N. B. Kenigfest

A LACERTILIAN DORSAL RETINORECIPIENT THALAMUS : A RE-INVESTIGATION IN THE OLD-WORLD LIZARD PODARCIS HISPANICA

The aim of this work is to delineate the retinorecipient cell groups of the dorsal thalamus of lizards and to study some of the differential connections in order to help to understand the evolution of the visual system in tetrapods. Tract-tracing and immunohistochemical techniques were applied to the retinorecipient dorsal thalamus of the lizard Podarcis hispanica. The retina of Podarcis projects to four areas of the dorsal thalamus: nucleus ovalis (Ov), intergeniculate leaflet (IGL), dorsal lateral geniculate nucleus (GLD) and dorsolateral anterior nucleus (DLA). Nucleus ovalis shows a clear cell plate/neuropile organization and projects to the ventral thalamus. Thus, it seems to belong to the ventral rather than to the dorsal thalamus. The IGL contains large cells reactive for GABA and/or NPY immunohistochemistry. It is interconnected with the supra/retrochiasmatic hypothalamus and projects to the opposite thalamus and to the ipsilateral tectum. The caudal DLA, which lacks both GABA- and NPY-like immunoreactive cells, is reached by a few thin retinal fibers, although distal dendrites of DLA cells enter the GLD, suggesting an important retinal input. The DLA projects to the medial and dorsal telencephalic cortices. The GLD is the main retinorecipient thalamic structure that projects to the telencephalon. It shows a crude laminar organization in which cell plate neurons project to the ipsilateral pallial thickening, but it does not receive a descending projection from the visual telencephalon and thus differs from the GLD of other amniotic vertebrates. In the context of present knowledge, these results suggest that an IGL homologue is present in all tetrapods studied, whereas Ov seems to be restricted to diapsid vertebrates. Moreover, our data suggest that a unimodal visual projection to the telencephalon (arising from the GLD) first appeared in reptiles by segregation from a limbic (multimodal) thalamo-telencephalic pathway.

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 cellular 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.

Tectothalamic visual projections in turtles: their cells of origin revealed by tracing methods.

In two species of turtle (Emys orbicularis and Testudo horsfieldi), retrograde and anterograde tracer techniques were used to study projections from the optic tectum to the nucleus rotundus (Rot) and to the dorsal lateral geniculate nucleus (GLd). The ipsilateral Rot received the most massive tectal projections, stemming from numerous neurons located in the stratum griseum centrale (SGC). These neurons varied in size and shape, many of them having a wide zone of dendritic arborization within both the (SGC) and the stratum griseum et fibrosum superficiale (SGFS). Projections from the tectum to the GLd were ipsilateral, were extremely scarce, and arose from a small number of neurons of various shapes situated in the SGFS; these cells were, as a rule, smaller than those projecting to the Rot. For the most part, these neurons were radially oriented, with rather restricted dendritic arborizations in the most superficial sublayers of the SGFS; smaller numbers of projection neurons were horizontally oriented, with long dendrites branching throughout the layer. Some neurons located in the stratum griseum periventriculare (SGP) projected to both the Rot and the GLd. Most of these neurons had dendritic arborizations within the retinorecipient zone of the SGFS. We were unable to rule out the possibility that some cells projecting to the GLd were situated in the SGC. Both the GLd and the main body of the Rot did not contain neurons projecting to the optic tectum. Thalamic neurons projecting to the tectum were observed in the ventral lateral geniculate nucleus, the intergeniculate leaflet and the interstitial nuclei of the tectothalamic tract, and the nucleus of the decussatio supraoptica ventralis. The question of whether variation in the laminar organization of the tectorotundal and tectogeniculate projection neurons in reptiles, birds, and mammals may be related to different degrees of differentiation of the tectal layers is discussed. J. Comp. Neurol. 457:37–56, 2003.

Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families

The present review describes the molecular evolution of two phylogenetically related families of neuropeptides, the urotensin II (UII) and somatatostatin (SS) families. The UII family consists of four paralogous genes called UII, URP, URP1 and URP2 and the SS family is composed of six paralogous genes named SS1, SS2, SS3, SS4, SS5 and SS6. All these paralogs are present in teleosts, while only four of them, UII, URP, SS1 and SS2 are detected in tetrapods. Comparative genomics showed that most of these genes, namely UII, URP, URP1 and URP2 on the one hand and SS1, SS2 and SS5 on the other hand arose through the 2R. In contrast, the teleost-specific 3R had a much more moderate impact since it only concerned the UII and SS1 genes, which once duplicated, generated a second UII copy and SS4, respectively. The two remaining genes, SS3 and SS6, arose through tandem duplications of the SS1 and SS2 genes respectively, probably in the stem lineage of actinopterygians, before the emergence of teleosts. The history of the UII and SS families has also been marked by massive gene lost, both in tetrapods and in teleosts, but only after the 3R in this latter lineage. Finally, ancestral UII and SS genes are thought to have arisen through tandem duplication of a single ancestral gene, largely before the 1R. An important challenge for the future will be to understand the physiological significance of the molecular diversity of these two families.

