Juan Pérez-Fernández

Evolutionarily conserved organization of the dopaminergic system in lamprey: SNc/VTA afferent and efferent connectivity and D2 receptor expression

The dopaminergic system influences motor behavior, signals reward and novelty, and is an essential component of the basal ganglia in all vertebrates including the lamprey, one of the phylogenetically oldest vertebrates. The intrinsic organization and function of the lamprey basal ganglia is highly conserved. For instance, the direct and indirect pathways are modulated through dopamine D1 and D2 receptors in lamprey and in mammals. The nucleus of the tuberculum posterior, a homologue of the substantia nigra pars compacta (SNc)/ventral tegmental area (VTA) is present in lamprey, but only scarce data exist about its connectivity. Likewise, the D2 receptor is expressed in the striatum, but little is known about its localization in other brain areas. We used in situ hybridization and tracer injections, both in combination with tyrosine hydroxylase immunohistochemistry, to characterize the SNc/VTA efferent and afferent connectivity, and to relate its projection pattern with D2 receptor expression in particular. We show that most features of the dopaminergic system are highly conserved. As in mammals, the direct pallial (cortex in mammals) input and the basal ganglia connectivity with the SNc/VTA are present as part of the evaluation system, as well as input from the tectum as the evolutionary basis for salience/novelty detection. Moreover, the SNc/VTA receives sensory information from the olfactory bulbs, optic tectum, octavolateral area, and dorsal column nucleus, and it innervates, apart from the nigrostriatal pathway, several motor‐related areas. This suggests that the dopaminergic system also contributes to the control of different motor centers at the brainstem level. J. Comp. Neurol. 522:3775–3794, 2014.

Dopamine Differentially Modulates the Excitability of Striatal Neurons of the Direct and Indirect Pathways in Lamprey

The functions of the basal ganglia are critically dependent on dopamine. In mammals, dopamine differentially modulates the excitability of the direct and indirect striatal projection neurons, and these populations selectively express dopamine D1 and D2 receptors, respectively. Although the detailed organization of the basal ganglia is conserved throughout the vertebrate phylum, it was unknown whether the differential dopamine modulation of the direct and indirect pathways is present in non-mammalian species. We aim here to determine whether the receptor expression and opposing dopaminergic modulation of the direct and indirect pathways is present in one of the phylogenetically oldest vertebrates, the river lamprey. Using in situ hybridization and patch-clamp recordings, we show that D1 receptors are almost exclusively expressed in the striatal neurons projecting directly to the homolog of the substantia nigra pars reticulata. In addition, the majority of striatal neurons projecting to the homolog of the globus pallidus interna/globus pallidus externa express D1 or D2 receptors. As in mammals, application of dopamine receptor agonists differentially modulates the excitability of these neurons, increasing the excitability of the D1-expressing neurons and decreasing the excitability of D2-expressing neurons. Our results suggest that the segregated expression of the D1 and D2 receptors in the direct and indirect striatal projection neurons has been conserved across the vertebrate phylum. Because dopamine receptor agonists differentially modulate these pathways, increasing the excitability of the direct pathway and decreasing the excitability of the indirect pathway, this organization may be conserved as a mechanism that biases the networks toward action selection.

The lamprey blueprint of the mammalian nervous system

The basic features of the vertebrate nervous system are conserved throughout vertebrate phylogeny to a much higher degree than previously thought. In this mini-review, we show that not only the organization of the different motor programs underlying eye, orienting, locomotor, and respiratory movements are similarly organized, but also that the basic structure of the forebrain engaged in the control of movement is conserved. In the lamprey, which diverged already 560 million years ago from the vertebrate line of evolution leading up to primates, the basic components of the basal ganglia are similar to those of mammals in considerable detail. Moreover, the properties of the synaptic input are similar as well as transmitters/peptides in the direct and indirect pathway throughout the basal ganglia. The membrane properties of the striatal projection neurons with D1 and D2 receptors, respectively, are also similar, as are those of the pallidal output neurons. Our evidence suggests that the basal ganglia can be subdivided into functional modules controlling different motor programs, like locomotion and eye movements. What has happened during evolution is that the number of modules has increased in parallel with a progressively more complex behavioral repertoire. For value-based decisions, the circuitry through the lateral habenulae to the dopaminergic modulator neurons is also conserved, as well as the relay inhibitory interneurons involved. The habenular input is from a pallidal glutamatergic nucleus in lamprey as well as mammals, and this nucleus in turn receives input from the striosomal compartment within striatum and also from pallium (cortex in mammals).

