HRP-labeled Neurons Research Articles

Hypothalamic descending afferents to cells of origin of the greater petrosal nerve in the rat, as revealed by a combination of retrograde HRP and anterograde autoradiographic techniques

Projections from the hypothalamus to cells of origin of the greater petrosal nerve were studied by the HRP and autoradiographic techniques in the rat. After an injection of [ 3H]amino acids into the hypothalamus and an application of HRP to the greater petrosal nerve, we found that a compact group of HRP-labeled neurons was located within a dense accumulation of silver grains in the nucleus reticularis parvocellularis. The present results show that the hypothalamus projects directly to cells of origin of the greater petrosal nerve.

The posteromedial ventral nucleus of the thalamus (VPM) of the cat: direct ascending projections to the cytoarchitectonic subdivisions.

The posteromedial ventral nucleus (VPM) of the cat is divided cytoarchitectonically into the magnocellular (VPMmc), lateral parvocellular (VPMpcl), and medial parvocellular (VPMpcm) divisions. Cell bodies of neurons in the VPMpcm are small, while those in the VPMpcl are small to medium-sized. The VPMmc contains large neurons. Direct projections from the lower brain stem structures to each of the three divisions of the VPM were examined by the retrograde horseradish peroxidase (HRP) method. When HRP injection was done into the VPMmc, labeled neurons were mainly located contralaterally in the ventral division of the principal sensory trigeminal nucleus (Vp), in the rostral part of the oral subnucleus in the spinal trigeminal nucleus (Vsp), and in the interpolar subnucleus of the Vsp; a few labeled neurons were also found contralaterally in lamina I of the caudal subnucleus of the Vsp. When HRP injection was restricted to the VPMpcl or VPMpcm, HRP-labeled neurons were mainly observed ipsilaterally, respectively, in the dorsal division of the Vp, or in the parabrachial nucleus (PBN) regions dorsomedial and ventromedial to the brachium conjunctivum. After HRP injection into the parvocellular part of the VPM (VPMpc), labeled neurons were also seen contralaterally in the Vsp, but these were far less numerous than those seen after HRP injections into the VPMmc. Thus, each of the three divisions of the VPM receives main ascending afferent fibers from different brain stem structures; the VPMpcm, VPMpcl, or VPMmc receives afferent fibers, respectively, from the PBN ipsilaterally, from the dorsal division of Vp ipsilaterally, or from the ventral division of the Vp and the Vsp contralaterally.

A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods

The location of the superior salivatory nucleus and terminal labelings of the hypothalamic descending fibers were demonstrated in the nucleus reticularis parvocellularis using HRP and the autoradiographic techniques, respectively. When both techniques were used in the same animals, some HRP-labeled neurons were seen among the accumulations of silver grains, suggesting pericellular terminations. The present study demonstrates that the hypothalamic efferents project directly to the superior salivatory nucleus innervating salivary and lacrimal glands.

Topographic arrangement of motoneurons innervating the suprahyoid and infrahyoid muscles. A horseradish peroxidase study in cats.

The topographic arrangement of motoneurons innervating the muscles which participate in the rostrocaudal movement of the larynx was studied in cats by means of the horseradish peroxidase (HRP) method. After HRP injection into these muscles, all labeled neurons were found in the nuclei of the brainstem and cervical nerves (C1, C2) ipsilaterally. The mylohyoid and anterior digastric motoneurons were seen in a cluster in the medial part of the rostrocaudal area of the motor trigeminal nucleus. Within the medial part of the nucleus, the mylohyoid motoneurons were located ventrally and the anterior digastric motoneurons dorsally. The posterior digastric neurons were situated in the accessory facial nucleus, the stylohyoid neurons in the ventral aspect of the facial nucleus, the geniohyoid motoneurons in the ventral aspect of the hypoglossal nucleus, and the thyrohyoid motoneurons in the lateral aspect of the hypoglossal nucleus and the dorsomedial part of the ventral horn cells of C1. The HRP-labeled neurons of the sternohyoid and sternothyroid muscles were observed in the central portion of the ventral horn cells of C1 and C2.

