Pharmacologic evidence for NMDA, APB and kainate/quisqualate retinotectal transmission in the isolated whole tectum of goldfish

The optic tectum of goldfish with intact optic and toral marginal fiber tracts was isolated in a perfusion chamber where the effectiveness of antagonists was tested on synaptic field potential responses to stimulation of each afferent system. There were 3 main conclusions about excitatory synapses. First, monosynaptic activation of retinotectal synapses was not detectably antagonized by D-tubocurarine, implying there is no nicotinic cholinergic component to optic transmission nor strong cholinergic gating of optic terminals. Second, a significant component of retinotectal transmission was shown to be mediated by kainate and quisqualate receptors since 6,7-dinitroquinoxaline-2,3-dione and kynurenate strongly suppressed the optic field potential. In addition, activation of these synapses involves two previously undescribed N-methyl-D-aspartate (NMDA) and APB receptor subtypes since optic field potentials were partially suppressed by 2-amino-5-phosphonovalerate (APV), 2-amino-4-phosphonobutyrate (APB) and MK-801. This is the first evidence that APB receptors exist in the visual system central to the retina. Together, these results are consistent with the possibility that retinal ganglion cells use multiple glutamate receptor subtypes. Third, the optic tectum contains a population of intrinsic glutaminergic synapses activated by a non-optic input, the marginal fibers, which can be suppressed by both APV and kynurenate. The existence of tectal NMDA receptors which are not at primary optic synapses implies that APV used to interfere with rearrangement of optic fibers during development may act not only at afferent synapses but also at a more central component of the circuit.     

Comparison of acetylcholinesterase and choline acetyltransferase staining patterns in the optic tectum of the goldfish Carassius auratus. A histochemical and immunocytochemical analysis

Although the optic tectum of nonmammalian vertebrates has been extensively studied anatomically, there is little information about the identification of neurotransmitters and the enzymes critical to their synthesis. Choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis, is presently regarded as the most reliable marker for cholinergic neurons, and its localization within putative cholinergic neurons has been made possible by the development of antibodies specific to ChAT. We have compared the immunocytochemical localization of ChAT to the histochemical staining of acetylcholinesterase (AChE) in the goldfish optic tectum. Goldfish brains reacted with the monoclonal antibody AB8 to ChAT have revealed that: (1) type XIV neurons are the only ChAT-positive cells in the tectum, and there are approximately 15,000 such cells per tectal hemisphere; (2) these neurons and other ChAT-containing afferent fibers form bands of label which correspond to those seen after AChE staining, and (3) many AChE-stained neurons do not contain ChAT. The immunohistochemical localization of ChAT has provided a direct method for determining the localization and organization of putative cholinergic structures in the optic tectum of goldfish. Future studies may elucidate the relationship of these cholinergic systems to the retinotectal projections, as there is close correspondence between AChE and ChAT location and the retinotectal termination patterns.     

Recovery of tectal nicotinic-cholinergic receptor sites during optic nerve regeneration in goldfish

The concentration of cholinergic nicotinic-like sites as measured by alphabungarotoxin (αBuTX) binding, decreased in the goldfish (Carassius auratus) optic tectum after optic nerve disconnection. Initially, the rate of loss of sites is greater than the rate of tissue or protein degradation in experiments where disconnection was achieved either by unilateral optic nerve crush or by enucleation of one eye. When the crushed optic nerve is allowed to regenerate and form behaviorally potent connections, the number and concentration of these sites appears restored. Pharmacological studies indicate that the αBuTX binding site in the goldfish optic tectum has a drug binding profile similar to that seen at central or peripheral αBuTX sites in other species.     

Electron microscope histochemistry of acetylcholinesterase distribution in the optic tectum of teleosts.

