Cytoarchitecture,fiber connections,and ultrastructure of nucleus isthmi in a teleost (Navodon modestus) with a special reference to degenerating isthmic afferents from optic tectum and nucleus pretectalis

Cytoarchitecture and fiber connections of the nucleus isthmi in a teleost (Navodon modestus) were studied by means of Nissl, Bodian, toluidine blue, Golgi, and Fink-Heimer methods. Synaptic terminals were classified by the ultrastructural characteristics, and their origins were determined by electron microscopic degeneration experiments. The nucleus isthmi is composed of an outer cellular area or shell and an inner noncellular area or core. The shell covers anterior, dorsal, and ventral aspects of the core. The cell bodies in the shell are oval (15 × 20 μm) with an anteroposterior long axis, and have many somatic spines. Spines are also seen on the initial segment of the axon. Primary dendrites extend postermedially and branch out in the core. The core contains thin and thick myelinated fibers, which originate in the optic tectun and in the nucleus pretectalis, respectively. At least two types of axons terminal were distinguished in the nucleus isthmi: S type, containing spherical vesciles, and F type, containing flattened vesicles. S terminals are derived from thin myelinated fibers and are only seen in the core where they form asymmetric synapses with dendrites. Frequently a portion of the S terminal membrane near the usual synaptic cleft is in close apposition with the membrane of an adjacent small dendrite or spine. F terminals, which derived from thick myelinated fibers, make symmetric synaptic contacts with both cell bodies in the shell and dendrites in the core. S terminals degenerate after ipsilateral ablation of the optic tectum, whereas F terminals degenerate after destruction of the nucleus pretectalis.     

Retinal afferents to the tectum opticum and the nucleus opticus principalis thalami in the pigeon.

The retinal afferents of the tectum opticum and the n. opticus principalis thalami (OPT) were studied with fluorescent tracers in pigeons. Injections into the tectum opticum revealed topographically related areas of high density labelling in the contralateral retina. In these areas up to 15,000 cells/mm2 were labelled. After tectal injections the soma sizes of labelled retinal ganglion cells in the area centralis ranged from 5 to 23 microns with a mean of 7.5 microns. Afferents from the ipsilateral retina could not be demonstrated. Injections into the OPT labelled neurons throughout the retina without a clear topographical relation to the locus of injection. The density never exceeded 150 cells per mm2. The soma size range was 8 to 35 microns with a mean of 14.6 microns. Independently of the injection area within the OPT, the red field in the dorsotemporal retina was always extremely sparsely labelled. The number of labelled ganglion cells in this area never exceeded 25 neurons/mm2. After OPT injections the average density of labelling per unit area was six times higher in the yellow than in the red field. The results confirm previous reports of a massive and topographically organized retinal projection onto the optic tectum. The projection onto the OPT was clearly smaller and with the retrograde tracing techniques in use, an orderly topography has not been demonstrated. The paucity of red field projections onto the OPT suggests that the role of the thalamofugal pathway in binocular integration is very limited.     

Synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi in Rana pipiens

The nucleus isthmi is reciprocally connected to the ipsilateral optic tectum. Ablation of the nucleus isthmi compromises visually guided behavior that is mediated by the tectum. In this paper, horseradish peroxidase (HRP) histochemistry and electron microscopy were used to explore the synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi. After localized injections of HRP into the optic tectum, there are retrogradely labeled isthmotectal neurons and orthogradely labeled fibers and terminals in the ipsilateral nucleus isthmi. These terminals contain round. Clear vesicles of medium diameter (40–52 nm). These terminals make synaptic contact with dendrites of nucleus isthmi cells. Almost half of these postsynaptic dendrites are retrogradely labeled, indicating that there are monosynaptic tectoisthmotectal connections. Localized HRP injection into the nucleus isthmi labels terminals primarily in tectal layers B, E, F, and 8. The terminals contain medium-sized clear vesicles and they form synaptic contacts with tectal dendrites. There are no instances of labeled isthmotectal terminals contacting labeled dendrites. Retrogradely labeled tectoisthmal neurons are contacted by unlabeled terminals containing medium-sized and small clear vesicles. Fifty-four percent of the labeled fibers connecting the nucleus isthmi and ipsilateral tectum are myelinated fibers (average diameter approximately 0.6 μm). The remainder are unmyelinated fibers (average diameter approximately 0.4 μm). © 1994 Wiley-Liss, Inc.     

