The Independent Evolution of Dorsal Pallia in Multiple Vertebrate Lineages.

Comparative neurobiologists have long wondered when and how the dorsal pallium (e.g., mammalian neocortex) evolved. For the last 50 years, the most widely accepted answer has been that this structure was already present in the earliest vertebrates and, therefore, homologous between the major vertebrate lineages. One challenge for this hypothesis is that the olfactory bulbs project throughout most of the pallium in the most basal vertebrate lineages (notably lampreys, hagfishes, and lungfishes) but do not project to the putative dorsal pallia in teleosts, cartilaginous fishes, and amniotes (i.e., reptiles, birds, and mammals). To make sense of these data, one may hypothesize that a dorsal pallium existed in the earliest vertebrates and received extensive olfactory input, which was subsequently lost in several lineages. However, the dorsal pallium is notoriously difficult to delineate in many vertebrates, and its homology between the various lineages is often based on little more than its topology. Therefore, we suspect that dorsal pallia evolved independently in teleosts, cartilaginous fishes, and amniotes. We further hypothesize that the emergence of these dorsal pallia was accompanied by the phylogenetic restriction of olfactory projections to the pallium and the expansion of inputs from other sensory modalities. We do not deny that the earliest vertebrates may have possessed nonolfactory sensory inputs to some parts of the pallium, but such projections alone do not define a dorsal pallium.

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

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.

Variation in reptilian brains and cognition.

The class Reptilia is monophyletic, if all synapsid tetrapods are excluded and birds are included. The phylogenetic position of turtles within the reptilian clade is still problematic, but recent microRNA data suggest that turtles are the sister group to lepidosaurians. Brain-body data for approximately 60 reptilian taxa indicate that the relative brain size for a given body weight varies some six-fold among reptiles, with some turtles and lizards having relatively large brains and other turtles and lizards having relatively small brains. Snakes appear to be characterized by relatively small brains, and crocodilians appear to possess the largest brains among living reptiles, with the exception of birds. Data on the relative size of major brain divisions among tetrapods are limited, but the telencephalic and cerebellar hemispheres account for much of the variation. Telencephalic hemispheres in reptiles are approximately twice as large as those in amphibians, and the relative size of the telencephalic hemispheres in monitor lizards and crocodilians approaches that in basal birds and mammals. New data on the relative volumes of telencephalic pallial divisions in tetrapods reveal that the dorsal ventricular ridge, a ventral pallial derivative, accounts for much of the increase in pallial size that characterizes reptiles. Studies of spatial and visual cognition in nonavian reptiles reveal that they learn mazes and make visual discriminations as rapidly as most birds and mammals. Studies of social cognition and novel behavior, including play, reveal levels of complexity not previously believed to exist among nonavian reptiles. Given this level of neural and cognitive complexity, it is possible that consciousness has evolved numerous times, independently, among reptiles.

Comparative analysis of the organization of the cholinergic system in the brains of two holostean fishes, the Florida gar Lepisosteus platyrhincus and the bowfin Amia calva.

The cholinergic system in the brain has been widely studied in most vertebrate groups, but there is no information available about this neurotransmission system in the brains of holostean fishes, a primitive and poorly understood group of actinopterygian fishes. The present study provides the first detailed information on the distribution of cholinergic cell bodies and fibers in the central nervous system in two holostean species, the Florida gar, Lepisosteus platyrhincus, and the bowfin, Amia calva. Immmunohistochemistry against the enzyme choline acetyltransferase (ChAT) revealed distinct groups of ChAT-immunoreactive (ChAT-ir) cells in the habenula, isthmic nucleus, laterodorsal tegmental nucleus, octavolateral area, reticular formation, cranial nerve motor nuclei and the motor column of the spinal cord, all of which seem to be highly conserved among vertebrates. Some ChAT-ir cells were detected in the basal telencephalon that appear in actinopterygians for the first time in the evolution of this neurotransmission system, whereas the remarkable cholinergic population in the optic tectum is a peculiar characteristic, the presence of which varies throughout evolution, although it is present in all teleosts studied. Abundant cholinergic fibers were found in the pretectal region and optic tectum, where they probably modulate vision, and in the hypothalamus and the interpeduncular neuropil. Some interspecific differences were also observed, such as the presence of ChAT-ir cells in the supraoptoparaventricular band only in Lepisosteus and in in the nucleus subglomerulosus only in Amia. In addition, ChAT-ir fibers in the olfactory bulb were detected only in Amia. Comparison of these results with those from other classes of vertebrates, and a segmental analysis to correlate cell populations, reveal that the pattern of the cholinergic system in holosteans is very close to that in ancestral actinopterygian fishes, as recently described in the bichir (Cladistia), although an important evolutionary novelty in holosteans is the presence of cholinergic cells in the basal telencephalon.

