[Gaba- and glycine-immunoreactive synapses in the spinal cord of the frog Rana temporaria].

Postembedding immunogold method was used to examine the distribution of gamma-aminobutyric acid- and glycine-immunoreactives synapses on the motoneurons and primary afferent axons in frog spinal cord. Analysis of all labeled boutons on dendrites and somata of motoneurons showed that 7% were labeled for GABA, 23% only for glycine and approximately 70% were immunoreactive for both GABA and glycine. These results confirm the predominant role of glycine in postsynaptic inhibition of motoneuronal activity. Three populations of synaptic boutons were found on primary afferent axons: GABA-immunoreactive (25%), glycine-immunoreactive (5%) and the majority of the immunoreactive synapses exhibited colocalization of two inhibitory transmitters. Greater proportion of axo-axonal synases was organized in synaptic triads. The possible roles of glycine in the axo-axonal synapses on the primary afferent fibers are discussed.

[Immunoreactivity of the synapses on the primary afferent axons and sensory neurons of the spinal cord Lampetra fluviatilis].

The existence of GABA-like immunoreactivity in the synapses on the primary afferent axons and GABA- and glutamate immunoreactive synapses on the dorsal cell somatic membrane was shown using double postembedding immunogold cytochemistry. These morphological findings suggest that control of the sensory information in the lamprey spinal cord is realized by means of presynaptic inhibition through the synapses on the primary afferent axons as well as directly through the synapses on the somata of the sensory neurons.

The evolution of the centrifugal visual system of vertebrates. A cladistic analysis and new hypotheses.

In a recent review of the available data concerning the centrifugal visual system (CVS) of vertebrates [Repérant, J., Ward, R., Miceli, D., Rio, J.P., Médina, M., Kenigfest, N.B., Vesselkin, N.P., 2006. The centrifugal visual system of vertebrates: a comparative analysis of its functional anatomical organization, Brain Res. Rev. 52, 1-57], we have shown that this feature of the visual system is not a particularity of birds, but is a permanent component of the vertebrate central nervous system which nevertheless shows considerable morphological and functional variation from one taxonomic group to another. Given these findings, the primary objective of the present article is an attempt to specify the evolutionary significance of this phylogenetic diversity. We begin by drawing up an inventory of this variation under several headings: the intracerebral location of the retinopetal neurons; the mode of intra-retinal arborizations of the centrifugal fibres and the nature of their targets; their neurochemical properties; and the afferent supplies of these neurons. We subsequently discuss these variations, particularly that of the intracerebral location of the retinopetal neurons during development and in adult forms, using the neuromeric terminology and in the framework of cladistic analysis, and seek to interpret them in a phylogenetic context. From this analysis, it becomes evident that the CVS is not a homogeneous entity formed by neurons with a common embryological origin, but rather a collection of at least eight distinct subsystems arising in very different regions of the neuraxis. These are the olfacto-retinal, dorsal thalamo-retinal, ventral thalamo-retinal, pretecto-retinal, tecto-retinal, tegmento-mesencephalo-retinal, dorsal isthmo-retinal and ventral isthmo-retinal systems. The olfacto-retinal system, which is probably absent in Agnatha, appears to be a pleisiomorphic characteristic of all Gnathostomata, while on the other hand the tegmento-mesencephalo-retinal system appears to be present only in Agnatha. Our cladistic analysis also shows that the remaining six subsystems are polyphyletic in origin and have arisen independently on several occasions in different radiations of Gnathostoma. In conclusion, we suggest that, in the course of the palaeontological history of vertebrates, these different retinopetal pathways have been selected on the basis of widely different environmental pressures which remain to be identified.

The postnatal development of the optic nerve of a reptile (Vipera aspis): A quantitative ultrastructural study.

The number of axons in the optic nerve of the ovoviviparous reptile Vipera aspis was estimated from electron micrographs taken during the first 5 weeks of postnatal life. One to two days after birth, the optic nerve contains about 170,000 fibres, of which about 9% are myelinated. At the end of the fifth postnatal week, the number of optic fibres has fallen to about 100,000, of which about 42% are myelinated. This fibre loss continues after the fifth postnatal week, since in the adult viper the nerve contains about 60,000 fibres, of which 85% are myelinated; overall, about 65% of the optic nerve fibres present at birth disappear before the number of axons stabilises at the adult level. This study shows, for the first time, that the mode of development of the visual axons of reptiles is not that of anamniote vertebrates but similar to that of birds and mammals.

