Fluorescent double‐label study of lateral reticular nucleus projections to the spinal cord and periaqueductal gray in the rat

Following injections of WGA‐HRP into either the spinal cord or periaqueductal gray, labeled neurons were 7observed bilaterally along the periphery of the lateral reticular nucleus (LRN) magnocellular division. The possibility that some of these neurons in the LRN provide collateral axonal branches to both the periaqueductal gray and the spinal cord was investigated in rats using a retrograde double‐labeling method employing two different fluorescent tracers, True Blue and Nuclear Yellow. Following sequential injection of the two fluorescent axonal tracers into the spinal cord and periaqueductal gray in the same animal, a modest number of double‐labeled neurons were observed bilaterally near the medial and dorsal perimeter of the magnocellular division of the LRN. The labeled neurons were distinctly multipolar in shape and measured approximately 15–18 μ in their greatest transverse diameter. No double‐labeled neurons were observed in the parvocellular or subtrigeminal divisions of the LRN. Based upon these observations, it is suggested that collaterals of the LRN‐spinal pathway provide feedback information to the periaqueductal gray that might then be used to modulate the participation of the latter cell group in a variety of pain processing and cardiovascular regulatory functions. Anat Rec 256:91–98, 1999. © 1999 Wiley‐Liss, Inc.     

Spinal projections to the lateral reticular nucleus in the rat

Summary The synaptic relationships and the distribution of the afferent terminals of the spinal pathway to the lateral reticular nucleus (LRN) of the rat were examined following induced degeneration. After high cervical hemisections, the spino-LRN projection was first examined with the Fink-Heimer silver impregnation method. Degeneration was confined primarily to the ipsilateral LRN and all three divisions of the nucleus were involved. Maximum degeneration was observed in the caudal regions of the parvocellular division. The magnocellular division, except for the extreme dorsomedial area, showed substantial degeneration as well. The subtrigeminal division throughout its entire length contained only sparse degeneration.Electron microscopic examination following spinal cord lesions revealed both round and pleomorphic-vesicle terminals in various stages of electron dense degeneration. The majority of the degenerating terminals were of the round-vesicle variety. Both types of terminals contacting somata were also observed to degenerate but their number was small in comparison to those on dendritic profiles. Terminals in synaptic contact with two dendritic profiles were also observed to degenerate. Some of the large terminals belonging to synaptic configurations (glomeruli) underwent degeneration and were therefore of spinal origin as well.     

Evidence that dopamine-beta-hydroxylase immunoreactive neurons in the lateral reticular nucleus project to the spinal cord in the rat.

The existence of noradrenergic projections from the lateral reticular nucleus (LRt) to the dorsal quadrant of cervical, thoracic, or lumbar spinal cord was investigated using a combined method of WGA-apo-HRP-gold retrograde tracing and dopamine-beta-hydroxylase (DBH) immunocytochemistry. Preliminary retrograde tracing studies indicated that LRt neurons projecting to cervical, thoracic, or lumbar spinal cord were characteristically located near the perimeter of the LRt. Double-labeling experiments demonstrated that a portion of these peripherally-located, spinal-projecting neurons were DBH-immunoreactive. Double-labeled neurons were also located at the parvocellular division of the contralateral LRt in the thoracic injection cases. Double-labeled neurons were not observed at the subtrigeminal division in cervical, thoracic, or lumbar injection case. The results suggest the possibility that the noradrenergic LRt-spinal pathway might be involved in a variety of pain processing and cardiovascular regulatory functions in the rat.     

Collateral projections from the midbrain periaqueductal gray to the nucleus raphe magnus and nucleus accumbens in the rat. A fluorescent retrograde double-labelling study.

Collateral projections of single neurons in the midbrain periaqueductal gray (PAG) to the nucleus accumbens (ACB) and nucleus raphe magnus (NRM) were observed in the rat by a fluorescent retrograde double-labelling technique. After injecting propidium iodide into the ACB and bisbenzimide into the NRM, doubly labelled PAG neurons were most frequently seen in the ventrolateral subnucleus and ventral part of the medial subnucleus at the middle and caudal levels of the PAG.     

