Cholecystokinin in the central nervous system of the sea lamprey Petromyzon marinus: precursor identification and neuroanatomical relationships with other neuronal signalling systems.

Cholecystokinin (CCK) is a neuropeptide that modulates processes such as digestion, satiety, and anxiety. CCK-type peptides have been characterized in jawed vertebrates and invertebrates, but little is known about CCK-type signalling in the most ancient group of vertebrates, the agnathans. Here, we have cloned and sequenced a cDNA encoding a sea lamprey (Petromyzon marinus L.) CCK-type precursor (PmCCK), which contains a CCK-type octapeptide sequence (PmCCK-8) that is highly similar to gnathostome CCKs. Using mRNA in situ hybridization, the distribution of PmCCK-expressing neurons was mapped in the CNS of P. marinus. This revealed PmCCK-expressing neurons in the hypothalamus, posterior tubercle, prethalamus, nucleus of the medial longitudinal fasciculus, midbrain tegmentum, isthmus, rhombencephalic reticular formation, and the putative nucleus of the solitary tract. Some PmCCK-expressing neuronal populations were only observed in adults, revealing important differences with larvae. We generated an antiserum to PmCCK-8 to enable immunohistochemical analysis of CCK expression, which revealed that GABA or glutamate, but not serotonin, tyrosine hydroxylase or neuropeptide Y, is co-expressed in some PmCCK-8-immunoreactive (ir) neurons. Importantly, this is the first demonstration of co-localization of GABA and CCK in neurons of a non-mammalian vertebrate. We also characterized extensive cholecystokinergic fibre systems of the CNS, including innervation of habenular subnuclei. A conspicuous PmCCK-8-ir tract ascending in the lateral rhombencephalon selectively innervates a glutamatergic population in the dorsal isthmic grey. Interestingly, this tract is reminiscent of the secondary gustatory/visceral tract of teleosts. In conclusion, this study provides important new information on the evolution of the cholecystokinergic system in vertebrates.

The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

Organization of the visual system in larval lampreys: an HRP study.

The organization of the visual system of larval lampreys was studied by anterograde and retrograde transport of HRP injected into the eye. The retinofugal system has two different patterns of organization during the larval period. In small larvae (less than 60-70 mm in length) only a single contralateral tract, the axial optic tract, is differentiated. This tract projects to regions in the diencephalon, pretectum, and mesencephalic tegmentum. In larvae longer than 70-80 mm, there is an additional contralateral tract, the lateral optic tract, which extends to the whole tectal surface. In addition, ipsilateral retinal fibers are found in both small and large larvae. Initially, the ipsilateral projection is restricted to the thalamus-pretectum, but it reaches the optic tectum in late larvae. Changes in the organization of the optic tracts coincide with the formation of the late-developing retina and consequently, the origin of the optic tracts can be related to specific retinal regions. The retinopetal system is well developed in all larvae. Most retinopetal neurons are labeled contralaterally and are located in the M2-M5 nucleus of the mesencephalic tegmentum, in the caudolateral mesencephalic reticular area and adjacent ventrolateral portions of the optic tectum. Dendrites of these cells are apparent, especially those directed dorsally, which in large larvae extend to the optic tectum overlapping with the retino-tectal projection. These results indicate that in lampreys, visual projections organize mainly during the blind larval period before the metamorphosis, their development being largely independent of visual function.