Retrograde Transport Of Horseradish Peroxidase Research Articles

Localization of enkephalinergic neurons in the dorsolateral pontine tegmentum projecting to the spinal cord of the cat

The dorsolateral pontine tegmentum of the cat is known to contain enkephalinergic neurons, with most of the enkephalin co-contained in the catecholaminergic neurons; however, enkephalinergic cells projecting to the spinal cord have not been identified. This study employs retrograde transport of horseradish peroxidase in combination with methionine-enkephalin or tyrosine hydroxylase immunocytochemistry to 1) determine the locations of pontospinal enkephalinergic neurons and 2) compare these with the locations of pontospinal catecholaminergic neurons. Pontospinal enkephalinergic neurons were observed in the nuclei locus coeruleus and subcoeruleus and the Kölliker-Fuse nucleus. A high concentration of these neurons was evident in the Kölliker-Fuse nucleus when compared to the nuclei locus coeruleus and subcoeruleus (P less than .01). Both the enkephalinergic and catecholaminergic neurons projecting to the spinal cord were located in the same general areas of the dorsolateral pontine tegmentum and there was no significant difference in the mean diameters of these two neuronal types (P greater than .05). Quantitative data concerning the pontospinal enkephalinergic neurons correlated well with previous data on pontospinal catecholaminergic neurons (Reddy et al., Brain Res. 491:144-149, '89). A majority of the descending neurons from the dorsolateral pontine tegmentum contain enkephalin (72-80%) and catecholamine (80-87%). The observations suggest that enkephalin is contained in many of the pontospinal catecholaminergic neurons.

Fine Structural Characteristics and Synaptic Connections of Trigeminocerebellar Projection Neurons in Rat Trigeminal Nucleus Oralis

In order to classify the presynaptic terminals contacting trigeminocerebellar projection neurons (TCPNs) in rat trigeminal nucleus oralis (Vo), electron-microscopic examination of sequential thin sections made from TCPNs located in the border zone (BZ) of Vo, labeled by the retrograde transport of horseradish peroxidase, was undertaken. The use of BZ TCPNs, labeled in Golgi-like fashion so that many of their dendrites and axons were visible, allowed for the determination of the distribution of each bouton type along the soma and dendrites, as well as for the characterization of the morphology and synaptic relations of the labeled axon and its terminals. Three types of axon terminals contacting labeled BZ TCPNs have been recognized, depending upon whether they contain primarily spherical-shaped, agranular synaptic vesicles (S endings); predominantly flattened, agranular synaptic vesicles (F endings); or a population of pleomorphic-shaped, agranular synaptic vesicles (P endings). The S endings represent the majority of axon terminals contacting labeled BZ TCPNs and establish asymmetrical axosomatic and axodendritic synaptic contacts. Many S endings are situated in one of two types of synaptic glomeruli. One type of glomerulus has a large S ending at its core, whereas the other contains a small S ending. Large-S-ending glomeruli include only labeled distal dendrites of BZ TCPNs; small-S-ending glomeruli contain either a labeled soma, proximal dendrite, or distal dendritic shaft. The remaining S endings are extraglomerular, synapsing on distal dendrites. P endings are less frequently encountered and establish intermediate axosomatic and axodendritic synapses. These endings exhibit a generalized distribution along the entire somatodendritic tree. F endings make symmetrical axodendritic synapses with distal dendrites, are only found in glomeruli containing small S endings, and are the least frequently observed ending contacting labeled BZ TCPNs. The majority of axonal endings synapsing on labeled BZ TCPNs are located along distal dendrites, with only a relatively few synapsing terminals situated on proximal dendrites and somata. The axons of labeled BZ TCPNs arise from the cell body and generally give rise to a single short collateral near their points of origin. This collateral remains unbranched and generates several boutons within BZ, while the parent axon acquires a myelin sheath and, without branching further, travels dorsolaterally toward the inferior cerebellar peduncle. The collateral boutons resemble extraglomerular S endings. They contain agranular, spherical-shaped synaptic vesicles and make asymmetrical axodendritic synapses with small-diameter unlabeled dendritic shafts in the BZ neuropil.

