Further studies on the development of the isthmo-optic nucleus with special reference to the occurrence and fate of ectopic and ipsilaterally projecting neurons

Using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) and the fluorescent dyes true blue and nuclear yellow, we have reex-amined the time of arrival at the retina of the centrifugal fibers from the contralateral isthmo-optic nucleus (ION), and have followed, quantitatively, the appearance and fate of other neurons that can be retrogradely labeled from the ipsilateral and contralateral eyes. The earliest age at which ION neurons can be retrogradely labeled is on the ninth day of incubation; the cells labeled at this stage are located in the ventrolateral part of the nucleus on the contralateral side. Over the course of the next 48 hours of development more and more cells can be labeled in a distinct ventrolateral to dorsomedial progression within the nucleus. Since this labeling sequence parallels the time course of generation of the ION neurons it is suggested that the axons of the first ION cells to be generated are the first to reach the contralateral retina, and that in this system there is a close relationship between the time that neurons withdraw from the cell cycle and the time that their axons reach their target area. In addition to the neurons in the ION, about 1,500 cells outside of the nucleus can be retrogradely labeled by WGA-HRP injected into the contra-lateral eye in posthatched chicks. This is slightly less than half the number of “ectopic isthmo-optic neurons” that can be similarly labeled on the 13th day of incubation (when the ION is numerically complete). The reduction in the number of ectopic ION neurons occurs over the same period as the phase of naturally occurring cell death in the ION itself–between the 13th and 17th days of incubation. Long-term labeling experiments with true blue indicate that the disappearance of about 53% of the ectopic ION cells is due to their death rather than the elimination of axon collaterals. In their morphology the ectopic neurons resemble the cells in the ION at early stages in their development, and there is indirect evidence that the ectopic ION neurons which survive also receive an input from the optic tectum through the-tecto-isthmal tract. On these and other grounds it is suggested that the ectopic neurons are indeed “misplaced” ION cells. Between the tenth and 13th days of incubation a small and rather variable number of neurons (58–178) was retrogradely labeled from the ipsilateral eye. Of these neurons with aberrant, ipsilaterally projecting axons, an average of just under 30 lay within the interior of the ION, about 33 were along its borders, and 38 were observed scattered among the ectopic ION cells. Double labeling experiments indicate that early in their development some of these neurons have axon collaterals which project to the contralateral eye. In a large number of animals injected with WGA-HRP after the 17th day of incubation, only a single neuron was seen in the interior of the ipsilateral ION, but, on average, about nine labeled neurons were found along its borders and about 20 were ectopically placed. The numbers of ectopic ION neurons and ION cells with ipsilaterally projecting axons that we have observed are substantially higher than those reported in a previous study (Clarke and Cowan, '76) in which it was also suggested the both classes of cells were effectively eliminated between the 13th and 17th days of incubation. The observed differences are attributable to the greater sensitivity of the WGA-HRP method when used with the chromogen tetramethyl benzidine. It is now clear that although cells in the ION with aberrantly projecting axons (or axon collaterals) may be selected against during the later stages of development, the fate of the ectopic ION neurons is not significantly different from that of the cells in the ION itself, but since they lie outside the nucleus their dendritic processes are not subject to the same morphogenetic influences.     

The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons.

The development of the centrifugal projection to the chick retina in the isthmo-optic tract (IOT) has been studied by the retrograde transport of the enzyme marker horseradish peroxidase (HRP) injected into the eye at various times during the incubation period. Some neurons in the isthmo-optic nucleus (ION--the cells of origin of the IOT) can first be labeled from the eye following injections on the tenth day of incubation; after injections on the twelfth day or later, about 95% of the neurons can be so labeled. It follows from this that the axons of virtually all the neurons in the ION (including the 60% which normally degenerate between the thirteenth and seventeenth days of incubation) reach the contralateral eye. Since in 12-day old embryos the IOT is between six and seven millimeters in length and HRP can be identified in the perikarya of ION neurons within three and one-half hours, the rate of retrograde transport in the system must be of the order of 48 mm/day. A similar time is required for HRP to appear in the perikarya of ION neurons in post-hatched chicks in which the length of the IOT is estimated to be about 14 mm. This suggests that at some time during the latter half of the incubation period there is a significant acceleration in the rate of retrograde transport, similar to that found for anterograde axonal transport in the chick and rabbit visual systems.     