Neuroanatomical organization of the cholinergic system in the central nervous system of a basal actinopterygian fish, the senegal bichir Polypterus senegalus.

Polypterid bony fishes are believed to be basal to other living ray-finned fishes, and their brain organization is therefore critical in providing information as to primitive neural characters that existed in the earliest ray-finned fishes. The cholinergic system has been characterized in more advanced ray-finned fishes, but not in polypterids. In order to establish which cholinergic neural centers characterized the earliest ray-finned fishes, the distribution of choline acetyltransferase (ChAT) is described in Polypterus and compared with the distribution of this molecule in other ray-finned fishes. Cell groups immunoreactive for ChAT were observed in the hypothalamus, the habenula, the optic tectum, the isthmus, the cranial motor nuclei, and the spinal motor column. Cholinergic fibers were observed in both the telencephalic pallium and the subpallium, in the thalamus and pretectum, in the optic tectum and torus semicircularis, in the mesencephalic tegmentum, in the cerebellar crest, in the solitary nucleus, and in the dorsal column nuclei. Comparison of the data within a segmental neuromeric context indicates that the cholinergic system in polypterid fishes is generally similar to that in other ray-finned fishes, but cholinergic-positive neurons in the pallium and subpallium, and in the thalamus and cerebellum, of teleosts appear to have evolved following the separation of polypterids and other ray-finned fishes.

Forebrain organization in elasmobranchs.

It has long been known that many elasmobranch fishes have relatively large brains. The telencephalon, in particular, has increased in size in several groups, and as a percent of total brain weight, it is as large as in some mammals. Little is known, however, about the organization, connections, and functions of the telencephalon in elasmobranchs. Early experimental studies indicated that olfaction does not dominate the telencephalon and that other sensory modalities are represented, particularly in the pallium. We have investigated the intrinsic and extrinsic connections of the telencephalon in two elasmobranch species: the thornback guitarfish, Platyrhinoidis triseriata, and the spiny dogfish, Squalus acanthias. Tracers were injected into various parts of the forebrain and olfactory pathways were found to be extensive and were seen to involve the pallium. Injections into various parts of the pallium revealed a major input from the area basalis, which receives secondary and tertiary olfactory fibers. Nonolfactory input from the diencephalon appeared relatively minor and seemed to converge with olfactory information in the dorsal pallium and area superficialis basalis. Major descending projections were seen to originate in the dorsal pallium and terminate in the hypothalamus and - in the case of Platyrhinoidis - massively in the lateral mesencephalic nucleus. Descending pathways appeared mainly crossed in Platyrhinoidis, but not in Squalus. Our data indicate that the concept of the dorsal pallium as a nonolfactory area in elasmobranchs must be reconsidered, and we suggest that many telencephalic centers, including the dorsal pallium, are involved in olfactory orientation.

Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes.

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

Evolution of centralized nervous systems: two schools of evolutionary thought.

Understanding the evolution of centralized nervous systems requires an understanding of metazoan phylogenetic interrelationships, their fossil record, the variation in their cephalic neural characters, and the development of these characters. Each of these topics involves comparative approaches, and both cladistic and phenetic methodologies have been applied. Our understanding of metazoan phylogeny has increased greatly with the cladistic analysis of molecular data, and relaxed molecular clocks generally date the origin of bilaterians at 600-700 Mya (during the Ediacaran). Although the taxonomic affinities of the Ediacaran biota remain uncertain, a conservative interpretation suggests that a number of these taxa form clades that are closely related, if not stem clades of bilaterian crown clades. Analysis of brain-body complexity among extant bilaterians indicates that diffuse nerve nets and possibly, ganglionated cephalic neural systems existed in Ediacaran organisms. An outgroup analysis of cephalic neural characters among extant metazoans also indicates that the last common bilaterian ancestor possessed a diffuse nerve plexus and that brains evolved independently at least four times. In contrast, the hypothesis of a tripartite brain, based primarily on phenetic analysis of developmental genetic data, indicates that the brain arose in the last common bilaterian ancestor. Hopefully, this debate will be resolved by cladistic analysis of the genomes of additional taxa and an increased understanding of character identity genetic networks.

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.

Olfactory projections in the lepidosirenid lungfishes.