Intrinsic circuitry: Synapses involving the local axon collaterals of corticocortical projection neurons in the mouse primary somatosensory cortex

Pyramidal neurons in the mouse SmI cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) injected into the ipsilateral MsI cortex. Terminals of the local axon collaterals of these neurons (CC terminals) were identified in SmI, and their distribution and synaptic connectivity were examined. To avoid confusion, terminals in SmI cortex labeled by the anterograde transport of HRP injected into MsI were eliminated by lesion-induced degeneration. Lesions of MsI were made 24 hours after the injection of HRP; postlesion survival time was 4 days. Most CC axon terminals occurred in layers III and V where they formed asymmetrical synapses. Of 139 CC synapses in layer III and 104 in layer V, approximately 13% were formed with dendritic shafts. Reconstruction of 19 of these dendrites from serial thin sections showed them to originate from both spiny and nonspiny neurons. Most synapses of CC terminals (about 87%) were onto dendritic spines. In contrast, White and Keller (1987) demonstrated that terminals belonging to the local axon collaterals of corticothalamic (CT) projection cells synapse mainly with dendritic shafts of nonspiny neurons: 92% onto shafts, the remainder onto spines. The distribution of asymmetical synapses onto spines and dendritic shafts was analyzed for neuropil in layers III, IV, and V. Depending on the layer, from 34 to 46% of the asymmetrical synapses in the neuropil were onto dendritic shafts. Results showing that CC and CT terminals form proportions of axodendritic vs. axospinous synapses that differ from each other, and from the neuropil, indicate that local axon collaterals are highly selective with regard to their postsynaptic elements.

Morphological classification and retinal distribution of large ganglion cells in the retina ofBufo marinus

The retrograde transport of horseradish peroxidase (HRP) and cobaltic-lysine complex (CLC) was used to morphologically characterize large ganglion cells (GCs) and to determine their distribution in retinal wholemounts and in sectioned material in the retina of Bufo marinus. Large GCs, amounting to about 0.5% of total GC population, were defined to be those with very large dendritic field sizes varying between 0.1 mm2 to 0.6 mm2 and cell soma sizes of between 100 microns 2 to 400 microns 2. These cells were subdivided into 3 major groups, Types I, II and III, on the basis of their dendritic field sizes, aborization patterns and the strata of dendritic branching within the inner plexiform layer (IPL). The majority of large neurons (about 90%) were classified as Type I GCs with symmetrical dendritic arbor. These cells had either bistratified branching in the scleral and vitreal sublamina of the IPL (65% of Type I Cells) or unistratified branching in the scleral (26%) or in the vitreal (9%) sublamina. Their dendritic field sizes increased linearly from the retinal centre from 0.13 mm +/- 0.02 mm2 (mean and S.D.) to 0.58 +/- 0.11 mm2 in the retinal periphery. Type II GCs (about 9% of large GC population) were characterized by an asymmetrical dendritic aborization directed towards the ciliary margin with unistratified branching in the scleral sublamina of the IPL. The mean dendritic field sizes of these cells were 0.26 +/- 0.09 mm2. Type III GCs, the least frequent (about 1%) category of large GCs had sparsely branching, elongated dendritic branching aligned approximately parallel with the nasotemporal axis of the retina. The unistratified dendritic branches of these neurons were located in the vitreal sublamina of the IPL with a mean dendritic field size of 0.42 +/- 0.11 mm2. The dendritic field sizes of Types II and III GCs did not increase with retinal eccentricity. Type I GCs were distributed unevenly across the retina, the density being greatest in the visual streak, along the nasotemporal meridian of the retina. The dendritic field sizes of these cells increased towards the retinal periphery, resulting in a constant dendritic field coverage factor across the retina. Each retinal point was covered by the dendritic fields of 4-5 adjacent GCs. In contrast, Types II and III GCs had only discontinuous dendritic coverage. The identification of morphological types of large GCs with previously described functional classes of GCs in the anuran retina is discussed.

