An ultrastructural study of the segmental uptake of horseradish peroxidase in the endplate region of denervated skeletal muscle fibres

The segmental uptake of horseradish peroxidase (HRP) in the endplate region of denervated skeletal muscle fibres has been studied ultrastructurally using a method for selecting single muscle fibres with high segmental peroxidase staining from denervated mouse tibialis anterior muscle. Segments containing large peroxidase positive phagosomes could already be seen 10-15 min after i.v. injection of HRP. Such segments were still present 24 h after HRP injection. The localization of phagosomes, deep in the fibres rather than immediately under the sarcolemma, suggests that the uptake occurs from t-tubuli. Vivid proliferation of t-tubuli, consisting of vesiculation, enlargement and encircling of cytoplasmic components, was also observed. The HRP accumulates in phagosomes of varying size and shape. Similar membrane-limited bodies without or with very weak peroxidase staining were also observed. The peroxidase-positive phagosomes participate in autophagic processes as suggested by their content of undegraded cellular material. Golgi profiles, which occurred deep in the muscle fibres, and enlarged components of the sarcoplasmic reticulum were frequently encountered in the segments. Myofibrillar degeneration occurs in the segments and progresses with time after denervation. The described segments may be related to the increased membrane turnover in denervated muscle fibres and/or they may be related to processes aimed at establishing new synaptic contacts.     

The organization of the facial nucleus of the brush-tailed possum (Trichosurus vulpecula).

The facial nucleus of the brush-tailed possum has been studied using Nissl staining and the horseradish peroxidase (HRP) retrograde tracing technique. In Nissl stained sections the nucleus is seen to comprise five distinct subnuclei. Injections of HRP into individual facial muscle groups have shown that these subnuclei reflect the peripheral innervation pattern of efferents from this nucleus. Although in most cases, injection of HRP into a single facial muscle group resulted in the labelling of neurons in more than one facial subnucleus, the following subnuclei were most completely labelled subsequent to intramuscular injection of HRP: the dorsal intermediate subnucleus was labelled with HRP reaction product following injection of m. auricularis anterior; the middle intermediate subnucleus was labelled following injection of the muscle underlying the malar vibrissae; the ventral intermediate subnucleus was labelled following injection of the m. mentalis; the medial subnucleus was labelled following injection of the m. auricularis posterior; the lateral subnucleus was labelled following injection of the m. nasolabialis with HRP. In general there is a mediolateral representation in the facial nucleus of neurons innervating facial muscle groups which are found in anteroposterior succession along the head of the animal. Muscle groups found in dorsoventral succession on the animal are represented dorsoventrally in the facial nucleus.     

Localization of retractor bulbi motoneurons in the rabbit

Motoneurons innervating the rabbit retractor bulbi muscle have been identified by retrograde transport of horseradish peroxidase (HRP). Following injection of HRP into single slips or all 4 slips of the retractor bulbi muscle, labeled motoneurons were consistently observed in the abducens (ABD) nucleus and in the accessory abducens (ACC) nucleus located ventral, lateral and rostral to the ABD. Axons from the ACC motoneurons could be seen to enter the VIth nerve. Injection of HRP into the lateral rectus muscle produced consistent labeling of motoneurons in the ABD nucleus overlapping the distribution of retractor bulbi motoneurons, but labeling was never observed in the ACC nucleus. The number of labeled ABD neurons after lateral rectus injections was far less (36%) than after injection into all 4 slips of the retractor bulbi muscle (72%). Injection of HRP into the superior oblique, superior rectus or medial rectus muscle produced labeling of motoneurons in the corresponding subdivisions of the oculomotor nucleus or trochlear nucleus but no labeled motoneurons were observed in either the ABD or ACC nuclei. Some highly inconsistent labeling of oculomotor nucleus was observed after retractor bulbi or lateral rectus muscle injections and this was judged to be due to intraorbital diffusion of the HRP. It was concluded that the retractor bulbi muscle is innervated by motoneurons located in both the ABD and ACC nuclei.     

