Interpeduncular nucleus afferents in the rat

Afferents to the interpeduncular nucleus (IPN) of the rat were studied with the horseradish peroxidase (HRP) retrograde transport method. HRP was deposited microelectrophoretically in the IPN of adult rats. Major projections to the IPN originate in the medial habenular nucleus, the region surrounding the dorsal tegmental nucleus (accessory dorsal tegmental nucleus and the so-called dorsal tegmental nucleus pars lateralis), and the midbrain raphe (nucleus centralis superior and nucleus raphe dorsalis). Also, minor projections originate in the central gray and nucleus locus coeruleus. Our results indicate that the habenulointerpeduncular projection originates solely from the medial habenular nuclei and is topographically organized; medial regions of the medial habenular nuclei project to ventral portions of IPN and lateral regions project to the dorsal IPN.     

The dorsal tegmental nucleus: an axoplasmic transport study

The afferent and efferent connections of the dorsal tegmental nucleus (DTN) were studied in the rat using axoplasmic transport techniques. Horseradish peroxidase (HRP) and Fast Blue were injected stereotaxically into either pars centralis or pars ventromedialis of the DTN, two subdivisions of the nucleus with distinctive connected with the ipsilateral lateral mammillary and interpeduncular neclei; these projections constitute the major afferent and efferent systems of the DTN. Commissural fibers from the corresponding pars centralis and intrinsic fibers systems are massive and form a complex fiber meshwork within the subnucleus. The prepositus hypoglossi nuclei (bilateral) also project to the pars centralis. Smaller numbers of afferent fibers arise from the lateral habenular nucleus, the posterior hypothalamus and the brainstem reticular formation.The pars ventromedialis of the DTN receives diverse inputs which include the septal nuclei, diagonal band of Broca, preoptic area, anterior and lateral hypothalamus, lateral and medial habenular nuclei, medial mammillary nucleus and many nuclei of the brainstem reticular formation. Based on the differences of connections and cytoarchitecture between the pars and the pars ventromedialis, the pars ventromedialis may be an entity separate from the dorsal tegmental nucleus.     

Efferent projections of the lateral dorsal nucleus in the rat

The present study was undertaken to determine the efferent projections of the lateral dorsal nucleus of the rat thalamus using procedures based on anterograde degeneration as well as anterograde and retrograde axoplasmic transport. Discrete lesions or injections of [³H]leucine were placed at separate anterior and posterior loci in the lateral dorsal nucleus to determine if these portions of the nucleus project to different terminal fields. From both loci, labeled or degenerating axons coursed laterally from the nucleus, passed through the superior thalamic radiation, and entered the caudate-putamen complex. From there, a large number of fibers coursed rostrally in the album cerebri and terminated in layers I and III of anterior cingulate cortical areas 23 and 24. The remaining fibers passed dorsally and entered the posterior cingulate cortex where terminations were observed in areas 29b and 29c. Within area 29b, terminations were arranged parallel to the surface in layers I and III at its border with area 29c. In animals with anteriorly placed lesions or injections, area 29c exhibited input in layers I, III, and IV at the lip of the hemisphere. After injections in the posterior part of the lateral dorsal nucleus, a denser distribution was observed within the same layers at the border of areas 29b and 29c. Projections and areas of termination were also observed from both anterior and posterior lesions or injection sites to the laminae dissecans and externa of the pre- and parasubiculum. These projections were confirmed by retrograde axoplasmic transport of horseradish peroxidase after its injection in the cingulate and subicular cortices. These results indicate that the lateral dorsal nucleus, through its prominent connections with the anterior and posterior parts of the cingulate cortex and subicular region may have significant connections with the limbic system.     

