Horseradish Peroxidase Reaction Product Research Articles

The ultrastructural morphology of the subthalamic-nigral axon terminals intracellularly labeled with horseradish peroxidase

The labeled axons of neurons intracellularly injected with horseradish peroxidase (HRP) in the rat subthalamic nucleus (STH) were studied with electron microscopy. The main axons and the afferent daughter branches were all myelinated. The morphology of the intrinsic axon terminals within STH was obscured by the dard HRP reaction products, but the labeled efferent STH terminals in the substantia nigra (SN) were revealed to contain small oval vesicles and formed asymmetrical synapses with dendrites of SN neurons.

Ultrastructural correlates of electrical-chemical synaptic transmission in goldfish cranial motor nuclei.

The output connections of the cranial relay neurons, part of the Mauthner cell network, were examined in goldfish with light and electron microscopic techniques. Either lucifer yellow or horseradish peroxidase (HRP) was injected into cranial relay neuron axons to demonstrate that they diverge to several motor nuclei and to many motoneurons within one nucleus. Retrograde transport of the enzyme from injections of mandibular muscles was used to label the trigeminal motoneurons. In the electron microscope, cranial relay neuron processes were distinguished by the granular appearance of the electron-opaque polymer formed enzymatically by HRP, while the retrogradely labeled motoneurons had the polymer enclosed in lysosomes. The cranial relay neuron terminals contained many presynaptic vesicles which concentrated the HRP reaction product. Active zones and synaptic clefts were evident. At some synapses, both gap junctions and presynaptic vesicles were found. The mechanism of synaptic transmission was investigated by simultaneous recording with two intracellular microelectrodes from cranial relay neuron-motoneuron pairs. Composite postsynaptic potentials in a trigeminal motoneuron were evoked by intracellular stimulation of a cranial relay neuron axon. The earliest excitatory postsynaptic potential (EPSP) component had a latency of 0.25 msec and had a peak amplitude that was not depressed by repetitive stimulation. A second component had larger peak amplitudes which were reduced easily by repetitive stimulation. Antidromic action potentials were not transmitted from motoneurons to the cranial relay neuron axons. Thus, both electrical and chemical transmission probably occur at the cranial relay neuron-motoneuron synapses. Since the cranial relay neurons fire synchronously and receive excitatory chemical synapses, the function of the gap junctions and electrical transmission is unclear. Perhaps the importance of these gap junctions is more for transport of small molecules than for impulse transmission.

Increased cerebrovascular permeability to protein during systemic kainic acid seizures.

Cerebrovascular permeability to protein (CVP-p) was assessed during limbic seizures by injecting unrestrained rats intraperitoneally with kainic acid followed by intravenous horseradish peroxidase (HRP); animals survived approximately 1 h after seizure onset. Brains were processed for the blue HRP reaction product followed by light microscopic examination of sequential sagittal sections. In all cases kainate-induced seizures caused increased CVP-p within the thalamus, temporal hippocampal formation, and neocortex. Somewhat less frequently other limbic structures and the striatum were HRP-positive. A lamina-specific extravasation occurred in the dorsal hippocampus; reaction product occupied the mossy fiber zone of field CA3, a likely focus of kainate action. Extravasation of HRP also occurred within, or juxtaposed to, certain myelinated fiber bundles. Brains from animals treated as blanks (kainate but no HRP) were devoid of peroxidase activity, and in nonseizing animals HRP gained access only to circumventricular organs. Although regions of increased CVP-p partially covary with areas of increased electrical activity and glucose metabolism, neuronal activation occurs over a much greater volume of brain tissue than does CVP-p. A close relationship may exist between these circumscribed areas of protein extravasation and seizure foci. Both vasogenic and cytotoxic edema appear to be simultaneously present during kainate seizures.

Fixation of horseradish peroxidase reaction products with ammonium molybdate

Reaction products of the tetramethylbenzidine method for horseradish peroxidase demonstration were preserved, unchanged, for a long time after fixation with ammonium molybdate.

Effect of ethanol and chlorpromazine on transhepatic transport and biliary secretion of horseradish peroxidase.