Dopamine receptor antagonists increase markedly the quantity of retrograde transport of HRP in the rat masseteric motoneuron.

Horseradish peroxidase (HRP) was injected, bilaterally, into the rat masseter muscle, subsequent to an intramuscular or intraperitoneal injection of one of five dopamine antagonists (chlorpromazine and haloperidol as the D1 and D2 receptor antagonist, SCH 23390 as the specific D1 receptor antagonist, sulpiride and domperidone as the specific D2 receptor antagonist). Control rats received an injection of a corresponding vehicle solution. After a survival period of 16 h, the brainstem was cut into 60 microns cryosections and processed with the TMB technique. The amount of retrogradely transported HRP was quantitatively measured in terms of the amount of HRP reaction product present in the motoneuron by the method which we have developed using an image processing system combined with a light microscope and a TV camera. Chlorpromazine, haloperidol, SCH 23390 and sulpiride significantly raised the quantity of retrograde transport of HRP. On the contrary, domperidone which can not penetrate the blood-brain barrier showed no significant change in the amount of the retrograde transport. In addition, an intravenous injection of chlorpromazine (8 mg/kg) was found to increase the amplitude of monosynaptic masseteric reflex EMG activity evoked by stimulations of the mesencephalic trigeminal nucleus. These results suggest that a possible regulatory system involving the dopamine receptor in the uptake and retrograde transport of HRP from axon terminals to cell bodies of the masseteric motoneuron exists in higher order neurons which make synaptic contact with the motoneuron.     

Transsynaptic retrograde transport of fragment C of tetanus toxin demonstrated by immunohistochemical localization

Injection of the non-toxic fragment C of tetanus toxin into the superior oblique muscle of the eye results in strong direct retrograde labelling of the motoneurons in the contralateral trochlear nucleus and clear transsynaptic labelling of neurons in the ipsilateral and contralateral vestibular nuclei. Standard immunohistochemical procedures using a monoclonal antibody localize fragment C in the brain with high sensitivity and excellent resolution. An injection of fragment C into the superior oblique muscle labels the same pool of trochlear motoneurons as an HRP injection into the superior oblique muscle. A comparison of the fragment C labelling of trochlear motoneurons with intracellular injections of HRP into trochlear motoneurons suggests that fragment C stains not just the soma, but also the distal dendrites of motoneurons. Moreover, a fragment C injection into the superior oblique muscle labels transsynaptically more neurons in each vestibular nucleus than an injection of HRP into the trochlear nucleus labels directly.     

Characterization of the pigeon isthmo-tectal pathway by selective uptake and retrograde movement of radioactive compounds and by Golgi-like horseradish peroxidase labeling.

Horseradish peroxidase (HRP) injected into developing limb buds of Xenopus laevis tadpoles is carried by retrograde axonal transport to the somata of motoneurones in the ventral horn. Small injection of 10% HRP were found to remain well localised to specified sites in the limb bud. Two types of labelled cells were found: diffusely labelled and granular labelled. Diffusely labelled cells result from axonal damage in the presence of HRP. Granular labelled cells result only from uptake of HRP from the region of the axon endings. No gradular uptake was found from axon shafts. It is concluded that the distribution of granular labelled cells accurately reflects the region of the ventral horn projecting to the site of injection in the limb.     

Central location of the motoneurons supplying the thyrohyoid and the geniohyoid muscles as demonstrated by horseradish peroxidase method

After HRP injection into the geniohyoid muscle labeled neurons were found in the ventral part of the hypoglossal nucleus, extending in the rostral two-thirds (about) of this nucleus. After HRP injection into the thyrohyoid muscle, the labeled neurons were identified in the lateral part of the caudal one-third (about) of the hypoglossal nucleus and in the dorsomedial part of the ventral horn of C1.     

Retrograde labelling of retinogeniculate neurons in the cat by HRP uptake from the diffuse injection zone

The effective uptake region for retrograde HRP labelling was investigated in the retinogeniculate system in the cat. Contrary to previous reports, labelling of ganglion cells was found not only in the retinal area projecting to the dense central zone of the injection site, but also in areas projecting to extensive regions of its surrounding diffuse zone in the lateral geniculate nucleus. It is concluded that the widely accepted view that retrograde HRP labelling occurs only by uptake of HRP from the dense zone of the injection site is erroneous.     