On the presence of nucleus ruber in the urodele Salamandra salamandra and the caecilian Ichthyophis kohtaoensis

The presence of nucleus ruber in urodeles and caecilians (amphibia) was investigated. For that purpose, horseradish peroxidase was applied to the rostral spinal cord, the medulla oblongata at various levels and the dorsolateral funiculus. Whereas Salamandra salamandra possesses a rubrospinal tract, it is absent in the limbless caecilian Ichthyophis kohtaoensis.     

Topographic projections between the nucleus isthmi and the tectum of the frog Rana pipiens.

The connections between the nucleus isthmi and the tectum in the frog have been determined by several anatomical techniques: iontophoresis of horseradish peroxidase into the tectum, iontophoresis of 3H-porline into the nucleus isthmi and the tectum, and Fink-Heimer degeneration staining after lesions of the nucleus isthmi. The results show that the nucleus isthmi projects bilaterally to the tectal lobes. The ipsilateral isthmio-tectal fibers are distributed in the superficial layers of the tectum, coincident with the retionotectal terminals. The contralateral isthmio-tectal fibers travel anteriorly adjacent to the lateral optic tract and cross the midline in the supraoptic ventral decussation, where they turn dorsally and caudally; upon reaching the tectum, the fibers end in two discrete layers, layers 8 and A of Potter. The tectum projects to the ipsilateral nucleus isthmi and there is a reciprocal topographic relationship between the two structures. Thus, a retino-tecto-isthmio-tectal route exists which may contribute to the indirect ipsilateral retinotectal projection which is observed electrophysiologically. The connections between the nucleus isthmi and the tectum in the frog are strinkingly similar to the connections between the parabigeminal nucleus and the superior colliculus of mammals.     

Topographic projections between the nucleus isthmi and the optic tectum in a teleost,Navodon Modestus

Following horseradish peroxidase injections into the optic tectum of a teleost,Navodon modestus, reciprocal and topographic projections between the nucleus isthmi and the ipsilateral optic tectum were determined. The isthmo-tectal fibers diverge to the optic tectum while maintaining the spatial arrangements of the isthmic cells from which the fibers originate. The tecto-isthmic projections also keep the spatial arrangements in the optic tectum. The tectal fibers converge near the nucleus isthmi and terminate in the non-cellular portion of the nucleus. The reciprocal topography is apparent in the combined results of 9 experiments with one tectal injection in each region. No labeled cells and fibers were found in the contralateral nucleus isthmi.     

Caudal topographic nucleus isthmi and the rostral nontopographic nucleus isthmi in the turtle, Pseudemys scripta

Isthmotectal projections in turtles were examined by making serial section reconstructions of axonal and dendritic arborizations that were anterogradely or retrogradely filled with HRP. Two prominent tectal-recipient isthmic nuclei--the caudal magnocellular nucleus isthmi (Imc) and the rostral magnocellular nucleus isthmi (Imr)--exhibited strikingly different patterns of organization. Imc cells have flattened, bipolar dendritic fields that cover a few percent of the area of the cell plate constituting the nucleus and they project topographically to the ipsilateral tectum without local axon branches. The topography was examined explicitly at the single-cell level by using cases with two injections at widely separated tectal loci. Each Imc axon terminates as a compact swarm of several thousand boutons placed mainly in the upper central gray and superficial gray layers. One Imc terminal spans less that 1% of the tectal surface. Imr cells, by contrast, have large, sparsely branched dendritic fields overlapped by local axon collaterals while distally, their axons nontopographically innervate not only the deeper layers of the ipsilateral tectum but also ipsilateral Imc. Imr receives a nontopographic tectal input that contrasts with the topographic tectal input to Imc. Previous work on nucleus isthmi emphasized the role of the contralateral isthmotectal projection (which originates from a third isthmic nucleus in turtles) in mediating binocular interactions in the tectum. The present results on the two different but overlapping ipsilateral tecto-isthmo-tectal circuits set up by Imc and Imr are discussed in the light of physiological evidence for selective attention effects and local-global interactions in the tectum.     