Morphology and somatotopic organization of the central terminals of hindlimb hair follicle afferents in the rat lumbar spinal cord

The morphology of the central collateral arborizations of 24 A-beta hair follicle afferents (HFAs) innervating different regions of the skin of the hindlimb were studied by the intra-axonal injection of horseradish peroxidase (HRP) in adult rats. A total of 236 collaterals were recovered. These fell into three classes--complex, simple, and blind-ending--based on numbers of boutons and terminal branch patterns. The morphology of the HFA central arbors innervating the lateral and medial leg and dorsum of the foot was flame-shaped. Afferents with receptive fields on the glabrous-hairy skin border consistently had extra terminal branches running ventromedially into laminae IV/V. Differences in the width of terminal arbors were found. HFA terminals innervating the lateral leg formed narrower sheets than those innervating the dorsum of the foot and toes. The somatotopic organization of the collaterals and terminal arborizations of individual afferents were analyzed both by considering all the collaterals along an axon's rostrocaudal extent and by only examining arbors with boutons (the complex and simple arbors). Thirty-seven percent of blind-ending and 18% of simple collaterals were found to overlap in the rostrocaudal direction with the complex arborizations of afferents whose receptive fields were in a different cutaneous nerve territory. There was no overlap between complex arborizations of afferents from different nerve territories. However, the complex arbors of afferents with receptive fields within a particular nerve territory showed considerable terminal overlap even if they had nonadjacent peripheral receptive fields. The topographical organization of the central terminals of HFAs, forms a coarse somatotopic map of overlapping terminals whereby a particular region of dorsal horn has a maximal, but not exclusive, input from a particular area of skin.     

Topographic commissural and descending projections of the habenula in the rat

The habenular complex of the epithalamus connects the limbic basal forebrain with numerous neuromodulatory centers in the midbrain. The habenula consists of the medial and lateral nuclei, each of which is speculated to contain multiple subdivisions. Such anatomical arrangements raise the possibility that the habenula accounts for multiple channels of information flow from the limbic forebrain to the midbrain. For understanding whether and, if so, how the multiple streams are organized via the habenula, knowledge of the precise input–output connectivity of each habenular subdivision is essential. In the present study, biotinylated dextran amine and cholera toxin subunit B were used to delineate the differential outputs of various subregions of the medial and lateral nuclei of the habenula in the rat. Both anterograde and retrograde tracing uncovered a heavy commissural connection between the two habenulae on the ipsilateral and contralateral sides. The commissural projection arises primarily in the lateral nucleus and exhibits a fine topography in that a local commissural efferent terminates primarily in the corresponding subregion on the contralateral side. The subregions of the medial and lateral nuclei also give rise to distinct projections to midbrain areas such as the interpeduncular nucleus, the median/paramedian nuclei, and the central gray. These projections produce terminal fields centered in different areas of the targets, supporting the topographically organized descending projections from the habenula. These data together support the organization of multiple channels in the habenula that convey parallel streams of information to the contralateral habenula, midbrain, and brainstem. J. Comp. Neurol. 513:173–187, 2009. © 2009 Wiley‐Liss, Inc.     

Direct amygdaloid projections to the dorsal motor nucleus of the vagus nerve: a light and electron microscopic study in the rat

The projections from the central nucleus of the amygdala to the dorsal vagal complex were examined in the rat by means of anterograde and retrograde axonal transport of wheat germ agglutinin-horseradish peroxidase and anterograde degeneration. Light microscopic findings confirmed that the amygdala projects to the dorsal motor nucleus (DMV) and the nucleus of the solitary tract. Electron microscopic experiments demonstrated degenerating axosomatic and axodendritic terminals in the DMV following electrolytic lesions in the central nucleus of the amygdala.     

Afferent and efferent innervation patterns of the cochlear nucleus (dorsal medullary nucleus) of the leopard frog

The afferent and efferent innervation patterns of the frog dorsal medullary nucleus (DMN; anuran homolog of the cochlear nucleus) were examined by studying the anterograde and retrograde transport patterns of horseradish peroxidase injected focally into the nucleus. It was found that this structure projected bilaterally to the superior olivary nuclei (SON) and dorsal midbrain tegmental nuclei, and contralaterally to the opposite DMN, the lateral lemniscus nucleus (LLN) and the torus semicircularis (TS). The termination sites in the TS were restricted to the laminar and principal nuclei. The DMN in turn received projections from these structures with the exception of the TS and dorsal tegmental nuclei. The projection to the ipsilateral LLN and TS was not pronounced. In addition to the above findings, the ascending projection to the DMN, SON and TS, as well as the centrifugal projection from the SON, were found to be organized tonotopically.     