In order to demonstrate the effect to the acute administration of ethanol and chlorpromazine (CPZ) on bile flow and transhepatic transport of horseradish peroxidase (HRP) into bile, male rats were administered either 5 gm per kg ethanol intragastrically (E-rats) or 3 mg per kg CPZ intraperitoneally (CPZ rats). Control rats (C-rats) received saline. Two hours after ethanol feeding or 90 min after CPZ injection HRP was injected into the portal vein, and bile samples were collected at 10-min intervals for 2 hr. Tissue samples were removed at 1, 10, 60, and 120 min to study HRP transport using electron microscopic cytochemical localization. Bile flow was reduced (p less than 0.001) both in E- and CPZ-rats compared to C-rats. In E-rats HRP secretion was significantly decreased at 30 and 40 min post-HRP injection (p less than 0.05) and the peak rate of HRP secretion was delayed by 10 min compared to C-rats. Uptake and transhepatic transport of HRP were similar to controls. These results suggest that bile secretion and flow were impaired by ethanol. CPZ inhibited secretion of HRP significantly (p less than 0.001) during the first hr after HRP injection and by 25% after 2 hr. In CPZ rats studied cytochemically HRP reaction product decreased in the cytoplasm of hepatocytes 10 min after HRP injection (p less than 0.01). These findings suggest that acute CPZ administration caused an inhibition of the uptake of HRP as well as secretion and bile flow.

Distribution of HRP in the inner ear after injection into the middle ear cavity.

The distribution patterns of horseradish peroxidase (HRP) reaction products in the inner ears of guinea pigs were studied after injections into the middle ear cavities and perilymphatic and subarachnoid spaces. The normal round window membrane resisted HRP penetration from the middle ear side, but when it became pathological after repeated applications, its permeability increased. HRP deposits were found in the cochlear and vestibular sensory cells and in the lumen of the endolymphatic sac. HRP reaction products were minimal at the cochlear apex even after long survival times, suggesting that perilymph flow, if it exists, is rather weak toward this direction. Whereas the stria vascularis is impermeable to HRP, the vestibular dark cells were accessible; thus, the metabolic activity of the dark cells can be more readily controlled by drug applications through the middle ear cavity. The finding of HRP deposits on the scala vestibuli surface of Reissner's membrane and the absence of HRP in the upper portion of the spiral ligament at the basal turn suggests that the oval window is a secondary route of passage for these particles from the middle ear cavity to the inner ear. In order to determine the route of HRP into the endolymphatic sac from the middle ear cavity or scala tympani, the cochlear and/or vestibular aqueducts were obliterated singly or together. The route of HRP was determined to be the vestibular aqueduct. HRP is believed to enter the sac lumen through Reissner's and saccular membranes and the sac epithelium. Drugs and other large molecular substances instilled in or gaining access to the middle ear cavity may reach the endolymphatic sac causing its functional alteration.

血小板凝集起因脳血管傷害発生機序の電顕的解析

When platelets were aggregated with an injection of ADP into cerebral artery, cerebrovascular injuries were observed. For analysis of the process of this vascular injury, electron microscopic observation was performed using horseradish peroxidase (HRP) as a marker of vascular permeability change. Two different types of changes; increased vesicular transport and vacuole formation were recognized. Vesicules (0.05-0.2μm) contained HRP reaction product but vacuoles (1.2-2.0μm) did not. In middle cerebral artery, vacuole formation was mainly revealed. Extravasation of HRP reaction products was not seen. In capillaries, arterioles or venules, increased vesicular formation was rare. Extravasation and perivascular edematous changes were remarkable. Namely endothelial cell damage was more prominent in large artery but extravasation and perivascular edematous changes were mainly seen in smaller vessels. And extravasation was more frequently seen in cerebral cortex and hippocampus comparing with basal ganglia. Following the ADP injection, blood levels of TXB2 and 6-keto PGF1α revealed marked temporarily increase at 3min after the injection. The results suggest that these vasoactive substances from platelets may play an important role in producing these vascular injuries.These phenomenon seems to be important to understand the mechanism of vascular damage in various cerebrovascular lesions.