The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates.

Development and organization of the lamprey telencephalon with special reference to the GABAergic system.

Lampreys, together with hagfishes, represent the sister group of gnathostome vertebrates. There is an increasing interest for comparing the forebrain organization observed in lampreys and gnathostomes to shed light on vertebrate brain evolution. Within the prosencephalon, there is now a general agreement on the major subdivisions of the lamprey diencephalon; however, the organization of the telencephalon, and particularly its pallial subdivisions, is still a matter of controversy. In this study, recent progress on the development and organization of the lamprey telencephalon is reviewed, with particular emphasis on the GABA immunoreactive cell populations trying to understand their putative origin. First, we describe some early general cytoarchitectonic events by searching the classical literature as well as our collection of embryonic and prolarval series of hematoxylin-stained sections. Then, we comment on the cell proliferation activity throughout the larval period, followed by a detailed description of the early events on the development of the telencephalic GABAergic system. In this context, lampreys apparently do not possess the same molecularly distinct subdivisions of the gnathostome basal telencephalon because of the absence of a Nkx2.1-expressing domain in the developing subpallium; a fact that has been related to the absence of a medial ganglionic eminence as well as of its derived nucleus in gnathostomes, the pallidum. Therefore, these data raise interesting questions such as whether or not a different mechanism to specify telencephalic GABAergic neurons exists in lampreys or what are their migration pathways. Finally, we summarize the organization of the adult lamprey telencephalon by analyzing the main proposed conceptions, including the available data on the expression pattern of some developmental regulatory genes which are of importance for building its adult shape.

Distribution of a Y1 receptor mRNA in the brain of two lamprey species, the sea lamprey (Petromyzon marinus) and the river lamprey (Lampetra fluviatilis)

The neuropeptide Y system consists of several neuropeptides acting through a broad number of receptor subtypes, the NPY family of receptors. NPY receptors are divided into three subfamilies (Y1, Y2, and Y5) that display a complex evolutionary history due to local and large‐scale gene duplication events and gene losses. Lampreys emerged from a basal branch of the tree of vertebrates and they are in a key position to shed light on the evolutionary history of the NPY system. One member of the Y1 subfamily has been reported in agnathans, but the phylogenetic tree of the Y1 subfamily is not yet clear. We cloned the sequences of the Y1‐subtype receptor of Petromyzon marinus and Lampetra fluviatilis to study the expression pattern of this receptor in lampreys by in situ hybridization and to analyze the phylogeny of the Y1‐subfamily receptors in vertebrates. The phylogenetic study showed that the Y1 receptor of lampreys is basal to the Y1/6 branch of the Y1‐subfamily receptors. In situ hybridization showed that the Y1 receptor is widely expressed throughout the brain of lampreys, with some regions showing numerous positive neurons, as well as the presence of numerous cerebrospinal fluid–contacting cells in the spinal cord. This broad distribution of the lamprey Y1 receptor is more similar to that found in other vertebrates for the Y1 receptor than that of the other members of the Y1 subfamily: Y4, Y8, and Y6 receptors. Both phylogenetic relationship and expression pattern suggest that this receptor is a Y1 receptor. J. Comp. Neurol. 521:426–447, 2013.

Spatiotemporal interplay between multisensory excitation and recruited inhibition in the lamprey optic tectum

Animals integrate the different senses to facilitate event-detection for navigation in their environment. In vertebrates, the optic tectum (superior colliculus) commands gaze shifts by synaptic integration of different sensory modalities. Recent works suggest that tectum can elaborate gaze reorientation commands on its own, rather than merely acting as a relay from upstream/forebrain circuits to downstream premotor centers. We show that tectal circuits can perform multisensory computations independently and, hence, configure final motor commands. Single tectal neurons receive converging visual and electrosensory inputs, as investigated in the lamprey - a phylogenetically conserved vertebrate. When these two sensory inputs overlap in space and time, response enhancement of output neurons occurs locally in the tectum, whereas surrounding areas and temporally misaligned inputs are inhibited. Retinal and electrosensory afferents elicit local monosynaptic excitation, quickly followed by inhibition via recruitment of GABAergic interneurons. Multisensory inputs can thus regulate event-detection within tectum through local inhibition without forebrain control. DOI: http://dx.doi.org/10.7554/eLife.16472.001

