Differential localization of rabbit's flocculus Purkinje cells projecting to the medial and superior vestibular nuclei,investigated by means of the horseradish peroxidase retrograde axonal transport

The retrograde axonal transport of horseradish peroxidase (HRP) was used to study distribution of those Purkinje cells in the flocculus which project to the medial and superior vestibular nuclei of albino rabbits. HRP was injected electrophoretically into those nuclei and after survival for 24–48 h the flocculus was examined microscopically. It was found that Purkinje cells projecting to the medial nucleus were located relatively rostrally, while those projecting to the superior nucleus lay relatively caudally. These two groups of Purkinje cells, however, were distributed in such a way as to yield an interdigitated striped pattern over the surface of the flocculus.     

Identification of different size motoneurons labeled by the retrograde axonal transport of horseradish peroxidase

Summary Three main groups of motoneurons of different size have been labeled in adult cats by using the method for retrograde axonal transport following injection of horseradish peroxidase in the medial gastrocnemius and soleus muscles. In particular small, medium-size and large neurons which probably correspond respectively to gamma, small alpha and large alpha motoneurons innervating the calf muscles, have been identified and the corresponding area measured.     

Pallido-cortical projections in the cat studied by means of the horseradish peroxidase retrograde transport technique

Using a multi-microelectrode, in 5 animals, orientation tuning was measured simultaneously in 30 closely spaced parallel penetrations perpendicular to the surface of the striate cortex. Actual penetration angles were determined by three-dimensional track reconstruction. Above and below layer IVc, two columnar systems were found whose orientation angles were independent.     

The use of retrograde transport of horseradish peroxidase for studying the dendritic trees and axonal courses of particular groups of tract cells in the spinal cord

Summary Modifications have been made in Mesulam's method for labelling neurons by retrograde transport of horseradish peroxidase, with tetramethylbenzidine as chromogen, with the object of increasing the extent of labelling of dendrites and axons. A procedure was devised specifically for studying spinomedullary and medullospinal tract systems, involving implanting easily-made HRP-agar pellets into areas of controlled damage in particular spinal fascicles, and sealing the site of implant with cyanoacrylate glue. Lesions of other fascicles were often made to limit transport to the implanted fascicle. Fourth-order dendrites were regularly labelled over long (30 cm or more) transport distances: axons were also labelled over this whole distance, often allowing exact study of the initial course of particular axons. Controls in both cat and rat showed that the uptake of HRP under these circumstances occurred almost wholly from the region of axonal damage at the site of implant which can be characterized histologically.     

Various types of corticotectal neurons of cats as demonstrated by means of retrograde axonal transport of horseradish peroxidase

Summary The retrograde labeling of cortical neurons with horseradish peroxidase (HRP) was used to investigate the morphological features of neurons in various cortical areas projecting to the superior colliculus in the cat.Corticotectal cells were found to be labeled in layer V of the entire cerebral cortex. The number of labeled cells and their locations varied according to the sites of injections of HRP in the colliculus. Most of the Corticotectal cells identified in the present study were small (9–20 m in diameter, 66%) and medium (20–40 urn, 30%) pyramidal neurons and only 4% of them were large (more than 40 m). The labeled cells, 261 in total number, had somal diameters of 20.8±8.0 m (mean and SD). The range of sizes of the labeled neurons was different in different cortical areas. For example, the labeled neurons in the Clare-Bishop area had a greater proportion of large diameter cells than in other areas.The present findings are largely in agreement with the previous data of anterograde degeneration methods with respect to the topographical correlation of the Corticotectal projections. However, in some cortical areas, e.g., the sensorimotor and the first visual (area 17) cortex of the lateral surface of the hemisphere, relatively small numbers of Corticotectal neurons appear to have been labeled by retrogradely transported HRP. The sparsity of the labeled neurons in certain cortical areas may reflect the existence of Corticotectal neurons with axon collaterals supplying brain structures other than the superior colliculus.Abbreviations A.c. Aqueductus cerebri - AEct Gyrus ectosylvius anterior - AEs Sulcus ectosylvius anterior - AI Stratum album intermediale - AL Gyrus lateralis anterior - AP Stratum album profundum - AS Gyrus sylvius anterior - Cd Nucleus caudatus - F.l.m. Fasciculus longitudinalis medialis - GI Stratum griseum intermediale - GP Stratum griseum profundum - GS Stratum griseum superficiale - Ic Inferior colliculus - L Left - MEct Gyrus ectosylvius medius - MS Gyrus sylvius medius - MSup Gyrus suprasylvius medius - N.r. Nucleus ruber - O Stratum opticum - P Pontine nuclei - P.c. Pedunculus cerebri - PEct Gyrus ectosylvius posterior - P.g. Periaqueductal gray matter - PSigm Gyrus sigmoideus posterior - PSup Gyrus suprasylvius posterior - R Right - Sc Superior colliculus - S.n. Substantia nigra - Z Statum zonale - II Optic nerve - III and IV Motor nuclei of cranial nerves     