Vagal afferent innervation in regenerated rat liver

The possible presence of neural sprouting in the afferent neurons of regenerated rat liver after hepatectomy was investigated by retrograde transport of horseradish peroxidase. This experiment was carried out to see if the increase in hepatic parenchyma could provide an adequate stimulus for the sprouting process. The study was limited to the vagal afferents, particularly the left ones, because they are the principal contributors to hepatic afferent innervation in the rat. The results show that neural sprouting does not occur in regenerated rat liver after 3 weeks. In fact, the number of intensely labeled neurons in the left nodose ganglia of hepatectomized rats was significantly smaller than in controls. This could be due to a lessened availability of horseradish peroxidase to nerve terminals because of the increased non-innervated hepatic mass. There was no difference between right nodose ganglia neurons in hepatectomized and control animals. This could be a consequence of their possible distribution in hepatic areas not involved in the regenerative process.

Postnatal development of phrenic motoneurons in the cat

The postnatal growth of phrenic motoneurons in the cat was studied using retrograde transport of horseradish peroxidase (HRP). The mean somal surface area of these developing motoneurons increased 2.5 times from day 3 to adult while the mean somal volume increased four-fold. This change in mean somal surface area during postnatal development was found to be correlated with the change in mean axonal conduction velocity measured from phrenic motoneurons.

Sources of thalamic afferents projecting to the suprasylvian gyrus of the black sea porpoise

Neurons sending fibers to different loci of the suprasylvian gyrus (SSG) of the porpoise(Phocaena phocaena) cortex were located in the thalamus by retrograde horseradish peroxidase transport and fluorescent tracing techniques. Horseradish peroxidase injection into the anterior section of the suprasylvian gyrus led to retrograde labelling of neurons in the lateral portion of the ventrobasal complex of nuclei and the ventroposteroinferior nucleus. A group of labelled cells was found in the ventral section of the main medial geniculate nucleus. Injecting bisbenzimide into different loci of the medial suprasylvian gyrus also led to retrograde labelling of neurons belonging to the ventral division of the main medial geniculate nucleus. Somewhat lower numbers of labelled cells were found in the inferior nucleus of the pulvinar. Small groups of labelled neurons were also found in the lateral nucleus of the pulvinar, the medioventral nucleus of the medial geniculate body, and the posterior complex of nuclei. A similar distribution of labelled cells was also observed after injecting bisbenzimide into the more caudal portion of the gyrus, although the location of labelled cells in the ventral division of the main medial geniculate nucleus and the lower pulvinar nucleus were shifted in a lateral direction.

Effects of prepubertal castration on the spinal motor nucleus of the ischiocavernosus muscle of the rat

The location, number and size of the motoneurons innervating the ischiocavernosus muscle, identified by means of horseradish-peroxidase (HRP) retrograde transport, were studied (1) in adult untreated male rats, (2) in adult male rats castrated before puberty, and (3) in adult male rats castrated before puberty and injected with testosterone from the day of castration. After injection of HRP into the ischiocavernosus muscle, labeled motoneurons were found in the dorsolateral and dorsomedial columns of the lamina IX, at the level of L6 and S1 segments of the spinal cord. Morphometric analysis demonstrated that prepubertal castration induces a statistically significant reduction in the somatic and nuclear areas (40% and 35%, respectively, if compared to those of the control rats) of both the dorsolateral and dorsomedial motoneurons, but does not affect their number. The effects of castration are prevented by exogenous testosterone.

Topographic and zonal pattern of olivocerebellar projection to the paramedian lobule in the rabbit: an experimental study with an HRP retrograde tracing method

Distribution of neurons in the inferior olive (IO) projecting to the paramedian lobule (PML) was studied in rabbits by means of retrograde transport of horseradish peroxidase (HRP). HRP was injected into various regions of different folia of the PML. Findings indicate zonal and to some extent topographic organization in olivocerebellar projections to PML folia. The neurons in corresponding areas of dorsal (dlPO) and ventral lamina (vlPO) of the principal olive (PO) project to composite zones D (D 1 + D 2) in sublobule f: medial, intermediate and lateral. In addition, the caudal part of the medial accessory olive (MAO) projects to the most laterally located zone (C 2-lateral) and the caudal part of the dorsal accessory olive (DAO) to the most lateral zone (C 3) in sublobule f. The middle part of the DAO sends projections to zone C 1 located medially in sublobules d-a. The rostral and caudal parts of the DAO send projections to zone C 3 in sublobules a and f, respectively. The rostral, middle and adjacent caudal parts of MAO, with no clear topographic organization, project to broad zone C 2 in sublobules e-b. The neurons in restricted areas of the caudomedial part of the dlPO and vlPO, probably intermingled with those supplying the composite medial zone D in sublobule f, project to sublobules e-b to terminate in zones D 1 and D 2, respectively.