Ultrastructure of HRP-Labelled Neurons: A comparison of two sensitive techniques

Rat sciatic nerves were transected and placed in a solution of HRP. Following perfusion, L4-6 spinal segments and sensory ganglia were cut into 60 μm sections. Alternate sections were processed with the glucose oxidase (GOD) or the benzidine dihydrochloride (BDHC) procedure. Sections were then either mounted on glass slides for light microscopic (LM) examination or embedded in epoxy resin for sequential light and electron microscopic (EM) analysis. At the LM level, GOD reaction product was granular and confined to the soma and proximal dendrites of motoneurons, while BDHC reacted tissue was characterized by a diffuse labelling of the cytoplasm, extending as far as tertiary dendrites. At the EM level, neurons in tissue processed with the GOD technique contained lysosomal and vesiculotubular elements that were extremely electron dense. Label in BDHC reacted tissue appeared as dense patches of reaction product dispersed throughout the cytoplasm. Possible reasons for the qualitative differences in the reaction product provided by the two techniques as well as suggested applications of the techniques are discussed.

Movement of horseradish peroxidase after its entry into intact and damaged peripheral nerve axons

After application of a solution of horseradish peroxidase (HRP) around intact sciatic nerve axons in the rat, numerous HRP-labeled neurons were found in the ipsilateral L5 dorsal root ganglion and spinal cord ventral horn. These findings indicate that HRP enters intact peripheral nerve axons, and is transported retrogradely from its site of entry to their cell bodies of origin.

Supramedullary afferents of the nucleus raphe magnus in the rat: a study using the transcannula HRP gel and autoradiographic techniques.

Afferents of the nucleus raphe magnus (NRM) were retrogradely labelled by using a transcannula HRP gel technique in conjunction with tetramethylbenzidine neurohistochemistry to determine the sources of inputs to the nucleus which could potentially influence the descending antiociceptive raphe-spinal system. Large numbers of HRP-labelled neurons were seen in the frontal cortex, dorsomedial nucleus of the hypothalamus, zona incerta, nucleus parafascicularis prerubralis (NPfPr), pretectum, dorsal and lateral periaqueductal gray, nucleus cuneiformis (NC), deep superior colliculus (dSC), a paraoculomotor cell group which may be the medial accessory nucleus of Bechterew, dorsal column nuclei, and spinal trigeminal nucleus. Smaller numbers of labelled cells were also observed in the preoptic area, nucleus of Darkschewitsch, ventral peri(third)ventricular gray, nucleus reticularis pontis oralis and caudalis, medial and lateral vestibular nuclei, and a subdivision of the hypoglossal nucleus. Confirmational anterograde autoradiographic studies were performed by injecting tritiated leucine into two of the principal sources of afferents to NRM: NPfPr, and dSC/NC. The results are compared with control HRP gel implants in the inferior olive, spinal cord, nucleus reticularis paragigantocellularis, and medial facial nucleus. Comments are also made concerning the parcellation of the ventromedial medulla and the possible role of both NRM and its afferents in central analgesic mechanisms.

Retrograde transport of horseradish peroxidase by regenerating preganglionic axons in the cat's cervical sympathetic trunk

Horseradish peroxidase (HRP) was applied to the proximal stumps of regenerating (7 days postsectioned) and freshly cut cervical sympathetic trunks of cats. Counts of HRP-labeled neurons in the spinal cord showed that regenerating sympathetic axons were just as capable, if not better able, of taking up and retrogradely transporting HRP, as freshly cut normal sympathetic axons.