The centrifugal visual system of vertebrates: a comparative analysis of its functional anatomical organization.

The present review is a detailed survey of our present knowledge of the centrifugal visual system (CVS) of vertebrates. Over the last 20 years, the use of experimental hodological and immunocytochemical techniques has led to a considerable augmentation of this knowledge. Contrary to long-held belief, the CVS is not a unique property of birds but a constant component of the central nervous system which appears to exist in all vertebrate groups. However, it does not form a single homogeneous entity but shows a high degree of variation from one group to the next. Thus, depending on the group in question, the somata of retinopetal neurons can be located in the septo-preoptic terminal nerve complex, the ventral or dorsal thalamus, the pretectum, the optic tectum, the mesencephalic tegmentum, the dorsal isthmus, the raphé, or other rhombencephalic areas. The centrifugal visual fibers are unmyelinated or myelinated, and their number varies by a factor of 1000 (10 or fewer in man, 10,000 or more in the chicken). They generally form divergent terminals in the retina and rarely convergent ones. Their retinal targets also vary, being primarily amacrine cells with various morphological and neurochemical properties, occasionally interplexiform cells and displaced retinal ganglion cells, and more rarely orthotopic ganglion cells and bipolar cells. The neurochemical signature of the centrifugal visual neurons also varies both between and within groups: thus, several neuroactive substances used by these neurons have been identified; GABA, glutamate, aspartate, acetylcholine, serotonin, dopamine, histamine, nitric oxide, GnRH, FMRF-amide-like peptides, Substance P, NPY and met-enkephalin. In some cases, the retinopetal neurons form part of a feedback loop, relaying information from a primary visual center back to the retina, while in other, cases they do not. The evolutionary significance of this variation remains to be elucidated, and, while many attempts have been made to explain the functional role of the CVS, opinions vary as to the manner in which retinal activity is modified by this system.

Dual innervation of the lamprey retina by GABAergic and glutamatergic retinopetal fibers. A quantitative EM immunogold study.

A quantitative electron microscopic analysis of glutamate and GABA immunocytochemistry, using the postembedding immunogold technique, was undertaken in the lamprey retina to determine the proportion of glutamate-immunoreactive (GLU-ir) centrifugal visual terminals which were identified by anterograde axonal transport after an iontophoretic deposit of HRP in the sectioned optic nerve. Single immunogold labeling carried out with two different GABA and GLU antibodies showed that about 45% of the retinopetal axon terminals were GABAergic, and that two types of GLU-ir terminals (GLU-ir/1 and GLU-ir/2) were observed in the inner plexiform layer. The former type showed a high density of gold particles (Neosystem: 19.38+/-0.74; Sigma: 106.26+/-5.70) which statistically differed from the GLU-ir/2 (Neosystem: 3.23+/-0.31; Sigma: 31.73+/-5.61). Subcellular estimates showed that gold particles were concentrated over the vesicular pool in the GLU-ir/1 terminals and over mitochondria in the second terminal type. Consecutive sections alternately processed for GABA and GLU revealed that the GABA-immunonegative terminals corresponded to the GLU-ir/1, whereas the GABA-ir terminals only contained few GLU-ir particles, mostly concentrated in mitochondria. The occurrence of GABAergic and glutamatergic retinopetal terminals in the adult lamprey retina is discussed in functional terms of their differential inhibitory and excitatory effects on ganglion cell activity and the possible role of the centrifugal visual system in visually-guided behavior.

Ultrastructural study of glutamate- and GABA-immunoreactive terminals contacting the primary afferent fibers in frog spinal cord. A double postembedding immunocytochemical study.

The in vitro HRP application to the dorsal root of the frog spinal cord produced an intensive staining of primary afferent fibers. A double postembedding GABA and glutamate immunocytochemical study revealed GABA- or glutamate-immunopositive presynaptic boutons establishing axo-axonic synapses onto HRP-stained primary afferent fibers in the spinal cord intermedial zone.

Evolutionary significance of different neurochemical organisation of the internal and external regions of auditory centres in the reptilian brain: an immunocytochemical and reduced NADPH-diaphorase histochemical study in turtles.