Morphology of developing motoneurons innervating the medial gastrocnemius of the cat

The morphology of medial gastrocnemius (MG) motoneurons labeled by retrograde transport of horseradish peroxidase was quantified in 5 postnatal ages (3 to 79–86 days) and in adults. A bimodal distribution of somal volumes was evident at birth which permitted separating the motoneurons into alpha and gamma subpopulations for analysis. There was a significant increase in the axial dimensions, surface area and volume calculated for both alpha and gamma cell bodies between each of the age-groups studied. A greater relative growth of the major over minor axis for the gammas produced a significant decrease in the form factor (i.e. greater eccentricity) between the youngest and oldest age-groups. The number of primary dendrites observed remained constant throughout postnatal development. The surface area of alpha somata more than tripled while that of the gammas doubled from 3 days to the adult. The mean somal volume of an alpha motoneuron at birth was only 17% of its adult value while the gamma cell bodies were 33% of their adult volume. A positive correlation was found for both alpha and gamma motoneurons when their somal surface area was plotted against postnatal age and weight. The rate of growth of the MG somal surface area is compared to the changes found in axonal conduction velocity and axonal diameter for MG in the literature.

The postnatal growth of motoneurons at three levels of the cat neuraxis

The postnatal growth of motoneuron cell bodies located in the brainstem, cervical and lumbosacral spinal cord was investigated using retrograde transport of horseradish peroxidase in kittens ages 2, 12, 30, 55, 82 and 114 postnatal days and in an adult. The motoneurons innervating an extrinsic tongue muscle, the genioglossus, reached their adult size by eight weeks after birth. In contrast, the phrenic motoneurons innervating the diaphragm achieved adult size by 12 weeks and the motoneurons innervating the medial gastrocnemius muscle continued to grow beyond the twelfth postnatal week. The sizes of these motoneurons relative to one another remained constant during periods of development.

Changes in EEG spindle activity induced by ibotenic acid lesions of medialis dorsalis thalamic nuclei in the cat

The injection of an excitotoxin into medialis dorsalis thalamic nuclei (MD) elicited a short-term increase followed by a depression on EEG spindle waves in chronically implanted cats. This biphasic action provides further evidence to the hypothesis that MD plays a crucial role in transferring and inducing spindling on frontal cortex. In addition, retrograde horseradish peroxidase transport from previously lesioned MD labeled subcortical structures such as basal forebrain, anterior hypothalamus, reticular thalamic nucleus, ventral tegmental area, and locus coeruleus.

Triads: a synaptic network component in the cerebral cortex

The objective of this study was to examine synaptic relationships among 3 neuronal elements in the cerebral cortex: thalamocortical afferents (TC), corticothalamic projection cells (CT), and GABAergic neurons. TC axon terminals in the barrel cortex of the mouse were labeled by lesion induced degeneration; local axon collaterals belonging to CT cells were labeled by the retrograde transport of horseradish peroxidase; and GABAergic neurons were identified using immunocytochemistry. CT and GABAergic neurons form synapses with each other and both receive synapses from TC afferents. These findings indicate the existence in the cerebral cortex of a triadic circuit involving afferent input both to projection and to local inhibitory neurons, and reciprocal synaptic interactions among these neuronal populations.

Isthmo-optic connections in congenitally monophthalmic chicks.

By means of retrograde transport of horseradish peroxidase, isthmo-optic projections were investigated in 1 to 9-day-old posthatching chickens. Some of the specimens were congenitally anophthalmic, others were monophthalmic. In contrast to a recent study, we did not detect significant differences in the one-eyed chicks. In particular, we find no indication for a retarded elimination of ectopic and aberrantly projecting isthmo-optic neurons during ontogeny. We tentatively suggest that more than one mechanism leading to monophthalmic deformities may exist.