Putative cholinergic projections from the nucleus isthmi and the nucleus reticularis mesencephali to the optic tectum in the goldfish (Carassius auratus)

The nucleus isthmi of fish and amphibians has reciprocal connections with the optic tectum, and biochemical studies suggested that it may provide a major cholinergic input to the tectum. In goldfish, we have combined immunohistochemical staining for choline acetyltransferase with retrograde labeling of nucleus isthmi neurons after tectal injections of horseradish peroxidase. Seven fish received tectal horseradish peroxidase injections, and brain tissue from these animals was subsequently processed for the simultaneous visualization of horseradish peroxidase and choline acetyltransferase. In many nucleus isthmi neurons the dense horseradish peroxidase label obscured the choline acetyltransferase reaction product but horseradish peroxidase and choline acetyltransferase were colocalized in 54 cells from nine nuclei isthmi. The somata of nucleus reticularis mesencephali neurons stained so intensely for choline acetyltransferase that we could not determine whether they were labelled also with horseradish peroxidase. However, the large choline acetyltransferase-immunoreactive axons of nucleus reticularis mesencephali neurons stained intensely enough for us to follow them rostrally; the axons are clustered together until the level of the rostral tectum where two groupings form: one travels into the tectum and the other travels rostroventrally to cross the midline and enter the contralateral diencephalic preoptic area. We conclude therefore that cholinergic neurons project to the optic tectum from the nucleus isthmi as well as nucleus reticularis mesencephali in goldfish.     

Connections of the commissural nucleus of Cajal in the goldfish,with special reference to the topographic organization of ascending visceral sensory pathways

The primary general visceral nucleus of teleosts is called the commissural nucleus of Cajal (NCC). The NCC of goldfish has been divided into the medial (NCCm) and lateral (NCCl) subnuclei that receive inputs from subdiaphragmatic gastrointestinal tract and the posterior pharynx, respectively. Fiber connections of the NCC were examined by tract‐tracing methods in the goldfish Carassius auratus. Tracer injections into the NCC suggested that the NCC projects directly not only to the secondary visceral sensory region in the rhombencephalic isthmus and other brain stem centers, but also to the forebrain, similar to the situations in mammals, birds, and the Nile tilapia. Although fiber connections of the NCCm and NCCl were basically similar, the NCCm was the more important source of ascending general visceral fibers to the forebrain. Topographic organization was recognized regarding projections to the isthmic secondary visceral sensory zone; input from the NCCm is represented in the secondary general visceral sensory nucleus, while input from the NCCl in the lateral edge of the secondary gustatory nucleus. Moreover, specific injections into different regions of the vagal lobe revealed that the dorsomedio–ventrolateral axis of the lobe is represented in the lateromedial axis of the secondary gustatory nucleus. These observations suggest fine topographic organization of ascending visceral sensory pathways to the isthmic secondary centers. It should also be noted that the reception of primary afferents from the posterior pharynx and projections to the secondary gustatory nucleus suggest that the NCCl may be regarded as a gustatory rather than a general visceral sensory structure. J. Comp. Neurol. 523:209–225, 2015. © 2014 Wiley Periodicals, Inc.     

