Horseradish Peroxidase Method Research Articles

Central distribution of afferent and efferent components of the chorda tympani in the cat as revealed by the horseradish peroxidase method

Central distribution of afferent and efferent components of the chorda tympani (CT) in the cat was examined by using the anterograde and retrograde tracing techniques of horseradish peroxidase (HRP). HRP was applied to the CT in the tympanic cavity. HRP-labeled CT fibers were traced to the brain stem along the ventral surface of the vestibular nerve. The afferent CT fibers were divided into ascending and descending components. The rostrally directed ascending fibers ended within and around the dorsomedial portions of the principal sensory trigeminal nucleus. The descending fibers entered the solitary tract to run caudally as far as the levels slightly rostral to the obex, giving terminals to the solitary nucleus. A cluster of HRP-labeled neurons were seen ipsilaterally in the lateral reticular formation medial to the spinal trigeminal nucleus; it was observed from the caudalmost levels of the exiting root of the facial nerve to the caudal levels of the facial nucleus. HRP-labeled axons arising from the HRP-labeled neurons firstly ran dorsomedially and then medially under the genu of the facial nerve to form a small genu at the region medial to the genu of the facial nerve. Subsequently the labeled axons ran laterally and ventrolaterally to join other CT fibers at the dorsomedial aspect of the spinal trigeminal tract.

A direct projection from the hypothalamus to the area postrema in the rat, as demonstrated by the HRP and autoradiographic methods

A direct hypothalamic afferent to the area postrema was found in the rat. After an injection of tritiated amino acids into the hypothalamus, labeled fibers and terminals were observed in the entire part of the area postrema, with dense patches of silver grains present in places. Cells of origin of this novel projection were identified with the retrograde horseradish peroxidase (HRP) method in the dorsal hypothalamic area at the most rostral level of the dorsomedial hypothalamic nucleus.

Extrageniculate projections to the visual cortex in the macaque monkey: an HRP study

Extrageniculate projections to the visual cortex were examined in the macaque monkeys by the horseradish peroxidase (HRP) method. Extrageniculate neurons sending fibers to the visual cortex were found in the lateral and inferior pulvinar nuclei, paracentral thalamic nucleus, claustrum, basal nucleus of Meynert, lateral part of the basal amygdaloid nucleus, lateral hypothalamus, locus coeruleus, and dorsomedial and midline regions of the pontine tegmentum.

The distribution of descending brain stem projections to spinal cord in the guinea pig. A horseradish peroxidase study

In the guinea pig troncoencephalic neurons projecting to the spinal cord were identified by the horseradish peroxidase (HRP) method. Following lumbar injection labelled neurons were found in the RF, VL, R, MNV, TMNV, RTP, NR, PVG, LH and DMH. The distribution of HRP-positive neurons following cervical injection was roughly the same as that observed after lumbar injection except for the presence of labelled cells in the NSNV and their absence in the MNV and TMNV.

Reciprocal connections between the lateral hypothalamus and the frontal cortex in the rat: Electrophysiological and anatomical observations

Inputs to rat lateral hypothalamus (LHA) from prefrontal cortex (FC), and vice versa, were studied by intracellular recording, and by retrograde horseradish peroxidase (HRP) method. Stimulation of the FC evoked 3 types of response: a polysynaptic EPSP-IPSP sequence, IPSPs alone, or antidromic response in LHA neurons. Forty-four per cent of IPSPs were considered to be monosynaptic since the latencies were constant when stimulus intensity was changed. The neurons labeled in the FC following electrophoretic injections of HRP into LHA were located in the medial and sulcal FC. In these cortical areas, not only pyramidal neurons in layer V, but also non-pyramidal neurons in layer VI were labeled. Stimulation of the LHA evoked an EPSP-IPSP sequence, or antidromic response in FC neurons. Some of the fast EPSPs were considered to be monosynaptic. The neurons labeled in the LHA following HRP injection into the FC were either relatively large spherical neurons or small ovoid-shaped neurons. These were distributed diffusely throughout the LHA.