An ultrastructural analysis was made on acetylcholinesterase (AChE) localization in the optic tectum of two teleosts, the goldfish and the catfish. Electron microscope histochemistry reveals several details on synthesis, distribution and possible sites of utilization of the enzyme in the different tectal layers. The results show that AChE is synthesized by all the neuronal types present in the optic tectum. The final localization of the enzyme is the result of its synthesis in cell bodies, its storage and transport along dendrites and its release in extracellular spaces. The differences in AChE localization between the two teleosts examined mainly derive from differential enzyme release in the corresponding layers of the optic tectum. Cholinergic synapses cannot be precisely identified by means of AChE histochemistry, but the layers in which maximum release of enzyme in the extracellular spaces occurs most likely correspond to areas where cholinergic mechanisms are operating. In this connection some interesting differences of AChE localization in the superficial tectal layers (stratum marginale, stratum opticum and stratum fibrosum et griseum superficiale) are discussed. Electron microscopic histochemistry of AChE confirms its usefulness in better understanding some links between the anatomical and functional organization of complex neural structures.     

Histological localization of binding sites of α-bungarotoxin and of antibodies specific to acetylcholine receptor in goldfish optic nerve and tectum

Goldfish optic nerve as well as ganglion cell neurites grown in culture selectively bind rhodamine-labeled α-bungarotoxin following tissue fixation. Binding is competed for by unlabeled bungarotoxin, by carbamylcholine and tubocurarine, but not by atropine. In cross-sections, the label is seen confined to axonal bundles. The binding is not detectable without prior fixation and is very faint in brain sections, even after fixation.To further establish the nature of the binding, immunocytochemical studies were performed, taking advantage of a high cross-reactivity found between goldfish brain and antibodies against eel acetylcholine receptor (AChR). Antigenic sites were detected by an indirect unlabeled antibody complexed to horseradish peroxidase. Anti-AChR antibody binding to optic nerve and neurites in culture correlated with that seen with α-bungarotoxin. Binding of anti-AChR was observed in the brain, and was reduced in the denervated tectum following unilateral optic nerve crush or enucleation.The results are discussed in relation to functions of receptor proteins in the retinotectal system.     

Nicotinic depolarization of optic nerve terminals augments synaptic transmission

The role of acetylcholine (ACh) as the neurotransmitter at the vertebrate neuromuscular junction has served as a model in the study of synaptic physiology throughout the nervous system, but its function in the brain has remained obscure. Nicotinic ACh receptors are found on afferent nerve terminals in several regions of vertebrate brain, and nicotinic agonists can cause transmitter release (as measured biochemically). Yet there is no direct evidence that activation of these receptors modulates synaptic function. Here I report that nicotinic agonists directly depolarize optic nerve terminals in goldfish tectum recorded via sucrose gap and simultaneously enhance synaptic transmission recorded via tectal field potentials. I also show that a recurrent cholinergic circuit in tectum, acting on these terminals, initiates antidromic impulses that cause repeated transmission at the retinotectal synapses.     

Evidence for shifting connections during development of the chick retinotectal projection

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.     

α-Bungarotoxin binding in relation to functional organization of the rat suprachiasmatic nucleus

[¹²⁵I]α-Bungarotoxin (α-Btx) binding was quantified in autoradiographs of in vitro labeled sections through the rat suprachiasmatic nucleus (SCN). Recent evidence suggests that acetylcholine and α-Btx binding sites in the SCN region²⁵ may mediate effects of light on circadian functions. In contrast to the ventrolateral termination of retinal axons, there was a consistent pattern of relatively high α-Btx binding levels in the dorsolateral SCN and low levels in the ventromedial SCN. In addition, enucleation had little or no effect. We also investigated the possibility that phase-dependent influences of light on circadian functions could reflect the presence of light-induced or daily rhythms in α-Btx binding. Animals were tested in a light-dark cycle or after 1–2 days in constant light. The dorsolateral SCN showed a small but significant decline of α-Btx binding in animals in light at night compared to darkness at night. α-Btx binding in the SCN showed no statistically significant daily variation in animals in the light-dark cycle.     