A correlative quantitative study comparing the nerve fibers in the cervical sympathetic trunk and the locus of the somata from which they originate in the rat

Correlative quantitative analyses were performed on the rat comparing the number of fibers in the cervical sympathetic trunk (CST) and the horseradish peroxidase (HRP)-labeled neurons in the superior cervical sympathetic ganglion (SCG), stellate ganglion, as well as in the spinal cord. The total number of nerve fibers in the left CST was 4180 ± 169 (mean ± S.E.M.) among which 92 ± 3 (mean ± S.E.M.) were myelinated. The diameter of unmyelinated fibers was 0.68 ± 0.22 (mean ± S.D.) μm and showed single-peaked distribution. After the application of HRP to the proximal cut end of the CST, labeled neurons were found in the stellate ganglion as well as in the ipsilateral spinal cord from C7 to T4 segments. The total number of HRP-labeled neurons in the spinal cord was 1334 ± 45 (mean ± S.E.M.) with the range between 844 and 1808. Ninety-nine percent of labeled neurons were located in the intermediolateral column and in the lateral funiculus while 1% were in the intercalated region and central autonomic area. Labeled neurons were encountered only sporadically in the dorsal root ganglia (DRG) from C8 to T3 level. After the application of HRP to the distal cut end of the CST, about 200 labeled neurons were observed in the caudal part of the SCG. The present results were discussed with special reference to the organization of the CST of the rat.

Identification of motoneurons supplying the tensor veli palatini muscle in the guinea pig and cat: An horseradish peroxidase study

Identification of motoneurons supplying the tensor veli palatini muscle was attempted by the horseradish peroxidase (HRP) method in the guinea pig and cat. After HRP injection into the tensor veli palatini muscle, HRP-labeled neurons were seen in the regions closely medial to the cluster of the lateral pterygoid motoneurons in the dorsolateral division of the trigeminal motor nucleus. In the guinea pig the tensor veli palatini motoneurons were observed at the level of the whole rostrocaudal extent of the trigeminal motor nucleus, while in the cat they were seen at the level of the rostral two-thirds of the nucleus.

Localization of motoneurons innervating the tensor tympani muscles: An horseradish peroxidase study in the guinea pig and cat

Motoneurons innervating the tensor tympani muscle were identified in the adult guinea pig and cat by the horseradish peroxidase (HRP) method. After HRP injection into the tensor tympani muscle, HRP-labeled neurons were seen in the regions outside the cytoarchitectonically-defined confines of the trigeminal motor nucleus; in the regions rostral to the rostral pole of the nucleus, as well as in the regions ventral and ventrolateral to the nucleus at the levels of the rostral half (guinea pig) or the rostral two-thirds (cat) of the nucleus. The tensor tympani motoneurons were generally smaller than the masticatory motoneurons.

Non-hippocampal cortical projections from the entorhinal cortex in the rat and rhesus monkey

The entorhinal cortices are known to give rise to powerful projections that terminate in the hippocampus and dentate gyrus. Collectively, these link the hippocampal formation to many parts of the cortex and to subcortical structures like the amygdala. Non-hippocampal projections from the entorhinal cortices are understood poorly. Such projections to neighboring temporal areas in the rat and rhesus monkey have been investigated using the autoradiographic and horseradish peroxidase (HRP) tracing procedures. In the rat, HRP-labeled neurons were observed in the intermediate and lateral fields of the entorhinal cortices after injections of temporal cortical areas 20, 35, 36 and 41. They were located predominantly in layers II, III and IV. In the monkey , HRP-labeled neurons were observed in the entorhinal cortices after injections of the rostral superior temporal gyrus (area TA or 22); the temporal polar cortex (area TG or 38); the inferior temporal cortex (area TE or 20); the perirhinal cortex (area 35) and the posterior parahippocampal cortices (areas TF and TH). Unlike the rat, labeled entorhinal neurons in the monkey were located in layer IV. Autoradiographic experiments in the monkey yielded complimentary results. In view of the fact that layer IV of the entorhinal cortex in both the rat and monkey receives a powerful projection from the subicular-CA1 fields of the hippocampal formation, the results imply that this layer mediates an indirect non-fornical connection between the hippocampal formation and the temporal cortex.