Cholinergic and non-cholinergic septo-hippocampal projections: a double-label horseradish peroxidase-acetylcholinesterase study in the rabbit

The existence of a massive cholinergic projection from cells in the medical septal nucleus (MS) and nucleus of the diagonal band (DB) to the hippocampal formation has been recognized for some time. However, the actual percentages of cholinergic and non-cholinergic neurons in the MS and DB which project to the hippocampus have not been reported. A procedure which combines horseradish peroxidase (HRP) and acetylcholinesterase (AChE) histochemistry in the same tissue was used to determine these percentages in the rabbit. Less than 50% of the neurons in the MS and DB which were labeled with reaction product following an HRP injection into the dorsal hippocampus also stained for AChE. Moreover, 70% of all neurons containing HRP reaction product were located in the DB, but neurons in the DB could not be differentiated from those in the MS on the basis of size or morphology. These data are taken to indicate that much of the MS-DB hippocampal projection is not cholinergic. Substance P is suggested as another possible transmitter within this anatomical system.

Double labelling of retinofugal projections in the cat: a study using anterograde transport of 3H-proline and horseradish peroxidase.

A new method is described for combining 3H-proline and horseradish peroxidase (HRP) as anterograde neuronal tracers. By this method the presence of both substances can be demonstrated in the same histological section. We developed this method to investigate the retinofugal projections from the two eyes in the cat. One eye was injected with 3H-proline the other with HRP. Cryostat sections of the brain were mounted on emulsion coated slides in the dark. Sections were first exposed to the emulsion for 2-3 weeks at -40 degrees C and developed for autoradiography. Only then they were reacted for HRP-activity with tetramethylbenzidine (TMB). Keeping to this sequence autoradiographic procedures could not abolish the HRP-reaction product and silver grains and TMB can be visualized on the same slide. The wellknown projection pattern in the lateral geniculate nuclei was confirmed as a control for the new method. In the superior colliculus and in the pretectum a clear overlap of retinal terminals from the two eyes could be demonstrated for the first time.

The development and postnatal organization of primary afferent projections to the rat thoracic spinal cord.

Primary afferent projections to the thoracic spinal cord in fetal and postnatal rats were labelled by applying horseradish peroxidase (HRP) to the central stumps of cut peripheral nerves. Diaminobenzidine (DAB) and tetramethyl benzidine (TMB) histochemical processing procedures were used to reveal the HRP reaction product. In postnatal rats, individual muscle nerves were labelled to reveal the organization of muscle afferent projections to the motor nuclei. The terminals of muscle afferents were distributed widely across the dendritic arbors of motoneurons supplying the same muscles. No spatial segregation of the terminations of different populations of muscle afferents was discernable. Afferents supplying different regions of the skin were labelled by applying HRP to the dorsal and ventral primary rami of the spinal nerves. Afferents in the dorsal rami projected to lateral portions of both the ipsilateral and contralateral dorsal horns while afferents in the ventral rami projected to the medial portions of both dorsal horns. The projections of the dorsal rami were shifted caudally relative to those of the ventral rami. This relationship reflects the fact that the regions of skin innervated by the dorsal rami are displaced caudally relative to those innervated by the corresponding ventral rami. In fetuses, dorsal rami were labelled alone or in combination with ventral rami. These experiments disclosed the time course of development of the projections to different laminae of the spinal gray matter and revealed that afferents in the two primary rami project to appropriate regions in the ipsilateral and contralateral dorsal horns from the very outset.

Retinal projections to the inferior and medial pulvinar nuclei in the old-world monkey

Horseradish peroxidase (HRP) and wheat germ agglutinin conjugated to HRP (WGA-HRP) were used as anterograde tracers to study the macaque monkey retinofugal pathways. Labeled axons were mapped beyond the lateral geniculate nucleus to the inferior (PI) and the medial (PM) pulvinar nuclei, where they terminated. Retinofugal fibers project to the medial part of PI, adjacent to the brachium of the superior colliculus. A smaller projection to PM leaves the ventrolateral aspect of the lateral geniculate nucleus and travels parallel to the stria terminalis to reach the dorsal surface of the thalamus and PM. HRP reaction product was bilateral in PI and predominantly contalateral in PM.