The vestibulothalamic projections in the cat studied by retrograde axonal transport of horseradish peroxidase

Summary Horseradish peroxidase (HRP) was injected or iontophoretically ejected in various thalamic nuclei in 63 adult cats. In 11 other animals HRP was deposited outside the thalamic territory. The number and distribution of labelled cells within the vestibular nuclear complex (VC) were mapped in each case. To a varying degree all subgroups of VC appear to contribute to the vestibulothalamic projections. Such fibres are distributed to several thalamic areas. From the present investigation it appears that generally speaking, there exist three distinct vestibulothalamic pathways with regard to origin as well as to site of termination of the fibres. One projection appears to originate mainly in caudal parts of the medial (M) and descending (D) vestibular nuclei and in cell group z. This pathway terminates chiefly in the contralateral medial part of the posterior nucleus of the thalamus (POm) including the magnocellular part of the medial geniculate body (Mgmc), the ventrobasal complex (VB) and the area of the ventral lateral nucleus (VL) bordering on VB. A second projection originates mainly in the superior vestibular nucleus (S) and in cell group y and terminates mainly in the contralateral nucleus centralis lateralis (CL) and the adjoining nucleus paracentralis (Pc). A third, more modest, pathway originates chiefly in the middle M and D, with a minor contribution from S and cell group y, and terminates in the contralateral ventral nucleus of the lateral geniculate body (GLV). There is some degree of overlap between the origin of these three vestibulothalamic pathways.Abbreviations B.c. brachium conjunctivum - CeM nucleus centralis medialis thalami - CL nucleus centralis lateralis thalami - CM nucleus centrum medianum - D nucleus vestibularis descendons - f cell group f - g cell group g - GLD corpus geniculatum laterale dorsalis - GLV corpus geniculatum laterale ventralis - i.e. nucleus intercalatus - L nucleus vestibularis lateralis - LD nucleus lateralis dorsalis thalami - LIM lamina medullaris interna - Lim nucleus limitans - LP nucleus lateralis posterior thalami - M nucleus vestibularis medialis - MD nucleus medialis dorsalis thalami - MGmc corpus geniculatum mediale, pars magnocellularis - MGp corpus geniculatum mediale, pars principalis - N.cu.e. nucleus cuneatus externus - N.f.c. nucleus fasciculi cuneati - N.mes. V nucleus mesencephalicus nervi trigemini - NR nucleus ruber - N.tr.s. nucleus tractus solitarius - N. VII nervus facialis - N. VIII nervus statoacusticus - PC pedunculus cerebri - Pc nucleus paracentralis thalami - Pf nucleus parafascicularis - p.h. nucleus prepositus hypoglossi - PO posterior thalamic group - PO1 lateral part of PO - POm medial part of PO - Prt nucleus pretectalis - Pul pulvinar - R nucleus reticularis thalami - S nucleus vestibularis superior - Sg nucleus suprageniculatus - SN substantia nigra - Sv nucleus supravestibularis - Tr.s. tractus solitarius - VA nucleus ventralis anterior thalami - VL nucleus ventralis lateralis thalami - VPL nucleus ventralis posterior lateralis - VPL1 lateral part of VPL - VPLm medial part of VPL - VPM nucleus ventralis posterior medialis - x cell group x - y cell group y - z cell group z - V nucleus motorius nerve trigemini - X nucleus dorsalis nerve vagi - XII nucleus nervi hypoglossi     

Amygdaloid projections to the motor,premotor and prefrontal areas of the cat's cerebral cortex: A topographical study using retrograde transport of horseradish peroxidase