A new approach to intramuscular placement of horseradish peroxidase

A new method of intramuscular placement of a semisolid paste of horseradish peroxidase (HRP) using a root canal file is described. This method has been demonstrated to produce consistent and reproducible numbers of labeled motoneurons in the mouse extensor digitorim longus muscle (EDL). While the number of labeled cells is similar to that found with optimal intramuscular injection of liquid HRP, this new technique provides more reproducible results by eliminating the reflux often associated with intramuscular injection of liquid HRP.     

Location of the cutaneous trunci motor nucleus in the dog

Using the methods of muscle injection of horseradish peroxidase (HRP) and application of HRP to the proximal stump of the cut lateral thoracic nerve (LTN), the motor nucleus of the cutaneous trunci muscle (CTM) has been located predominantly in C8 and T1 spinal cord segments in the ventral and ventrolateral nuclei of Rexed's Lamina IX. Cells of the nucleus are somatotopically arranged rostro-caudally to correspond to the rostro-caudal segmentation. There was a higher average number of HRP-filled cells per section and greater rostro-caudal extent of the nucleus for the cut nerve technique than for the muscle injection technique. Evidence is presented that the CTM is an appendicular muscle in spite of its obvious axial position on the trunk.     

Uptake of horseradish peroxidase in denervated skeletal muscle occurs primarily at the endplate region

The spatial distribution of horseradish peroxidase (HRP) uptake has been studied by light- and electron microscopy in the denervated hemidiaphragm of the mouse. Segments with high HRP uptake were observed in a band centrally located in the denervated muscle. This distribution is similar to the well-known innervation pattern of the diaphragm. Ultrastructural studies demonstrated a high incidence of postsynaptic folds in close proximity of fibre areas with high intracellular content of HRP. 8-12 days after denervation a large number of fibres showed segments of high HRP uptake. 2-4 days after denervation very few such segments were observed. Biochemical studies also demonstrated an increase in HRP uptake after denervation occurring primarily in the endplate region. The activities of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase, acid phosphatase and cathepsin D all increased after denervation, most prominently in the endplate region. It is suggested that the observed segmental uptake of HRP and lysosomal activation reflects a process for rapid membrane turnover in denervated muscle.     

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

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

The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study

Retrograde transport of horseradish peroxidase (HRP) from the region of retinal genglion cell axon terminals back to the cell bodies has been studied by light and electron microscopy. After injection of HRP into the chick optic tectum, it was taken up by axon terminals and unmyelinated axons as well as by other processes and cell bodies of the outer tectal layers. Subsequently the HRP was obseved in vesicles, multivesicular bodies, cup-shaped organelles and small tubules within axons in the stratum opticum, optic tract, optic nerve and optic fiber layer of the retina with accumulation in the retinal ganglion cell bodies. Pinocytosis of extracellular HRP along the axon shaft was rare. After a short postinjection interval, HRP was found in organelles within the axons of the optic nerve but not in the extracellular spaces. After larger injections or longer postinjection intervals, extracellular HRP diffused from the injection site to the back of the eye, but none was found in the extracellular spaces of the retina; ganglion cells were the only cells of the retina which contained HRP product. HRP disappeared from the cell bodies 3–4 days after transport. These findings suport the concept of intraaxonal retrograde movement of HRP. Within axons the vesicles carrying HRP frequently were partially or completely surrounded by a regualr array of microtubules. Doses of colchicine greater than 5–10 µ/eye administered 4 days before tectal injection of HRP interfered with the uptake and/or transport of HRP. HRP also moved in an anterograde direction in membrane-bound vesicles within the ganglion cell axons, although apparently more slowly and in smaller quantities than in the retrograde direction. The localization of HRP in neurons of the isthmo-optic nucleus following intravitreal injections has also been studied. The marker enzyme was found in neuronal cell bodies 4 hours after injection, indicating a rate of retrograde transport of at least 84 mm/day in these neurons.     