Nicotine exposure refines visual map topography through an NMDA receptor-mediated pathway

The precise mapping of one surface onto another is fundamental to visual system organization and depends upon adequate stimulation of postsynaptic targets to stabilize correctly placed synapses. As exogenous nicotine alters neuronal activity, we investigated whether it would affect the visual map created by retinal ganglion cell terminals in the frog optic tectum. Chronic exposure of the tectum to nicotine decreased the retinal area from which cells project to a given tectal site. This map refinement was also produced by exposure to either the alpha-bungarotoxin sensitive nicotinic receptor agonist, anatoxin-a or the alpha-bungarotoxin-insensitive nicotinic receptor agonist epiboxidine. Immunocytochemical studies using mAb306 and mAb22 demonstrated that alpha-bungarotoxin-sensitive and -insensitive nicotinic receptors, respectively, occupied different tectal sites. Choline acetyltransferase immunoreactivity overlapped with mAb306, but not mAb22, staining. The developing optic tectum was more sensitive to nicotine than the adult tectum and nicotine induced both map refinements and map disruptions in a concentration-dependent manner. Blockade of the N-methyl-D-aspartate (NMDA) receptor with D(-)-2-amino-5-phosphonopentanoic acid (D-APV) prevented nicotine from refining the map in the adult tectum. Exposure to the use-dependent NMDA antagonist MK801 alone had no effect on retinotectal topography but in combination with either NMDA or nicotine it disrupted the map. Exposure to NMDA alone produced refinement. We conclude that the map refinement induced by chronic nicotine treatment has as its basis an increase in the level of NMDA receptor activity. The data are consistent with a model whereby map topography can be bidirectionally affected by either increasing or decreasing NMDA receptor activity.     

Kainate-induced lesion in the optic tectum: dependency upon optic nerve afferents or glutamate

Kainic acid is known to induce characteristic lesions in neurons receiving an intact input with presumed glutamate-mediated neurotransmission. There are indications for glutamate as a transmitter of retinal afferent terminals in the pigeon optic tectum. After tectal injection of kainic acid (0.5–2.0 μg in 0.5 μl) the optic tectum was studied by light and electron microscopy and the following changes were observed: (a) within 1–48 h important neuropil vacuolization predominantly in lower part of layer 5. Such vacuoles were sometimes postsynaptic to identified retinal afferent terminals: (b) within 1 h to 21 days progressive neuronal cell loss throughout the tectal layers. These toxic effects were not observed 2–12 weeks after contralateral retinal ablation but could partially be restored by combined glutamate (0.2 mg) and kainate injection. Thus in the pigeon tectum, kainic acid neurotoxicity is dependent upon an intact retinal input, a finding consistent with a special role for glutamate — possibly as a transmitter — in retinal terminals.     

Tests for neuroplasticity in the anuran retinotectal system

Mapping of retinotectal projections in the tree frog Hyla regilla was carried out by both behavioral and electrophysiological recording techniques following tectal ablations, with and without optic nerve regeneration. Scotomata produced by unilateral and bilateral half tectum ablations and by unilateral rectangular midtectal excisions were found to remain essentially unaltered in all cases through recovery periods up to 334 days. Similarly, electrophysiological mapping of the rostral half tectum separated by Gelfilm implants from the caudal tectum for up to 191 days yielded a normal rostral visual field. The stability of the retinotectal projection in adult Hylidae observed in these experiments contrasts with the plastic readjustments obtained in young goldfish in which the retinotectal system is still probably growing and presumably capable of field regulation. The results are taken to support the original explanatory model for developmental patterning of retinotectal connections in terms of selective cytochemical affinities between retinal and tectal neurons.     

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-Btx). Biochemical studies on binding of [¹²⁵I]α-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, d-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [¹²⁵I]α-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [¹²⁵I]α-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

Goldfish retina and tectum influence each other's growth activity during regrowth of the retinotectal projection

Variations in retinal and tectal growth activity, during regrowth of the goldfish retinotectal projection, were monitored by measuring the rates of incorporation of [14C]leucine into soluble protein and tubulin-enriched fractions at different times after crushing the optic nerves. Other experiments tested for growth-modulating interactions between tectum and retina. Here we studied how the absence of one of these structures (i.e. tectal ablation or eye removal) affected the profile of biosynthetic activity in the other. Experiments were also conducted on groups of fish in which the tectum was reinnervated by a half-retina (either half-nasal (1/2 N) or half-temporal (1/2 T) retina). This was done to ascertain if growth interactions between retina and tectum display any position-dependent differences that may be relevant to retinotopic ordering during regeneration. Our studies have revealed that: the retina and tectum of 1/2 T and 1/2 N groups differ in their growth responses during regeneration of the visual pathway: the tectum may exert a stimulatory and at other times an inhibitory influence on retinal protein synthesis; and retina and tectum display a bimodal profile of biosynthetic activity during regeneration that coincides with two stages of increased cell division (primarily glia) which other workers have found occurs in the tectum and tract during regeneration of the retinotectal projection. Indeed it seems there may be a link between this glial proliferation and the neurotrophic and guiding influences which tectum and retina exert upon one another during regeneration.     

