Afferent projections to the interpeduncular nucleus in the rat, as studied by retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase

We examined the afferent projections to the subnuclei of the interpeduncular nucleus (IPN) in the rat by means of retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). We observed locations of retrogradely labeled cells following injections of WGA-HRP into the IPN, and distributions of anterogradely labeled fibers and terminals within the IPN following injections into the areas that contain cells of origin of afferents. Results of the retrograde and anterograde experiments have clarified the detailed organization of the IPN afferents. A part of the nucleus incertus, located dorsomedial to the dorsal tegmental nucleus, projects to the contralateral half of the rostral subnucleus of the IPN; the pars caudalis of the dorsal tegmental nucleus projects sparsely to the rostral lateral, dorsal lateral, lateral, caudal, and apical subnuclei predominantly contralaterally; the laterodorsal tegmental nucleus, to most of the subnuclei predominantly contralaterally; the ventromedial central gray rostral to the dorsal tegmental nucleus and lateral to the dorsal raphe nucleus projects to the rostral lateral and dorsal lateral subnuclei predominantly contralaterally; the median raphe nucleus, substantially to all subnuclei; the medial habenular nucleus, in a topographic manner, to the rostral, central, and intermediate subnuclei, to the rostral lateral and lateral subnuclei predominantly ipsilaterally, and to the dorsal lateral subnucleus predominantly contralaterally; the supramammillary nucleus and areas around the origin of the mammillothalamic tract and near the third ventricle project sparsely to the ventral part of the rostral subnucleus and to the central, lateral, caudal and apical subnuclei; the nucleus of the diagonal band, sparsely to the rostral, central, dorsal lateral, caudal, and apical subnuclei. These differential projections of the afferents to the subnuclei of the IPN may reflect its complex functions within the limbic midbrain circuit.     

Innervation of the hard palate in the rat studied by anterograde transport of horseradish peroxidase conjugates

The innervation of the rat hard palate and the bordering part of the soft palate was studied after anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) and to choleragenoid (B-HRP) in separate experiments. WGA-HRP labeling showed leakage from several types of nerve endings, whereas B-HRP did not. Both conjugates gave rise to heavy labeling of a variety of nerve endings. Intragemmal and, especially, perigemmal fibers were labeled in chemosensory corpuscles, which were most common in the medial wall of the incisive canal and in the most anterior part of the soft palate. Ruffini endings of different sizes were labeled in the incisive papilla. Other subepithelial endings forming elongated expanded profiles with medium-to large-caliber source fibers were most common in protruding parts of the palate. Labeled intraepithelial endings included Merkel endings, which were most frequent in the incisive papilla and the rugae. Other labeled profiles were medium-caliber afferents giving rise to irregular, beaded, and sometimes branched endings often located far superficially in the epithelium. Such endings were present both within and between protruding parts of the palate. Fine-caliber intraepithelial endings were labeled almost exclusively in WGA-HRP experiments. © 1995 Willy-Liss, Inc.     

Spinal cord projections from hindlimb muscle nerves in the rat studied by transganglionic transport of horseradish peroxidase, wheat germ agglutinin conjugated horseradish peroxidase, or horseradish peroxidase with dimethylsulfoxide