Sources of projections to subdivisions of the inferior colliculus in the rat

Brainstem and forebrain projections to major subdivisions of the rat inferior colliculus were studied by using retrograde and anterograde transport of horseradish peroxidase. Retrograde label from injection into the external cortex of the inferior colliculus appears bilaterally in cells of the inferior colliculus, as well as in other brainstem auditory groups including the ipsilateral dorsal nucleus of the lateral lemniscus and contralateral dorsal cochlear nucleus. The external cortex is the only collicular subdivision where an injection labels cells in the contralateral cuneate nucleus, gracile nucleus, and spinal trigeminal nucleus. Other projecting cells to the external cortex are found in the lateral nucleus of substantia nigra, the parabrachial region, the deep superior colliculus, the midbrain central gray, the periventricular nucleus, and area 39 of auditory cortex. Injection of the dorsal cortex of inferior colliculus heavily labels pyramidal cells of areas 41, 20, and 36 of the ipsilateral neocortex. Anterograde label from a large injection of auditory cortex is densely distributed in the dorsal cortex, lesser so in the external cortex, and only slightly in the central nucleus. Labelled cells appear in the central nucleus, dorsal cortex, and external cortex, primarily ipsilaterally, following dorsal cortex injection. Relatively few cells from other brainstem auditory groups show projections to the dorsal cortex. Injection of the central nucleus of the inferior colliculus results in robust labelling of nuclei of the ascending auditory pathway including the anteroventral, posteroventral, and dorsal cochlear nuclei (mainly contralaterally), and bilaterally the lateral superior olive, lateral nucleus of the trapezoid body, dorsal nucleus of the lateral lemniscus, and the central nucleus, dorsal cortex, and external cortex of the colliculus. The medial superior olive, superior paraolivary nucleus, and ventral nucleus of the trapezoid body essentially show ipsilateral projections to the central nucleus. The differential distribution of afferents to the inferior colliculus provides a substrate for functional parcellation of collicular subdivisions.     

Termination in trigeminal nucleus oralis of ascending intratrigeminal axons originating from neurons in the medullary dorsal horn: an HRP study in the rat employing light and electron microscopy

The anterograde horseradish peroxidase (HRP) technique was used to identify ascending intratrigeminal axons originating from neurons in the medullary dorsal horn (MDH) which terminate in trigeminal nucleus oralis (Vo). HRP injections into the MDH labeled two populations of axons ascending ipsilaterally within the spinal trigeminal nucleus. The first population was composed of parent branches which each gave off a single branching collateral strand to Vo as they ascended. These collaterals were characterized by boutons filled with small, round synaptic vesicles and forming asymmetrical synaptic contacts with large diameter dendritic shafts. The second axonal population was made up of parent branches which terminated directly in Vo. Their short terminal strands were distinguished by axonal endings containing pleomorphic synaptic vesicles and forming symmetrical synaptic junctions with small diameter dendritic shafts and spines.     

Excitatory projection from the interpeduncular nucleas to central superior raphe neurons

The projection from the interpeduncular nucleus (IP) to the central superior raphe nucleus (CS) was studied using intracellular electrophysiologic methods. IP stimulation generates monosynaptic EPSPs in a large number of CS neurons studied with latency of 1–2 ms. Intracellular peroxidase injections into CS neurons responding to IP shock confirmed the location and somatic origin of intracellular potentials. These findings document the existence of a direct excitatory projection from IP onto CS neurons.     

