Anterograde axoplasmic transport of peroxidase in the rat visual system after intraocular neurotoxin injection

The effect of intraocular injection of the neurotoxin, kainic acid, on the retinofugal pathway has been examined by anterograde transport of horseradish peroxidase. Although less peroxidase was present in all the primary optic centers after the intraocular injection of kainic acid that in the controls, the area occupied by the retinal terminals remained fairly constant except for the pretectal region where there is a smaller area of peroxidase precipitate. These results suggest that certain ganglion cells are killed by kainic acid but some others survive and their terminals are present in the majority of the terminal fields.     

Ultrastructure of transganglionic HRP transport in cat trigeminal system

The ultrastructure of transganglionic transport of horseradish peroxidase (HRP) from the inferior alveolar (IA) nerve to the brainstem is being studied in the cat. The IA nerve was soaked in an HRP solution and following a two-day survival the animal was perfused transcardially with a paraformaldehyde-glutaraldehyde solution. The tissue was immediately dissected and postfixed for 1-3 h in perfusate. Sections of 75 micron thickness were cut with a Vibratome and reacted utilizing tetramethyl benzidine (TMB) as the chromagen. Optimum results for electron microscopy were obtained by osmication in a pH 6.0, 1% osmium tetroxide solution for 45 min at 45 degrees C, followed by rapid dehydration and embedment in Epon. The resulting HRP-TMB reaction product was characterized and identified ultrastructurally in ganglion cells, peripheral and central axons and in brainstem terminals. The HRP-TMB reaction product varied in density but had consistent crystalline-like laminations of a repeating unit and characterized by a membrane 4-5 nm in diameter. Some of the HRP-TMB reaction product found in terminals and axons was below the limit of resolution of the light microscope.     

Transneuronal labeling with horseradish peroxidase in the visual system of the house fly

Horseradish peroxidase (HRP) is taken up by lesioned neurons in the fly CNS and passes from these into certain sets of adjoining neurons which are in synaptic contact. This transneuronal labeling resolves neurons in fine detail. Electron microscopy shows reaction product in secondary neurons associated with plasmalemma and microtubules. In some cases also vesicles were found containing reaction product. It is suggested that the transneuronal passage takes place in vivo.     

Organization of the visual system in larval lampreys: an HRP study.

The organization of the visual system of larval lampreys was studied by anterograde and retrograde transport of HRP injected into the eye. The retinofugal system has two different patterns of organization during the larval period. In small larvae (less than 60-70 mm in length) only a single contralateral tract, the axial optic tract, is differentiated. This tract projects to regions in the diencephalon, pretectum, and mesencephalic tegmentum. In larvae longer than 70-80 mm, there is an additional contralateral tract, the lateral optic tract, which extends to the whole tectal surface. In addition, ipsilateral retinal fibers are found in both small and large larvae. Initially, the ipsilateral projection is restricted to the thalamus-pretectum, but it reaches the optic tectum in late larvae. Changes in the organization of the optic tracts coincide with the formation of the late-developing retina and consequently, the origin of the optic tracts can be related to specific retinal regions. The retinopetal system is well developed in all larvae. Most retinopetal neurons are labeled contralaterally and are located in the M2-M5 nucleus of the mesencephalic tegmentum, in the caudolateral mesencephalic reticular area and adjacent ventrolateral portions of the optic tectum. Dendrites of these cells are apparent, especially those directed dorsally, which in large larvae extend to the optic tectum overlapping with the retino-tectal projection. These results indicate that in lampreys, visual projections organize mainly during the blind larval period before the metamorphosis, their development being largely independent of visual function.     

Prestriate afferents to inferior temporal cortex: an HRP study

The inferior temporal (IT) cortex of 6 macaques was injected with horseradish peroxidase. HRP-labeled cells were found throughout IT cortex itself (outside the injection area) but were not found in the polysensory areas that surround IT dorsally, anteriorly and ventrally. Posterior to IT, labeled cells were found in the anterior parts of prestriate cortex. In one animal, the anterior prestriate region was injected with HRP. Labeled cells were then found in the regions of posterior prestriate cortex that receive direct projections from striate cortex. These results suggest that IT cortex receives information from striate cortex after at least two stages of processing in prestriate cortex.     