The spinal cord projections of four different groups of hindlimb muscle nerve branches--the medial and lateral gastrocnemius nerves, muscle branches of the deep peroneal nerve, muscle branches of the femoral nerve, and a nerve to the hamstring muscles--were studied with transganglionic transport of horseradish peroxidase (HRP) in the rat. The influence of varying the postoperative survival (3, 6, and 10 days) and of using wheat germ agglutinin-HRP conjugate (WGA-HRP), or HRP with dimethylsulfoxide (DMSO) instead of free HRP was studied for the gastrocnemius nerves. After 3 days' survival following application of HRP to the gastrocnemius nerves, fine granular labeling was found mainly in lamina V in L4-5, and coarse granular labeling was found in Clarke's column as far caudally as L2, and in laminae VI and VII predominantly in Th12-L2. After 6 or 10 days' survival, the fine labeling in lamina V was sparse or absent, whereas the coarse labeling appeared to remain or to be only slightly reduced in Clarke's column and in laminae VI and VII. No labeling suggestive of terminals was observed in laminae I-III from the gastrocnemius nerves. Except for sparse labeling in lamina I in some of the cases and some minor differences rostrocaudally, the spinal distribution of labeling was similar to that from the other nerves investigated. The distribution of labeling obtained after application of WGA-HRP or HRP with DMSO to the gastrocnemius nerves was very similar to that obtained with free HRP after 3 days' survival. The results indicate that the spinal cord projections of hindlimb muscle nerves in the rat distribute mainly in the deep part of the dorsal horn and in the intermediate zone. Furthermore, the lack of labeling suggestive of terminals in laminae I-III from the gastrocnemius nerves suggests, in conflict with earlier findings in the cat, that primary afferent fibers from muscles do not necessarily terminate in these laminae in the rat. The results suggest, furthermore, that fine granular labeling found in lamina V represents fine-calibered afferent fibers. Finally, the similar spinal projection patterns of the different muscle nerves investigated suggest either a less developed or an essentially different somatotopic organization for muscle afferents compared to cutaneous afferents, as revealed in earlier studies.     

Transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase into trigeminal interneurones of the rat

Intramuscular injections of either horseradish peroxidase (HRP) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) were made into the masseter muscle of rats. Both tracers labeled primary sensory neurones in the V mesencephalic nucleus, motoneurones in the V motor nucleus, and some motoneurones in the facial motor nucleus. WGA-HRP labeled additional neurones in the V main sensory nucleus and the rostral pole of the V nucleus oralis. These were classed as interneurones because they lay in areas outside those known to contain either first-order afferent or motoneurone somata. We argue that these were labeled by retrograde transport of tracer because they lay close to the V motor nucleus, and from some of them processes could be followed into the region of the V motor nucleus.     

Long lasting inhibition of horseradish peroxidase (HRP) transport in sensory nerves induced by capsaicin pretreatment of the receptive field

Capsaicin, when injected into the scrotal skin of adult rats was seen to produce a long-lasting desensitization. To investigate the possible effect of this treatment on the sensory neurones of the pudendal nerve, rats were injected with horseradish peroxidase (HRP) at different time intervals following pretreatment with capsaicin. The HRP was applied in two different ways. In one group of rats HRP was injected into both sides of the scrotal skin between 2 and 12 days after capsaicin pretreatment of one side of the scrotal skin. More HRP labelled neurones were seen in the S1 and L6 dorsal root ganglia (DRG) on the non-treated side than on the pretreated side. Similar results were obtained when the HRP was injected directly into the pudendal nerve. A marked decrease in the number of HRP labelled neurones on the pretreated side was observed even when HRP was injected into the nerve up to 1 year after the capsaicin pretreatment. It appears therefore that at least part of the effect of capsaicin on peripheral sensory neurones is due to an effect of capsaicin on the axoplasmic transport processes, and that this effect is long lasting.     

Contralateral termination of primary afferent axons in the sacral and caudal segments of the cat, as studied by anterograde transport of horseradish peroxidase

Contralateral termination of primary afferent axons in the sacral and caudal segments was studied with the anterograde horseradish peroxidase technique in the cat. The distribution of terminals and branching pattern of terminal fibers were examined on sections incubated in a diaminobenzidine-cobalt chloride medium. The crossed primary afferent axons passing in the dorsal commissure projected to (1) the dorsal gray commissure, (2) small, dorsomedial and ventrolateral parts of laminae III and IV, (3) lamina I, and (4) the Xth area (region dorsal to and around the central canal). In the dorsal gray commissure primary afferent axons formed a dense plexus after taking a tortuous course in mediolateral and rostrocaudal directions. Single axons terminated on both sides, but rarely did they extend outside of this region. The axons terminating in the dorsomedial and ventrolateral parts of laminae III and IV passed through the dorsal gray commissure without giving off collaterals. After reaching laminae III and IV they ascended and/or descended within these laminae and terminated at more rostral or caudal levels. The crossed primary afferent axons passing in the ventral commissure terminated in lamina VIII of the contralateral ventral horn. Some axons terminated in a region ventral to the central canal, and on both sides of lamina VIII.     