The topographic organization of the projections from the amygdaloid complex to the frontal (motor, premotor and prefrontal) cortex has been investigated in the cat by means of the horseradish peroxidase retrograde transport technique. While most of these projections arise from the magnocellular component of the basal nucleus, some arise also from other nuclei, such as the parvocellular basal nucleus, the corticoamygdaloid transition area and the cortical nucleus. The projections from the latter nuclei are directed to the central portions of the prefrontal cortex, both laterally and medially. No clear-cut topographic segregation appeared to exist in the distribution within the magnocellular basal nucleus of the cells of origin of projections to the motor, premotor and prefrontal cortex.The gross topographic arrangement of the amygdalocortical projections seems to reciprocate, to some extent at least, the organization of corticoamygdaloid projections from high-order sensory and polymodal association areas.     

Afferent and efferent connections of brainstem locomotor regions: study by means of horseradish peroxidase transport technique

Afferent and efferent connections of the hypothalamic and mesencephalic locomotor regions and also the bulbar locomotor strip were studied in cat using retrograde (horseradish peroxidase) transport technique. To study the sources of afferent projections, the enzyme microinjections were performed exactly into the same brain sites eliciting treadmill locomotion by means of electrical stimulation. When studying efferent projections horseradish peroxidase labeled neurons were revealed within locomotor regions after enzyme microinjections into different brain structures. Experimental data have shown that the hypothalamic and mesencephalic locomotor regions have mutual afferent and efferent projections with numerous brain areas including interconnections. Apart from the entopeduncular nucleus, the great number of different sensory nuclei are noted: among the sources of afferent projections are the nucleus tractus spinalis nervi trigemini, nucleus cuneatus, nucleus tractus solitarius and vestibular nuclei. In addition, after horseradish peroxidase injection into the mesencephalic locomotor region labeled neurons were found in the cochlear nuclei. Direct descending neuronal projections of the hypothalamic and mesencephalic locomotor regions are distributed mainly in the ipsilateral brainstem. Only a few of them reach the lumbar spinal cord. The most marked efferent projections of given regions are those to the brainstem reticular formation. After horseradish peroxidase injection into a functionally identified bulbar locomotor strip, labeled neurons were revealed in different stem regions mainly caudal to the enzyme injection site. The existence of a locomotor strip as an independent structural formation is called into question. When studying the locomotor region connections, the structural heterogeneity of these regions is revealed. Transitory fibers of ascending tracts are presumably within their limits side by side with neurons. The role of these fibers in stepping initiation by electrical stimulation of locomotor regions remains uncertain.     

Projection of thalamic neurons to cat primary vestibular cortical fields studied by means of retrograde axonal transport of horseradish peroxidase

Summary The two vestibular cortical projection areas in the anterior suprasylvian sulcus and post-cruciate dimple regions were defined by evoked potential technique in anaesthetized cats. The thalamic location of neurons with axon terminals in these fields was determined using the method of retrograde axonal transport of horseradish peroxidase. The ascending vestibular pathway appeared to be separated also at the thalamic level, where cells in the ventro-posterolateral nucleus were found to project to the post cruciate dimple and cells in the posterior nuclear group to the anterior suprasylvian vestibular cortical fields.     

A re-evaluation of the retrograde axonal transport of horseradish peroxidase isoenzymes by semi-quantitative cytochemical analysis

Retrograde axonal transport of 2 horseradish peroxidase (HRP) isoenzymes, the cationic isoenzyme C (HRP-C) and anionic isoenzyme A (HRP-A), was compared using the trigeminal innervation of the cornea in the rabbit. The effects of various fixatives, cytochemical reaction solutions and intracellular decay on the enzymatic activity and cytochemical visualization of both HRP-C and HRP-A were first evaluated quantitatively. Spectrophotometric assays showed that HRP-C and HRP-A respectively retained 92% and 84% of their initial activity after exposure to a fixative containing 1% paraformaldehyde and 0.5% glutaraldehyde for 1 h at 20°. The specific activity of HRP-A was 26% that of HRP-C when assayed in a solution containing 0.5 mg/ml diaminobenzidine and 0.01% H₂O₂ in 0.15 m citrate buffer, pH 5.1. By killing the rabbits at different times following injection of HRP into the corneas, the intracellular half-life of HRP-C was estimated to be 40–52 h and HRP-A 8–12 h. After taking all of these differences into consideration, corrected concentrations of the 2 isoenzymes were compared for retrograde axonal transport. Results showed that both label intensity (scored according to 4 classes) and total number of labeled cell profiles in animals injected with HRP-C was significantly greater than in animals injected with HRP-A.These data are consistent with evidence from previous in vivo and in vitro studies demonstrating a certain selectivity of both uptake and retrograde axonal transport of HRP isoenzyme C that does not appear to be simple ‘fluid-phase’ internalization. The methodology and precautionary steps taken in this study in obtaining a semi-quantitative evaluation of HRP cytochemical visualization should be of general use in a variety of histochemical studies.     

