Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the brain of the European green frog Rana esculenta.

A novel neuropeptide of the RFamide peptide family was isolated in pure form from a frog (Rana esculenta) brain extract by using reversed-phase high performance liquid chromatography in combination with a radioimmunoassay for mammalian neuropeptide FF (NPFF). The primary structure of the peptide was established as Ser-Leu-Lys- Pro-Ala-Ala-Asn-Leu-Pro-Leu- Arg-Phe-NH(2). The sequence of this neuropeptide, designated Rana RFamide (R-RFa), exhibits substantial similarities with those of avian LPLRFamide, gonadotropin-inhibitory hormone, and human RFRP-1. The distribution of R-RFa was investigated in the frog central nervous system by using an antiserum directed against bovine NPFF. In the brain, immunoreactive cell bodies were primarily located in the hypothalamus, i.e., the anterior preoptic area, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. The most abundant population of R-RFa-containing neurons was found in the periependymal region of the suprachiasmatic nucleus. R-RFa- containing fibers were widely distributed throughout the brain from the olfactory bulb to the brainstem, and were particularly abundant in the external layer of the median eminence. In the spinal cord, scattered immunoreactive neurons were found in the gray matter. R-RFa-positive processes were found in all regions of the spinal cord, but they were more abundant in the dorsal horn. This study provides the first characterization of a member of the RFamide peptide family in amphibians. The occurrence of this novel neuropeptide in the hypothalamus and median eminence and in the dorsal region of the spinal cord suggests that, in frog, R-RFa may exert neuroendocrine activities and/or may be involved in the transmission of nociceptive stimuli.

Cerebrospinal fluid-contacting neurons, sensory pinealocytes and Landolt's clubs of the retina as revealed by means of an electron-microscopic immunoreaction against opsin.

Opsin-immunoreactive sites of hypothalamic cerebrospinal fluid (CSF)-contacting neurons, pinealocytes and retinal cells were studied in various vertebrates (Carassius auratus, Phoxinus phoxinus, Triturus cristatus, Bombina bombina, Rana esculenta) by means of postembedding immuno-electron microscopy with the use of the protein A-gold labeling method. The retina of the rat served as a general reference tissue for the quality of the immunocytochemical reaction. A strong opsin immunoreaction (rat-antibovine opsin serum) was obtained in the rod-type outer segments of photoreceptors in the retina of all species studied. Cone-type outer segments exhibited only very few antigenic binding sites. In the pineal organ of the goldfish and the frog, outer segments of the photoreceptor cells displayed strong immunoreactivity. No immunoreaction was found in hypothalamic CSF-contacting neurons and Landolt's clubs of nerve cells of the bipolar layer of the retina. The morphological similarity between the ciliated dendritic terminal of the Landolt's club and the intraventricular dendritic ending of the CSF-contacting neurons is emphasized.

Neuroanatomical identification of the frog habenular connections using peroxidase (HRP).

A study of the habenular nuclei connections by means of horseradish peroxidase (HRP) has never been carried out in amphibia. In the present paper we have investigated the afferent projections of the left and right habenular nuclei of the frog Rana esculenta using this technique. Cells, labelled by HRP, were either in a Golgi-like pattern or in a granular pattern. It appears that the habenular nuclei on the two sides of the epithalamus do not show different connections even though they are morphologically asymmetric. In fact, each habenula is connected bilaterally with the septal area and the bed nucleus of the hippocampal commissure, and ipsilaterally with the hypothalamic areas, the entopeduncular nucleus, the periventricular gray of the third ventricle and the interpeduncular nucleus. However, the habenular commissure has typical commissural fibres which apparently do not involve the medial portion of the left habenula. The habenular connections in the frog are generally similar to those reported in the literature for mammals. In addition, our results show the possibility that HRP is transported both retrograde and anterograde.

[Primary visual centers in the frog (Rana esculenta L.) and the problem of ipsilateral projections. Radioautographic study]. / Les centres visuels primaires chez la grenouille (Rana esculenta L.) et le problème des projections ipsilatérales. Etude radioautographique.

The topographic organization of the frog's primary visual system has been reexamined in Rana esculenta using autoradiographic technique. 10 to 24 hrs following an intraocular injection of tritiated proline, an intense labelling was observed contralaterally in the various retinal projection centres (preoptic area); neuropils of Bellonci; corpus geniculatum of the thalamus; posterior thalamus; posterodorsal preoptic area; magnocellular pretectal nucleus and basal optic nucleus; tectal laminae A to G of Potter), as already described. Furthermore, the presence of a ipsilateral projection within the hypothalamus (preoptic area), the thalamus (neuropils of Bellonci and corpus geniculatum) and the pretectium (posterior thalami neuropil, pretectal optic area) is confirmed. On the other hand, the existence of ipsilateral retinotectal (to Potter's lamina F) and retino-tegmentomescencephalic projections (to the basal optic nucleus), which were thusfar questioned, has been demonstrated.

Comparative ultrastructure of cerebrospinal fluid-contacting neurons and pinealocytes.

The pinealocytes of fishes, amphibians, reptiles, birds and mammals have been compared with cerebrospinal fluid (CSF) contacting neurons. We found that the intraventricular dendrite terminal of the latter resembles the pinealocytic inner segment and that the atypical cilium (9x2+0 tubules) of the CSF contacting neurons is analogous with the outer segment of the pinealocytes, even though the outer segment bears photoreceptor lamellae in lower vertebrates. Regular, but small-sized photoreceptor outer segments were also found on pinealocytes of the chicken. In mammals, too, primitive outer segments are present in the form of 9x2 to cilia similar to those of CSF contacting dendritic terminals. In the Golgi areas of the perikarya of both cell types there are granulated vesicles which may contain transmitter substances and/or neurohormones. The synaptic junctions of the pinealocytes differ from those in the CSF contacting neurons. Many synapses occur on the latter, but they appear only rarely on pinealocytes. The axons of the CSF contacting neurons form synaptic connections with other cells, or terminate as neurohormonal synaptic hemidesmosomes on the basal lamina of the brain surface. The pinealocyte axons give rise to terminals containing synaptic ribbons. Such ribbons do not occur in CSF contacting neurons. In Lacertilians, we found pinealocytic terminals without ribbons on dendrite-like profiles. On the basis of the ultrastructural comparisons, we consider the CSF contacting neurons and pinealocytes to be very similar, but not to represent precisely the same cell type.