The origins of cholinergic and other subcortical afferents to the thalamus in the rat

The origins of the cholinergic and other afferents of several thalamic nuclei were investigated in the rat by using the retrograde transport of wheat germ agglutinin conjugated-horseradish peroxidase in combination with the immunohistochemical localization of choline acetyltransferase immunoreactivity. Small injections placed into the reticular, ventral, laterodorsal, lateroposterior, posterior, mediodorsal, geniculate, and intralaminar nuclei resulted in several distinct patterns of retrograde labelling. As expected, the appropriate specific sensory and motor-related subcortical structures were retrogradely labelled after injections into the principal thalamic nuclei. In addition, other basal forebrain and brainstem structures were also labelled, with their distribution dependent on the site of injection. A large percentage of these latter projections was cholinergic. In the brainstem, the cholinergic pedunculopontine tegmental nucleus was retrogradely labelled after all thalamic injections, suggesting that it provides a widespread innervation to the thalamus. Neurons of the cholinergic laterodorsal tegmental nucleus were retrogradely labelled after injections into the anterior, laterodorsal, central medial, and mediodorsal nuclei, suggesting that it provides a projection to limbic components of the thalamus. Significant basal forebrain labelling occurred only with injections into the reticular and mediodorsal nuclei. Only injections into the reticular nucleus resulted in retrograde labelling of the cholinergic neurons in the nucleus basalis of Meynert. The results provide evidence for an organized system of thalamic afferents arising from cholinergic and noncholinergic structures in the brainstem and basal forebrain. The brainstem structures, especially the cholinergic pedunculopontine tegmental nucleus, appear to project directly to principal thalamic nuclei, thereby providing a possible anatomical substrate for mediating the well-known facilitory effects of brainstem stimulation upon thalamocortical transmission.     

Ultrastructure of the dorsal motor nucleus of the vagus nerve in monkey with a comparison of synaptology in monkey and cat

Neurons of the dorsal motor nucleus of the vagus nerve were studied following injections of horseradish peroxidase into the vagus nerve in a monkey (Macaca fascicularis). In frozen sections, the dorsal motor nucleus appeared to be completely filled by labeled medium-sized (20-30 micron in long axis) neurons. Labeled dendrites from these neurons often extended outside the borders of the nucleus into the nucleus of the tractus solitarius. In 1 micron thick plastic sections and ultrathin sections of the dorsal motor nucleus, two distinct types of neurons were observed with the light and electron microscope. Medium-sized neurons with abundant cytoplasm and an oval nucleus were retrogradely labeled with HRP, while small (10-15 micron in long axis) neurons with a paucity of organelles and an invaginated nucleus remained unlabeled. Medium-sized neurons outnumbered the small neurons by approximately five to one. The synaptic organization of the dorsal motor nucleus in monkey was studied and compared with that in cat. The porportions of different types of axosomatic synapses were similar in both species. Terminals containing round vesicles and making symmetrical or asymmetrical contact with the postsynaptic structure were more common than synaptic terminals containing pleomorphic vesicles. In both species, there was a slightly greater synaptic density on the medium-sized neurons than on the small neurons. The synaptic density in the monkey dorsal nucleus was greatest on the smallest dendrites in the neuropil and least on the somata.     

Connections of the nucleus incertus

The nucleus incertus (NI) is a distinct cell group in caudoventral regions of the pontine periventricular gray, adjacent to the ventromedial border of the caudal dorsal tegmental nucleus. Recent interest in the NI stems from evidence that it represents one of the periventricular sites with the highest expression levels of mRNA encoding the type 1 corticotropin-releasing hormone (CRH) receptor, which has a high affinity for naturally occurring CRH, perhaps accounting for some of the extrapituitary actions of the peptide on autonomic and behavioral components of the stress response. However, almost nothing is known about NI function and hodological relationships. In this paper, we present the results of a systematic analysis of NI inputs and outputs using cholera toxin B subunit as a retrograde tracer and Phaseolus vulgaris-leucoagglutinin as an anterograde tracer. Our retrograde tracer experiments indicate that the NI is in a strategic position to integrate information related to behavioral planning (from the prefrontal cortex), lateral habenular processing, hippocampal function, and oculomotor control. Based on its efferent connections, the NI is in a position to exert significant modulating influences on prefrontal and hippocampal cortical activity, and the nucleus is also in a position to influence brain sites known to control locomotor behavior, attentive states, and learning processes. Overall, the present results support the idea that the NI is a distinct region of the pontine periventricular gray, and together with the superior central (median raphé) and interpeduncular nuclei the NI appears to form a midline behavior control network of the brainstem.     