Reinnervation of the gastrocnemius muscle by the contralateral S1 nerve root

Functional regeneration after transposition of a ventral nerve root was established in the adult cat. Reconstruction of the ventral root, using microsurgical methods, directed the right S1 ventral nerve root to innervate the left gastrocnemius muscle. Stimulus-induced unit responses were recorded from the left gastrocnemius muscle 5 to 8 months after the root cross, demonstrating the reestablishment of neuromuscular connections. The innervation of the left gastrocnemius muscle by neurons in the right vantral horn of the spinal cord was verified by injecting horseradish peroxidase into the muscle. Horseradish peroxidase reaction product was located in alpha and gamma motor neurons in the right S1 segment of the spinal cord. Computer-assisted determination of the soma area of the labeled neurons was compared with a normal S1 innervation of the gastrocnemius muscle. Analysis of the percentage of cells of a given soma area demonstrated an overall decrease in soma area in the operated animals. Because ventral root reconstruction can result in innervation of a foreign muscle, studies such as this may encourage repair or reconstruction of nerve roots to gain some functional recovery after spinal cord or nerve root injuries.

Parasympathetic preganglionic neurons and visceral primary afferents in monkey sacral spinal cord revealed following application of horseradish peroxidase to pelvic nerve.

AbstractHorseradish peroxidase (HRP) applied to the central stump of the transected pelvic nerve of rhesus monkeys labeled afferent and efferent neurons innervating the pelvic viscera. Preganglionic neurons forming the sacral parasympathetic nucleus (SPN) were located in a narrow band 150–200 μm wide on the border of the ipsilateral intermediate gray matter. The SPN contained an average of 820 neurons and characteristically extended over two or three sacral segments (average length: 14.5 mm) with the majority of cells in S2. It was composed of medium sized, elongated somata (15.7 × 30.7 μm) with their major axes in the transverse plane and oriented parallel to the lateral border of the gray matter. Dendrites extended deep into the lateral funiculus, horizontally into the medial dorsal gray commissure, and dorso‐laterally along the lateral edge of the dorsal horn. Preganglionic axons followed the lateral border of the ventral horn to the ventral rootlets.Pelvic nerve afferent axons carried HRP to neurons in the dorsal root ganglia (DRG) and by transganglionic transport to the spinal cord. The average number of labeled DRG cells was 2, 992 (range: 2, 193–4, 363) with the largest fraction (80%) in S2. The numbers of DRG cells correlated with the numbers of SPN cells in corresponding spinal cord segments. Within the spinal cord labeled primary afferent fibers were prominent in the sacral segments and were also observed in decreasing numbers within two to three segments beyond the rostral and caudal limits of the SPN. In transverse sections, primary afferents were found throughout Lissauer's tract (LT) and in the lateral half of the dorsal columns. A prominent collateral fiber bundle from LT was located in lamina I along the lateral edge of the dorsal horn. This bundle extended into the SPN and a few collaterals continued medially into the dorsal gray commissure and the contralateral side of the cord. A much less prominent collateral fiber group from LT passed medially over the apex of the dorsal horn in lamina 1 and descended into the dorsal gray commissure. Horizontal sections revealed that these two collateral pathways were organized into periodically spaced bundles of fibers about 250 μm apart. HRP reaction product in terminal fields related to these afferent collateral projections were found in the lateral marginal zone and bilaterally in the dorsal gray commissure.

Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death

Horseradish peroxidase (HRP) was injected into the retino-recipient nuclei of each hemisphere in newborn rats. The animals were perfused 3–9 days after the injections; the number of retinal ganglion cells in retinal wholemounts was estimated by counting cells containing granules of HRP reaction product. The mean number (150,500) of labelled cells in 3-day-old rats was significantly higher than those in older animals (117,000, 121,000, 113,000 respectively on 6th, 8th and 9th postnatal days). However, in animals of any of the ages studied, the estimated numbers of ganglion cells were virtually the same as those in the animals of the same age but injected with HRP only 15–20 h before the perfusion. Thus, the reduction in the number of retinal cells projecting to the central visual nuclei observed during the first few postnatal days is due to a wave of retinal ganglion cell death; ganglion cell death induced by the neonatal removal of the contralateral superior colliculus has a similar time course.