A comparative radioautographic study of the bidirectional axonal and transcellular transport of different amino acids and sugars in the lamprey visual system

Following recent radioautographic evidence for the bidirectional axonal transport of [³H]proline from the eye of Lampetra fluviatilis, the present study examined the anterograde and/or retrograde labeling properties of 11 other amino acids and two sugars after intraocular injections of these tritiated substances in the lamprey. The intraocular injections of aspartic acid, glutamic acid, γ-aminobutyric acid, arginine, phenylalanine, fucose and acetyl glucosamine resulted in a weak labeling of the primary visual centers; there was no evidence of either the transcellular transport of these tracers or the retrograde labeling of the retinopetal neurons. The primary visual system was found to be heavily labeled 24 h after eye injections of alanine, leucine, glycine, lysine, serine and valine. Among the latter only glycine produced a retrograde somatic labeling of retinopetal neurons. With a longer survival period (7 days), a specific transneuronal labeling was also noted after glycine injections. The significance of the differential uptake and transport of these different tracers in the lamprey visual system and possible mechanisms involved in the axonal retrograde transport of [³H]glycine and [³H]proline in the centrifugal pathways are discussed.     

Anterograde tracing of horseradish peroxidase (HRP) with the electron microscope using the tetramethylbenzidine reaction

A technique is described for anterograde horseradish peroxidase (HRP) tracing with the electron microscope using tetramethylbenzidine (TMB) histochemistry. Following HRP injection into the primary visual cortex or one eye of rabbits the superior colliculus was studied with the light and electron microscope in adjacent sections. In target preparations from the colliculus examined with the electron microscope labelled axons and axon terminals were found. The TMB reaction product was identified as crystal like electron-dense structures. In control sections from the opposite colliculus the neuropil was free from label.     

Organization of the callosal connections of visual areas V1 and V2 in the macaque monkey

The interhemispheric efferent and afferent connections of the V1/V2 border have been examined in the adult macaque monkey with the tracers horseradish peroxidase and horseradish peroxidase conjugated to wheat germ agglutinin. The V1/V2 border was found to have reciprocal connections with the contralateral visual area V1, as well as with three other cortical sites situated in the posterior bank of the lunate sulcus, the anterior bank of the lunate sulcus, and the posterior bank of the superior temporal sulcus. Within V1, callosal projecting cells were found mainly in layer 4B with a few cells in layer 3. Anterograde labeled terminals were restricted to layers 2, 3, 4B, and 5. In extrastriate cortex, retrograde labeled cells were in layers 2 and 3 and only very rarely in infragranular layers. In the posterior bank of the lunate sulcus, labeled terminals were scattered throughout all cortical layers except layers 1 and 4. In the anterior bank of the lunate sulcus and in the superior temporal sulcus, anterograde labeled terminals were largely focused in layer 4. Callosal connections in all contralateral regions were organized in a columnar fashion. Columnar organization of callosal connections was more apparent for anterograde labeled terminals than for retrograde labeled neurons. In the posterior bank of the lunate sulcus, columns of callosal connections were superimposed on regions of high cytochrome activity. The tangential extent of callosal connections in V1 and V2 was found to be influenced by eccentricity in the visual field. Callosal connections were denser in the region of V1 subserving foveal visual field than in cortex representing the periphery. In V1 subserving the fovea, callosal connections extended up to 2 mm from the V1/V2 border and only up to 1 mm in more peripheral located cortex. In area V2 subserving the fovea, cortical connections extended up to 8 mm from the V1/V2 border and only up to 3 mm in peripheral cortex.     