A projection from the entorhinal cortex to the nucleus accumbens in the rat

A study was performed in which both anterograde ([³H]leucine radioautography) and retrograde (horseradish peroxidase (HRP) histochemistry) tracing methods were employed to identify the origin of the fimbrial projection to the nucleus accumbens in the rat. The data reveal that this pathway arises predominantly from layers II–III of the anterior two-thirds of entorhinal cortex rather than from any part of hippocampal formation.     

Single fiber studies of ascending input to the cuneate nucleus of cats: II. Postsynaptic afferents.

The morphology of single postsynaptic afferent fibers terminating in the feline cuneate nucleus was investigated by using transport of Phasolus vulgaris leucoagglutinin from the cervical spinal cord and intraaxonal injections of horseradish peroxidase into identified postsynaptic fibers in the cuneate fasciculus. Injections of Phaseolus in C5 and C6 of both rhizotomized and non-rhizotomized cats gave similar results and confirmed previous observations with other techniques. In one animal with the smallest injection and the fewest labeled fibers in the cuneate nucleus, ten individual collaterals were reconstructed from serial sections. Most of these collaterals were at middle levels of the cuneate (from obex to about 4 mm caudal to it); they were largely confined to the rim and ventral regions of the nucleus, and their terminal fields were restricted rostrocaudally. Electrophysiologically identified fibers stained with horseradish peroxidase had large receptive fields on the ipsilateral forepaw, and latencies suggesting an oligosynaptic link to the periphery. Most of the collaterals from these fibers were also at middle cuneate levels and terminated mainly at the periphery of the nucleus but gave rise to larger terminal arbors, including sparse terminal branches to the core of the nucleus. Individual postsynaptic fibers differed in several respects from primary afferent fibers. While the spacing of collaterals of postsynaptic fibers was intermediate between that of G hair and Ia fibers, their arbors were larger than either, and could extend through the dorsoventral extent of the cuneate nucleus. The pattern of bifurcation of postsynaptic fibers resulted in stringier arbors which encompassed larger and less dense terminal fields than those of primary afferents. The number of boutons per collateral was intermediate between G hair and Ia fibers, but boutons of postsynaptic fibers were substantially smaller. These morphological differences are consistent with distinct functional roles for the two main ascending afferent systems, as suggested by electrophysiological data.     

Mode of termination of afferents from the thalamic reticular nucleus in the dorsal lateral geniculate nucleus of the rat

The terminals of axons projecting to the dorsal lateral geniculate nucleus from the thalamic reticular nucleus were identified by electron microscopy 8–24 h after placing small lesions in the ipsilateral reticular nucleus. The terminals contained flattened synaptic vesicles and made Gray type II axo-dendritic synaptic contacts with geniculate neurons. Their identification as F-axons accords well with physiological evidence for a powerful monosynaptic inhibitory input to geniculocortical projection cells from reticular nucleus neurons.     

Sources of noradrenergic afferents to the hypoglossal nucleus in the rat

The sources of noradrenergic (NA) innervation to the hypoglossal nucleus (nXII) in the rat were investigated with double-labeling histochemical/immunocytochemical and lesion/degeneration techniques. Following injection of wheat germ-agglutinin conjugated to horseradish peroxidase into nXII, brain stem sections were reacted with tetramethylbenzidine, stabilized, and incubated in antiserum to tyrosine hydroxylase (TH). Double-labeled neurons were observed in three pontine sites bilaterally, although mainly ipsilaterally, that included the nucleus subceruleus (nSC; 68.75%) and the A7 (21.09%) and A5 (10.15%) cell groups. Confirmation of the above results and identification of the course taken by descending NA-nXII projections was accomplished by lesioning the rostral pons, the nSC, or the medullary catecholamine bundle (MB), the suspected route by which NA afferents reach nXII. Quantitative estimates of the reduction of TH immunoreactivity on the lesioned compared to nonlesioned side of nXII were made densitometrically. In each case, TH immunostaining was significantly decreased (75%) in the ipsilateral caudoventromedial district of nXII, the predominant target area of NA input. The results from this study establish that multiple NA sources in the pons project to nXII in the rat, the majority of NA-nXII afferents are derived from the nSC, and descending NA-nXII projections course in the MB. These data are discussed relative to tongue control.     