Opiocortin and catecholamine projections to raphe nuclei

Afferent projections to the nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) were identified using retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA). Neurons were labeled in important nociceptive regions including periaqueductal gray (PAG), arcuate nucleus, lateral hypothalamus and medial thalamic nuclei following both injections. We have immunocytochemically identified opiocortin/WGA neurons in the arcuate nucleus following NRM and DRN injections. Dual stained catecholamine/WGA perikarya were found in zona incerta, locus coeruleus, substantia nigra, nucleus tractus solitarius and adjacent A2, C2 and C3, lateral paragigantocellular reticular nucleus/C1 and lateral reticular nucleus/A1 following DRN injections and in zona incerta, substantia nigra, nucleus tractus solitarius/A2 and lateral reticular nucleus/A1 after NRM injections. These results provide further evidence for opiocortin and catecholamine modulation of analgesia.

Ultrastructural anf functional evidence for the survival of corticogeniculate neurons in kainic acid-lesioned lateral geniculate nucleus

After a kainic acid lesion in the dorsal lateral geniculate nucleus of rat, retrograde axonal transport of fluorescent dyes is blocked in corticogeniculate but not in retinogeniculate neurons. This inhibition, however, can be reversed by electrical stimulation in the subcortical white matter (Woodward and Coull, Brain Research 454 (1988) 106–115). These observations suggest that retrograde axonal transport in corticogeniculate neurons is impulse-dependent and that neuronal activity in this pathway is reduced as a consequence of the lesions. To test this we examined retrograde transport of horseradish peroxidase (HRP) and cytochrome oxidase activity in the cortex of lesioned animals. Unilateral kainic acid lesions in the geniculate inhibit the retrograde transport of HRP, but this inhibition is reversed by electrical stimulation of white matter. Moreover, histochemical staining for cytochrome oxidase activity is less intense over visual cortex on the lesioned side, implying that cortical activity in intrinsic and efferent pathways is reduced as a consequence of removal of geniculate afferents. Inasmuch as the retrograde transport of HRP is dependent upon impulse activity in neurons and is thought to be mediated by synaptic vesicle recycling, these results suggest that terminals of corticogeniculate fibers survive the kainic acid lesions in the geniculate and are capable of releasing synaptic vesicles. Ultrastructural examination of lesioned geniculates strongly supports this conclusion and reveals the presence of axon terminal profiles which are filled with small round synaptic vesicles and have membrane specializations reminiscent of synaptic contacts. These terminal profiles are presumed to be of retinal and cortical origin.

Ultrastructural and neurochemical analysis of synaptic input to trigemino‐thalamic projection neurones in lamina I of the rat: A combined immunocytochemical and retrograde labelling study

The synaptology of lamina I thalamic projection neurones in the spinal trigeminal nucleus of the rat was investigated by combining electron microscopic immunocytochemistry with the retrograde transport of horseradish peroxidase. Fifteen retrogradely labelled neurones were serially sectioned and their dendrites were traced for up to 160 microns in order to characterise the synaptic input to their cell bodies and proximal dendrites. Projection neurones receive synapses from dome-shaped substance P and enkephalin immunoreactive terminals, which make simple axosomatic or axodendritic synapses. In addition, the cells receive synapses from numerous nonimmunoreactive terminals including a wide range of different dome-shaped terminals and various scalloped or glomerular terminals. Dome-shaped terminals synapse with small stubby spines in addition to cell bodies or dendritic shafts and they are probably derived from lamina II interneurones and from descending bulbospinal pathways. Glomerular terminals occur in two main classes: small type A terminals with dark axoplasm and larger type B terminals. Type B terminals participate in synaptic triads in which a peripheral terminal synapses both axoaxonically with the glomerular terminal and axodendritically with the projection neurone. Type A and type B terminals closely resemble the central terminals of spinal cord lamina II glomeruli and are probably derived from C and A delta I degrees afferent fibers. The results indicate that lamina I projection neurones are under pre- and postsynaptic control from diverse sources. Their complex synaptic organisation highlights the key role that such cells play in the rostrad transmission of somatosensory information.