Corticotectal terminals in the superior colliculus of the rabbit: a light- and electron microscopic analysis using horseradish peroxidase (HRP)-tetramethylbenzidine (TMB)

The projection from the striate cortex to the superior colliculus was studied light- and electron microscopically by means of anterogradely transported horseradish peroxidase and tetramethylbenzidine histochemistry. Labeled boutons were found in the stratum zonale (SZ) and in the stratum griseum superficiale (SGS), not in stratum opticum (SO). There are two maxima of frequency of labeled boutons, one in middle SGS at about 500 microns depth, and a smaller one in upper SGS at about 200 microns depth. Such a bimodal distribution of corticotectal terminals has not been described in any species before. Labeled myelinated axons were found in SGS and SO with a maximal frequency in middle SGS at about 400 microns depth. The myelinated axons in SZ, which are commonly considered to be of cortical origin, were not labeled. The labeled cortical terminals contained numerous round synaptic vesicles and predominantly dark mitochondria. They formed usually asymmetrical synapses and contacted dendrites, some of which contained synaptic vesicles. Occasionally, labeled boutons were observed which definitely did not belong to the type that is generally considered to be of cortical origin.     

Orthograde axonal and transcellular transport of different fluorescent tracers in the primary visual system of the rat

The differential labeling properties of various fluorescent tracers injected intraocularly were investigated using as a model the rat primary visual system. All of the tracers tested (Fast Blue, FB; True Blue, TB; Nuclear Yellow, NY; bisbenzimide, BB; Evans Blue, EB; propidium iodide, PI) produced a retrograde neuronal labeling of oculomotor neurons. However, no such labeling was observed in the medial pretectal nucleus (NPM) considered to be the site of origin of the rat centrifugal visual pathway. Orthograde transport within the axons of the optic tract and their terminal arborizations were visualized directly with FB and TB. No evidence of EB or PI orthograde transport was demonstrated. Furthermore, FB, TB, NY and BB displayed varying degress of leakage from the optic axons and terminals into the extracellular space, there to be taken up by glial (FB, TB, NY, BB) and neuronal (NY, BB) somas of the primary optic system or in adjacent structures including NPM (BB). The neuronal labeling with NY or BB does not result from the retrograde axonal transport but appears to involve an orthograde transneuronal process transport. Some limitations in the use of different fluorescent tracers for determining neuronal connections are discussed.     

Vesicular transport of horseradish peroxidase by ependymal cells of the medulla oblongata

Methionine-enkephalin (ME) released from superfused slices of rat corpus striatum was estimated by radioimmunoassay (RIA). The basal release of2.5 ± 0.2pmol/g/min (0.15% of content per min) was increased approximately 3-fold upon exposure of tissue to 30 mM K⁺ for 5 min. This increase in release was not observed in the absence of Ca²⁺. Both morphine (10^?5 M) and (?)-naloxone (10?⁵ and 10?⁶ M) significantly depressed the release of ME evoked by 30 mM K⁺ but did not alter basal release. The +-isomer of naloxone, which lacks opiate antagonist activity, did not affect basal or evoked release. A consistent depression of release was not observed when 47 mM K⁺ was used to evoke the release of ME. The issue of whether a feedback mechanism controls the release of ME from the striatum cannot be resolved until it is known whether the effect of morphine and naloxone on ME release are mediated by opiate or non-opiate mechanisms.     

The hypothalamic ‘tuberoinfundibular’ system of the rat as demonstrated by horseradish peroxidase (HRP) microiontophoresis

Microiontophoresis of horseradish peroxidase (20%) into the median eminence of the rat has allowed visualization of perikarya and axon projections of the tuberoinfundubular system after retrograde transport. Cells projecting to the median eminence were found in the periventricular regions of the hypothalamus and were particularly pronounced in dorsal portions of the rostral arcuate nucleus, the medial division of the paraventricular nucleus, and within the anterior periventricular nucleus. Labeling of perikarya within the ventromedial nucleus was rarely found. No labeling by HRP was found within cells of the dorsomedial, anterior, suprachiasmatic, preoptic, lateral hypothalamic nuclei or within the septal and amygdaloid nuclei. Axons from identifiable cells were located within the periventricular neuropil and contained within the baso-lateral portions of the hypothalamic-hypophysial tract.     