Ascending projections to the mammillary nuclei in the rat: a study using retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase

Cells of origin of ascending afferents to the mammillary nuclei and the afferents' fields of termination within these nuclei were studied by using retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase in the rat. The pars compacta of the superior central nucleus projects bilaterally to the median region of the medial mammillary nucleus. The ventral tegmental nucleus projects ipsilaterally to the medial mammillary nucleus, except for its median region, in a topographic manner such that the rostrodorsolateral part of the ventral tegmental nucleus projects to the medial quadrant of the medial mammillary nucleus; the rostroventromedial part projects to the dorsal quadrant; the caudodorsolateral part projects to the ventral quadrant; and the caudoventromedial part projects to the lateral quadrant. These projection fields extend throughout the longitudinal axis of the medial mammillary nucleus, except for its most caudal region, to which only the dorsolateral part of the ventral tegmental nucleus projects. This nucleus also projects topographically to the ipsilateral dorsal premammillary nucleus; the rostral part of the ventral tegmental nucleus projects to the dorsal part of the dorsal premammillary nucleus, whereas the caudal part projects to the ventral part. The periaqueductal gray around the dorsal tegmental nucleus projects bilaterally to the supramammillary nucleus. The pars alpha of the pontine periaqueductal gray projects bilaterally to the peripheral part of the lateral mammillary nucleus, whereas the pars ventralis of the dorsal tegmental nucleus projects ipsilaterally to the lateral mammillary nucleus. The results show that the tegmentomammillary projections are organized in a gradient fashion, with the rostral to caudal position of cells of origin within the tegmental nuclei of Gudden being reflected by the medial to lateral position of fields of termination within the mammillary nuclei.     

On retro- and anterograde transport of horseradish peroxidase in the pontocerebellar fibers as studied with the Mesulam TMB technique

In cats where injections of HRP have been made in the cerebellar cortex, pontocerebellar fibers are visualized throughout their length. Likewise, injections of HRP into the pontine nuclei visualize the pontocerebellar fibers and these anterogradely stained axons can in fortunate sections be followed into the granular layer. A visualization of retrogradely filled pontocerebellar fibers was unexpected and its another example of the sensitivity of the Mesulam8 technique.     

Sympathetic innervation of the rat cornea as demonstrated by the retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin

The sympathetic innervation of the rat cornea was studied by using the method of intraaxonal transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA). In the first set of experiments, the relative number of superior cervical ganglion neurons that innervate the rat central cornea was estimated by the method of retrograde HRP-WGA transport. Following tracer application to the scarificed central corneal surface, 49-198 labeled neurons were observed in the ophthalmic region of the ipsilateral trigeminal ganglion and zero to four cells in the rostral pole of the ipsilateral superior cervical ganglion. In the second set of experiments, the three-dimensional distribution and termination sites of the corneal sympathetic nerve fibers were investigated by the technique of HRP-WGA anterograde transport from the superior cervical ganglion. HRP-WGA-labeled axons in corneal whole mounts were identified by the presence within their axoplasm of linear arrays of HRP-TMB reaction product, and their distribution was plotted faithfully onto line drawings made with a drawing tube attachment. Large numbers of HRP-labeled fibers were found in all animals within the corneoscleral limbus where the majority were associated with blood vessels. Fewer fibers (zero to 14 per animal) entered the cornea proper. The latter fibers entered the peripheral cornea in the deep to middle layers of the stroma and ascended into progressively more superficial layers as they coursed centrally. The majority of fibers branched infrequently in the peripheral cornea and increased in branching complexity near the central cornea. HRP-labeled axonal varicosities suggestive of terminal and preterminal expansions were located preferentially in the subepithelial layer of the corneal stroma and in the basal epithelium. Approximately 75% of the axonal varicosities were located in the central half of the cornea. The results of the current investigation reveal that the rat cornea is innervated sparsely by sympathetic nerve fibers derived from the superior cervical ganglion. These data provide additional support to current theories that corneal sympathetic nerve fibers may influence select aspects of corneal physiology, including ion transport and hydration, mitogenesis and wound healing, and sensitivity.     