Expression of a Novel D4 Dopamine Receptor in the Lamprey Brain. Evolutionary Considerations about Dopamine Receptors

Numerous data reported in lampreys, which belong to the phylogenetically oldest branch of vertebrates, show that the dopaminergic system was already well developed at the dawn of vertebrate evolution. The expression of dopamine in the lamprey brain is well conserved when compared to other vertebrates, and this is also true for the D2 receptor. Additionally, the key role of dopamine in the striatum, modulating the excitability in the direct and indirect pathways through the D1 and D2 receptors, has also been recently reported in these animals. The moment of divergence regarding the two whole genome duplications occurred in vertebrates suggests that additional receptors, apart from the D1 and D2 previously reported, could be present in lampreys. We used in situ hybridization to characterize the expression of a novel dopamine receptor, which we have identified as a D4 receptor according to the phylogenetic analysis. The D4 receptor shows in the sea lamprey a more restricted expression pattern than the D2 subtype, as reported in mammals. Its main expression areas are the striatum, lateral and ventral pallial sectors, several hypothalamic regions, habenula, and mesencephalic and rhombencephalic motoneurons. Some expression areas are well conserved through vertebrate evolution, as is the case of the striatum or the habenula, but the controversies regarding the D4 receptor expression in other vertebrates hampers for a complete comparison, especially in rhombencephalic regions. Our results further support that the dopaminergic system in vertebrates is well conserved and suggest that at least some functions of the D4 receptor were already present before the divergence of lampreys.

Cloning, phylogeny, and regional expression of a Y5 receptor mRNA in the brain of the sea lamprey (Petromyzon marinus)

The NPY receptors known as Y receptors are classified into three subfamilies, Y1, Y2, and Y5, and are involved in different physiological functions. The Y5 receptor is the only member of the Y5 subfamily, and it is present in all vertebrate groups, except for teleosts. Both molecular and pharmacological studies show that Y5 receptor is highly conserved during vertebrate evolution. Furthermore, this receptor is widely expressed in the mammalian brain, including the hypothalamus, where it is thought to take part in feeding and homeostasis regulation. Lampreys belong to the agnathan lineage, and they are thought to have branched out between the two whole‐genome duplications that occurred in vertebrates. Therefore, they are in a key position for studies on the evolution of gene families in vertebrates. Here we report the cloning, phylogeny, and brain expression pattern of the sea lamprey Y5 receptor. In phylogenetic studies, the lamprey Y5 receptor clusters in a basal position, together with Y5 receptors of other vertebrates. The mRNA of this receptor is broadly expressed in the lamprey brain, being especially abundant in hypothalamic areas. Its expression pattern is roughly similar to that reported for other vertebrates and parallels the expression pattern of the Y1 receptor subtype previously described by our group, as it occurs in mammals. Altogether, these results confirm that a Y5 receptor is present in lampreys, thus being highly conserved during the evolution of vertebrates, and suggest that it is involved in many brain functions, the only known exception being teleosts. J. Comp. Neurol. 522:1132–1154, 2014.

Developmental changes of calretinin immunoreactivity in the lamprey spinal cord

We studied the distribution of calretinin immunoreactivity (CR-ir) in the rostral and intermediate levels of the spinal cord of lampreys from embryonic to adult periods. CR-ir was first observed at hatching in motoneurons and primary sensory neurons of the spinal cord, the dorsal cells. During the prolarval period two new cell types showed CR-ir: ganglion cells and interneurons. Motoneurons, dorsal cells, and ganglion cells were strongly positive, whereas interneurons were weakly stained in late prolarvae. The intensity of CR-ir in these four types of cells changed during the larval period. Increase of CR-expression was found in interneurons but a decrease in dorsal cells and in ganglion cells. These changes were more evident in premetamorphic larvae. Postmetamorphic lampreys showed almost no CR-ir in dorsal cells. In adult lampreys, the interneurons showed the highest CR-ir, whereas motoneurons were more weakly stained than in earlier stages of development. Moreover, in adults the dorsal cells and the ganglion cells showed no CR-ir. The present study shows that CR-ir changes during lamprey spinal cord development in different types of neurons, sometimes in opposite ways. This plasticity of CR-expression may indicate different needs from subsets of lamprey spinal cord cells involved in the different locomotor behaviors along its life cycle.