Focal cortical seizures prevent HRP and HRP-WGA labeling only in neurons bidirectionally connected to the cortex

Intracortical implants of polyacrylamide gel containing horseradish peroxidase labeled cortical efferents and perikarya in some cortical areas and a number of subcortical formations. When epileptogenic penicillin was added to the gel, no labeling was seen in the efferents and cell bodies of the cortex, thalamus, or claustrum, whereas the magnocellular nuclei of the basal forebrain, raphe nuclei and locus coeruleus did contain the label.     

Anatomy and physiology of a binocular system in the frograna pipiens

The locations of tectal neurons projecting to nucleus isthmi (n. isthmi) were found by iontophoretic injection of horseradish peroxidase (HRP) into n. isthmi. After retrograde transport, stained tectal somata are found to lie almost exclusively in layer 6 and below of the ipsilateral tectum. Many cells are colored throughout the extent of their dendrites into the fine rami, giving the appearance of a Golgi stain. Nucleus isthmi receives projections from the ipsilateral tectum and from no other region. Nucleus isthmi units recorded electrically respond to visual stimuli and are arranged in a topographic map of the visual field. There are two types of receptive fields, those with small centers and those with large centers. The small centers are about 3-5 degrees in diameter, similar to type 2 optic nerve fibers. Their response is to many of the same geometric features of stimulus as excite type 2 fibers. The large centers are at least 7-10 degrees in diameter and respond to many of the same features as excite types 3 and 4 optic nerve fibers. The responsiveness of small and large center n. isthmi units is very similar to the elements of the ipsilateral visual field projection onto tectum, i.e. the neuropilar units recorded in layers A and 8 of the tectum when the contralateral eye is occluded. These are in strong contrast to those of tectal cells of layer 6 and below, which have large receptive fields, show far less vivacious response, adapt extremely rapidly to repeated stimuli and are hard to describe in terms of characteristic stimuli because they are unresponsive most of the time. We suggest, therefore, that the axons of tecto-isthmic cells are quite active and that their cell bodies, located in layer 6 and below, only fire occasionally on the firing of their axons.     

The accessory optic system in a prosimian primate (Microcebus murinus): evidence for a direct retinal projection to the medial terminal nucleus

The accessory optic system (AOS) was studied in the prosimian primate, Microcebus murinus, by using intraocular injections of the anterograde tracers 3H-proline and horseradish peroxidase (HRP). Retinal fibers were found to terminate bilaterally in all three mesencephalic AOS nuclei as defined by Hayhow ('66, J. Comp. Neurol. 126:653-672). In contrast to previous reports in primates, we find that both the ventral and dorsal divisions of the medial terminal nucleus (MTN) receive projections from the retina. The ventral MTN is composed of a compact triangular group of cells, situated at the medial base of the cerebral peduncle, rostral to the rootlets of the third cranial nerve. The dorsal MTN extends dorsomedial to the substantia nigra and is composed of characteristic fusiform cells embedded in a fibrous neuropil. Although the cells of the dorsal MTN intermingle somewhat with the nigral cells, the nucleus is clearly distinguished by cyto- and myeloarchitectural features. The large lateral terminal nucleus (LTN) receives a dense projection from the retina and forms a prominent bulge on the lateral surface of the cerebral peduncle. The dorsal terminal nucleus (DTN) is located between the brachia of the superior and inferior colliculi, near the origin of the superior fasciculus of the accessory optic tract (AOT). This fasciculus is composed of anterior, middle, and posterior branches. In addition, a ventral group of fibers, corresponding to the inferior fasciculus of the AOT previously described in nonprimates, was identified in all planes of section. The results confirm the existence of a common plan of AOS organization in mammals.     

Development and characterization of glucocorticoid receptors in rat superior cervical ganglion

The effect of intraocular injection of the neurotoxin, kainic acid, on the retinofugal pathway has been examined by anterograde transport of horseradish peroxidase. Although less peroxidase was present in all the primary optic centers after the intraocular injection of kainic acid that in the controls, the area occupied by the retinal terminals remained fairly constant except for the pretectal region where there is a smaller area of peroxidase precipitate. These results suggest that certain ganglion cells are killed by kainic acid but some others survive and their terminals are present in the majority of the terminal fields.     

