New insights on the neuropeptide Y system in the larval lamprey brain: neuropeptide Y immunoreactive neurons,descending spinal projections and comparison with tyrosine hydroxylase and GABA immunoreactivities

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.     

Somatostatin- and enkephalin-like immunoreactivities are frequently colocalized in neurons in the caudal brain stem of rat

Summary The medulla oblongata and pons of colchicine treated rats were analyzed with a doublestaining technique using mouse monoclonal antibodies to somatostatin and rabbit polyclonal antibodies raised against methionine-enkephalin. Numerous cells reacted with both antisera but cells reacting with only one antiserum were also observed. Double-stained cells were most frequently encountered at all levels of the nucleus tractus solitarii, in a well defined group in the caudal medullary reticular formation, along the lateral ventral surface of the medulla oblongata, dorsolateral to the inferior olive and in the nucleus raphe magnus. These findings provide further examples of coexistence of two peptides and indicate the possibility that somatostatin-and enkephalin-like peptides are co-released.     

GABA neurotransmitter signaling in the developing mouse lens: Dynamic regulation of components and functionality

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate nervous system, serves as a signaling molecule modulating diverse processes during embryonic development. Earlier we have demonstrated that different forms of glutamic acid decarboxylase (GAD) are differentially regulated during mouse lens development. Here we show that the developing lens expresses also components of GABA signaling downstream of GAD. Multiple GABA_A and GABA_B receptor subunits as well as the GABA transporters show expression profiles highly correlated with the expression of different GADs. GABA receptors (GABAR) and the vesicular GABA transporter localize at the apical/basal membranes of the lens epithelia and differentiating fibers and may be involved in conventional GABAR‐mediated signaling, while the membrane GABA transporters may also function as Na⁺/Cl^?/GABA carriers. The functionality of GABAR was verified by calcium imaging in whole lenses. Our data suggest that GABA synthesized locally by GAD, acts through GABA receptors by modulating the intracellular calcium levels. Developmental Dynamics 237:3830–3841, 2008. © 2008 Wiley‐Liss, Inc.     

Retinal and non-retinal inputs upon retinopetal RMA neurons in the lamprey: a light and electron microscopic study combining HRP axonal tracing and GABA immunocytochemistry

A light and electron microscopic study, combining HRP axonal tracing or degeneration and GABA immunocytochemistry, was performed in the lamprey Lampetra fluviatilis in order to analyze retinal and non-retinal inputs upon the retinopetal neurons localized in the reticular mesencephalic area (RMA). The iontophoretic deposit of HRP onto the central stump of the cut optic nerve produced a dense anterograde labeling in the retino-recipient strata marginale and cellulare externum of the optic tectum as well as the retrograde labeling of retinopetal neurons in the mesencephalic tegmentum. The large ascending proximal dendrites of the retinopetal neurons constituted a distinct bundle coursing first dorso-laterally in the dorsal mesencephalic tegmentum, and then dorso-medially in the strata fibrosum centrale and cellulare et fibrosum internum of the optic tectum before their distal portions penetrated the retino-recipient tectal layers. The distribution of GABA immunoreactivity was also investigated in the tectal layers and dorsal mesencephalic tegmentum with both pre- and post-embedding methods. The retinal terminals, identified either following HRP iontophoresis in the optic nerve or in early phases of degeneration after short-term survivals following retinal lesion, contained rounded-shaped synaptic vesicles and were always GABA immunonegative. They established asymmetrical synaptic contacts on the distal dendrites of RMA neurons and represented 11.4% of all terminals contacting such neurons (15% of these neurons were GABA immunopositive). The dense extra-retinal input upon the retinopetal RMA neurons was composed of five types of axon terminal profiles, either GABA-immunopositive or -immunonegative. Considering the different cytochemical types of axon terminals contacting RMA neurons, as well as the characteristics of the retinal targets of these neurons, we suggest that, globally, the effects of RMA neurons upon the retina are mainly inhibitory.     