Transient up-regulation of the rostrocaudal gradient of ephrin A2 in the tectum coincides with reestablishment of orderly projections during optic nerve regeneration in goldfish

During development, a graded expression of ephrin A2 has been implicated in retinotectal map formation. Here we have examined ephrin A2 expression during optic nerve regeneration in the mature goldfish. In the tecta of normal animals, a gradient of ephrin A2 expression is detected in cell bodies within the stratum fibrosum et griseum superficiale with more immunopositive cells caudally than rostrally. The gradient in the mature animal presumably reflects the plasticity associated with continued retinal and tectal neurogenesis. During optic nerve regeneration, expression throughout the tectum is increased by 1 month as a strong rostrocaudal gradient. The gradient declines to normal by 3 months. The up-regulation of ephrin A2 during optic nerve regeneration is likely to be instrumental in reestablishing the retinotectal map.     

Effect of enucleation on choline acetyltransferase activity in layers of goldfish optic tectum

Choline acetyltransferase (ChAT) activity was determined in layers of optic tectum in control goldfish and in goldfish 4-20 days following unilateral enucleation. Significant changes in activity were found in the periventricular (PV) and superficial gray and white (SGW) layers. Within 4 days, ChAT activity in the PV layer on the lesioned side was about 75% of that on the control side. By 20 days, ChAT specific activity in the SGW layer on the lesioned side was about 150-160% of that on the control side. This increase in specific activity in the SGW layer was accounted for by the decrease in volume and in density of the layer after enucleation, so that the total amount of activity in the layer did not change significantly, indicating that the optic terminals contain little to no ChAT activity. ChAT activity in the optic tract was very low and did not decrease after enucleation. These data strongly indicate that the retinotectal pathway in goldfish is not cholinergic and, therefore, that the ChAT activity in the SGW layer is related to sources other than retinal ganglion cells. It is suggested that one such source might be neurons with somata in the PV layer.     

Selective retinal reinnervation of a surgically created tectal island in goldfish. I. Light microscopic analysis

Through anatomical and physiological studies of the regenerating retinotectal projection of goldfish, we sought to determine whether the establishment of a topographic projection is attained through a refinement of an initially less precise pattern of innervation. A 1-mm-wide mediolateral strip of caudal tectum was removed so that a small island of tectal tissue was spared at the caudal pole, and the contralateral nerve was either crushed (TIX) or left intact (TI). The presence of regenerated axons in the ablated zone and the reinnervation of the caudal island were assessed with anterograde and retrograde labeling methods in the following postoperative intervals: early, 20-50 days; middle, 50-110 days; and late, more than 170 days. The anterograde radioautographic method revealed that the appropriate layers of the tectal island became reinnervated by optic axons during the early period. During the middle and late periods, one to several large, discrete bundles bridging the lesion zone along the surface of exposed subtectal structures were readily identified both by radioautography and by anterograde or retrograde labeling following application of horseradish peroxidase to the transected optic nerve or tectal island, respectively. In contrast, the anterograde horseradish peroxidase method did not reveal axon bundles extending caudal to the half-tectum in the absence of a tectal island. Among TIX cases, retrograde horseradish peroxidase labeling of the contralateral nasal retina was more widespread in the middle period than in the late period, a result we interpret as reflecting an improvement in topographical precision with time. The area of retinal labeling among TIX cases in the late period was similar to that following caudal tectal injection in cases with simple nerve crush, although it was still elevated above normal control values. Physiological maps indicated a focal representation of the nasal retina in the tectal island in both periods and did not reveal a transient extreme convergence of retinal input. These findings are discussed in relation to Sperry's chemoaffinity theory.     