Infrared sensory neurons in the trigeminal ganglia of crotaline snakes: Transganglionic HRP transport

Trigeminal neurons were labeled by inserting HRP into holes cut in the pit receptor membranes of a crotaline snake, Agkistrodon blomhoffi brevicaudus. Neurons were labeled in the opthalamic ganglion and the maxillary division of the maxillo-mandibular ganglion, and the HRP was further transported across the ganglia and through the lateral descending trigeminal tract (dlv) to label axon terminals exclusively in the dlv nucleus (DLV). In 6 successful preparations, 7.1–19.3% of totals of 5568–5986 cells in the maxillary division of the ganglion were labeled, but none at all were labeled in the mandibular division. Only a few or none at all were labeled in the opthalmic ganglion. Cells in the two ganglia ranged in size from 10 to 55 μm, but large cells(⩾ 40 μm) were scarce (4.9% of the total population). All HRP-labeled neurons fell in the median range of 20–39 μm. We concluded that these ganglion cells were infrared neurons, and were therefore the origin of the Aδ fibers in the pit membrane. There were no HRP-labeled neurons above or below this range, in spite of the fact that smaller cells(⩽ 19 μm) made up 35.8% of the total population. In normal Nissl preparations we found both light- and dark-staining cells, but the size range of neither corresponded to the size range of infrared neurons.

Cerebellar afferents to paramedian lobule from the trigeminal complex in Tupaia glis: a horseradish peroxidase (HRP) study.

Projections from the trigeminal complex to paramedian lobule (PML) were studied in the tree shrew (Tupaia glis) by means of retrograde transport of horseradish peroxidase (HRP). Neurons which project to both dorsal and ventral folia of PML are located primarily in those areas of the trigeminal nuclear complex interpreted as nucleus interpolaris (Vi) and caudal areas of the nucleus oralis (Vo). The majority of HRP-labeled neurons lie in ventral and ventrolateral regions of Vi/Vo. No HRP-reactive cells are present in the principal (Vp), mesencephalic, or motor nuclei nor in nucleus caudalis or rostral portions of oralis. The majority of trigeminocerebellar (TC) cells are found in ipsilateral Vi; however, sparse numbers of labeled somata are present in this subnucleus on the contralateral side. Within Vi/Vo, small fusiform and medium-and large-sized multipolar neurons contain HRP-reaction product. Large multipolar cells are found primarily in ventrolateral portions of Vi/Vo, while medium and small neurons are scattered throughout the ventral half of the nucleus. Small-sized neurons are also present dorsally within Vi/Vo. Axons of labeled TC cells course laterally through the spinal trigeminal tract, enter medial aspects of the restiform body, and arch dorsally into the cerebellum.

Central distribution of afferent and efferent components of the glossopharyngeal nerve: An HRP study in the cat

Central distribution of efferent and afferent components of the glossopharyngeal (IX) nerve in the cat was studied by applying horseradish peroxidase (HRP) to the lesser petrosal nerve (LPN), carotid sinus nerve (CSN) or cervical trunk (CT) distal to the branching point of the CSN. After applying HRP to the LPN or CT, HRP-labeled neurons were distributed ipsilaterally in the lateral regions of the reticular formation from the caudalmost levels of the facial nerve genu to the rostral levels of the solitary nucleus. HRP-labeled axons of these neurons ran dorsomedially and then medially along the ventral borders of the medial vestibular nucleus and the facial nerve genu, to form a small genu rostrally at the regions medial to the facial genu and caudally at the medial tip of the medial vestibular nucleus. Subsequently, the labeled axons coursed ventrolaterally to exit from the brain stem with other IX nerve components. After HRP application to the CT, labeled neurons were also seen ipsilaterally within the rostralmost levels of the nucleus ambiguus, and labeled fibers were traced ipsilaterally to the medial and interstitial subnuclei of the solitary nucleus, dorsalateral portions of the spinal trigeminal nucleus at the caudalmost levels of the alaminar parts, and ventrolateral regions of the medial cuneate nucleus. Afferent components of the CSN ended ipsilaterally in the medial subnucleus, and bilaterally in the commissural subnucleus of the solitary nucleus; within the medial subnucleus, the CSN terminals located more dorsolaterally than the CT terminals. No efferent components were found in the CSN.