Distribution of lumbar dorsal root fibers in the lower thoracic and lumbosacral spinal cord of the rat studied with choleragenoid horseradish peroxidase conjugate

Spinal cord projections from lumbar dorsal root ganglia (DRGs) were investigated in adult rats following injections of choleragenoid horseradish peroxidase (B-HRP) into each of the six lumbar DRGs. This method is known to label primarily thick fibers. Labeling was found in all laminae except in the outer part of lamina II. Labeled fibers and terminal-like structures were found 8-13 segments rostral and 1-5 segments caudal to the injected DRGs. A somatotopic organization was revealed in laminae III, where the labeling seemed to be organized in mediolateral zones. Some of these protruded as fingerlike processes through segments rostral and caudal to the root entry level. An interdigitating pattern for these fingerlike processes was seen between some DRGs, while an extensive overlap was found between other DRGs. Many zones were found to correspond to the known central projections of peripheral sensory nerves supplied by the injected ganglion. This suggests that the central projection of a DRG is highly related to the projections of the peripheral nerves included in the DRG. The projections to lamina IV were organized in a similar manner as in lamina III, even though the projections showed a higher degree of overlap than in lamina III. No clear somatotopic organization was found in laminae V-IX. Provided that the topographical relationship between central projections of single peripheral nerves and of DRGs correspond to their peripheral projections, the results of this study, together with results of earlier studies suggest that the outlines of dermatomes are highly related to the territories of peripheral nerves included in the dermatomes.     

The use of wheat germ agglutinin--horseradish peroxidase conjugates for studies of anterograde axonal transport

Injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), into the hemisected spinal cord of the rat, cat and monkey consistently resulted in the intense anterograde labeling of ascending spinal projections such as the spinothalamic tract and spinocerebellar tracts and their terminal fields. Injections of WGA-HRP in the dorsal column nuclei resulted in the anterograde labeling of the medial lemniscus and its terminal fields in the thalamus. Injection of similar amounts of horseradish peroxidase alone (HRP) in hemisected animals or the dorsal column nuclei resulted in little anterograde labeling. The rate of the anterograde transport of WGA-HRP in cut axons appears to be greater than 200 mm/day. Small amounts of transneuronal labeling appeared to occur after injection of WGA-HRP in both cut axons and undamaged cell bodies. These results suggest that the amount of anterograde labeling observed after injection of WGA-HRP into both cut axons and undamaged cell bodies is significantly greater than the anterograde labeling observed after injections of HRP alone. Therefore, in the central nervous system WGA-HRP appears to be a far more effective anterograde tracer than HRP alone.     

Retrograde axonal transport of horseradish peroxidase from cornea to trigeminal ganglion

Summary Horseradish peroxidase (HRP) was dripped on the scarified left cornea of adult mice. Twentyfour hours later the animals were fixed by vascular perfusion and frozen sections cut from both trigeminal ganglia.After incubation for peroxidase activity labelled nerve cells were restricted to the medial ophthalmic part of the ganglion ipsilateral to HRP administration. If the scarification was omitted no neuronal labelling was observed. This labelling of the neurons is most probably the result from axonal uptake and subsequent retrograde axonal transport of the tracer.The similarity in distribution of peroxidase labelled nerve cells and the first ganglionic lesions occurring after instillation of herpes simplex virus in the cornea is pointed out.     