Olfactory nerve and olfactory bulb projections in lepidosirenid lungfishes were experimentally determined with neural tracers. Unilateral injections of DiI into the olfactory nerve labeled the accessory and main olfactory bulbs as well as fibers of the anterior root of the terminal nerve, which terminates extensively in cell groups of the medial hemispheric wall, the dorsal and lateral pallia, and the preoptic nuclei and posterior tubercle. Lepidosirenid lungfishes do not exhibit separate vomeronasal nerves, but previous data indicate that calbindin-positive receptors within basal crypts of the olfactory epithelium are homologous to the vomeronasal organ of tetrapods. Unilateral injections of DiI into the accessory olfactory bulb reveal an accessory olfactory tract which terminates primarily if not solely in the ipsilateral medial amygdalar nucleus as in amphibians. Unilateral injections of tracers into the main olfactory bulb reveal extensive projections to all cell groups in the ipsilateral telencephalic hemisphere, except for the medial amygdalar nucleus, as well as secondary olfactory projections (decussating in the habenular commissure) to the contralateral dorsal pallium and main olfactory bulb. Secondary olfactory projections also terminate bilaterally in diencephalic and midbrain centers after partial decussation in the anterior and postoptic commissures, as well as in the ventral hypothalamus and posterior tubercle. Cladistic analysis of the extensive secondary olfactory projections indicates that this pattern is primitive for all bony fishes whereas the reduction in secondary olfactory projections in amphibians, particularly anurans, is a derived, simplified pattern.

Electrosensory ampullary organs are derived from lateral line placodes in bony fishes.

Electroreception is an ancient subdivision of the lateral line sensory system, found in all major vertebrate groups (though lost in frogs, amniotes and most ray-finned fishes). Electroreception is mediated by 'hair cells' in ampullary organs, distributed in fields flanking lines of mechanosensory hair cell-containing neuromasts that detect local water movement. Neuromasts, and afferent neurons for both neuromasts and ampullary organs, develop from lateral line placodes. Although ampullary organs in the axolotl (a representative of the lobe-finned clade of bony fishes) are lateral line placode-derived, non-placodal origins have been proposed for electroreceptors in other taxa. Here we show morphological and molecular data describing lateral line system development in the basal ray-finned fish Polyodon spathula, and present fate-mapping data that conclusively demonstrate a lateral line placode origin for ampullary organs and neuromasts. Together with the axolotl data, this confirms that ampullary organs are ancestrally lateral line placode-derived in bony fishes.

Olfactory projections in the white sturgeon, Acipenser transmontanus: an experimental study.

Telencephalic evolution in ray-finned fishes shows increasing complexity from polypteriform fishes through sturgeons to teleosts. Telencephalic organization in sturgeons is thus critical to our understanding of ray-finned fish evolution, but it is poorly understood, particularly as regards the roof or pallium. Two major hypotheses exist regarding the medial part of area dorsalis (Dm): that Dm is extended; and that Dm is restricted. The extent and topography of secondary olfactory projections to the pallium are critical in evaluating these hypotheses, but there is little agreement regarding these projections. Olfactory projections in the white sturgeon were therefore examined by using the carbocyanine probe DiI, biocytin, and biotinylated dextrin amine (BDA). Both DiI and BDA revealed primary olfactory projections to the olfactory bulb and primary extrabulbar projections widely in the telencephalon and to more restricted regions of the diencephalon. Myelinated secondary olfactory fibers caused DiI to be less effective in labeling secondary olfactory projections, which terminate in all subpallial nuclei and in the pallium: sparsely in the medial pallial division (Dm); heavily in the posterior pallial division (Dp); and more lightly in the lateral pallial division (Dl). In the diencephalon, substantial secondary olfactory projections were seen to the habenular nuclei, the rostral pole of the inferior lobe, and several nuclei of the posterior tubercle. All secondary olfactory projections were bilateral but heavier ipsilaterally. Bulbopetal neurons were located in both pallial and subpallial centers and were more numerous ipsilaterally. These results corroborate an earlier experimental study on the shovelnose sturgeon and indicate a restricted Dm in sturgeons.

A reinterpretation of the cytoarchitectonics of the telencephalon of the comoran coelacanth.

The cytoarchitecture of the telencephalon of the Comoran coelacanth, Latimeria chalumnae, was analyzed in the context of recent advances in our understanding of telencephalic organization in lungfishes and amphibians, which constitute the sister group to coelacanths. In coelacanths, the telencephalon is divided into pedunculated olfactory bulbs, paired hemispheres, and an unevaginated telencephalon impar. The hemispheres consist of a ventrally located subpallium and, dorsally, a greatly expanded pallium. Traditionally, the subpallium in coelacanths has been divided into a medial septal area and a lateral striatum. Re-examination of the lateral subpallial wall, however, suggests that the striatum is more restricted than previously believed, and it is replaced dorsally by a more scattered plate of cells, which appears to represent the ventral pallium. The putative ventral pallium is continuous with a ventromedial pallial formation, which appears to receive input from the lateral olfactory tract and should be considered a possible homolog of the lateral pallium in tetrapods. The putative lateral pallium is replaced by a more dorsomedial pallial formation, which may represent the dorsal pallium. This formation is replaced, in turn by an extensive lateral pallial formation, which appears to be homologous to the medial pallium of tetrapods. An expanded medial pallium in coelacanths, lepidosirenid lungfishes, and amphibians may be related to well developed spatial learning. Traditionally, the telencephalon impar of coelacanths, has been interpreted as an enlarged preoptic area, but reanalysis indicates that the so-called superior preoptic nucleus actually consists of the medial amygdalar nucleus.