The development of the nucleus isthmi in Xenopus laevis. I. Cell genesis and the formation of connections with the tectum

The nucleus isthmi (NI) of the amphibian relays visual input from one tectum to the other tectum and thus brings a visual map from the eye to the ipsilateral tectum. This isthmotectal visual map develops slowly; it is first detected electrophysiologically at stages 60-62, the age at which the eyes begin their dorsalward migration and the region of binocular overlap beings to increase in extent. During this critical period of life, normal binocular visual input is required for establishment of normal topographic isthmotectal projections. In this study, we have used anatomical methods to trace cell birth, cell death, and formation of connections by the nucleus isthmi during the critical period. Tritiated thymidine labelling demonstrates that cells in the nucleus isthmi are generated throughout most of tadpole life (stages 29-62). Most cells conform to an orderly ventrodorsal gradient starting from stage 29 and extending to stages 56; later cells are inserted at apparently random locations in the nucleus. We have re-examined the hypothesis of Tay and Straznicky ('80) that the order of cell genesis in the NI and tectum could help establish proper isthmotectal connections, and we find that a timing mechanisms does not explain the two-dimensional topography of the isthmotectal map but that timing may aid in proper mediolateral positioning of isthmotectal axons at the points where they first enter the tectum. Horseradish peroxidase labelling was used to investigate whether anatomical projections from tectum to NI and from NI to tectum are present prior to the onset of eye migration. The results show that there are tectoisthmotectal projections by stage 52. Moreover, isthmotectal axons grow into as yet monocular tectal regions prior to the onset of eye migration. At stage 60, when binocular overlap begins, isthmotectal axons are visible throughout the tectum but are densely branched only at the rostral tectal margin, the location where they are predicted to occur on the basis of electrophysiological maps.     

Circadian rhythms and energy metabolism with special reference to the suprachiasmatic nucleus

The role of the hypothalamic suprachiasmatic nucleus (SCN) was examined in rats and obtained following results: (a) The time-dependent (light > dark) hyperglycemic response to intracranial injection of 2-deoxy- -glucose (2DG) disappeared in rats with bilateral lesions of the SCN, in rats on weeks 4–6 after surgical blinding, and in congenitally blind (hereditary microphthalmic) rats; (b) The hyperglycemia induced by electrical stimulation of the SCN was not observed in weeks 4–8 after surgical blinding; (c) Change in the blood glucose concentration after insulin injection into the SCN was eliminated by SCN lesions; (d) Alterations in activity of autonomic efferents to peripheral organs on light exposure disappeared after SCN lesions; (e) SCN lesions decreased the blood glucagon level and increased the blood insulin level; (f) SCN lesions decreased protein intake, and glucagon increased it; (g) Increases in the plasma renin activity and vasopressin concentration after water-deprivation were suppressed in hereditary microphthalmic rats with abnormal SCN. These findings suggest that the SCN is involved in the mechanism of blood glucose and body fluid intake as well as that of circadian rhythm.     

Circadian rhythms and energy metabolism with special reference to the suprachiasmatic nucleus

The role of the hypothalamic suprachiasmatic nucleus (SCN) was examined in rats and obtained following results: (a) The time-dependent (light > dark) hyperglycemic response to intracranial injection of 2-deoxy-d-glucose (2DG) disappeared in rats with bilateral lesions of the SCN, in rats on weeks 4–6 after surgical blinding, and in congenitally blind (hereditary microphthalmic) rats; (b) The hyperglycemia induced by electrical stimulation of the SCN was not observed in weeks 4–8 after surgical blinding; (c) Change in the blood glucose concentration after insulin injection into the SCN was eliminated by SCN lesions; (d) Alterations in activity of autonomic efferents to peripheral organs on light exposure disappeared after SCN lesions; (e) SCN lesions decreased the blood glucagon level and increased the blood insulin level; (f) SCN lesions decreased protein intake, and glucagon increased it; (g) Increases in the plasma renin activity and vasopressin concentration after water-deprivation were suppressed in hereditary microphthalmic rats with abnormal SCN. These findings suggest that the SCN is involved in the mechanism of blood glucose and body fluid intake as well as that of circadian rhythm.     