Central projections of muscle afferents from the sternomastoid nerve in the rat

Perikarya and central endings of muscle afferents of the sternomastoid nerve of the rat were investigated using the horseradish peroxidase (HRP) method in its modification by Mesulam. Application of an aqueous solution of HRP to the cut sternomastoid nerve was followed by heavy labeling of cell bodies in ipsilateral spinal ganglia C3 and C4. In addition, a number of peripheral and central processes of spinal ganglion cells clearly showed reaction product. Labeled structures in the spinal cord and medulla oblongata were regarded as axons and/or terminals of sternomastoid nerve primary afferents because the motor root to this nerve had been interrupted before the application of HRP. Analysis of serial sections and mapping of all labeled nervous structures in the CNS revealed many stained axons and terminals in the medial parts of dorsal and ventral horns as well as in the medial part of zona intermedia in the segments C1–C3. Clearly labeled zones were also seen in the central and lateral areas of the ventral horn. These zones correspond to the location of the motor nuclei for the sternomastoid and infrahyal muscles, partly also for the longus colli and splenius capitis muscles. The labeled area in the zona intermedia includes the field of the nucleus cervicalis centralis, which was shown by other authors to receive input from primary afferents of the neck region as well as from vestibular nuclei via fasciculus longitudinalis medialis, and which in turn sends many fibers into the cerebellum. Label was also found in many axons of the fasciculus cuneatus and in terminals spreading over the whole nucleus cuneatus lateralis. Here, close relations of those terminals to perikarya were seen. Only weak labeling was found in laminae I–III of the upper three spinal cord segments.

A light microscopic investigation of the afferent connections of the lateral reticular nucleus in the cat

The topographical organization of the projections to the lateral reticular nucleus (LRN) of the cat was investigated using the horseradish peroxidase (HRP), silver-impregnation and autoradiographic tracing methods. Following injection of HRP into the LRN, labelled cells were found mainly within Rexed's laminae VII and VIII of the spinal cord, the contralateral red nucleus, the ventro-rostral aspect of the contralateral fastigial nucleus and the contralateral anterior sigmoid and coronal gyri of the cerebral cortex. Animals with injections of tritiated amino acids placed within the pericruciate cortex, red nucleus or fastigial nucleus were processed for autoradiography. In a corresponding series of animals, electrolytic lesions were placed selectively into the above sources of reticular afferents, and terminal degeneration within the LRN was studied by light microscopy. An extensive input from the spinal cord was found to terminate predominantly on the ipsilateral side throughout the rostrocaudal extent of the LRN, except for a small ventromedial area of the rostral parvocellular division and a small rostromedial area of the magnocellular division. The cortical projection terminated diffusely within the middle one-half of the contralateral magnocellular division, while the rubral projection terminated extensively within the contralateral subtrigeminal division and the dorsolateral region of the rostral magnocellular and neighbouring parvocellular divisions. The rubral projection did not overlap the cortical projection. The fastigial nucleus projected sparsely to the contralateral LRN, mainly to the medial aspect of the rostral two-thirds of the magnocellular division, with less to the parvocellular and subtrigeminal divisions. The LRN therefore receives spinal and supraspinal projections within at least its rostral one-half, and these terminate within specific areas in a partially overlapping fashion, whereas the caudal one-half is primarily a spinal receiving region. No convergence of the rubral and sensorimotor cortical projections was evident.

Connections of the olfactory bulb in the piranha (Serrasalmus nattereri).

The connections of the olfactory bulb were studied in the piranha using the Nauta and horseradish-peroxidase methods. Three olfactory tracts project to seven terminal fields in the telencephalon and one in the diencephalon, all of them bilaterally. The contralateral olfactory bulb also receives a small input. All contralateral projections decussate in the anterior commissure and are relatively weak compared to the ipsilateral projections. HRP-containing cells were found in all of the ipsilateral telencephalic aggregates receiving an olfactory tract projection; the contralateral side was free of labeled cell bodies. Although only about one fourth of the entire telencephalon receives a direct olfactory input, the high degree of differentiation of the olfactory system suggests that the piranha depends substantially on the sense of olfaction and that this species may be a good model for further studies on olfactory mechanisms.

Localization of efferent neurons innervating the pharyngeal constrictor muscles and the cervical esophagus muscle in the cat by means of the horseradish peroxidase method

Following horseradish peroxidase (HRP) injection into the cephalopharyngeal muscle (CeP), the hyopharyngeal muscle (HP), the thyropharyngeal muscle (TP), the cricopharyngeal muscle (CP) and the cervical esophagus muscle (CE) of the cat, labeled neurons were identified in the nucleus retrofacialis and the rostral part of the nucleus ambiguus ipsilaterally. The rostral end of the labeled cell column was located more rostrally for CeP and HP than for TP, CP and CE. No difference was noted within the former two or within the latter three. The level of the caudal end of the labeled cell column became more caudal in the order of CeP, HP, TP and CP. The caudal end was located more rostral for CE than for TP. The neurons for CE were located more ventro-laterally than those for the other muscles.