An immunocytochemical and histochemical study was undertaken of the torus semicircularis and nucleus reuniens, the mesencephalic and diencephalic auditory centres, in two chelonian species, Testudo horsfieldi and Emys orbicularis. The nucleus centralis of the torus semicircularis receives few 5-HT-, TH-, substance P-, and menkephalin-immunoreactive fibres and terminals, in marked contrast to the external nucleus laminaris of the torus semicircularis, in which 5-HT-, TH-, substance P-, and menkephalin-immunoreactive elements and cell bodies show a laminar distribution. Dense NPY-positive terminal-like profiles and cell bodies were observed in both the nuclei centralis and laminaris, and many NADPH-d-positive cell bodies were observed in the cell layers of the latter. In the nucleus reuniens, the distribution of 5-HT-, TH-, substance P-, and menkephalin-immunolabelling resembles that seen in the torus semicircularis, but at a lower density. The dorsorostral regions of the nucleus reuniens, as in the nucleus centralis, is insignificantly labelled, in contrast to the ventrocaudal regions in which labelled elements abound. NPY-positive elements are uniformly distributed throughout the nucleus, but no labelled cell bodies were observed. NADPH-d-positive fibres and terminals were observed in both dorsal and ventral regions of the nucleus reuniens, but the few labelled cell bodies to be observed were located in the peripheral regions of the nucleus. These findings are discussed in terms of the evolution of the core-and-belt organisation of sensory nuclei observed in other vertebrate species.

Serotonergic innervation of the isthmo-optic nucleus of the pigeon centrifugal visual system. An immunocytochemical electron microscopic study.

The ultrastructural features of serotonergic fibers, terminals and synaptic contacts were studied with the pre-embedding immunocytochemical method in the isthmo-optic nucleus of the pigeon centrifugal visual system. The 5-HT immunoreactive (-ir) profiles were diffusely distributed and their density was low. The labeled axons were thin and unmyelinated (mean diameter=0.21+/-0.03 microm) though a few larger myelinated axons were observed (mean diameter=0.51+/-0.07 microm). The 5-HT-ir terminals or varicosities were small (diameter=0.71+/-0.54 microm) and contained small agranular synaptic vesicles (diameter=28.5+/-6.9 nm) and large granular vesicles (diameter=102.2+/-19.5 nm). The latter only constituted approximately 1% of the total profiles containing synaptic vesicles in the isthmo-optic nucleus. In single thin sections, only 5% of the 5-HT-ir varicosities exhibited an active asymmetrical zone synapsing upon dendritic profiles of centrifugal visual neurons. Calculations indicated that 17% of these 5-HT-ir varicosities were actually engaged in junctional synaptic relationships, whereas the remaining (83%) were nonjunctional. The data suggest that, within the isthmo-optic nucleus, 5-HT acts both at synaptic junctions (wiring transmission) and at a distance via the extracellular space (volume transmission). These 5-HT afferents could thus modulate the activity of the retinopetal neurons and visual information processing.

Axo-axonic GABA-immunopositive synapses on the primary afferent fibers in frogs.

In three frog species Rana esculenta, Rana temporaria and Xenopus laevis, the contacts established by gamma-aminobutyric acid and glutamate decarboxylase immunoreactive (-ir) terminals upon primary afferent fibers were studied using confocal and electron microscopy. For confocal microscopy, the primary afferent fibers were labeled through the dorsal root with Dextran-Texas Red, whereas gamma-aminobutyric acid and glutamate decarboxylase immunoreactivity were revealed with fluorescein isothiocyanate. Appositions of gamma-aminobutyric acid and glutamate decarboxylase immunoreactive profiles onto primary afferent fibers were observed and were considered as putative axo-axonic contacts of GABAergic terminals upon primary afferents. The latter was confirmed by the ultrastructural finding of axo-axonic synapses from gamma-aminobutyric acid immunopositive boutons upon the HRP-labeled primary afferent fibers in postembedding immunoelectron microscopic study. Such synapses may represent the morphological basis of GABAergic presynaptic inhibition of primary afferent fibers.

The distribution of NPY-like immunoreactivity in the chameleon brain.