Axonal neurofilament-H immunolabeling in the rabbit retina

A monoclonal antibody directed at the multiphosphorylated epitope of axonal neurofilament-H (NF-H) was used to label axon-like fibers in the rabbit retina. NF-H-immunopositive fibers were found in the outer plexiform layer (OPL), inner plexiform layer (IPL), and optic fiber layer (OFL). The morphological characteristics of the labeled processes identified those in the OPL as horizontal cell axons and axon terminals and fibers in the OFL as axons of ganglion cells. The NF-H-positive profiles in the OPL formed a subset of horizontal cell processes labeled for calbindin. In the IPL, NF-H-immunoreactive profiles lay at all levels but were detected most often in the middle strata, 2-4. Occasionally, we observed NF-H-immuoreactive processes emerging from the IPL and entering either the GCL or the inner nuclear layer (INL). The labeled fibers in the IPL were typically very thin, less than 1 microm in diameter, and could often be followed for over 1 mm as they ran laterally across the retina. Cell bodies were never labeled by the immunoserum. To identify the NF-H-immunopositive fibers in the IPL, standard immunocytochemical double-labeling techniques were applied, using antibodies directed against several neurotransmitters or modulators thought to be expressed by axon-bearing amacrine cells. The NF-H-positive processes in the IPL were found to correspond to those labeled for tyrosine hydroxylase, somatostatin, substance P, and NADPH diaphorase activity. However, the NF-H labels did not colocalize with those against the vasoactive intestinal peptide-associated protein PHM27. Our results indicate that putative axons in the retina possess the multiphosphorylated NF-H protein found within classic axons in the central nervous system. These results thus support the idea that certain subtypes of amacrine and horizontal cells maintain true axons in the mammalian retina.     

The development of the isthmo-optic nucleus.

The nucleus of origin of centrifugal fibers to the retina (the so-called isthmo-optic nucleus - ION) has been used as a model for the study of the major features of neural development, from the period of cell proliferation until after the formation of its afferent and efferent connections. 3H-thymidine autoradiography has established that in the chick cells of the ION are generated (i.e., become post-mitotic) between the middle of the 5th and the end of the 7th days of incubation. The first-formed cells are found in the ventrolateral part of the nucleus, while those that are generated at successively later stages come to occupy progressively more medial and dorsal positions within the nucleus. The anlage of the ION can be identified on the 8th day of incubation, and by the 11th day, when it is numerically complete, it occupies a prominent position in the caudo-dorsal part of the midbrain tegmentum at the level of the IVth nerve nucleus. At this stage the nucleus contains about 22,000 neurons, and shows no signs of cytoarchitectonic differentiation. Between the 13th and 17th days of incubation, about 60% of the neurons in the nucleus degenerate; as a result of this degeneration, the arrival of afferent fibers, and the growth of the cells' processes, the nucleus comes to have its characteristic adult form of a complex, folded, bilaminar sheet, in which each part of the retina is precisely represented. Experiments based on the retrograde transport of horseradish peroxidase (HRP) from the eye indicate that the first centrifugal fibers, in the isthmo-optic tract (IOT), reach the retina on the 10 day of incubation, and by the 12th day all but about 5% of the neurons in the ION can be retrogradely labeled in this way...     