Ultrastructure of visual callosal neurons in cat identified by retrograde axonal transport of horseradish peroxidase

Summary The ultrastructure of neurons at the border of areas 17 and 18 of the visual cortex of the cat was studied by the combined use of the retrograde transport of horseradish peroxidase (HRP) and electron microscopy. Callosal neurons were retrogradely labelled by injecting HRP at the 17/18 border region of the contralateral hemisphere. They were found mainly in layer III but also in IV and VI. They were most commonly pyramidal cells and less often large, spiny stellate cells. Pyramidal callosal neurons received only symmetrical synapses on their soma and mainly symmetrical (but a few asymmetrical) synapses on their dendritic shafts. Their abundant spines received asymmetrical synapses. The stellate cells were contacted by moderate numbers of symmetrical and asymmetrical axodendritic and axosomatic synapses and also had asymmetrical axospinous contacts. We propose that the callosal stellate neurons consist of a class of large spiny stellates, recognizable by light and electron microscopic criteria.The work described in this paper forms part of a study for a doctoral dissertation in the University of Lausanne by J. P. Hornung.     

The pontine projection to the flocculonodular lobe and the paraflocculus studied by means of retrograde axonal transport of horseradish peroxidase in the rabbit

Summary The occurrence and distribution of labeled cells in the pontine nuclei were mapped following injections of small amounts of horseradish peroxidase (0.05–0.5 l, 50% suspension) in the flocculus, nodulus and the dorsal and ventral paraflocculus in adult albino rabbits. While no labeled cells were found in the pontine nuclei following injections in the nodulus, some were present following injections in the flocculus and a great number following injections in the paraflocculus. The projections onto the flocculus and paraflocculus are precisely organized. Following injections in the paraflocculus labeled neurons are arranged in four columns (E and G in the paramedian pontine nucleus, F in the peduncular and H in the dorsolateral nucleus). Following injections in the ventral paraflocculus labeled cells are present only in parts of column E and F, while columns G and H and parts of E and F project onto the dorsal paraflocculus. Following injections in the flocculus labeled cells occur in the rostral part of column E only.A comparison between the sites of termination of pontine afferents and the areas giving origin to floccular and parafloccular fibers shows that only few fibers mediating visual impulses end in these pontine areas, while they receive numerous fibers from gyrus cinguli and areas 18 and 19 of the cerebral cortex.     

Topographical representation in rabbit cerebellar flocculus for various afferent inputs from the brainstem investigated by means of retrograde axonal transport of horseradish peroxidase.

A small amount of horseradish peroxidase (HRP) was injected into several small areas of the flocculus and adjacent ventral paraflocculus of albino rabbits. Labeled cells were surveyed through the inferior olive, vestibular nuclear complex, nucleus reticularis tegmenti pontis, pontine nucleus and abducens nucleus. Distribution of labeled neurons in these structures varied depending on the location of injection sites, indicating the existence of a fine topographical organization for afferent inputs in the flocculus and adjacent ventral paraflocculus.     