Presynaptic neurotrophin-3 increases the number of tectal synapses,vesicle density,and number of docked vesicles in chick embryos

To determine whether presynaptically derived neurotrophins may contribute to synaptic plasticity, we examined whether neurotrophin-3 (NT-3) changed the number, size, vesicle content, or vesicle distribution of synapses within the retinorecipient layers of the chick optic tectum. In this system, endogenous NT-3 derives presynaptically from retinal ganglion cell axons. Retinotectal synapses comprise the majority of synapses in superficial tectal layers, as demonstrated by destruction of retinotectal input by intraocular application of the drug monensin. To examine the effect of increased or decreased levels of NT-3, either exogenous NT-3 or monoclonal NT-3 blocking antibodies were injected into the optic tectum of 19-day-old chick embryos, spiked with radiolabeled protein to verify the success of injections and estimate effective concentrations. After 48 hours, the ultrastructure of superficial tectal layers was analyzed and compared with samples from control tecta injected with cytochrome C. NT-3 increased the number of synapses, synaptic vesicles/profile, synaptic vesicle densities, the number of docked vesicles, and the length of the synaptic profile. Deprivation of anterogradely transported endogenous NT-3 with NT-3 antibodies resulted in the opposite effect: decreased numbers of synapses, decreased vesicle densities, and decreased numbers of docked vesicles. Brain-derived neurotrophic factor (BDNF) had a largely different effect than NT-3. BDNF increased the density of vesicles and deprivation of endogenous TrkB ligands with TrkB fusion protein reduced the density of vesicles in the synapses, without effects on synapse number or docked vesicles. We conclude that anterogradely transported NT-3 affects synapse strength in a way that differs from that of presumably postsynaptic-derived BDNF.     

A critical period in the development of tectal neurons in the chick, as revealed by early enucleation

To further study the existence of a critical trophic period in the development of the chick optic tectum¹⁷, during which the presence of retinal synapses is essential to the continued growth of tectal neurons, we have unilaterally enucleated embryos between stages 14–20 and allowed survival until stages 35–43. If the critical trophic period is between stages 40–44, as previously reported¹⁷, then we reasoned that early removal of the eye might not have any effect on tectal development until the critical period. We assessed tectal neuron survival by staining for degeneration in the efferent projections of tectal neurons. In early enucleates, degeneration was present from stages 37–43, and the severity of the degeneration was much reduced in comparison to animals enucleated during the critical period.These findings substantiate the proposition that there is a critical period late in chick tectal development. However, because the degeneration in tectal projections is less intense than in animals enucleated during the critical period, we suggest that the early enucleation has permitted axons from the remaining eye to be routed to the deafferented tectum, where they may help to sustain a portion of the tectal neurons through the critical period. Moreover, the somewhat earlier appearance of degeneration in tectal efferent pathways of early enucleates suggests that a subtle trophic relationship between retina and tectum may exist prior to stage 40, even though this relationship is not revealed when enucleations are performed later, as between stages 35–40 (ref. 17).     

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.     

Projection of optic fibers to visual centers in a turtle (Emys orbicularis)

Studies of retinal projections to the thalamus and midbrain of the turtle were based on a personal modification of the Nauta-Laidlaw technique (modified Nauta method) after unilateral enucleation. Decussation of optic fibers in the chiasma is incomplete. In the thalamus, optic fibers are found to terminate in three nuclei – with greater density in the corpus geniculatum laterale (lateral geniculate body) (LGB) and more sparsely in the nucleus suprapeduncular and nucleus ovalis. Retinal projections to the LGB assume a focal pattern, being somewhat more compact in the lateral neuropil region. Optic fibers are also shown to end in a group of pretectal nuclei: n. pretectalis dorsalis, n. lentiformis mesencephalis, n. comissurae posterior. In addition, terminations of optic fibers have been revealed in the three upper layers of the tectum. Peculiarities of preterminal and terminal degeneration of retinal fibers have been distinguished in the different tectal layers. In the second stratum, terminations of large fibers are mostly seen with a characteristic appearance of lumpy pericellular (preterminal) degeneration. In the third stratum, both large and finer fibers degenerate, showing fine debris of preterminal degeneration. Different patterns of terminal degeneration have been revealed in tectum and thalamus. The maximal size of optic fibers in the tectum proves to be larger than in thalamus. Available evidence is discussed with particular reference to comparison of the phylogenetically more recent retinothalamic system and more ancient retinotectal system.