Target-derived BDNF (brain-derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo-optic nucleus

Neurons in the peripheral nervous system depend on single neurotrophic factors, whereas those in the brain are thought to utilize many different trophic factors. This study examined whether some neurons in the brain critically depend on a single trophic factor during development. Neurons in the isthmo-optic nucleus (ION) of chick embryos respond to exogenous brain-derived neurotrophic factor (BDNF). Relatively high concentrations of endogenous BDNF were present in the ION of 14-18-day-old chick embryos. ION target cells in the retina were immunolabeled for BDNF but showed surprisingly low levels of BDNF mRNA. These data suggest that ION target cells derive some BDNF from other retinal sources. No BDNF mRNA was detected in the ION itself. ION neurons had a very efficient retrograde transport system for BDNF and exogenous BDNF arrived in the ION intact. When the ION was deprived of endogenous trkB ligands by injection of trkB fusion proteins in the eye, cell death of ION neurons was enhanced, and this effect was mimicked by BDNF-specific blocking antibodies in the eye. TrkB fusion proteins in the retina induced cell death of ION neurons prior to visible effects on ION target cells in the retina. Immunolabel for endogenous BDNF was sparse in pyknotic ION neurons, suggesting that ION neurons with low BDNF content were eliminated by apoptosis. These data show that BDNF is an essential target-derived trophic factor for developing ION neurons and thereby validate the neurotrophic hypothesis for at least one neuronal population in the brain.     

Olivocerebellar and cerebelloolivary connections of the oculomotor region of the fastigial nucleus in the macaque monkey

Anatomical connections of the caudal portion of the fastigial nucleus (FN) with the inferior olive (IO) were studied in macaque monkeys with wheat-germ-agglutinin-conjugated horseradish peroxidase (WGA/HRP) and HRP. When injected HRP was confined to a caudal portion of the FN, retrogradely labeled Purkinje cells (P cells) appeared in the oculomotor vermis. We defined the area that receives the projection from vermal lobule VII as the fastigial oculomotor region. The same HRP injection resulted in retrograde labeling of IO neurons in an area of group b (of Bowman and Sladek: J. Comp. Neurol. 152:299-316, '73) of the contralateral medial accessory olive (MAO). This area was designated as the Z-portion because in the coronal section it appears like the letter "Z." Retrogradely labeled IO neurons were also found in the Z-portion when HRP was injected into the oculomotor vermis, indicating that neurons in this portion project to both the fastigial and vermal oculomotor regions. Anterogradely labeled axons from the contralateral fastigial oculomotor region also terminated in the Z-portion. When the effective site included a region anterior to the fastigial oculomotor region, labeled P cells appeared in lobule V and labeled IO neurons appeared in group a. Labeled terminals of fastigial fibers were also found in group a. When the effective site included a region ventral to the oculomotor region, labeled P cells appeared in vermal lobules VIII and IX and labeled IO neurons appeared in caudal parts of a and b, in addition to group c. HRP injection into the posterior interposed nucleus (PIN) resulted in labeling of P cells in the paravermal zone and of IO neurons in the rostral two-thirds of the MAO and the dorsal accessory olive (DAO). The location of the labeled terminals coincided with the region where the densest labeling of IO neurons was found. Thus, the olivary projections to both the cerebellar cortex and deep cerebellar nuclei and the nucleoolivary projection exhibited a closely related topographical organization.     

Serotonin immunoreactivity in the retinal projecting isthmo-optic nucleus and evidence of brainstem raphe connections in the pigeon

Serotonin (5-HT) immunoreactive (-ir) profiles within the isthmo-optic nucleus (ION) of the centrifugal visual system (CVS) were studied in the pigeon using light microscopic immunohistofluorescent and electron microscopic immunocytochemical pre-embedding techniques. The brainstem origin of the 5-HT input upon the ION was determined by combining 5-HT immunohistofluorescence (FITC) and retrograde transneuronal tracing after intraocular injection of Rhodamine beta-isothiocyanate. The light microscopic results showed that 5-HT endings were mainly localised within the neuropillar zones of the ventral ION. The 5-HT-ir cell bodies, belonging to a lateral extension of the dorsal raphe system, were observed within the same region as the centrifugal ectopic neurons (EN) underlying the ION and some displayed dendritic processes which penetrated the nucleus. Double-labeled neurons, representing 5-HT-ir afferents to the ION, were identified only within the n. linearis caudalis region of the ventral raphe. The electron microscopic results confirmed the presence of 5-HT-ir dendritic processes within the ventral part of the nucleus and showed that they were contacted by axon terminals belonging to intrinsic interneurons. The functional organisation of the ION and the possible contribution of serotonergic raphe afferents and efferents are discussed in relation to present hypotheses linking the avian CVS to mechanisms of visual attention.     