Uptake and retrograde axonal transport of horseradish peroxidase in regenerating facial motor neurons of the mouse

Summary Uptake and retrograde axonal transport of intravenously injected horseradish peroxidase (HRP) was studied during regeneration after a crush injury of the facial nerve of the mouse. The circulation time of HRP was 12 to 24 h. HRP injected immediately after the crush diffused into injured axons in the crushed region and accumulated subsequently in perikarya of facial neurons in the brain stem. After a time interval of 1 h or 5 days between the crush and the injection only a faint HRP accumulation occurred in a few facial neurons. After an interval of 7 days a moderate number of neurons had incorporated the tracer, while after more than 9 days the HRP activity in the regenerating neurons was more pronounced than in the contralateral neurons. Ultrastructurally, muscles of the vibrissae showed denervated subneural apparatuses 6 days after the crush. 8 days after the crush regenerating axon terminals containing small clusters of synaptic vesicles, dense cored vesicles and some HRP-labelled vesicles, were found over some gutters and after 10 to 13 days all examined gutters contained axon terminals with large numbers of synaptic vesicles and some HRP-containing vesicles. More than one axon terminal profile was seen in the same synaptic gutter. 32 and 64 days after the crush the neuromuscular junctions had regained a more mature appearance. The calibre spectra of the crushed facial nerves still showed a shift towards smaller diameters 134 days after the crush, at a time when a slight increase in HRP activity in the facial neurons persisted.     

The origin of the reticulo-olivary projection in the rat: a retrograde horseradish peroxidase study

Electrophoretic injections of horseradish peroxidase were made in different parts of the rat inferior olivary complex using a ventral approach. Data from these injections provide anatomical evidence for the existence of a projection to the inferior olive which takes origin from reticular nuclei in the brainstem. The majority of reticulo-olivary neurons are located in the nucleus raphe obscurus and nucleus raphe pallidus. Other reticular nuclei which contribute to this projection include the nucleus reticularis ventralis and nucleus reticularis gigantocellularis. Analysis of injections confined to specific parts of the olivary complex reveals a topographical pattern in the reticulo-olivary projection. Caudal parts of the complex receive input primarily from the nucleus reticularis ventralis. As more rostral and medial parts of the inferior olive are included in the injection, there is concomitant shifting of labeled neurons to the nucleus reticularis gigantocellularis and the raphe nuclei. The reticulo-olivary neurons may serve several non-mutually exclusive roles in olivary circuitry. They may be the source of serotonin and/or substance P to the nucleus. Physiologically, they may provide the inhibitory input observed in the nucleus. Finally, some of these neurons may be the brainstem relay of the lateral funiculus and dorsolateral funiculus spino-olivo-cerebellar pathway proposed by Larson and his co-workers (J. Physiol., Lond. 203, 611-640, 641-649).     

Interamygdaloid connections in the rat studied by the horseradish peroxidase method

Neurons of the rat amygdaloid body were labeled with horseradish peroxidase following its injection into contralateral nuclei of the amygdala. The results strongly suggest that there is a contralateral amygdaloid projection from the basal (dorsal and ventral) nuclei of amygdala; it terminates in the medial, central and lateral nucleus. True commissural connections were found only between posterior parts of the cortical nuclei of amygdala and between homonymous areas of the piriform cortex.     

Afferents to the cerebellar cortex of turtles studied by means of the horseradish peroxidase technique

Summary Origins of afferents to the cerebellar cortex from the brainstem were explored in turtles by means of the horseradish peroxidase (HRP) technique. Following relatively large injections involving all cortical layers, HRP label was observed in neural perikarya of the following structures: 1) contralateral reticular formation just lateral and ventral to the hypoglossal nucleus; 2) a few cells in the central gray of the cervical spinal cord; 3) neurons scattered in the dorsolateral, ventromedial and descending vestibular nuclei, mainly ipsilaterally; 4) a few solitary cells in the mesencephalic and medulalry tegmentum; 5) the nucleus isthmi magnocellularis caudalis on the ipsilateral side; 6) a group of small cells in the isthmic tectum; 7) the ipsilateral nucleus of the optic tract; 8) a prominent group of small cells in the isthmic region just rostral to the vestibular complex ipsilaterally. Most of these cells were localized within the so called nuclei gustatorius secundarius,-lemnisci lateralis and-isthmi parvocellularis. This parvocellular isthmic complex (PIC) was the only region containing labelled cells when small injections restricted to the molecular layer were achieved. We interpret the PIC as a source of climbing fibers, possibly corresponding to the mammalian inferior olive which migrates from the alar plate to its' ventral destination during ontogenesis. Connecting axons were sometimes homogeneously stained which permitted the tracing of connecting pathways. Contorted axon branches stained by anterograde HRP transport were found concentrated in cerebellar and superior vestibular nuclei and sparsely distributed in other vestibular nuclei.