TRH regulation of tracheal tension through vagal preganglionic motoneurons

TRH-immunoreactive nerve terminals innervate the ambiguous nucleus in the rabbit. Vagal preganglionic motoneurons that innervate the trachea, were revealed by HRP histochemistry in the rostral part of the ambiguous nucleus and the dorsal motor nucleus of the vagus. HRP histochemistry plus TRH immunocytochemistry revealed that TRH-immunoreactive axon terminals synapsed on ambiguous nucleus neurons retrogradely labeled by HRP injection into tracheal smooth muscle and the superior laryngeal nerve. Microinjection of 50 ng TRH into the rostral ambiguous nucleus caused slight dilation followed by constriction, which was inhibited by atropine and vagotomy. Results show that central TRH-containing neurons regulate tracheal tension through synapses on vagal preganglionic motoneurons that innervate tracheal smooth muscle.     

Absence of postnatal death among motoneurones supplying the inferior gluteal nerve of the rat

Motoneurones innervating the caudal part of the gluteus maximus muscle of 0-2 day, 10-12 day and 2-3-month-old rats were labelled by a half-hour application of a solution of 30% horseradish peroxidase (HRP) and 2% lysophatidylcholine delivered by suction electrode to the cut inferior gluteal nerves. The numbers of motoneurones labelled 24-48 h later were not significantly different in the 3 age groups (mean = 58.75, 54.0, 57.5, respectively). When a simple 30% HRP solution was used in adult rats, the number of motoneurones labelled was significantly less (mean = 48.75). In contrast, application of 0.5 microliter of HRP in a pledget of gelfoam to either the cut or uncut inferior gluteal nerve of neonates labelled large numbers of motoneurones, presumably by diffusion into nearby muscles. It is concluded that no death of motoneurones innervating the gluteus maximus muscle occurs postnatally, and that spread of HRP to neighbouring muscles can give rise to spuriously high motoneurone counts in neonates, and that incomplete uptake or transport of HRP in adults can lead to incorrectly low counts.     

Amygdaloid and pontine projections to the ventromedial nucleus of the hypothalamus

Amygdaloid and pontine projections to the feline ventromedial nucleus of the hypothalamus (HVM) were studied with retrograde transport of horseradish peroxidase (HRP) and anterograde transport of tritiated amino acids. Following injections of HRP into HVM, amygdaloid neurons were labeled in the ipsilateral cortical and medial nuclei and the ventral portion of the parvocellular part of the basal nucleus. In experiments in which HRP was injected into the tuberal hypothalamus following stria terminalis lesions, it was determined that amygdaloid neurons projecting to HVM by way of the stria terminalis were located in the cortical and medial nuclei while those projecting through another route, presumably the ventral amygdalofugal pathway, were found in the rostral part of the medial nucleus and the parvocellular basal nucleus. Following HRP injection into lateral hypothalamus at the level of HVM, labeled neurons were seen in the magnocellular basal nucleus. After preoptic injections, neurons containing the HRP reaction product were in cortical and medial nuclei and magnocellular and parvocellular parts of the basal nucleus. In addition to cells in the amygdala, rostral pontine neurons were labeled after HRP injections into HVM. The cells were located ipsilateral to the injection, mostly in the dorsal nucleus of the lateral lemniscus, lateral and dorsolateral to the brachium conjunctivum. The pontine cells labeled following HVM injections of HRP were different from those labeled following lateral hypothalamic and preoptic region injections. The pontine projection to HVM was confirmed using axoplasmic transport autoradiography. A mixture of tritiated leucine and tritiated proline was injected into the lateral pontine region labeled after HRP injections into HVM. Labeled axons ascending in the medial forebrain bundle terminated throughout the rostro-caudal extent of HVM.     

The anatomical evidence of recurrent axonal collaterals of the thalamus projecting neurons of the rostral pole of the trigeminal sensory nuclear complex in the rat.

