CUBIC-f: An optimized clearing method for cell tracing and evaluation of neurite density in the salamander brain.

Background: Although tissue clearing and subsequent whole-brain imaging is now possible, standard protocols need to be adjusted to the innate properties of each specific tissue for optimal results. This work modifies exiting protocols to clear fragile brain samples and documents a downstream pipeline for image processing and data analysis.

New Method: We developed a clearing protocol, CUBIC-f, which we optimized for fragile samples, such as the salamander brain.

Model systems for regeneration: salamanders.

Salamanders have been hailed as champions of regeneration, exhibiting a remarkable ability to regrow tissues, organs and even whole body parts, e.g. their limbs.

Homeostatic and regenerative neurogenesis in salamanders.

Large-scale regeneration in the adult central nervous system is a unique capacity of salamanders among tetrapods. Salamanders can replace neuronal populations, repair damaged nerve fibers and restore tissue architecture in retina, brain and spinal cord, leading to functional recovery. The underlying mechanisms have long been difficult to study due to the paucity of available genomic tools.

Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration.

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research.

Cellular basis of brain maturation and acquisition of complex behaviors in salamanders.

The overall bauplan of the tetrapod brain is highly conserved, yet significant variations exist among species in terms of brain size, structural composition and cellular diversity. Understanding processes underlying neural and behavioral development in a wide range of species is important both from an evolutionary developmental perspective as well as for the identification of cell sources with post-developmental neurogenic potential. Here, we characterize germinal processes in the brain of and during both development and adulthood.

Studying newt brain regeneration following subtype specific neuronal ablation.

The realization that neuronal injury does not result in permanent functional or cellular loss in all vertebrates has fascinated regenerative biologists. Neuronal regeneration occurs in a subset of species, including lizards, teleost fish, axolotls, and newts. One tool for studying neuronal regeneration in the adult brain is intraventricular injection of selective neuronal toxins, which leads to loss of subpopulations of neurons.

Husbandry of Spanish ribbed newts (Pleurodeles waltl).

Research on urodele amphibians, such as newts, is constantly contributing to our understanding of fundamental biological processes. In the present chapter, we present detailed husbandry protocols for the Spanish ribbed newt (Pleurodeles waltl ). We describe the main phases of their life cycle, with emphasis on the progressive development of sensory, motor, and integration systems, which lead to the acquisition of specific stereotyped (and conditioned) behaviors.

Conserved localization of Pax6 and Pax7 transcripts in the brain of representatives of sarcopterygian vertebrates during development supports homologous brain regionalization.

Many of the genes involved in brain patterning during development are highly conserved in vertebrates and similarities in their expression patterns help to recognize homologous cell types or brain regions. Among these genes, Pax6 and Pax7 are expressed in regionally restricted patterns in the brain and are essential for its development. In the present immunohistochemical study we analyzed the distribution of Pax6 and Pax7 cells in the brain of six representative species of tetrapods and lungfishes, the closest living relatives of tetrapods, at several developmental stages.

Fundamental differences in dedifferentiation and stem cell recruitment during skeletal muscle regeneration in two salamander species.

Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl).

Spatiotemporal patterns of Pax3, Pax6, and Pax7 expression in the developing brain of a urodele amphibian, Pleurodeles waltl.

The onset and developmental dynamics of Pax3, Pax6, and Pax7 expressions were analyzed by immunohistochemical techniques in the central nervous system (CNS) of embryos, larvae, and recently metamorphosed juveniles of the urodele amphibian Pleurodeles waltl. During the embryonic period, the Pax proteins start being detectable in neuroepithelial domains. Subsequently, they become restricted to subsets of cells in distinct brain regions, maintaining different degrees of expression in late larvae and juvenile brains.

Expression patterns of Pax6 and Pax7 in the adult brain of a urodele amphibian, Pleurodeles waltl.

Expression patterns of Pax6, Pax7, and, to a lesser extent, Pax3 genes were analyzed by a combination of immunohistochemical techniques in the central nervous system of adult specimens of the urodele amphibian Pleurodeles waltl. Only Pax6 was found in the telencephalon, specifically the olfactory bulbs, striatum, septum, and lateral and central parts of the amygdala. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3.

Regional distribution of calretinin and calbindin-D28k expression in the brain of the urodele amphibian Pleurodeles waltl during embryonic and larval development.

The sequence of appearance of calretinin and calbindin-D28k immunoreactive (CRir and CBir, respectively) cells and fibers has been studied in the brain of the urodele amphibian Pleurodeles waltl. Embryonic, larval and juvenile stages were studied. The early expression and the dynamics of the distribution of CBir and CRir structures have been used as markers for developmental aspects of distinct neuronal populations, highlighting the accurate extent of many regions in the developing brain, not observed on the basis of cytoarchitecture alone.

Characterization of the bed nucleus of the stria terminalis in the forebrain of anuran amphibians.

Major common features have been reported for the organization of the basal telencephalon in amniotes, and most characteristics were thought to be acquired in the transition from anamniotes to amniotes. However, gene expression, neurochemical, and hodological data obtained for the basal ganglia and septal and amygdaloid complexes in amphibians (anamniotic tetrapods) have strengthened the idea of a conserved organization in tetrapods. A poorly characterized region in the forebrain of amniotes has been the bed nucleus of the stria terminalis (BST), but numerous recent investigations have characterized it as a member of the extended amygdala.

Distribution of orexin/hypocretin immunoreactivity in the brain of the lungfishes Protopterus dolloi and Neoceratodus forsteri.

Lungfishes are currently considered the closest living relatives of tetrapods and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. The brains of lungfishes have received little attention in comparative studies probably due to the difficulty of obtaining these unique animals. In the present study the distribution of orexin (hypocretin)-like immunoreactivity was studied in the brain of the African lungfish Protopterus dolloi and the Australian lungfish Neoceratodus forsteri by using antibodies directed against the mammalian orexin-A and orexin-B peptides.

Immunohistochemical localization of orexins (hypocretins) in the brain of reptiles and its relation to monoaminergic systems.

With the aim of gaining more insight into the evolution of the orexinergic systems in the brain of vertebrates we have conducted a comparative analysis of the distribution of orexin-immunoreactive cell bodies and fibers in two reptiles, the lizard Gekko gecko and the turtle Pseudemys scripta elegans. In both species most immunoreactive neurons were found in the periventricular hypothalamic nucleus and in the infundibular hypothalamus. Only in the gecko, orexinergic cell bodies were present in the dorsolateral hypothalamic nucleus and the periventricular preoptic nucleus.