Spinal and medullary lamina I projection to nucleus submedius in medial thalamus: a possible pain center.

1. Results obtained with the anterograde horseradish peroxidase (HRP) and autoradiographic techniques have revealed a dense and compact spinal and trigeminal projection to the dorsal portion of the nucleus submedius (Sm) in the medial thalamus of the cat. A homologous projection was observed in two cases in the monkey and in three cases in the rat with the anterograde HRP method. 2. The projection to Sm in topographically organized rostrocaudally, with trigeminal terminations located caudally and spinal terminations rostrally. The rostrocaudal extent of the trigeminal field in Sm (ca. 1 mm) is larger than that of the spinal field (ca. 0.8 mm). There is a considerable ipsilateral trigeminal projection, whereas there is a minimal uncrossed spinal projection to Sm. 3. Retrogradely labeled cells were analyzed in cases with small injections of HRP in medial thalamus in the cat. The results indicate that the projections to Sm identified in the anterograde experiments arise from lamina I cells of the spinal and medullary dorsal horm, probably exclusive of lamina V and other spinothalamic cells. The retrograde results corroborate the topographic observations made in the anterograde experiments. 4. These results suggest that Sm may be critically involved in specific nociception.

Topographical representation of the hypoglossal nerve branches and tongue muscles in the hypoglossal nucleus of macaque monkeys

Representation of the hypoglossal nerve branches and tongue muscles was examined in the hypoglossal nucleus of macaques by the horseradish peroxidase method. The nucleus was divided cytoarchitectonically into the medial and lateral divisions at rostral levels, and into the mediodorsal, medioventral, ventral and laterodorsal divisions at caudal levels. The medial, mediodorsal, medioventral and ventral divisions supplied the medial branch. The lateral and laterodorsal divisions supplied the lateral branch. The geniohyoid motoneurons (MN) composed the ventral division. The genioglossus MN were clustered dorsally in the medial division. The hyoglossus and styloglossus MN were located most laterally in the laterodorsal division.

Uncrossed retinal fibers terminating in the superior colliculus of the albino rat

The site of termination of uncrossed retinal fibers was examined in the superior colliculus of the albino rat. Sixty-eight male and female rats of Gifu, Donryu, Wistar and Sprague-Dawley strains from 9 days to 24 months of age were examined by means of a silver impregnation method for degenerating nerve fibers and the horseradish peroxidase method. In this study, terminals of uncrossed retinal fibers were observed in a small region from the posteromedial to the posterior part of the superficial gray stratum of the ipsilateral superior colliculus.

Isthmic afferent neurons identified by the retrograde HRP method in a teleost, Navodon modestus

Isthmic afferent neurons were investigated by the retrograde horseradish peroxidase (HRP) method in a teleost, Navodon modestus. Following HRP injections into the nucleus isthmi, large pyriform neurons are labeled in the ipsilateral optic tectum. Very large and multipolar neurons are also labeled in the ipsilateral nucleus pretectalis. No labeled neurons were found in other areas.

An ‘on the slide’ modification of the De Olmos-Heimer horseradish peroxidase method

The De Olmos-Heimer horseradish peroxidase method has been simplified without losing its sensitivity. The modification involves the use of cryostat sections and omitting several steps from the original procedure. The modification is especially suitable for small pieces of tissue that would otherwise require imbedding.