Restricted co‐localization of glutamate and dopamine in neurons of the adult sea lamprey brain

Co‐localization of dopamine with other classical neurotransmitters in the same neuron is a common phenomenon in the brain of vertebrates. In mammals, some dopaminergic neurons of the ventral tegmental area and the hypothalamus have a glutamatergic co‐phenotype. However, information on the presence of this type of dopaminergic neurons in other vertebrate groups is very scant. Here, we aimed to provide new insights on the evolution of this neuronal co‐phenotype by studying the presence of a dual dopaminergic/glutamatergic neuron phenotype in the central nervous system of lampreys. Double immunofluorescence experiments for dopamine and glutamate in adult sea lampreys revealed co‐localization of both neurotransmitters in some neurons of the preoptic nucleus, the nucleus of the postoptic commissure, the dorsal hypothalamus and in cerebrospinal fluid‐contacting cells of the caudal rhombencephalon and rostral spinal cord. Moreover, co‐localization of dopamine and glutamate was found in dopaminergic fibres in a few brain regions including the lateral pallium, striatum, and the preoptic and postoptic areas but not in the brainstem. Our results suggest that the presence of neurons with a dopaminergic/glutamatergic co‐phenotype is a primitive character shared by jawless and jawed vertebrates. However, important differences in the distribution of these neurons and fibres were noted among the few vertebrates investigated to date. This study offers an anatomical basis for further work on the role of glutamate in dopaminergic neurons.     

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals,but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum

Summary The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.     

In vivo detection and automatic analysis of GABA in the mouse brain with MEGA‐PRESS at 9.4 T

The goals of this study were to develop an acquisition protocol and the analysis tools for Meshcher–Garwood point‐resolved spectroscopy (MEGA‐PRESS) in mouse brain at 9.4 T, to allow the in vivo detection of γ‐aminobutyric acid (GABA) and to examine whether isoflurane alters GABA levels in the thalamus during anesthesia. We implemented the MEGA‐PRESS sequence on a Bruker 94/20 system with ParaVision 6.0.1, and magnetic resonance spectra were acquired from nine male wild‐type C57BL/6 J mice at the thalamus. Four individual scans were obtained for each mouse in a 2‐h time course whilst the mouse was anesthetized with isoflurane. We developed an automated analysis program with improved correction for frequency and phase drift compared with the standard creatine (Cr) fitting‐based method and provided automatic quantification. During MEGA‐PRESS acquisition, a single voxel with a size of 5 × 3 × 3 mm³ was placed at the thalamus to evaluate GABA to Cr (GABA/Cr) ratios during anesthesia. Detection and quantitative analysis of thalamic GABA levels were successfully achieved. We noticed a significant decrease in GABA/Cr during the 2‐h anesthesia (by linear regression analysis: slope < 0, p < 0.0001). In summary, our findings demonstrate that MEGA‐PRESS is a feasible technique to measure in vivo GABA levels in the mouse brain at 9.4 T.     

GABA,glycine, glutamate,aspartate and taurine in the perihypoglossal nuclei: an immunocytochemical investigation in the cat with particular reference to the issue of amino acid colocalization

Summary The differential distribution of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA) and taurine (Tau) was investigated in the cat's perihypoglossal nuclei. Serial semi-thin (0.5 m) sections through the perihypoglossal nuclei were incubated with antisera raised against the mentioned amino acids with the aim of studying possible co-localization. In each experiment different measures were undertaken in order to screen for possible cross-reactivities, and all sections were processed together with test conjugates in order to ascertain the specificity of the antisera used. A very high proportion of the neurons in the perihypoglossal nuclei (about 90%) shows strong immunostaining for Asp and also displays distinct immunoreactivity for Glu in neighbouring sections. About 25% of the cells in the perihypoglossal nuclei are intensely immunostained for Gly, but very few cells show immunoreactivity for GABA. Only glial cells appear to be immunostained for Tau. Neurons that are Gly(+) also display Glu and Asp immunoreactivities. The neuropil of the perihypoglossal nuclei shows a high density of GABA(+), Gly(+) and Glu(+) puncta mainly representing stained axons and terminals. Fewer Asp(+) puncta and very few Tau(+) nerve terminal-like puncta are seen. Details of the regional distribution of immunopositive neurons and puncta within the perihypoglossal nuclei are described. The findings are discussed with particular reference to the possible role of the mentioned amino acids as transmitter substances in the known synaptic circuitry of the perihypoglossal nuclei.Performed during leave of absence from Laboratory of Neurobiology, Center for Neuro-Behavioural Biology, Mahidol University, Salaya Campus, Bangkok, Thailand     