RETINOPETAL PROJECTIONS IN LAMPREYS

In the lamprey Lampetra fluviatilis the centrifugal visual system is well developed. Centrifugal fibers arise from cells of two tegmentomesencephalic nuclei-reticular mesencephalic area and nucleus M5 of Schober-as demonstrated by axonal tracing techniques and antidromic neuronal recordings after optic nerve stimulation. Amacrine and ganglion cells in the retina are targets of centrifugal innervation, as shown by electrophysiology and axonal tracing. Following postembedding immunogold labeling, 40% of centrifugal axon terminals in the retina express immunoreactivity to gamma-aminobutyric acid (GABA). About 65% of neurons in M5 and 15% of those in the reticular mesencephalic area have been observed to be immunoreactive for GABA. Combined horseradish peroxidase (HRP) labeling and double immunogold staining were used to investigate the central organization of the retinal feed-back loop. Some terminals of optic nerve fibers from the retina made direct synaptic contacts on dendrites of centrifugal cells of the reticular mesencephalic area and nucleus M5. Terminals of non-retinal origin, immunoreactive for GABA and glutamate, also made synapses on centrifugal neurons. Monosynaptic feed-back loops may be complemented by interneurons as well as by excitatory and inhibitory inputs of non-visual origin.

The turtle thalamic anterior entopeduncular nucleus shares connectional and neurochemical characteristics with the mammalian thalamic reticular nucleus.

Neurochemical and key connectional characteristics of the anterior entopeduncular nucleus (Enta) of the turtle (Testudo horsfieldi) were studied by axonal tracing techniques and immunohistochemistry of parvalbumin, gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). We showed that the Enta, which is located within the dorsal peduncle of the lateral forebrain bundle (Pedd), has roughly topographically organized reciprocal connections with the dorsal thalamic visual nuclei, the nucleus rotundus (Rot) and dorsal lateral geniculate nucleus (GLd). The Enta receives projections from visual telencephalic areas, the anterior dorsal ventricular ridge and dorsolateral cortex/pallial thickening. Most Enta neurons contained GABA and parvalbumin, and some of them were retrogradely labeled when the tracer was injected into the visual dorsal thalamic nuclei. Further experiments using double immunofluorescence revealed colocalization of GAD and parvalbumin in the vast majority of Enta neurons, and many of these cells showed retrograde labeling with Fluoro-gold injected into the Rot and/or GLd. According to these data, the Enta may be considered as a structural substrate for recurrent inhibition of the visual thalamic nuclei. Based on morphological and neurochemical similarity of the turtle Enta, caiman Pedd nucleus, the superior reticular nucleus in birds, and the thalamic reticular nucleus in mammals, we suggest that these structures represent a characteristic component which is common to the thalamic organization in amniotes.

Afferent and efferent projections of the dorsal anterior thalamic nuclei in the lizard Podarcis hispanica (Sauria, Lacertidae)

The aim of this study was to investigate the afferent and efferent connections of the anterior thalamic nuclei in the lizard Podarcis hispanica. To identify potential sources of sensory inputs and to determine the fine organization of the projections of these thalamic nuclei to the telencephalon, we injected the sensitive tracer biotinylated dextran amine (BDA) into different nuclei of the anterior dorsal thalamus. We also injected BDA into several telencephalic areas in order to corroborate the results of thalamic injections. Our results show that the anterior thalamic nuclei receive projections from multiple areas and nuclei distributed throughout most of the brain, from rhombencephalon to telencephalon, and project to several telencephalic areas. The nucleus dorsolateralis anterior receives somatic (visual, somatosensory, auditory) as well as visceral inputs, and it is thus an important gateway for the relay of multisensory information to the telencephalon.

Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts.

