanica (12,14) and distinct from the findings in I. iguana iguana, for which only three cell layers were described: external granular, mitral and internal granular layers (13) and in C. Vulgaris, where the cells are very little differentiated and appear to be more like granular cells, without distinct layers (17) .This divergence could be due to different staining methods, which may not have detailed the cytoarchitecture of the olfactory bulb in these species.

Telencephalon
Macroscopically, the brain hemispheres of T. torquatus presented cordiform shape (as in A. garmani, Ctenophorus decresii, G. gecko, I. iguana iguana, P. hispanica, T. teguixin and S. merianae) and were visually larger than the oval shaped optic tectum.Brain hemispheres in reptiles are composed of a superficial pallium and a subpallium (11-14, 16, 18-20) .The pallium component of T. torquatus was comprised of medial, dorsomedial, lateral and dorsal cortex, and the ventrolateral dorsal ventricular ridge.The dorsomedial cortex was continuous with the medial cortex, but its cells were larger and less densely packed.
Three layers are presented in the cortices of T. torquatus, external and internal plexiform layers with scarce cells and an organized cell layer in between.This disposition was also reported in A. garmani, A. grahami, A. lineatopus, C. Vulgaris, C. decresii, G. gecko, I. iguana iguana, T. teguixin, S. merianae (8,12,13,(16)(17)(18)(19)(20)(21)(22)(23) .The dorsal cortex of T. torquatus presented a less organized cell layer and it was partially overlapped by the dorsomedial and lateral cortices (Fig. 4) whereas in C. decresii, the lateral cortex presented the most indistinct plexiform layer (19) .In G. gecko and T. teguixin, the internal plexiform layer was described as a subcortical layer of scattered cells and a fiber layer (18,23) , we did not notice any distinct fiber layer in T. torquatus.A clear distinction between rostral and caudal parts of the dorsal ventricular ridge were not visible with H.E. staining, and it presented as uniform distributed cells in T. torquatus (Fig. 5-7).Some parts of the ventricular ridge were covered by the cortices but lateral parts of the ventricular ridge were covered by a layer of pia mater and ependyma called pallial membrane (Fig. 5A), which was also found in A. garmani, A. grahami and A. lineatopus (12) .Subpallium was composed of the septum, striatum, and pallidum, from which the first was located medially between medial cortex and striatum.The amygdaloid complex was identified in the subpallium of G. Gecko (18) , but it was not possible to distinguish it in T. torquatus.The lateral ventricle is associated with the telencephalon and was located between the cortices and dorsal ventricular ridge in T. torquatus (Fig. 5).The hippocampal and anterior commissures cross the hemispheres and were identified in T. torquatus (Fig. 1C).

Diencephalon
In T. torquatus, four regions composed the diencephalon: epithalamus, thalamus (dorsal and ventral), and hypothalamus, as in C. Vulgaris, G. gecko and T. teguixin (15,17,21) .The diencephalon is associated with the third ventricle (Fig. 5-7), a median ventricle located between both sides of the diencephalon, in T. torquatus.The diencephalon was almost completely covered by the hemispheres on the dorsal surface, with only part of the epithalamus being exposed (Fig. 2A).
Most of the epithalamus was composed of the habenula in T. torquatus, from which some fibers crossed over at the habenula commissure.Structures of the pineal complex projected from the epithalamus: the pineal organ, dorsal sac, paraphysis and parietal eye (Fig. 8).The pineal organ was oval shaped in T. torquatus and triangular shaped in S. merianae (11) .The pretectum lies at the transition of the diencephalon and the mesencephalon (15,17,21) and could not be distinct in T. torquatus.
The thalamus was only visible in the sagittal section.In its dorsal part the posterior commissure was identified in T. torquatus (Fig. 1C, 5C).The largest part of the diencephalon was composed by the hypothalamus, part of which was visible on the ventral surface, the protruding infundibulum was medially located in this region.Also, ventrally, thick optic nerves intersected at the optic chiasm and entered the brain through optic tracts, surrounding the infundibulum, where the hypophysis was connected to the brain (Fig. 2).A supraoptical commissure was present ventrally in the hypothalamus, caudal to the optic chiasm (Fig. 1B), that is in agreement with literature data (15,17,21) .