Trigeminocerebellar projections to the posterior lobe in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase.

Cerebellar projections of the nucleus interpolaris and oralis of the spinal trigeminal nucleus were studied in the cat by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into these nuclei labeled many mossy fiber terminals mainly ipsilaterally in the rostral folium of lobule IX (IXa or IXa + b), the simple lobule, the anterior part (sublobule A) of the paramedian lobule and the posterior part of crus II. Labeled terminals were also seen in the anterior lobe, lobules VI and VII, the anterior part of crus I, and the paraflocculus dorsalis. Projection fields in the horizontal plane of lobules were reconstructed from a series of transverse sections through each folium of lobule IX, the paramedian lobule, and the posterior part of crus II on the ipsilateral side. In sublobule IXa + b, labeled terminals were distributed in five longitudinal areas extending along the apicobasal axis of the sublobule. These five areas were located in the apical two-thirds of the ipsilateral half of the sublobule. Labeled terminals were distributed in five longitudinal areas in sublobule A (the rostral part) of the paramedian lobule. In the posterior part of crus II, four aggregations of labeled terminals were present in cross sections through a lobule. They were distributed in the apicobasal extent of the lobules. The present study indicates that the projection fields of trigeminocerebellar fibers are longitudinally arranged along the apicobasal axis of the cerebellar lobules.     

Central projections from cat suboccipital muscles: a study using transganglionic transport of horseradish peroxidase

Central projections of suboccipital muscle nerves were examined following exposure of cut peripheral nerves to the tracer horseradish peroxidase. Labelled fibers entered the C1 and C2 dorsal roots and accumulated in the dorsolateral part of the dorsal funiculus. Many labelled fibers entered the grey matter of C1 to C3 in ventrally directed bundles which passed medially to the base of the dorsal horn. No terminal labelling was apparent in superficial layers of the dorsal horn. However, labelled fibers ramified extensively throughout medial parts of the intermediate laminae, in and around the central cervical nucleus. Labelled fibers also projected toward the ventral horn. In cats subjected to ventral root section at the time of peripheral nerve exposure, a modest distribution of reaction product was observed deep in the ventral horn. In cats which did not undergo ventral root section, anterograde projections in the ventral horn were obscured by the simultaneous retrograde filling of motoneurons both in the ventromedial nucleus and on the medial and lateral borders of the gray matter. Labelled axons also coursed rostrally into the medulla where they formed a circumscribed bundle between the main cuneate nucleus and the spinal nucleus of V. Three consistent regions of HRP deposition could be identified at medullary levels. Dense accumulations of reaction product were present in circumscribed regions of the external cuneate nucleus (ECN) throughout its rostrocaudal extent. A second zone of dense labelling occurred in the intermediate nucleus of Cajal, where it appeared to form a continuing column rostral to the central cervical nucleus in C1-C3. Sparse labelling was restricted to a third zone in the ventrolateral part of the main cuneate nucleus.     

Origin of sympathetic and sensory innervation of the elbow joint in the rat: A retrograde axonal tracing study with wheat germ agglutinin conjugated horseradish peroxidase

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the elbow joint of adult rats, labeled neurons were found in the stellate and the T2-T4 ganglia of the ipsilateral sympathetic trunk, and also in dorsal root ganglia at the C4–T4 levels. Most labeled sympathetic cells, 90% or more, were located in the stellate ganglion. The sensory innervation to the joint originated mainly from the dorsal root ganglia at the levels of C7–T1.     

Rate of anterograde axonal transport of [I]wheat germ agglutinin from retina to optic tectum in the chick

We have investigated the rate of anterograde axonal transport of affinity-purified [¹²⁵I]wheat germ agglutinin (WGA) in chick retinal ganglion cells. By 2 lines of evidence, we found the rate to be 22–44 mm/day. This rate is slower than that reported for many other endogenously synthesized proteins or for horseradish peroxidase, the only other exogenously supplied protein studied as an anterograde transported marker. This difference in rate of transport of [¹²⁵I]WGA may reflect a novel pathway important in membrane recycling.     