A horseradish peroxidase study of the projections from the lateral reticular nucleus to the cerebellum in the rat

Summary The projection from the lateral reticular nucleus (LRN) to the cerebellar cortex was studied in the rat by utilizing the retrograde transport of horseradish peroxidase (HRP). In order to study the topographic features of this projection, small amounts of HRP were injected into various sites in the cerebellar cortex. The results demonstrated that the caudal lobules of the anterior lobe vermis tend to receive afferents from the medial LRN and the rostral lobules of the vermis receive afferents from more laterally situated cells. Lobules IV and V receive inputs primarily from the magnocellular division of the LRN of both the ventromedial and dorsolateral parts of the LRN, while lobules II and III receive inputs mainly from cells which lie in the border area between the parvocellular and magnocellular division of the ventromedial part. Following injections within various areas of the posterior lobe vermis, the results indicated that lobule VIII receives the most abundant projection from the LRN and that the cells of origin are present within the parvocellular and the adjacent part of the magnocellular division throughout the rostrocaudal extent of the LRN. Following injections within lobules VI and VII, few labelled cells were found and they tended to lie within the rostral two-thirds of the magnocellular division. Little or no projection from the LRN to lobule IX was evident. The hemispheres were found to receive a modest projection from the dorsal aspect of the LRN. The projection to lobulus simplex originates mainly from the caudal two-thirds of the magnocellular division, while the projection to the ansiform and paramedian lobules originates mainly from the dorsal aspect of the rostral two-thirds of the magnocellular division. Finally, there appears to be extensive overlapping of the orgins of all three projections to the cerebellar cortex studied, and this occurs within the central area of the magnocellular division throughout the rostrocaudal extent of the LRN.     

Visceral nociceptive input to the area of the medullary lateral reticular nucleus ascends in the lateral spinal cord

In halothane-anesthetized rats, neurons stereotaxically located in the region of the medullary lateral reticular nucleus (LRN) and responsive to urinary bladder distension (UBD) were characterized using extracellular electrodes. Most neurons excited by UBD were also excited by noxious stimuli applied to bilateral receptive fields comprising at least half of the body surface. These bilateral nociceptive specific (bNS) neurons exhibited graded responses to graded intensities of UBD. Neuronal responses to noxious UBD were highly positively correlated with responses to noxious colorectal distension, suggesting a convergence of visceral sensory information in the area of LRN. Bilateral lateral mid-cervical spinal cord lesions virtually abolished activity of bNS neurons evoked by noxious UBD, while dorsal midline lesions had no significant effect. These data support a role for neurons in the region of the LRN in visceral nociception and implicate traditional lateral spinal cord pain pathways in the transmission of visceral information to caudal ventrolateral medullary structures.     

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.     

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.     

A projection from the periaqueductal grey to the lateral reticular nucleus in the cat

Summary A projection from the periaqueductal grey (PAG) to the lateral reticular nucleus (NRL) in the cat was demonstrated by means of retrograde transport of the wheat germ agglutinin-horseradish peroxidase complex. The connection has its main origin ipsilaterally in the ventral part of the caudal PAG, but scanty projections from other parts of the PAG were also found. The neurons projecting to the NRL are of varying shapes and sizes, but most cells have a maximum diameter of less than 20 m. The findings are discussed in relation to the other afferent and efferent connections of the NRL.     

An ultrastructural study of the lateral reticular nucleus in the rat

Summary A systematic study of the normal synaptic patterns within the lateral reticular nucleus (LRN) of the rat revealed various synaptic relationships. Two types of axon terminals were identified according to the morphology of the synaptic vesicles contained within them. Axon terminals with round vesicles established asymmetrical synaptic contacts with the somata and all areas of the dendritic trees including somatic and dendritic appendages. Pleomorphic-vesicle terminals established symmetrical synaptic contacts on somata and their appendages and on all sizes of dendrites and their appendages. Both round and pleomorphicvesicle terminals were infrequently seen to synapse upon the somata and proximal dendrites. The round-vesicle terminals outnumbered the pleomorphic-vesicle terminals on the dendritic trees. Terminals of the en passant type were also common throughout the LRN. Both round and pleomorphic-vesicle terminals were observed simultaneously contacting the soma and one or more dendritic profiles, or two different dendritic profiles. Synaptic configurations (glomeruli) were also observed in all three divisions of the nucleus. They consisted of a large, central, round-vesicle terminal contacting a number of small-calibre dendritic processes. This arrangement was surrounded by one or more sheets of glial lamellae. Puncta adherentia were observed on the apposed membranes of adjacent cells, adjacent dendrites and adjacent axon terminals.     

A Golgi study of the lateral reticular nucleus in the rat

Summary The organization of the lateral reticular nucleus (LRN) of the rat was investigated by using the Golgi technique. Golgi-Cox preparations revealed neurons with shapes similar to those observed in Nissl-stained preparations. Fusiform cells possess rectilinear dendrites with secondary dendrites which are longer than the parent stem. The remaining cell types have short dendrites which branch for three or four generations and follow a tortuous course. These two types of neurons are similar to the isodendritic and allodendritic neurons which have been reported in the reticular formation. The neurons throughout the LRN form cell clusters. In Golgi preparations five to ten cells are seen in each cluster but counterstaining reveals that the clusters are made up of many more cells than the Golgi preparations suggest. Many cells lie in close apposition and the dendrites of the cells in each cluster intertwine to form dendritic plexuses. Dendritic input from both neighbouring and distant cell clusters also contributes to the plexus formations within each cell cluster. Under high magnification, the dendrites show irregularities in their contours, including warty excrescenses, bumps and an array of spines, some of which are pedunculated. The appendages are confined primarily to distal portions of the dendrites, with few spines observed on the somata and proximal dendrites. Varicosed dendrites are also in common occurrence throughout the nucleus.     