Mesencephalon
The mesencephalic tectum and tegmentum comprised the mesencephalon.The tegmentum was located ventrally, continuous with the hindbrain.Its tectum had an optic tectum and torus semicircularis (Fig. 5-7).Macroscopically, the optic tectum was oval shaped and noticeably smaller than the brain hemispheres and partially covered by the cerebellum (also described in S. merianae (11) ), while the torus semicircularis was a small median structure located caudoventrally to the optic tectum, completely covered by the cerebellum (Fig. 1B, 2) The torus semicircularis was funnel shaped and it was larger in its medial part, which in both antimeres are partly fused at the midline.It thins out gradually as it extends laterally in T. torquatus, as also seen in Gallotia galloti and T. teguixin (24,25) .A band of fibers crossed both parts of the tectum forming the tectal commissure (Fig. 1C,  5C).The cerebral aqueduct passed through the midbrain toward the fourth ventricle (Fig. 1B).

Hindbrain
The medulla oblongata and cerebellum composed the hindbrain.The ventral part of the medulla oblongata was composed of the tegmentum, which was continuous with the tegmentum of the midbrain.One structure of the tegmentum was the reticular formation, distinct for its appearance of loose fibers (Fig. 6).The medulla oblongata was large lateral-laterally and it caudally tapered toward its division with the spinal cord, which also appeared larger rostrally.Laterally the medulla oblongata had a curved appearance and several roots of nerves protruded from its surface, both laterally and ventrally.
Dorsally in the medulla oblongata, part of the rhomboid fossa was visible, being covered rostrally by the cerebellum, it was formed by the fourth ventricle, with the choroid plexus laying over it (Fig. 2).The rhomboid fossa in A. garmani, G. gecko and, T. teguixin (12,16,18) is longer (caudo-caudally) than in T. torquatus, and present a more cylindrical shape, while in the latter it is triangular shaped.It appeared to be more similar between I. iguana iguana, S. merianae (11,13) and T. torquatus.It was oblique in Anniella nigra, mainly covered by nerve roots, with only a vertical slit being visible in dorsal view (29) .In T. torquatus, the cerebellum protruded from the dorsorostral part of the medulla oblongata, connected to it through the cerebellar peduncle (Fig. 10B).It had a plate shape and it curved rostrally (Fig. 2), divided into two lateral flocculi (also observed in S. merianae (11) ) and hemispheres with a median part and two lateral parts (Fig. 10A).
In the lizard A. nigra, a legless lizard, the cerebellum was small in size, compared to the rest of the brain.It was almost hidden from view, lying in a depression formed by the midbrain, medulla oblongata and nerve roots.The cerebellum of Gerrhonotus principis presented interesting features.In dorsal view it presented a triangular shape, its median part had a tongue-like structure projecting backwards following the contour of the rhomboid fossa.The lateral parts were curved forward, similar to the cerebellum of other lizards mention above (29) .
The cerebellar cortex in T. torquatus was formed by three layers: granular layer, Purkinje cell layer and molecular layer.The most external and dorsocaudal layer was the granular layer, composed of small and densely packed granule cells and larger Golgi cells.The molecular layer was the most ventrorostral and inner layer, with the presence of many dendrites from the adjacent Purkinje cells and axons from granule cells, and few basket and stellate cells.The Purkinje cell layer was located between the other two layers, formed by a single line of cells (Fig. 10C).These layers and cells were very similar to the ones found in C. decresii, C. vulgaris, P. douglasii, S. biseriatus and S. graciosus (17,19,29) .
In A. nigra, the layers were more similar to that of anurans, with the granular layer located ventrocaudally and the molecular layer dorsorostrally.The Purkinje cell layer was also formed by a single line of cells.The median part of the cerebellum of G. principis presented the granular layer ventrocaudally, similar to A. nigra, while it bended rostrally as it extended laterally, like that of most lizards (e.g.T. torquatus).The author proposed that this lateral part is what predominated in other lizards with the common conformation to the cerebellum (29) .

Conclusions
Generally speaking, the brain of Tropidurus torquatus was similar to that of other lizard species, with their adaptations to live in the environment they are inserted.Their brain possessed a large area dedicated to processing olfactory and visual stimuli, which are necessary to survive, either foraging and hunting for prey or hiding from predators.