The distribution of neuropeptide Y (NPY) immunoreactivity was studied in the brain of the chameleon. Cell bodies and fibers displaying NPY-like immunoreactivity were widely dispersed throughout the brain and at the highest density in the telencephalon and diencephalon. Immunolabeled cell bodies were numerous in the medial and dorsomedial cortex and in the dorsal ventricular ridge, while the striatum and basal telencephalon only contained sparsely scattered NPY-positive somata. Immunopositive neurons were densely distributed in the dorsal thalamus (particularly in the perirotundal belt), the area triangularis, the nucleus geniculatus lateralis pars dorsalis, the periventricular hypothalamus and the medial eminence. In the pretectum, NPY-immunoreactive cell bodies were limited to the nucleus posterodorsalis, while in the mesencephalon immunolabeled somata were found in the stratum album centrale of the optic tectum and in the substantia nigra. Immunopositive fibers and terminals were particularly dense in the dorsomedial cortex, the periventricular hypothalamus, the nuclei accumbens, suprachiasmaticus and griseus tectalis, in the substantia nigra and in the torus semicircularis. These findings show that the NPY system in the chameleon has the same basic organization as in other vertebrate species, and indicate that this peptide could be also implicated in the regulation of several aspects of cerebral functions. In addition, and of particular interest, is the observation of numerous NPY-immunoreactive neurons and fibers in several visual nuclei, suggesting an important involvement of this substance in the visual function.

A quantitative ultrastructural study of the optic nerve of the chameleon.

The optic nerve of adult chameleons was investigated with an electron microscope. The total number of retinal ganglion cell axons, the proportion of myelinated axons, the frequency distributions of myelinated and unmyelinated axon diameters were estimated, together with the volume occupied by glial processes. These were distinguished from unmyelinated axons using an antibody directed against glial fibrillary acidic protein, in a post-embedding procedure. The total number of fibers was estimated to be 405,235 +/- 60,000 axons. The proportion of myelinated fibers varied with position between the eyeball and the chiasma; being 22-27% close to the eyeball, rising to 42-47% halfway along the optic nerve and to 56-62% close to the chiasma. Myelinated and unmyelinated fiber diameter distributions were unimodal and positively skewed, with modes of 0.7 microm and 0.2 microm, respectively. There was a significant regional variation in the size of optic nerve axons. Large myelinated axons were observed in the dorsal and ventral periphery, whereas smaller myelinated fibers and a high proportion of unmyelinated fibers were found in the center of the nerve.

Colocalization of neurotransmitters in presynaptic boutons of inhibitory synapses in the lamprey spinal cord.

Electron-microscopic immunocytochemical studies were performed to detect GABA and glycine immunoreactivity in presynaptic axon terminals in the central gray matter of the spinal cord of the lamprey Lampetra fluviatilis. The immunopositive presynaptic terminals contacting identified dendrites of motoneurons and unidentified postsynaptic profiles included terminals immunopositive for GABA only (44%) and glycine only (26%), as well as terminals containing GABA and glycine (30%). Glycine-immunopositive presynaptic terminals contained flattened synaptic vesicles. Large synaptic vesicles with dense cores were present along with classical synaptic vesicles in 74% of GABA-immunopositive boutons.

Pretectal connections in turtles with special reference to the visual thalamic centers: a hodological and gamma-aminobutyric acid-immunohistochemical study.

Projections of the pretectal region to forebrain and midbrain structures were examined in two species of turtles (Testudo horsfieldi and Emys orbicularis) by axonal tracing and immunocytochemical methods. Two ascending gamma-aminobutyric acid (GABA)ergic pathways to thalamic visual centers were revealed: a weak projection from the retinorecipient nucleus lentiformis mesencephali to the ipsilateral nucleus geniculatus lateralis pars dorsalis and a considerably stronger projection from the nonretinorecipient nucleus pretectalis ventralis to the nucleus rotundus. The latter is primarily ipsilateral, with a weak contralateral component. The interstitial nucleus of the tectothalamic tract is also involved in reciprocal projections of the pretectum and nucleus rotundus. In addition, the pretectal nuclei project reciprocally to the optic tectum and possibly to the telencephalic isocortical homologues. Comparison of these findings with previous work on other species reveals striking similarities between the pretectorotundal pathway in turtles and birds and in the pretectogeniculate pathway in turtles, birds, and mammals.

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [(3)H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT.

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [(3)H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.