The location of cerebellar-projecting neurons within the lumbosacral spinal cord in the cat. An anatomical study with HRP and retrograde chromatolysis.

The locations of lumbosacral spinocerebellar neurons were examined by two anatomical methods in kittens. In one group of animals chromatolytic changes were provoked by cerebellar lesions. In another group horseradish peroxidase (HRP) was injected into the cerebellum. Projection laterality was investigated by making unilateral spinal lesions prior to the cerebellar HRP injections. Diaminobenzidine (DAB) or tetramethylbenzidine (TMB) was used as substrate fo HRP. The morphological characteristics of HRP-labeled neurons in the TMB-processed material were examined. Neurons marked by the two methods were located within the same regions. A greater number of cells were marked with the HRP method, however, than with the retrograde chromatolysis method. Spinocerebellar neurons were found in laminae IV-IX with large differences with regard to specific locations depending on segmental level. Numerous marked neurons were found in the following areas: laminae IV-Vi in L3-L7, the column of Clarke in L3-L4, the medial part of lamina VII in L6-L7, the lateral part of lamina VII in L3-L4, the dorsolateral nucleus of lamina IX in L3-L6, the ventrolateral nucleus of lamina IX in L4-L5, and the ventromedial nucleus of lamina IX in S3 (and Ca1). Dorsally located neurons were in general more likely to project ipsilaterally than ventrally located neurons. Marked structural differences were frequently observed between spinocerebellar neurons in different locations. These results provide additional information on the anatomical complexity of the spinocerebellar pathways from the lumbosacral region in the cat. Together with results from some other recent anatomical studies on spinocerebellar tracts, they also form a basis for further anatomical and physiological investigations which could contribute to a better understanding of the organization of the spinocerebellar tracts.

Distribution of Sensory Ganglion Cells Innervating Facial Muscles in the Cat

The question of a possible sensory component in branches of the facial nerve innervating facial mimetic muscles in the cat was examined by the technique of retrograde axonal transport of horseradish peroxidase (HRP). HRP was applied to the proximal cut end of facial nerve branches innervating different facial muscle groups. Following survival periods of 71-75 h the animals were fixed by perfusion. Certain craniospinal sensory ganglia and the brain stem were processed histochemically for demonstration of HRP. HRP-labelled cell bodies, structurally resembling sensory neurons, were consistently observed ipsilaterally in the geniculate and proximal vagal ganglia and under certain conditions in the trigeminal ganglion. Measurements of HRP-labelled neurons in the geniculate and proximal vagal ganglia showed a wide size range but a unimodal size distribution with peaks in the small size range. These findings support the view that facial nerve branches innervating the mimetic muscles contain different types of sensory fibers.

Neural connections between the middle ear, eustachian tube and brain. Implications for the reflex control of middle ear aeration.