Laminar-related projection of primary trigeminal fibers in the caudal medulla demonstrated by transganglionic transport of horseradish peroxidase

The mode of termination of primary afferent fibers within the cat trigeminal nucleus caudalis was investigated by means of the transganglionic transport of horseradish peroxidase (HRP). Several types of laminar-related labeling were observed, depending upon the survival time after HRP application. At the earliest survival time (28–34 h) the highest density of labeling was found in laminae I and II. At 2 and 3 days survival laminae III and IV were heavily labeled, in addition to laminae I and II where the amount of labeling was greatly increased in lamina I, but not in lamina II. At 5 days survival time an abrupt drop of labeling occurred in laminae I and II, while this pattern was not predominant in laminae III and IV. In lamina V the pattern of labeling was less intense and not changeable through all survival times observed. These findings indicating a differentiation of the primary afferent terminals have good correspondence with a functional specialization of neuronal locations since the functional properties of neurons vary according to their locations.     

HRP study of the central components of the trigeminal nerve in the larval sea lamprey: organization and homology of the primary medullary and spinal nucleus of the trigeminus

The medullary and spinal connections of the trigeminal nerve of larval sea lampreys Petromyzon marinus were studied by anterograde and retrograde HRP transport after application into the orbit. Three components were found, all of them ipsilateral: 1) The motor nucleus was undivided in the larva, and its neurons possessed a rich dendritic tree. The single motor root was well separated from the sensory root. 2) The descending root was laterally located, and its fibers ran compactly to spinal levels. 3) Most medullary and many rostral spinal dorsal cells were labeled. Dorsal cells, which were mostly multipolar, had numerous mutual contacts. Some dorsal cell processes contacted the fourth ventricle. The name "primary medullary and spinal nucleus of the trigeminal nerve" (PMSV) is proposed for these dorsal cells. Medullary dorsal cells were not labeled by applying HRP at the level of spinal nerves, but application to the vagus nerve did label some. The possible relationship of this nucleus with the mesencephalic trigeminal nucleus of jawed vertebrates is discussed.     

Central projections and somatotopic organisation of trigeminal primary afferents in pigeon (Columba livia)

Injections of cholera toxin B-chain conjugated to horseradish peroxidase into individual peripheral branches of the trigeminal nerve or into the trigeminal ganglion showed that an ascending trigeminal tract (TTA) terminated in distinct ventral and dorsal divisions of the principal sensory nucleus (PrVv and PrVd, respectively), and a descending tract (TTD) terminated within pars oralis, pars interpolaris, and pars caudalis divisions of the nucleus of TTD (nTTD) and within the dorsal horn of the first six cervical spinal segments. In PrVD, mandibular, ophthalmic, and maxillary projections were predominantly located dorsally, ventrally, and medially, respectively. In nTTD, mandibular projections lay dorsomedially, ophthalmic projections lay ventrolaterally, and maxillary projections lay in between. At caudal medullary and spinal levels, mandibular projections were situated medially, ophthalmic projections were situated laterally, and maxillary projections were situated centrally. The terminations within the dorsal horn were most dense in laminae III and IV and were least dense in lamina II, with laminae III-IV also receiving topographically organised contralateral projections. Extratrigeminal projections were mainly to the external cuneate nucleus by way of a lateral descending trigeminal tract (ITTD; Dubbeldam and Karten [1978] J. Comp. Neurol. 180:661–678) and to the region of the tract of Lissauer and lamina I of the dorsal horn. Other projections were to a region medial to the apex of pars interpolaris, to the nuclei ventrolateralis anterior (Vla) and presulcalis anterior (Pas) of the solitary complex, and sparsely to the lateral reticular formation (plexus of Horsley) ventral to TTD. No projections were seen to the trigeminal motor nuclei or to the cerebellum. © 1996 Wiley-Liss, Inc.