The endocranial anatomy of Buriolestes schultzi (Dinosauria: Saurischia) and the early evolution of brain tissues in sauropodomorph dinosaurs

Our knowledge on the anatomy of the first dinosaurs (Late Triassic, 235–205 Ma) has drastically increased in the last years, mainly due to several new findings of exceptionally well-preserved specimens. Nevertheless, some structures such as the neurocranium and its associated structures (brain, labyrinth, cranial nerves, and vasculature) remain poorly known, especially due to the lack of specimens preserving a complete and articulated neurocranium. This study helps to fill this gap by investigating the endocranial cavity of one of the earliest sauropodomorphs, Buriolestes schultzi, from the Upper Triassic (Carnian—c. 233 Ma) of Brazil. The endocranial anatomy of this animal sheds light on the ancestral condition of the brain of sauropodomorphs, revealing an elongated olfactory tract combined to a relatively small pituitary gland and well-developed flocculus of the cerebellum. These traits change drastically across the evolutionary history of sauropodomorphs, reaching the opposite morphology in Jurassic times. Furthermore, we present here the first calculations of the Reptile Encephalization Quotient (REQ) for a Triassic dinosaur. The REQ of B. schultzi is lower than that of Jurassic theropods, but higher than that of later sauropodomorphs. The combination of cerebral, dental, and postcranial data suggest that B. schultzi was an active small predator, able to track moving prey.     

Developmental and evolutionary significance of the mandibular arch and prechordal/premandibular cranium in vertebrates: revising the heterotopy scenario of gnathostome jaw evolution

The cephalic neural crest produces streams of migrating cells that populate pharyngeal arches and a more rostral, premandibular domain, to give rise to an extensive ectomesenchyme in the embryonic vertebrate head. The crest cells forming the trigeminal stream are the major source of the craniofacial skeleton; however, there is no clear distinction between the mandibular arch and the premandibular domain in this ectomesenchyme. The question regarding the evolution of the gnathostome jaw is, in part, a question about the differentiation of the mandibular arch, the rostralmost component of the pharynx, and in part a question about the developmental fate of the premandibular domain. We address the developmental definition of the mandibular arch in connection with the developmental origin of the trabeculae, paired cartilaginous elements generally believed to develop in the premandibular domain, and also of enigmatic cartilaginous elements called polar cartilages. Based on comparative embryology, we propose that the mandibular arch ectomesenchyme in gnathostomes can be defined as a Dlx1-positive domain, and that the polar cartilages, which develop from the Dlx1-negative premandibular ectomesenchyme, would represent merely posterior parts of the trabeculae. We also show, in the lamprey embryo, early migration of mandibular arch mesenchyme into the premandibular domain, and propose an updated version of the heterotopy theory on the origin of the jaw.     

Mitochondrial DNA-like sequences in the nucleus (NUMTs): insights into our African origins and the mechanism of foreign DNA integration

Nuclear mitochondrial DNA sequences (NUMTs) are common in eukaryotes. However, the mechanism by which they integrate into the nuclear genome remains a riddle. We analyzed 247 NUMTs in the human nuclear DNA (nDNA), along with their flanking regions. This analysis revealed that some NUMTs have accumulated many changes, and thus have resided in the nucleus a long time, while others are >94% similar to the reference human mitochondrial DNA (mtDNA), and thus must be recent. Among the latter, two NUMTs, encompassing the COI gene, carry a set of transitions characteristic of the extant African-specific L macrohaplogroup mtDNAs and are more homologous to human mtDNA than to chimp. Screening for one of these NUMTs revealed its presence in all human samples tested, confirming that the African macrohaplogroup L mtDNAs were present in the earliest modern humans and thus were the first human mtDNAs. An analysis of flanking sequences of the NUMTs revealed that 59% were within 150 bp of repetitive elements, with 26% being within 15 bp of and 33% being within 15-150 bp of repetitive elements. Only 14% were integrated into a repetitive element. This association of NUMTs with repetitive elements is highly nonrandom (p<0.001). These data suggest that the vicinity of transposable elements influences the ongoing integration of mtDNA sequences and their subsequent duplication within the nDNA. Finally, NUMTs appear to preferentially integrate into DNA with different GC content than the surrounding chromosomal band. Our results suggest that chromosomal structure might influence integration of NUMTs.     