Thalamus projecting neurons and their recurrent axonal collaterals were observed in the dorsomedial part of the trigeminal principal sensory nucleus (Vpdm) and the caudolateral part of supratrigeminal nucleus (Vsup CL) after injection of horseradish peroxidase (HRP) into the contralateral ventrobasal complex of the thalamus (VBm) by using the HRP retrogradely tracing-Golgi-like staining method. About 7% (8/120) parent axons of the labeled cells gave rise to recurrent axon collaterals. However, no retrogradely labeled cells were observed in the VBm after injection of HRP into the Vpdm and Vsup CL. In an electron microscopic study, the terminals of recurrent axon collaterals made synapses with the dendrites of the thalamus projecting neurons or non-labeled neurons in the neuropil of the Vpdm and Vsup CL. It is suggested that the recurrent axon collaterals might play a role of negative feedback in transmission of the proprioceptive message from the jaw-closing muscle spindles to the thalamus.     

Muscle sensory neurons in the spinal ganglia in the rat determined by the retrograde transport of horseradish peroxidase

The method of retrograde transport of horseradish peroxidase (HRP) was used to identify muscle sensory neurons in the spinal ganglia in the rat. Experiments were conducted on 25 albino rats. Injections of 0.06 to 0.08 ml 2 to 20% Sigma type VIHRP were made unilaterally into anterior tibial muscle. Cells of origin of muscle receptors and motor endings in the same area where HRP was administered were demonstrated. The labeled cells, medium to large, were found in fourth and fifth lumbar ganglia ipsilateral to the site of injection. Simultaneously, labeled neurons were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia.     

Studies of age-related changes in intracerebral small vessels of rat--do all cerebral blood vessels get aging concurrently?

Age-related changes of intracerebral small blood vessels were studied with light and electron microscopes. In the first step of this investigation (Experiment 1), Wistar rats of 4-month-old were employed to determine the appropriate concentration of administrated HRP (horseradish peroxidase) for surveying the uptake capacity of HRP. Each rat was injected HRP intravenously under light anesthesia with ether. After 30 minutes, rat brains were excised and prepared for stretch specimen (Mato et al, 1979). Light microscopically, corresponding with decrease of the concentration of injected HRP, the reaction products of HRP in the cytoplasm of fluorescent granular perithelial (FGP) cells reduced linearly and the injection of 5 mg HRP revealed only FGP cells in the parietotemporal region of cerebral cortex. Referring to the results mentioned above, 10 mg of HRP was decided to be applicable dose for the following study. In the next experiment (Experiment 2), Wistar rats of 4-month-old, 1.7-year-old and 2.4-year-old were used for the study on the relation between morphological alteration of vascular cells and changes of uptake capacity of FGP cells in aging. At 30 minutes after the injection of 10 mg, rats were perfused and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde under light anesthesia. Then, rat brains were removed and divided coronally into three parts. After slicing with Vibratome, number and distribution of FGP cells were studied under light microscope. The other specimens were dehydrated and embedded in Epon 812. The ultrastructure of vascular cells (endothelial and smooth muscle cells) and FGP cells at each age was examined with JEM 2000 EX electron microscope. Supplementary, ultrastructure of middle cerebral and retinal arteries of 2.4-year-old rats was also studied for comparison with that of the intracerebral (cortical)small vessels. The findings obtained from Experiment 2 could be summarized as follows; 1. Light microscopically, in 4-month-old and 1.7-year-old rats, FGP cells including positive granules of HRP were often recognizable along small blood vessels of cerebrum, especially in cerebral cortices. In 2.4-year-old rats, the numbers of FGP cells with positive granules of HRP decreased significantly (p less than 0.01). It was confirmed that the uptake capacity of FGP cells reduced with aging. But, exceptionally, regardless from aging of animals, FGP cells belonging to a few special vessels in the parietotemporal area of cerebral cortices included many and intense reaction products. 2. Electronmicroscopically, the vascular cells changed in appearance and contents with aging.(ABSTRACT TRUNCATED AT 400 WORDS)