Behavioral and central visual correlates of inherited retinal degeneration in the domestic chicken ( Gallus domesticus)

A developmental, posthatch loss of cutaneous and ocular melanocytes in the mutant DAM (delayed amelanotic) chicken is accompanied by severe retinal degeneration. Using a food-location task, increased ocular pigment loss was associated with increased latency of response, both developmentally in young DAMS, and among adults with differing degrees of amelanosis. Analysis of optic nerve fiber composition in normals and DAMs showed a reduction in the small diameter optic axon population in severely amelanotic DAMs. With increasing severity of ocular amelanosis among animals, reduction in the density of anterogradely transported horseradish peroxidase (HRP) reaction product was seen first in retinorecipient thalamic nuclei, subsequently in the optic tectum, and ultimately in the accessory optic nucleus. An inverse relationship was also observed between the density of HRP reaction product occurring in retinal terminal fields and density of HRP label seen in the optic tracts of the same animal. No changes in either the volume or extent of central visual nuclei were apparent in partially sighted and blind DAMs.

Cholecystokinin-like immunoreactive neurons in rat cerebral cortex

The distribution and form of cholecystokinin immunoreactive neurons in neocortical areas within the posterior pole of the rat cerebral hemisphere was examined using the immunoperoxidase technique. Although cholecystokinin-positive neurons are present throughout the cortex, they are most frequent in the supragranular layers. These neurons are of three kinds: layer I neurons, bipolar cells, and other non-pyramidal cells with either multipolar or bitufted dendritic trees. In electron-microscopic preparations, the horseradish peroxidase reaction product is found to form a granular deposit which occurs throughout the cytoplasm and nucleoplasm and shows no predilection for any particular type of organelle. Electron-microscopy also shows cholecystokinin-positive neurons to have both symmetric and asymmetric synapses on their perikarya, which is additional evidence in favor of the interpretation that they are non-pyramidal cells. In the light-microscopic preparations three types of CCK-positive axons are encountered. These are vertically-oriented axons considered to arise from bipolar cells, a plexus in the superficial portion of layer II/III which is believed to arise from the multipolar and bitufted cells, and a deep plexus of unknown origin in layers VI and V. Since the axons of bipolar cells form asymmetric synapses they are thought to be excitatory neurons. In contrast, the bitufted and multipolar neurons are probably inhibitory, for previous studies have shown neurons with similar features to have axons which form symmetric synapses and to contain glutamic acid decarboxylase. Thus, although iontophoretically-applied cholecystokinin excites cortical neurons, it appears to be present in some neurons which are excitatory and others which are inhibitory.

Hypothalamic projection to the lateral septum in the guinea pig an HRP study

In the guinea-pig, a bilateral projection from the magnocellular dorsal nucleus (MDN) of the hypothalamus and the lateral hypothalamus (LH) to the lateral septum nucleus (LSN) was demonstrated by using horseradish peroxidase (HRP) histochemistry. The deposition of crystalline HRP into the LSN consistently resulted in labeling of cell bodies within the limits of the MDN. Labeled perikarya exhibited a typical granular appearance. The better labeling was obtained with HRP implants located in the posterior LSN. As it was previously described that most of the MDN cells were enkephalincontaining and that the posterior LSN contained enkephalinergic nerve terminals, it is suggested that the MDN may contribute to the enkephalinergic innervation of the LSN. In addition a group of labeled neurons was found in the LH. They were located ventrally and laterally in relation to the fornix column. HRP reaction product filled these neurons in a Golgi-like manner, providing information about their morphology.

The periaqueductal gray-raphe magnus projection contains somatostatin, neurotensin and serotonin but not cholecystokinin

The retrograde transport-HRP-immunocytochemical technique was employed to ascertain if the periaqueductal gray-raphe magnus projection arises from neurons containing somatostatin, neurotensi, serotonin or cholecystokinin. Following HRP injections into the raphe magnus (NRM) double-labeled cells containing HRP reaction product and somatostatin-, neurotensin- or serotonin-like immunoreactivity were identified in the midbrain periaqueductal gray (PAG). No cholecystokinin-like immunoreactive double-labeled neurons were found in the PAG. These results indicate that the PAG-NRM pathway contains somatostatin, neurotensin and serotonin but not cholecystokinin.