Central distribution of octavolateral afferents and efferents in a teleost (mormyridae)

The central distribution of afferents from individual eight nerve branches (N VIII) and mechanical lateral line end organs in mormyrid fish are described. Afferents were labeled with horseradish peroxidase (HRP) placed on the cut ends of the different N VIII branches and the anterior and posterior lateral line nerves. Descending, tangential, and magnocellular nuclei receive input almost exclusively from the utriculus and canals. Nucleus octavius receives afferents from the lateral line nerves and all N VIII branches, with one part receiving exclusive and bilateral input from the sacculus. Afferents from both lateral line nerves and all N VII branches, except the sacculus, end in eminentia granularis. Afferents from each of the two lateral line nerves and from each of three otolith branches of N VIII end in different regions of the anterior lateral line lobe, with some areas of overlap. Behavioral studies in other families of fish indicate that the utriculus and canals are critical for postural control, whereas the sacculus and possibly the lagena are concerned with hearing. Such findings, together with the results of this study, suggest that mormyrids and perhaps other fish possess separate auditory and vestibular centers within the octavolateral area. The HRP method also shows the cell bodies and axons of octavolateral efferents. N VIII and lateral line efferents arise from a common nucleus, and the central course of their axons parallels that of facial motoneurons. Axons of efferent cells divide to supply two or more branches of N VIII and some axons supply both lateral line and N VIII end organs.

A new method for application of horseradish peroxidase into a restricted area of the brain

A new method for horseradish peroxidase (HRP) application into the very restricted area of the brain was developed. Crystalline HRP was packed into the tips of fine micropipettes having tip size of about 50 micrometer in outer diameter. The HRP micropipettes were stereotaxically implanted into the globus pallidus of rats. After survival period of 6 to 48 hours, brain was excised and HRP distribution was investigated following conventional procedures. The diffusion area of HRP at the application site was restricted within the limits of 200--250 micrometer in diameter completely inside the globus pallidus. It was known that the slowly solubilized crystalline HRP was taken up by the nerve terminals in the globus pallidus, and transported retrogradely to the cell bodies in the pars compacta of substantia nigra, indicating the existence of nigro-pallidal projections.

Representation of the masticatory muscles in the motor trigeminal nucleus of the macaque monkey

The pattern of representation of the masticatory muscles in the motor trigeminal nucleus was examined in macaque monkeys by the horseradish peroxidase method. The motor trigeminal nucleus was divided cytoarchitectonically into the dorsolateral and ventromedial divisions. The temporalis, masseter and pterygoid muscles were represented in the dorsomedial, central and ventrolateral parts of the dorsolateral division, respectively. In the ventromedial division, which was located at the level of the caudal half of the nucleus, the anterior digastric or mylohyoid muscle was represented in the dorsomedial or ventrolateral part of the division, respectively.

Extraocular Muscles and their Relationship to the Accessory Abducens Nucleus in rats as Studied by the Horseradish Peroxidase Method

Extraocular Muscles and their Relationship to the Accessory Abducens Nucleus in rats as Studied by the Horseradish Peroxidase Method

Telencephalic efferents in channel catfish, Ictalurus punctatus: projections to the olfactory bulb and optic tectum.

Telencephalic projections to the olfactory bulb and optic tectum were examined with horseradish peroxidase (HRP) methods in catfish. Olfactory bulb afferents arise from the mitral cell layer of the contralateral olfactory bulb and the ipsilateral telencephalon. The majority of labelled cells appear within the ventral and dorsal nuclei of area ventralis telencephali and two divisions of the central zone of area dorsalis telencephali. Excepting one division of the dorsal central zone, all afferent sources are secondary olfactory targets. Tectal HRP injections reveal labelled cells in a third division of the dorsal central zone.

Distribution of horseradish peroxidase-labeled neurons giving rise to descending fiber systems in the basal ganglia and hypothalamus in cats

The distribution of neurons giving rise to various descending fiber systems to brain-stem structures in the basal ganglia (including amygdaloid nuclei) and hypothalamus of the cat was studied by the retrograde axonal transport of horseradish peroxidase method. Neurons in the medial part of the central nucleus and of the magnocellular part of the basal nucleus of the amygdaloid group were shown to send axons to the dorsal hippocampus, substantia nigra, lateral part of the central gray matter, and the mesencephalalic reticular formation and also to the region of the locus coeruleus and the lateral medullary reticular formation at the level of the inferior olives. The predominant source of projections to the hypothalamus and brainstem structures is the central amygdaloid nucleus, which also sends projections to the nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus nerve, and the superior cervical segments of the spinal cord. Uncrossed fiber systems descending from the basal ganglia terminate at the level of the pons, whereas uncrossed and crossed fiber systems descending from the dorsal and ventromedial hypothalamus can be traced into the spinal cord. The possible role of nuclei of the amygdaloid group, the hypothalamus, and their efferent projections in the regulation of somatic and vegetative functions and also of complex behavioral reactions is discussed.