Maintenance of targeting errors by isthmo-optic axons following the intraocular injection of tetrodotoxin in chick embryos.

We have studied the role of electrical activity in the elimination of axonal targeting errors, which is a normal process in brain development. The experiments were focused on the isthmo-optic nucleus (ION), which, in adults, projects in topographical order on the contralateral retina. During embryogenesis, however, a few isthmo-optic neurons project to the ipsilateral retina, and many project to topographically inappropriate parts of the contralateral one; both kinds of targeting error are known to be eliminated by the deaths of the parent neurons. We injected tetrodotoxin (TTX) intraocularly at embryonic days 13 and 15 and, on the latter, applied a retrograde label to the retina of the same eye. Embryos were fixed at embryonic day 17. In some embryos, the label was a peripherally placed fleck of the carbocyanine dye "diI"; the resulting retrogradely labeled neurons in the contralateral ION were much more widely scattered in the TTX-injected embryos than in controls (errors in topography). In other embryos, the label was a solution of rhodamine-B-isothiocyanate (RITC) injected into the vitreous body; this yielded several ipsilaterally labeled isthmo-optic neurons in the TTX-injected embryos, but virtually none in the controls. The numbers of both kinds of aberrantly projecting neuron approached those previously reported near the beginning of the ION's period of neuronal death. We conclude that electrical activity plays an important role in the elimination of axonal targeting errors in the chick embryo's isthmo-optic system.     

Morphological analysis of the hyperpolarization-activated cyclic nucleotide-gated cation channel 1 (HCN1) immunoreactive bipolar cells in the rabbit retina

Hyperpolarization-activated cation currents (I(h)) have been identified in neurons in the central nervous system, including the retina. There is growing evidence that these currents, mediated by the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN), may play important roles in visual processing in the retina. This study was conducted to identify and characterize HCN1-immunoreactive (IR) bipolar cells by immunocytochemistry, quantitative analysis, and electron microscopy. The HCN1-IR bipolar cells were a subtype of OFF-type cone bipolar cells and comprised 10% of the total number of cone bipolar cells. The axons of the HCN1-IR cone bipolar cells ramified narrowly in the border of strata 1 and 2 of the inner plexiform layer (IPL). These cells formed a regular distribution, with a density of 1,825 cells/mm(2) at a position 1 mm ventral to the visual streak, falling to 650 cells/mm(2) in the ventral periphery. Double-labeling experiments demonstrated that their axons stratified narrowly within and slightly proximal to the OFF-starburst amacrine cell processes. In the IPL, they were presynaptic to amacrine cell processes. The most frequent postsynaptic dyads formed of HCN1-IR bipolar cell axon terminals are pairs composed of both amacrine cell processes. These results suggest that these HCN1-IR cone bipolar cells might be the same as the DAPI-Ba1 bipolar population, and might therefore be involved in a direction-selective mechanism, providing inputs to the OFF-starburst amacrine cells and/or the OFF-plexus of the ON-OFF ganglion cells.     

Abrupt loss of dependence of retinopetal neurons on their target cells, as shown by intraocular injections of kainate in chick embryos

We have examined the capacity of neurons in the chick isthmo-optic nucleus (ION) to survive when their target neurons in the contralateral retinal are destroyed by intraocular injections of kainate (KA) at different stages in development. The retinal vulnerability to KA builds up progressively from embryonic day 10 (E10) until a plateau is reached at E15 (see accompanying paper); and the effects on the ION increase in parallel, almost all the ION neurons being rapidly lost after the E15 injections. KA injection before E15 lesioned only part of the retina and caused degeneration only in the topographically corresponding region of the ION. Near the end of the natural cell death period in the ION (E17), this initial dependence on the target cells is rapidly lost. Already at E16 the injections kill less ION neurons, and by E19 they kill none of them. The ION neurons have become completely insensitive to the KA injections and appear normal more than 4 months later, although axotomy (by eye removal) at a similar age would by then have killed them. The ectopic ION neurons, scattered outside the ION but projecting to the retina, are never affected by KA injections at any age.     

