Regional chemoarchitecture of the brain of lungfishes based on calbindin D‐28K and calretinin immunohistochemistry

Abstract

Lungfishes are the closest living relatives of land vertebrates, and their neuroanatomical organization is particularly relevant for deducing the neural traits that have been conserved, modified, or lost with the transition from fishes to land vertebrates. The immunohistochemical localization of calbindin (CB) and calretinin (CR) provides a powerful method for discerning segregated neuronal populations, fiber tracts, and neuropils and is here applied to the brains of Neoceratodus and Protopterus, representing the two extant orders of lungfishes. The results showed abundant cells containing these proteins in pallial and subpallial telencephalic regions, with particular distinct distribution in the basal ganglia, amygdaloid complex, and septum. Similarly, the distribution of CB and CR containing cells supports the division of the hypothalamus of lungfishes into neuromeric regions, as in tetrapods. The dense concentrations of CB and CR positive cells and fibers highlight the extent of the thalamus. As in other vertebrates, the optic tectum is characterized by numerous CB positive cells and fibers and smaller numbers of CR cells. The so-called cerebellar nucleus contains abundant CB and CR cells with long ascending axons, which raises the possibility that it could be homologized to the secondary gustatory nucleus of other vertebrates. The corpus of the cerebellum is devoid of CB and CR and cells positive for both proteins are found in the cerebellar auricles and the octavolateralis nuclei. Comparison with other vertebrates reveals that lungfishes share most of their features of calcium binding protein distribution with amphibians, particularly with salamanders.

Comparative Analysis of Nkx2.1 and Islet-1 Expression in Urodele Amphibians and Lungfishes Highlights the Pattern of Forebrain Organization in Early Tetrapods

“…However, Nkx2.1 cells are absent from pallial regions in anurans, birds and mammals (Sussel et al, 1999 ; Marín et al, 2000 ; Puelles et al, 2000 ; González et al, 2002 ), suggesting that cortical interneurons derived from the MGE down-regulate the expression of Nkx2.1 during their tangential migration to the cortex. The presence in the pallium of amphibians and lungfishes of GABAergic, nitrergic and calretinin containing cells postulate them as pallial interneurons (present results; Morona and González, 2008 ; González and Northcutt, 2009 ; Morona et al, 2018 ), and the lack of Nkx2.1 expression, except for the VP in the case of Pleurodeles (present results), would also reflect a down-regulation in its expression.…”

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish,Neoceratodus forsteri(Sarcopterygii: Dipnoi)

“…The nomenclature used is essentially the same followed in previous recent studies of lungfish brains (González, Morona, López, Moreno, & Northcutt, ; González & Northcutt, , ; López et al, , ; López & González, , ; López, Morona, Moreno, & González, ; Morona et al, ; Northcutt, , ), including the terminology of the prosomeric model for the forebrain (Puelles & Rubenstein, , ).…”

“…Thus, many features observed in living lungfishes may resemble those of ancestral tetrapods in the evolution from an aquatic fish ancestor (see Moreno et al, ). In spite of their important phylogenetic position, there are relatively few studies on the neuroanatomy of lungfishes using specific and more modern methods like immunohistochemistry, in situ hybridization, or tract‐tracing techniques (see López, Morona, Moreno, & González, ; Morona, López, Northcutt, & González, ). These studies are of special relevance because most brain regions that constitute clear divisions in amniotes are not distinguishable in lungfishes due to the reduced cell migration from the ventricular zone, which leaves most neuron perikarya crowded in a dense central layer where distinct brain structures can be recognized, at best, as local condensations (González & Northcutt, ; Nieuwenhuys, ; Northcutt, ).…”

“…Recently, the study of the neuroanatomy of lungfishes has experienced a new impulse with the application of immunohistochemical techniques that include the localization of neurotransmitters and other molecular markers, such as transcription factors and calcium binding proteins (see López, Morona, Moreno, & González, ; Morona et al, ). In particular, we have reported the organization of the cholinergic, serotonergic and catecholaminergic systems (López, Domínguez, Morona, Northcutt, & González, ; López & González, , ; López, Morona, & González, ) and these results allowed us to determine the extent to which this organization is comparable to that of tetrapods.…”

Pax6 expression highlights regional organization in the adult brain of lungfishes, the closest living relatives of land vertebrates

“…Studies on the neuroanatomy of lungfishes using modern methods such as immunohistochemistry, in situ hybridization, or tract‐tracing techniques (see López, Morona, Moreno, & González, ; Morona, López, Northcutt, & González, ) are of special relevance because with classical staining techniques most regions of the brain that constitute clear divisions in the amniotes are not distinguished in lungfishes. This is due to the very limited cell migration from the ventricular zone, which leaves most neuron perikarya in a central layer where distinct brain structures can be recognized, at best, as local condensations (González & Northcutt, ; Nieuwenhuys, ; Northcutt, ), Thus, the immunohistochemical detection of several markers, including neurotransmitters and other molecular markers, have revealed neuroanatomical features in the brain organization of lungfishes that allowed us to determine the extent to which it is comparable to that of tetrapods (see López, Morona, Moreno, & González, ; Morona et al, ; Reiner & Northcutt, ).…”