Origins of the renal innervation in the primate, macaca fascicularis

The origins of the renal efferent and afferent nerves in 5 cynomolgus monkeys ( Macaca fascicularis) were studied by using the retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase—wheat germ agglutinin (HRP WGA). The cut ends of the right renal nerves were soaked for 30–45 min in solutions consisting of 15% HRP and 1% HRP WGA. Three or four days later the animals were killed and the tissues examined for the presence of retrogradely labeled neurons, HRP-filled cells were observed, with rare exceptions, only in ganglia ipsilateral to the side of tracer application. Renal efferent neurons (4648–14565 cells per animal) were found in relatively equal numbers in prevertebral and paravertebral (sympathetic chain) ganglia. Labeled prevertebral cells were distributed among the renal (52%), aorticorenal (32%) and superior mesenteric (16%) ganglia, whereas labeled paravertebral neurons were mainly located in chain ganglia T 11–L 3, with 94% of these located in L 1–3.Labeled renal sensory neurons (31–543 per animal) constituted less than 5% of all labeled cells and were found in ipsilateral dorsal root ganglia T 10–L 3, with (80%) in T 12 and L 1. The labeled sensory neurons ranged from 18–64 μm in diameter (X = 32.4 μm). With the exception of a single cell in one animal, no labeled neurons were observed in the nodose ganglia. Many parallels were observed between the organization of the renal plexuses of macaques and humans, suggesting the utility of the non-human primate as an experimental model for functional studies of renal innervation in humans.

Afferent projections from the brainstem to the area hypothalamica dorsalis: a horseradish peroxidase study in the cat.

Experiments using the retrograde transport of horseradish peroxidase were performed in order to identify the cells of origin the ascending projections from different brainstem regions to the area hypothalamica dorsalis (aHd) in the cat. The afferent inputs to this area originate mainly from the midbrain and medulla oblongata regions. The main afferent source of the area hypothalamica dorsalis arises from the substantia grisea centralis, where a large number of labeled cells were observed bilaterally, although more abundant on the ipsilateral side. Substantial afferents reach the aHd from the nuclei vestibularis medialis and inferior and the formatio reticularis mesencephali. A modest number of peroxidase-labeled neurons were observed in the nuclei ruber, interpeduncularis, substantia nigra, reticularis gigantocellularis, vestibularis lateralis, cuneatus and gracilis. From the pons, the nucleus raphe magnus sends a weak projection to the aHd. These anatomical data suggest that such area could be involved in visceral, sexual, nociceptive somatosensorial, sleep-waking and motor mechanisms.

Postsynaptic dorsal column neurons in the cat: a study with retrograde transport of horseradish peroxidase.

The numbers, laminar position, perikaryal and dendritic morphology, and axonal trajectories of postsynaptic cells ascending the dorsal column have been studied after implantation of HRP pellets in either the dorsal columns or dorsal column nuclei after destruction of the dorsolateral fascicle on one side. Observations made throughout the spinal cord gave estimated figures of 800-1000 and 1700-2000 cells in lumbosarcal and brachial enlargements respectively on the side of the implant. The commonest type (C), centred on lamina IV, had dendritic trees greatly extended rostrocaudally and restricted mediolaterally in the lateral dorsal horn, the extension and restriction diminishing for more medial cells. Type B cells differed dramatically, with large straight dendrites in the transverse plane and large perikarya in medial lamina V. Type A cells, distinguished by both rostrocaudal and mediolateral restriction in dendritic trees, were only found medially in laminae III and IV. Outside the enlargements, in high lumbar and thoracic cord, many fewer cells were found, corresponding to Type C but with dendrites much elongated rostrocaudally and little mediolateral variation. Many small fusiform cells were found in medial lamina VI in the upper cervical cord, distinct from any of the above. A few cells were found in the cord enlargements in lamina VII of the contralateral ventral horn, with axons crossing through the ventral commissure. The axons of all cell types were tortuous, and some entered the dorsolateral fascicle before crossing into the dorsal column: collaterals were often seen but could not be followed far. A complementary study of cells with axons ascending in the dorsolateral fascicle is reported in the following paper.