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.     

A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons.

The phenomenon of retrograde intraaxonal transport of extracellular markers introduced at the level of the axon terminal has been suggested as a possible mechanism of communication between the axon terminal and the neuron cell body. We tested the hypothesis that communication after axotomy might consist of a change in the rate of uptake or of transport of material by injured neurons. Small lesions were made with a needle in one retinal quadrant of chicks and immediately afterwards horseradish peroxidase (HRP) was injected into the vitreous body of the eye. The amount of HRP accumulated by some of the neurons of the isthmo-optic nucleus (ION) which project to the damaged area was clearly different from that of nearby cells which project to the non-damaged portions of the retina. The uninjured cells accumulated enzyme marker beginning at 3.5 h after injection. The injured neurons did not accumulate significant amounts of HRP until between 4 and 6 h after injection. Between 6.75 h and 18 h the injured cells in the ION accumulated greater amounts of HRP than cells in other regions, but by 24 h the cells of the ION in the region of injury contained distinctly less label. This pattern of enzyme accumulation was confirmed by counts of the number of HRP-positive granules within cells of chicks fixed 4, 11.75, 12.25, 27.6 and 72 h after injury. In another series of experiments, the axon terminals of the ION were first exposed to HRP, and 1 h later some of the axons were damaged with a needle. In these cases, there was no difference between the injured and control neurons in the time of first appearance of labeled cells in the ION within the first 4 h after injection of HRP. These findings suggest that injury initially results in a decrease in the uptake of the marker rather than a decrease in the rate of retrograde transport. The amount of marker found in the injured neurons later is greater than that found in the control neurons. This subsequent difference may represent an increase in the rate of uptake, transport, or both or a decrease in the rate of degradation of HRP within the cell body as a response to injury of the axon.     

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light-induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin-immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin-expressing retinal ganglion cells.     

Evidence for a dopaminergic innervation of the subthalamic nucleus in the rat

The dopaminergic connection from the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) to the subthalamic nucleus in the rat was investigated using anterograde retrograde tracers. Iontophoretic injection of the retrograde tracer fluoro-gold (FG) into the subthalamic nucleus resulted in a substantial number of labeled neurons in the SNc. Immunohistochemistry of tyrosine hydroxylase (TH) confirmed the dopaminergic nature of these labeled neurons. Retrogradely labeled neurons were also found in the VTA. Injection of the anterograde tracer biocytin into the SNc produced biocytin-labeled terminals in the subthalamic nucleus hence providing clear evidence for a dopaminergic innervation of this nucleus. Quantitative analysis of labeled axons revealed that there were 15–38 terminal branches per axon, each branch being 50–150 μm long. The overall dimensions of one terminal arborization were 400 × 250 × 150 μm. There was no clear-cut topographical organization of the projection, but a slight mediolateral difference in the density of terminals. This direct dopaminergic projection is thought to interact with cortical and pallidal inputs in the subthalamic nucleus, which implies that the functions of the subthalamic nucleus are more complex than previously assumed.     