The CAG repeat at the Huntington disease gene in the Portuguese population: insights into its dynamics and to the origin of the mutation

Huntington disease (HD) is caused by an expansion of a CAG repeat. This repeat is a dynamic mutation that tends to undergo intergenerational instability. We report the analysis of the CAG repeat in a large population sample (2,000 chromosomes) covering all regions of Portugal, and a haplotype study of (CAG)_n and (CCG)_n repeats in 140 HD Portuguese families. Intermediate class 2 alleles represented 3.0% of the population; and two expanded alleles (36 and 40 repeats, 0.11%) were found. There was no evidence for geographical clustering of the intermediate or expanded alleles. The Portuguese families showed three different HD founder haplotypes associated with 7-, 9- or 10-CCG repeats, suggesting the possibility of different origins for the HD mutation among this population. The haplotype carrying the 7-CCG repeat was the most frequent, both in normal and in expanded alleles. In general, we propose that three mechanisms, occurring at different times, may lead to the evolution from normal CAGs to full expansion: first, a mutation bias towards larger alleles; then, a stepwise process that could explain the CAG distributions observed in the more recent haplotypes; and, finally, a pool of intermediate (class 2) alleles more prone to give rise to expanded HD alleles.     

Petrosal anatomy and inner ear structures of the Late Jurassic Henkelotherium (Mammalia, Cladotheria, Dryolestoidea): insight into the early evolution of the ear region in cladotherian mammals

The petrosal anatomy and inner ear structure of Jurassic cladotherian mammals represent the ancestral morphological conditions (groundplan) from which modern therian mammals (marsupials and placentals) have evolved. We present the reconstruction of the petrosal and inner ear features of the Late Jurassic dryolestoid mammal Henkelotherium guimarotae from high-resolution computed tomography and three-dimensional imaging analysis. This study of Henkelotherium revealed a combination of derived and primitive features, including: cladotherian apomorphies, such as the promontorial sulcus for the internal carotid artery and reduced lateral trough; trechnotherian characters, such as an enclosed cochlear canaliculus for the perilymphatic duct, post-promontorial tympanic sinus and caudal tympanic process; in addition to plesiomorphic mammalian features, such as the cavum supracochleare and prootic canal. The inner ear of Henkelotherium shows a division between the utricle and saccule, a cochlear canal coiled through at least 270°, a distinctive primary bony lamina for the basilar membrane, and a secondary bony lamina. The development of the primary and secondary bony laminae in the cochlear canal is suggested here to be correlated with the concurrent coiling of the bony canal and membranous duct of the inner ear cochlea, apomorphies of the more inclusive cladotherian clade that also represent the ancestral morphotype of modern therian mammals. Because these features are crucial for high-frequency hearing in extant therian mammals, their early appearance in Late Jurassic cladotherians suggests a more ancient origination for high-frequency hearing in mammalian history than previously thought.     

Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: Insights into the origin and evolution of type I IFNs in vertebrates

Type I IFNs are considered to be the core IFN species in vertebrates because of their predominant antiviral effects. But, a puzzling question remains to be answered, as to how intronless type I IFN genes in amniotes might have evolved from intron-containing type I IFN genes in fish and amphibians. In this study, intronless and intron-containing type I IFNs were found in the amphibian model, Xenopus tropicalis, with a total of sixteen and five genes, respectively. The intronless IFNs can be divided into three subgroups, and the intron-containing ones into two subgroups, implying that a retroposition event might have occurred in amphibians, resulting in the generation of intronless type I IFN genes. Two models were tentatively proposed to explain the evolution of type I IFNs in vertebrates: in model A, fish should possess the most primitive multi-exon-containing type I IFNs, and intronless type I IFN genes in amphibians are the ancestor of modern intronless type I IFNs in amniotes; in model B, intronless type I IFN genes in X. tropicalis may just represent an independent bifurcation in this species or probably in amphibians, and intronless type I IFN genes in amniotes may have arisen from another retroposition event occurred in a transition period even when reptiles were diverged from amphibians. It is considered that the model B can reflect the current knowledge on the occurrence of intronless and intron-containing type I IFN genes in vertebrate lineages. This study thus contributes to a better understanding of the origin and evolution of type I IFNs in vertebrates, and of the occurrence of intronless I IFNs in higher vertebrates.