Structure-function relationships in rat brainstem subnucleus interpolaris. I. Vibrissa primary afferents

Intra-axonal recording and horseradish peroxidase labelling techniques were used to examine structure-function relationships for vibrissa-sensitive primary afferent fibers (N = 40) in rat trigeminal brainstem subnucleus interpolaris (SpVi). All responded at short (mean-0.42 ms) latencies to trigeminal ganglion shocks and to innocuous stimulation of an individual vibrissa in a slowly adapting type I, slowly adapting type IIa, slowly adapting type IIb, low-velocity-sensitive rapidly adapting, or high-velocity-sensitive rapidly adapting fashion. As in the medullary dorsal horn (Hayashi, '82; Jacquin et al., '86a), functionally distinct mystacial vibrissae-related fibers were morphologically indistinguishable. Each gave rise to up to ten collaterals that entered interpolaris perpendicular to the long axis of the nucleus and often overlapped to form a densely packed, highly circumscribed, and largely continuous column of terminal arbors. While some morphological variability was observed both within and between individual axons, variance within a given functional class was no greater than that between classes. Nonmystacial vibrissae afferent arbors also formed similar ovoid, dense circumscribed terminal plexuses. Presumably, these individual arbors collectively form a longitudinal tubelike vibrissae representation in this and other components of the trigeminal brainstem nuclear complex. The relative locations of each fiber's terminal field could be accurately predicted by the particular vibrissa innervated. Contrary to previous data obtained with similar methods (Hayashi, '82), but consistent with the findings of earlier transganglionic tracing (Arvidsson, '82) and histochemical (Belford and Killackey, '79) studies, the arbors of these fibers terminated throughout the mediolateral extent of SpVi. Axons innervating rostral vibrissae terminated medially, and those that supplied caudal vibrissae innervated the lateral SpVi. Dorsal vibrissae were represented in the ventral SpVi, while ventral vibrissae were represented more dorsally. This transverse topography extended largely throughout the rostrocaudal extent of interpolaris. In summary, the vibrissae primary afferent map in SpVi is inverted, faces medially, and is rostrocaudally consistent. In its most caudal aspect, as the displaced substantia gelatinosa swings laterally, the map is less orderly and incomplete. These generalizations also apply to the nonmystacial vibrissae afferents.     

Synaptic organization of the rat parafascicular nucleus,with special reference to its afferents from the superior colliculus and the pedunculopontine tegmental nucleus

The synaptic organization of afferents to the parafascicular nucleus (Pf) of the thalamus was studied in rats. In the Pf, three types of axon terminals were identified: the first type was a small terminal with round synaptic vesicles forming an asymmetric synapse, the second type was a large terminal with round synaptic vesicles forming an asymmetric synapse, and the third type was a terminal with pleomorphic vesicles forming a symmetric synapse. They were named SR, LR and P boutons, respectively. In order to determine the origin of these axon terminals, biotinylated dextran amine (BDA) was injected into the main afferent sources of the Pf, the superior colliculus (SC) and the pedunculopontine tegmental nucleus (PPN). Axon terminals from the SC were both SR and LR boutons which made synaptic contacts with somata and dendrites. PPN afferents were SR boutons, which made synaptic contacts with somata and smaller dendrites. Double-labeled electron microscopic studies, in which a retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) was injected into the striatum and an anterograde tracer (BDA) into the SC revealed that SC afferent terminals made synapses directly with Pf neurons that projected to the striatum. Another experiment was performed to find out whether two different afferents converged onto a single Pf neuron. To address this question, two different tracers were injected into the SC and PPN in a rat. Electron microscopically, both afferent terminals from the SC and PPN made synaptic contacts with the same dendrite. Our results prove that a single neuron of the rat Pf received convergent projections from two different sources.