Morphological analysis of disabled-1-immunoreactive amacrine cells in the guinea pig retina

Disabled-1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by reelin, that controls cell positioning in the developing brain. It localizes to selected neurons in the nervous system, including the retina, and Dab1-like immunoreactivity is present in AII amacrine cells in the mouse retina. This study was conducted to characterize Dab1-labeled cells in the guinea pig retina in detail using immunocytochemistry, quantitative analysis, and electron microscopy. Dab1 immunoreactivity is present in a class of amacrine cell bodies located in the inner nuclear layer adjacent to the inner plexiform layer (IPL). These cells give rise to processes that ramify the entire depth of the IPL. Double-labeling experiments demonstrated that these amacrine cells make contacts with the axon terminals of rod bipolar cells and that their processes make contacts with each other via connexin 36 in sublamina b of the IPL. In addition, all Dab1-labeled amacrine cells showed glycine transporter 1 immunoreactivity, indicating that they are glycinergic. The density of Dab1-labeled AII amacrine cells decreased from about 3,750 cells/mm(2) in the central retina to 1,725 cells/mm(2) in the peripheral retina. Dab1-labeled amacrine cells receive synaptic inputs from the axon terminals of rod bipolar cells in stratum 5 of the IPL. From these morphological features, Dab1-labeled cells of the guinea pig retina resemble the AII amacrine cells described in other mammalian species. Thus, the rod pathway of the guinea pig retina follows the general mammalian scheme and Dab1 antisera can be used to identify AII amacrine cells in the mammalian retina.     

Localization of glycine-containing neurons in the Macaca monkey retina

Autoradiography following 3H-glycine (Gly) uptake and immunocytochemistry with a Gly-specific antiserum were used to identify neurons in Macaca monkey retina that contain a high level of this neurotransmitter. High-affinity uptake of Gly was shown to be sodium dependent whereas release of both endogenous and accumulated Gly was calcium dependent. Neurons labeling for Gly included 40-46% of the amacrine cells and nearly 40% of the bipolars. Synaptic labeling was seen throughout the inner plexiform layer (IPL) but with a preferential distribution in the inner half. Bands of labeled puncta occurred in S2, S4, and S5. Both light and postembedding electron microscopic (EM) immunocytochemistry identified different types of amacrine and bipolar cell bodies and their synaptic terminals. The most heavily labeled Gly+ cell bodies typically were amacrine cells having a single, thick, basal dendrite extending deep into the IPL and, at the EM level, electron-dense cytoplasm and prominent nuclear infoldings. This cell type may be homologous with the Gly2 cell in human retina (Marc and Liu: J. Comp. Neurol. 232:241-260, '85) and the AII/Gly2 of cat retina (Famiglietti and Kolb: Brain Res. 84:293-300, '75; Pourcho and Goebel: J. Comp. Neurol. 233:473-480, '85a). Gly+ amacrines synapse most frequently onto Gly- amacrines and both Gly- and Gly+ bipolars. Gly+ bipolar cells appeared to be cone bipolars because their labeled dendrites could be traced only to cone pedicles. The pattern of these labeled dendritic trees indicated that both diffuse and midget types of biopolars were Gly+. The EM distribution of labeled synapses showed Gly+ amacrine synapses throughout the IPL, but these composed only 11-23% of the amacrine population. Most of the Gly+ bipolar terminals were in the inner IPL, where 70% of all bipolar terminals were labeled. These findings are consistent with previous data from cats and humans and suggest that both amacrine and bipolar cells contribute to glycine-mediated neurotransmission in the monkey retina.     

Origins and targets of commissural connections between the cochlear nuclei in guinea pigs

Our objective was to identify the origins and targets of axons that project from one cochlear nucleus to the other. First, retrograde tracers were injected into one cochlear nucleus to label commissural cells in the opposite nucleus. In the dorsal cochlear nucleus, a few cells in the deep layers were labeled; they were not further classified according to type. In the ventral cochlear nucleus, all commissural cells that could be classified were multipolar cells. Second, an anterograde tracer was injected into one cochlear nucleus, and the distribution of boutons in the opposite cochlear nucleus was examined. Labeled boutons were present throughout the ventral cochlear nucleus, where they appeared to contact multipolar cells, spherical and globular bushy cells, and octopus cells. In the dorsal cochlear nucleus, labeled boutons were present in the fusiform cell and deep layers and appeared to contact fusiform cells and cells of unknown type. Many labeled terminals were also present in the granule cell regions. Injections into regions associated with high or low frequencies labeled boutons in corresponding regions in the contralateral ventral cochlear nucleus. Third, multiple tracers were used to determine whether cells that project to the inferior colliculus are contacted by commissural axons. Boutons labeled by anterograde transport of one tracer placed in the cochlear nucleus were frequently observed to be apposed to cells that were labeled by retrograde transport of a different tracer placed in the contralateral inferior colliculus. We conclude that commissural projections originate from multipolar cells throughout the ventral cochlear nucleus (and from a small number of cells in the dorsal cochlear nucleus) and make contact with all major cell types of the cochlear nuclei, including at least some of those that project to the inferior colliculus. © 1996 Wiley-Liss, Inc.     