This study was designed to investigate the neuroanatomic connections between the middle ear (ME), eustachian tube, and brain. The neural tracer, horseradish peroxidase (HRP), was injected into the promontory mucosa of the ME in six adult rabbits. Following a 24–48 hour survival, the brain was perfused with fixative and the HRP reacted by the tetramethylbenzidine (TMB) blue reaction process. Two groups of HRP-labeled neurons were noted in the ipsilateral brainstem: 1) a well-defined cluster of HRP-labeled neurons in the nucleus of the solitary tract, and 2) a more widely scattered group of labeled neurons in the inferior salivary nucleus. The labeled neurons in the solitary tract may represent the direct central sensory projections of the glomus tympanicum. In a second set of animals HRP was injected into both the tubal and palatal muscles to identify the motor innervation of the eustachian tube musculature; HRP-labeled motoneurons were noted in the trigeminal motor nucleus and nucleus ambiguus, respectively. Assuming that the glomus tympanicum cells can sense changes such as oxygen concentration in their immediate environment in the ME like the related glomus cells in the carotid and aortic bodies which also project to the solitary tract, then the neuroanatomic relationships of the HRP-labeled neurons in this study to other respiratory neurons in the medulla suggest the possibility of a polysynaptic ME aeration reflex. The relevance of these results to the pathogenesis of otitis media with effusion is discussed.

Brainstem afferents to the torus semicircularis of the Queensland cane toad (Bufo marinus).

The ascending projections to the major midbrain auditory center of anura, the torus semicircularis, have been examined using the technique of retrograde labeling with horseradish peroxidase (HRP). Localized iontophoretic injections of HRP were made in one torus semicircularis of Queensland cane toads (Bufo marinus). Visualization of HRP-labeled neurons with tetramethyl benzidine revealed that the torus semicircularis receives its major afferent input from the ipsilateral superior olive. Other prominent projections arise in the contralateral dorsal nucleus of the eighth nerve and the contralateral torus semicircularis. HRP-labeled neurons were also associated with the lateral lemniscus at a level ventral and lateral to the nucleus isthmi on both sides. It is suggested that these neurons constitute diffuse nuclei of the lateral lemnisci. Relatively minor projections to the torus semicircularis arise in the contralateral superior olive, ipsilateral dorsal nucleus of the eighth nerve, and the ventral division of both the ipsilateral and contralateral eighth nerve nuclei.

Afferents from the periaqueductal gray, medial hypothalamus and medial thalamus to the midbrain reticular core

On the basis of electrophysiological findings, a descending input from the midline diencephalic areas to the mesencephalic reticular formation has been envisaged to underlie a tonic, reinforcing control of the waking brain. In order to substantiate morphologically this hypothesis and to assess the exact sources of this descending input, unilateral injections (0.04 to 0.06 μl) of horseradish peroxidase (HRP: 50% solution in 2% DMSO) were made within the mesencephalic reticular formation (MRF) in 11 cats. The animals were allowed to survive from 24 to 48 hrs and the brain sections were treated according to the diamino-benzidine (DAB), the tetramethyl-benzidine (TMB), or the bezidine-dihydrochloride (BDHC) procedure. In most animals the injection site was rather small and confined to the nucleus cuneiformis and adjacent central tegmental field. Numerous positive cells, with HRP granules in the soma and proximal processes, were consistently found ipsilaterally at distances ranging from 0.2 to 1.5 mm from the wall of the aqueduct and the third ventricle. Thus, HRP-labelled neurons were seen (1) in the midbrain periaqueductal gray, mostly in its medio-dorsal aspect, (2) in the caudal diencephalic periventricular gray, medial to the retroflex bundle, (3) in the periventricular and medial territories of the preoptico-hypothalamic complex, (4) in the centrum medianum-parafascicularis thalamic nuclei, and (5) less consistently, in the paracentralis thalamic nucleus. A few positive cells were also scattered within the central gray, contralateral to the injection site. At hypothalamic levels, HRP-labelled neurons predominated particularly within and around the ventromedial, arcuate, suprachiasmatic and paraventricular nuclei, and in the medial preoptic region. These results demonstrate that, in addition to the massive subthalamic input to the midbrain reticular formation described recently, a well-developed neuronal system extending from the mesencephalon to the anterior preoptico-hypothalamic region also provides a significant number of afferent fibers to the upper brainstem reticular core.