Microvascular abnormalities in ethylnitrosourea (ENU)-induced rat brain tumors: structural basis for altered blood-brain barrier function.

The fine structure, histometric characteristics, and permeability of microvessels were studied by electron microscopy in normal and in ethylnitrosourea (ENU)-induced glioma tissue from rats, using horseradish peroxidase (HRP) as a tracer. The tumor vessels were classified into (1) capillary buds (Type I); (2) round small to large capillaries (Type II); (3) sinusoidal or venule-like microvessels (Type III), and (4) abnormal arteriole-like microvessels (Type IV). All endothelial cells, basement membranes and periendothelial cells in the tumor tissue demonstrated changes in structure. The most striking alterations occurred in the endothelial cells; there were abnormal endothelial tight junctions, altered pinocytotic activity, and thickening. In the tracer study, the reaction product of HRP was present around some sinusoidal or venule-like microvessels (Type III) and extended to the widened extracellular spaces around the microvessels. The endothelial cells of Type III microvessels showed decreased nuclear and mitochondrial fractions, and increased euchromatin content and a rough endoplasmic reticulum fraction. The pinocytotic vesicles with the HRP reaction product in the endothelial cells were not increased in number. Fenestrations and gaps of the endothelial cells were observed. These alterations of the endothelial cells of sinusoidal or venule-like microvessels (Type III) are considered to be the main cause of breakdown of the blood-brain barrier in this tumor.

Placental tissue concentration of pregnancy-associated proteins in early and term pregnancies

We examined the localization of five pregnancy-associated proteins such as pregnancy specific beta 1-glycoprotein (SP1), human chorionic gonadotropin (HCG), human placental lactogen (HPL), alpha 2-PA-glycoprotein (SP3) and pregnancy-associated plasma protein-A (PAPP-A) in the placentae of early and term pregnancies by means of the PAP method in immunohistochemical technology. It was found that the degree of staining of these proteins did not reflect their concentration in serum at each gestational age. Therefore, the concentrations of these proteins in the solution extracted from the placenta were measured. 1) Placental tissues were fixed with a 10% neutral formaldehyde solution and these were embedded into paraffin blocks. These specimens were used for the PAP method. The localizations of five pregnancy-associated proteins from 10 placentae at the gestational age of 7 to 10 weeks were compared with those at the gestation age of 38 to 41 weeks from 7 patients. The staining degree of SP1 in free villi was pale in both early and term pregnancies. HCG was stained deeply in early pregnancy but pale at term pregnancy. HPL was stained deeply at both gestational ages. Horseradish peroxidase reaction products from SP1, HCG and HPL were located chiefly in the syncytiotrophoblast. SP3 was not stained on the placental tissue. PAPP-A was stained to a greater extent in the cytotrophoblast in early pregnancy but pale in the syncytiotrophoblast at term pregnancy. 2) The concentrations of SP1, HCG, HPL, SP3 and PAPP-A were measured in the placental tissues in both early and term pregnancies. Placental tissues were obtained from 24 normal pregnancy patients aborted artificially at 8 to 11 weeks gestation and from 28 patients terminated by normal deliveries at 38 to 40 weeks gestation. The tissue was homogenated with 3 volumes of 1/2 PBS (0.005M phosphate-buffered 0.07M sodium chloride, pH 7.4). The supernatant was removed after centrifugation and stored at -20 degrees C until assayed. The concentration of SP1 was measured by single radial immunodiffusion. That of HCG was assayed by a directed Latex agglutination test (Gestate Slide Eiken). The concentrations of HPL, SP3 and PAPP-A were quantified by rocket immunoelectrophoresis. No significant difference in SP1 levels was shown between early and term pregnancies, and the SP1 level was 4.3 +/- 1.3 (mean +/- 1 S.D.) mg/dl at term pregnancy. The HCG level was 1,100 +/- 300 IU/ml in early pregnancy and at least 20-fold higher than that at term pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)