Changes in the topographically organized connections between the nucleus isthmi and the optic tectum after partial tectal ablation in adult goldfish

The projection of the nucleus isthmi to the ipsilateral optic tectum was examined in normal goldfish. This was compared to the projection in animals in which the entire visual field had been induced to compress onto a rostral half tectum by caudal tectal ablation. The isthmo-tectal projection was examined by making localized injections of horseradish peroxidase into the optic tecta and observing the patterns of labeled cells within the nucleus isthmi. The teleost nucleus isthmi consists of a cell sparse medulla covered by a cellular cortex, which is thick on the rostral, medial, and dorsal surfaces of the nucleus. Almost all isthmic cells projecting to the tectum were located in the area of thick cortex. In normal fish, rostral tectal injections labeled cells in the rostroventral portion of the thick cortex; injections midway in the rostrocaudal tectal axis labeled more caudodorsally located cells, and caudal tectal injections labeled cells a little further caudally in extreme dorsal cortex. The rostroventral to caudodorsal isthmic axis was therefore seen to project rostrocaudally along the tectum. This topography contrasts somewhat with the situation seen in amphibia where the rostrocaudal tectal axis receives projections from the rostrocaudal isthmic axis. In fish with half-tectal ablations, injections near the caudal edge of the half tectum (at a site that had originally been midtectal) labeled cells that had previously projected to caudal tectum. Rostral tectal injections in fish with compression of the visual field gave a normal pattern of labeled isthmic cells. The results indicate that a topographically ordered isthmo-tectal projection exists in goldfish that may be induced to compress onto a half tectum.     

Development of the oculomotor nucleus, with special reference to the time of cell origin and cell death

The developmental pattern of the oculomotor nucleus from day 7 of incubation through two weeks after hatching was studied in white Peking duck embryos. The neuroblasts comprising the nucleus complete their last phase of DNA synthesis on days 4 and 5 and the anlage first appears on day 7. The various subnuclei become identifiable as distinct cell groups on day 8 or 9. There is a cell migration between the ventral-most portions of the two ventromedial nuclei on days 9 through 11, and as a result a well-developed oculomotor commissure is established between these two subnuclei. The maximum number of cells in the nucleus is present on day 11. There is a normally occurring overall loss of approximately 43% of the cells during ontogenesis. Cell death appears to be random, without any gradient, and virtually all of it occurs between days 11 and 15. Although the duration of cell death is essentially similar in all subnuclei, great variations exist in its magnitude. For example, there is a cell loss of approximately 61% in the accessory nucleus, 38% in the dorsolateral nucleus, 40% in the dorsomedial nucleus and 33% in the ventromedial nucleus. Cell loss in the oculomotor nucleus is compared with that observed in the other two eye-muscle nuclei.     

Afferents of the lamprey optic tectum with special reference to the GABA input: combined tracing and immunohistochemical study

The optic tectum in the lamprey midbrain, homologue of the superior colliculus in mammals, is important for eye movement control and orienting responses. There is, however, only limited information regarding the afferent input to the optic tectum except for that from the eyes. The objective of this study was to define specifically the gamma-aminobutyric acid (GABA)-ergic projections to the optic tectum in the river lamprey (Lampetra fluviatilis) and also to describe the tectal afferent input in general. The origin of afferents to the optic tectum was studied by using the neuronal tracer neurobiotin. Injection of neurobiotin into the optic tectum resulted in retrograde labelling of cell groups in all major subdivisions of the brain. The main areas shown to project to the optic tectum were the following: the caudoventral part of the medial pallium, the area of the ventral thalamus and dorsal thalamus, the nucleus of the posterior commissure, the torus semicircularis, the mesencephalic M5 nucleus of Schober, the mesencephalic reticular area, the ishtmic area, and the octavolateral nuclei. GABAergic projections to the optic tectum were identified by combining neurobiotin tracing and GABA immunohistochemistry. On the basis of these double-labelling experiments, it was shown that the optic tectum receives a GABAergic input from the caudoventral part of the medial pallium, the dorsal and ventral thalamus, the nucleus of M5, and the torus semicircularis. The afferent input to the optic tectum in the lamprey brain is similar to that described for other vertebrate species, which is of particular interest considering its position in phylogeny.     

Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation

Monoaminergic innervation of a histamine-producing cell group, the tuberomammillary nucleus in the posterior hypothalamus, was investigated in the rat by light and electron microscopic immunohistochemical techniques. Immunohistochemical staining of sections of the posterior hypothalamus was demonstrated afferent fibers immunoreactive to tyrosine hydroxylase in ventral and medial subgroups of the tuberomammillary nucleus afferent fibers immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenyletanolamine-N-methyltransferase (PNMT), and serotonin (5-HT). TH- and DBH-immunoreactive fibers were similar and were evenly and densely distributed throughout the tuberomammillary nucleus. Fibers stained with 5-HT antibodies were also present throughout the tuberomammillary nucleus but exhibited the densest labeling in the dendritic layer adjacent to the glia limitans in the ventral subgroup. Innervation by PNMT-immunoreactive axons was sparse. Electron microscopic analysis of TH-, DBH-, and 5-HT-immunoreactive fibers in the tuberomammillary nucleus revealed vesicle-containing terminal boutons, which formed synapses with dendrites of varying size. Synaptic contacts with nerve cell bodies were not found. Retrograde transport of the fluorescent dye Fast Blue injected into the tuberomammillary nucleus, combined with immunofluorescent staining with anti-TH, anti-DBH, anti-PNMT, and anti-5-HT antibodies, showed that monoaminergic input to the tuberomammillary nucleus originated mainly from the adrenergic and noradrenergic cell groups C1-C3 and A1-A2, respectively, and from the serotoninergic cell groups B5-B9 as designated by Dahlstr?m and Fuxe ('65). Few double-labeled neurons were found in the nucleus locus coeruleus and the dopaminergic cell groups of the rostral brain stem. The present findings suggest that the activity of the histamine-producing neurons of the tuberomammillary nucleus is influenced by monoaminergic neurons in the ventrolateral and dorsomedial medulla oblongata and the raphe nuclei of the rostral brainstem.     

Synaptic organization of the rat parafascicular nucleus,with special reference to its afferents from the superior colliculus and the pedunculopontine tegmental nucleus

The synaptic organization of afferents to the parafascicular nucleus (Pf) of the thalamus was studied in rats. In the Pf, three types of axon terminals were identified: the first type was a small terminal with round synaptic vesicles forming an asymmetric synapse, the second type was a large terminal with round synaptic vesicles forming an asymmetric synapse, and the third type was a terminal with pleomorphic vesicles forming a symmetric synapse. They were named SR, LR and P boutons, respectively. In order to determine the origin of these axon terminals, biotinylated dextran amine (BDA) was injected into the main afferent sources of the Pf, the superior colliculus (SC) and the pedunculopontine tegmental nucleus (PPN). Axon terminals from the SC were both SR and LR boutons which made synaptic contacts with somata and dendrites. PPN afferents were SR boutons, which made synaptic contacts with somata and smaller dendrites. Double-labeled electron microscopic studies, in which a retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) was injected into the striatum and an anterograde tracer (BDA) into the SC revealed that SC afferent terminals made synapses directly with Pf neurons that projected to the striatum. Another experiment was performed to find out whether two different afferents converged onto a single Pf neuron. To address this question, two different tracers were injected into the SC and PPN in a rat. Electron microscopically, both afferent terminals from the SC and PPN made synaptic contacts with the same dendrite. Our results prove that a single neuron of the rat Pf received convergent projections from two different sources.