It has been recently established that the urotensin II (UII) family consists of four distinct paralogs in bony vertebrates, namely UII, and the three UII-related peptides (URPs) called URP, URP1 and URP2. These four peptides are encoded by genes which arose from the two rounds of tetraploidization (2R) which took place early during vertebrate evolution. Up to now, three of them, UII, URP1 and URP2, have been identified in teleosts, while only two, UII and URP, have been reported in tetrapods. The fact that fish URP has not been found in previous studies led to the suggestion that the corresponding gene had been lost in the teleost lineage. In the present study, we show that this view is not correct. A search of the most recent release of the Ensembl genome database led us to identify a novel UII/URP-like gene in teleosts. Using synteny analysis, we demonstrate that this gene corresponds to the true ortholog of the tetrapod URP gene. Molecular cloning of the corresponding cDNA in medaka revealed that URP gene encodes a putative peptide, with the primary structure GEPCFWKYCV. In stickleback, tilapia and takifugu, URP exhibited the same sequence while, in tetraodon, it differed by only one amino acid substitution Gly ↔ Ser. In zebrafish, URP appeared totally divergent at its N-terminus with the structure DDTCFWKYCV. In conclusion, the occurrence of a true URP in teleosts shows that the quartet of UII-related genes which arose from 2R has been integrally preserved in this lineage.

Comparative Distribution and In Vitro Activities of the Urotensin II-Related Peptides URP1 and URP2 in Zebrafish: Evidence for Their Colocalization in Spinal Cerebrospinal Fluid-Contacting Neurons

Urotensin II (UII) is an evolutionarily conserved neuropeptide initially isolated from teleost fish on the basis of its smooth muscle-contracting activity. Subsequent studies have demonstrated the occurrence of several UII-related peptides (URPs), such that the UII family is now known to include four paralogue genes called UII, URP, URP1 and URP2. These genes probably arose through the two rounds of whole genome duplication that occurred during early vertebrate evolution. URP has been identified both in tetrapods and teleosts. In contrast, URP1 and URP2 have only been observed in ray-finned and cartilaginous fishes, suggesting that both genes were lost in the tetrapod lineage. In the present study, the distribution of urp1 mRNA compared to urp2 mRNA is reported in the central nervous system of zebrafish. In the spinal cord, urp1 and urp2 mRNAs were mainly colocalized in the same cells. These cells were also shown to be GABAergic and express the gene encoding the polycystic kidney disease 2-like 1 (pkd2l1) channel, indicating that they likely correspond to cerebrospinal fluid-contacting neurons. In the hindbrain, urp1-expressing cells were found in the intermediate reticular formation and the glossopharyngeal-vagal motor nerve nuclei. We also showed that synthetic URP1 and URP2 were able to induce intracellular calcium mobilization in human UII receptor (hUT)-transfected CHO cells with similar potencies (pEC50=7.99 and 7.52, respectively) albeit at slightly lower potencies than human UII and mammalian URP (pEC50=9.44 and 8.61, respectively). The functional redundancy of URP1 and URP2 as well as the colocalization of their mRNAs in the spinal cord suggest the robustness of this peptidic system and its physiological importance in zebrafish.

Molecular cloning of the cDNAs encoding three somatostatin variants in the dogfish (Scylorhinus canicula)

It has been recently shown that the somatostatin gene family was likely composed of at least three paralogous genes in the common ancestor of all extant jawed vertebrates. These three genes, namely SS1, SS2 and SS5, are thought to have been generated through the two rounds of whole-genome duplications (2R) that took place early during the vertebrate evolution. In the present study, we report the cloning of three distinct somatostatin cDNAs from the dogfish Scylorhinus canicula, a member of the group of cartilaginous fish. We decided to call these cDNAs, at least provisionally, SSa, SSb and SSc, respectively. Two of them, SSa and SSb, encode proteins that both contain the same tetradecapeptide sequence at their C-terminal extremity (AGCKNFFWKTFTSC). This putative peptide is identical to that generated by the SS1 gene in other vertebrate species. The last cDNA, SSc, encodes a protein that contains at its C-terminal extremity the same peptide sequence as that generated by the SS2 gene in teleosts (APCKNFFWKTFTSC). Phylogenetic analysis showed that the SSa and SSc genes likely correspond to the dogfish counterparts of the SS1 and SS2 genes, respectively. In contrast, the phylogenetic status of the SSb gene is less clear. Several lines of evidence suggest that it could correspond to the SS5 gene, but this view will need to be confirmed, for example by synteny analysis. Finally, RT-PCR analysis revealed that SSa, SSb and SSc genes are differentially expressed in dogfish tissues, suggesting that the corresponding peptides may exert distinct functions.