An HRP study of the central projections of primary trigeminal neurons which innervate tooth pulps in the cat

Trigeminal ganglia and brain stem of adult cats were studied following HRP injections into tooth pulps or after exposure of the cut end of the inferior alveolar nerve to HRP. Ipsilateral ganglion cells within a wide range of sizes were labeled in both experimental situations, whereas no labeled cells were observed in the contralateral ganglion in any animal. Labeled central branches of tooth pulp and inferior alveolar neurons were observed in all subdivisions of the ipsilateral trigeminal sensory complex. Terminal labeling in the tooth pulp experiments was confined to the dorsomedial parts of the main sensory nucleus and subnuclei oralis and interpolaris. Caudal to the obex terminal labeling was restricted to the medial halves of laminae I, IIa and V of the medullary dorsal horn. In the inferior alveolar nerve experiments dense terminal labeling was observed in the dorsal parts of the main sensory nucleus and subnuclei oralis and interpolaris. Caudal to the obex terminal labeling was located throughout laminae I to V in contrast to the tooth pulp experiments. Neither of the two experimental situations offers any evidence for a bilateral or contralateral brain stem projection of primary trigeminal neurons.     

An anterograde HRP study of the retinotectal pathways in albino and pigmented guinea pigs

An uncrossed retinotectal projection was observed in albino guinea pigs using the anterograde HRP method, suggesting that the retinal projection in these animals, similar to that in the pigmented strain, is not strictly crossed as reported previously. It was also noted that a small retinal projection terminated in a restricted region in the most medial portion of the contralateral inferior colliculus, implying that it might be influenced directly by fibers arising from the contralateral eye.     

An anterogradely-labeled prefrontal cortico-oculomotor pathway in the monkey demonstrated with HRP gel and TMB neurohistochemistry

Implants of horseradish peroxidase gel⁶ in the sulcus principalis and/or area 8 “frontal eye field” cortex of macaque and cebus monkeys, processed with tetramethyl benzidine (TMD) neurohistochemistry⁹, resulted in anterograde labeling of a prominent “prefrontal oculomotor bundle” which tranversed the medial subthalamic region at the diencephalic-mesencephalic junction to terminate directly in accessory and principal oculomotor nuclear groups. This report provides clear evidence suggesting direct cortical involvement in ocular motility, and the location of the pathway contributes to our understanding of ophthalmoplegias resulting from lesions in the rostralmost mesencephalon.     

Intraocular tetrodotoxin reduces axonal transport and transcellular transfer of adenosine and other nucleosides in the visual system of goldfish

Intraocular injection of tetrodotoxin (TTX) in goldfish, which abolishes physiological activity in the optic axons, decreased by up to about 30% the amount of radioactively labeled adenosine, uridine and guanosine (and their nucleotide derivatives) that was axonally transported in the optic nerve. The amount of labeled nucleoside that reached the optic tectum and became incorporated into RNA in the postsynaptic tectal neurons and glial cells was reduced by up to about 50%. There was no change, however, in the amount of transported nucleoside that became incorporated into RNA in the optic nerve glia. The TTX-induced changes were eliminated when axonal transport was blocked with vincristine, indicating that this change did not involve material moving along the nerve by diffusion. If the TTX injection was delayed until several hours after labeling of the transported materials, the transported labeled nucleoside in the nerve was reduced very little, but the RNA labeling in the tectum was reduced just as much as when TTX was given prior to labeling. This indicates that the labeling of the tectal cells was affected more by the level of activity in the pathway than by the amount of transported nucleoside reaching the optic nerve terminals. It appears likely, therefore, that the process most affected by the decrease in physiological activity is the release of nucleoside from the terminals of the presynaptic neurons or its uptake into postsynaptic tectal neurons and glia. The fact that physiological activity may modify the amount of axonally transported nucleosides made available for metabolism (including RNA synthesis) in postsynaptic neurons may provide an explanation for activity-linked neurotrophic effects.