Connections of the medial telencephalic wall in the spotted African Lungfish.

The extent and boundaries of the roof, or pallium, of the telencephalon in lungfishes have been debated for over 30 years, and two hypotheses exist. Proponents of a restricted pallium claim that the medial border of the pallium occurs in a dorsal position and that the entire medial hemispheric wall is formed by the septal nuclei. Proponents of an extended pallium claim that the medial border of the pallium occurs in a more ventral position and that the medial hemispheric wall is divided into a dorsal medial pallium and ventral septal nuclei, as in amphibians. Immunohistochemical data have generally been interpreted to support the hypothesis of an extended pallium, but disagreement still exists. To clarify the extent of the pallium in lungfishes, the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish were examined using a number of neuronal tracers. In amphibians and other tetrapods, the afferent projections to the medial pallium and the septal nuclei differ extensively, as do the commissural routes taken by decussating interhemispheric connections. Although the descending projections of the medial pallium and septal nuclei are very similar to one another in amphibians and other tetrapods, they do differ in that the septal nuclei and the ventral thalamus are extensively interlinked, whereas the medial pallium lacks such connections. These differences also characterize the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish, which supports the hypothesis of an extended pallium. The telencephalic organization in lungfishes thus appears remarkably similar to that in amphibians and reflects a pattern that almost certainly existed in the last common ancestor of lungfishes and tetrapods.

Immunohistochemical localization of calbindin D28k and calretinin in the retina of two lungfishes, Protopterus dolloi and Neoceratodus forsteri: colocalization with choline acetyltransferase and tyrosine hydroxylase.

There are no previous studies of the distribution of calbindin-D28k (CB) and calretinin (CR) in retinal neurons in lungfishes. The present study aimed to characterize the distribution of these neurons in a lepidosirenid lungfish (Protopterus dolloi) and a ceratodontid lungfish (Neoceratodus forsteri) using the same immunohistochemical protocol, with specific poly- and monoclonal antibodies, used in a previous comparative study of amphibians. The pattern for both proteins was similar in the two lungfishes studied, but some differences were also found. CB was found in photoreceptors, in subpopulations of bipolar and amacrine cells, and in horizontal and ganglion cells. Photoreceptors were negative for CR, whereas subpopulations of horizontal, bipolar, and amacrine cells contained CR, as did cells in the ganglion cell layer. Colocalization of CB/CR occurred in some cells of the inner nuclear layer. Tyrosine hydroxylase (TH) immunoreactivity colocalized with CB in some amacrine cells and, less frequently, with CR, but colocalization of TH with CB or CR in cholinergic cells was never found. Our data suggest a specific pattern for CB and CR distribution in the retinal neurons of lungfishes, a pattern that is highly comparable to that seen in amphibians, with unique features not observed in other vertebrates.

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of lungfishes.

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.

Lungfishes, like tetrapods, possess a vomeronasal system.

The vomeronasal system (VNS) is an accessory olfactory system that in tetrapod vertebrates is composed of specific receptor neurons in the nasal organ and a set of centers in the forebrain that receive and relay the information consecutively towards the hypothalamus. Thus, only in tetrapods the VNS comprises a discrete vomeronasal (Jacobson's) organ, which contains receptor cells that are morphologically distinct from those of the olfactory epithelium and use different transduction mechanisms. The axons of the vomeronasal receptors in tetrapods project to the accessory olfactory bulb (AOB) in the rostral telencephalon. Secondary vomeronasal connections exist through the medial amygdala to the hypothalamus. Currently, the lungfishes are considered the closest living relatives of tetrapods. Here we show that the African lungfish, Protopterus dolloi, has epithelial crypts at the base of the lamellae of the olfactory epithelium that express markers of the vomeronasal receptors in tetrapods. The projections of these crypts allow us to identify an AOB on the lateral margin of the main olfactory bulb. The projections of this AOB reach a region that is topologically, hodologically, and immunohistochemically identical to the medial amygdala and could represent its homolog. Neurons of this putative medial amygdala were demonstrated to project to the lateral hypothalamus, as they do in tetrapods. All these features that lungfishes share with tetrapods indicate that lungfishes have the complete set of brain centers and connections involved in processing vomeronasal information and that these features were already present in the last common ancestor of lungfishes and tetrapods.