Role of nitric oxide in a fast retrograde signal during development

Two retrograde signals influence the chick embryo's isthmo-optic nucleus, which projects to the retina: a slow-acting survival signal due to uptake of neurotrophic factors, and a fast-acting death signal initiated by calcium entry into isthmo-optic terminals due to electrical activity. The latter signal also affects dendritic reorganization. Since nitric oxide synthase is present in isthmo-optic terminals and their retinal target cells, we have tested its possible role in the fast-acting signal. Intraocular injection of nitric oxide antagonists led within 6-12 h to a reduction in the number of dying isthmo-optic neurons and slowed dendritic reorganization. Surprisingly, nitric oxide agonists had a similar fast effect on neuronal death. Although the mechanism appears to be complex, nitric oxide is involved in mediating the fast-acting retrograde signal.     

Vasoactive intestinal polypeptide-containing cells in the rabbit retina: immunohistochemical localization and quantitative analysis

Vasoactive intestinal polypeptide (VIP) possesses neuroactive properties in the nervous system. In this study we characterized VIP immunoreactive neurons in the rabbit retina to provide a basis for a better understanding of the role of this peptide in retinal functions and to further define the morphology of wide-field amacrine cells. VIP immunoreactivity was demonstrated in colchicine-treated retinas. Immunolabeling was observed in amacrine cells located in the proximal inner nuclear layer and, occasionally, in the ganglion cell layer and inner plexiform layer (IPL). Varicose fibers were distributed in laminae 1, 3, and 5 of the IPL. The distribution of VIP immunoreactive cells showed a peak of approximately 50 cells/mm2 in the visual streak and minimum values of approximately 12 cells/mm2 in the peripheral retina. The total number of VIP immunopositive neurons was estimated to be about 11,000. Cell body diameters in the visual streak (8-9 microns) were slightly smaller than those measured in the dorsal or in the ventral retina (9-10 microns). The distribution of nearest neighbor distances (approximately 109 microns in the visual streak and approximately 99 microns in the peripheral retina) showed that VIP immunoreactive neurons were nonrandomly spaced. Labeled neurons emitted one to three thick primary processes, arborizing in secondary processes and collaterals rich in varicosities; these processes often crossed among different IPL laminae. Arborization fields of individual cells overlapped extensively. In the dorsal retina, estimated areas of single arborization fields were larger and processes had lower branching frequency than in the visual streak and in the ventral retina. On the whole, VIP immunoreactive amacrine cells gave rise to a loose meshwork of fibers in the IPL. These characteristics indicate VIP is contained in a class of wide-field amacrine cells and is likely to be involved in widespread regulatory or modulatory functions rather than in the direct transmission of visual information through the retina.     

Observations on the afferent and efferent connections of the avian isthmo-optic nucleus

The efferent and afferent connections of the avian isthmo-optic nucleus (ION) were studied using light microscopic techniques. Injections of [³H]proline into the nucleus resulted in labeling of centrifugal endings in the retina at the junction of the inner plexiform layer and inner nuclear layer, but produced no other transported label to any thalamic or mesencephalic nucleus. The origin of the tectal afferents to the ION was demonstrated by means of injections of [³H]proline into the most superficial layers of the optic tectum and by stereotaxic injections of horseradish peroxidase into the ION. The tectal efferent cell bodies were located in lamina h of the optic tectum and at the junction of laminae h and i.