Afferent projections to the periaqueductal gray in the rabbit

The afferents to the periaqueductal gray in the rabbit have been described following hydraulic pressure injection of horseradish peroxidase at various sites throughout this structure. Every third section was reacted with tetramethylbenzidine, for the localization of afferent neurons. At the site of the deposit alternate sections were reacted with tetramethylbenzidine, Hanker-Yates reagent, or diaminobenzidine, for comparative assessment of the injection site. A large number of retrogradely labelled cells, assessed by bright- and dark-field microscopy, were observed in a wide range of areas throughout the brain. Major labelled areas within the telencephalon were cortical areas 5, 20, 21, 32 and 40. Within the diencephalon, the hypothalamus contained quantitatively by far the largest number of labelled cells. Of these nuclei, the dorsal pre-mammillary nucleus contained the largest number of labelled cells. Considerable labelling was also found within medial and lateral preoptic nuclei, anterior hypothalamic area, and ventromedial hypothalamic nucleus. Another diencephalic region containing a significant number of retrogradely labelled neurons was the zona incerta. At midbrain, pontine and medullary levels, additional labelled regions were: the substantia nigra, cuneiform nucleus, parabigeminal nucleus, raphe magnus, and reticular areas. Heavy labelling was seen within the periaqueductal gray itself, rostral and caudal to deposits placed within each subdivision. In addition, a large number of other areas labelled throughout the brain (Tables 2A-D). Not only were some differences noted in the pattern of labelled cells with deposits placed rostrally or caudally within periaqueductal gray, but certain topographical differences with respect to the degree of labelling within nuclei were also seen with injection sites ventral, lateral or dorsal to the aqueduct. In addition, a further difference was noted, in that over one third of the areas labelled with deposits in just one or other of the "divisions" within periaqueductal gray. The results therefore suggest that the periaqueductal gray might be divisible to some extent on the basis of connectivity with intrinsic subdivisions of the complex. It is hoped that, with time, it might prove possible to resolve any such differential input in functional terms. The wide variety of afferent input to the periaqueductal gray, and its strategic location, would seem to place it in a unique position for integrating and modifying a diversity of motor, autonomic, hormonal, sensory and limbic influences.(ABSTRACT TRUNCATED AT 400 WORDS)     

The organization of afferent projections to the midbrain periaqueductal gray of the rat

The retrograde transport technique was utilized in the present study to investigate the afferent projections to the periaqueductal gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductal gray of 22 experimental animals and into regions adjacent to the periaqueductal gray in 6 control animals. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductal gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled cells occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornical nucleus and the area of the tuber cinereum. The largest mesencephalic input to the periaqueductal gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis, the ventral tegmental area, the locus coeruleus and the parabrachial nuclei. The medullary and pontine reticular formation provide the largest input to the periaqueductal gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain.The sources of afferent projections to the periaqueductal gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.     

Descending inhibitory influences from periaqueductal gray, nucleus raphe magnus, and adjacent reticular formation. I. Effects on lumbar spinal cord nociceptive and nonnociceptive neurons

1. This study examined the inhibitory effects of conditioning stimuli delivered to the periaqueductal gray (PAG), nucleus cuneiformis (CU), nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis (NGC), and nucleus reticularis magnocellularis (NMC) on functionally identified neurons of the lumbar spinal cord dorsal horn in chloralose-anesthetized or decerebrate cats. 2. Neurons were classified according to their responses to a variety of cutaneous stimuli as low-threshold mechanoreceptive (LTM), wide dynamic range (WDR), or nociceptive specific (NS). The major aim of this study was to determine whether there was a difference in the effectiveness of the brain stem stimulation-produced inhibition of nociceptive (noci) neurons (consisting of both WDR and NS neurons) and the LTM non-nociceptive (nonnoci) neurons. There were no statistical differences in the susceptibility of WDR and NS neurons to brain stem-induced inhibition. 3. Most neurons tested could be inhibited by stimulation of any of the brain stem regions tested. In all cases the percentage of noci neurons inhibited from a given region was higher than the percentage of nonnoci neurons; however, this difference was only statistically significant in the case of NMC stimulation. 4. Threshold current intensities necessary to produce inhibition were determined for each neuron from each stimulation site. Although there was a trend for noci neurons to require slightly lower current intensities, there was in fact no statistically significant difference in the inhibitory thresholds between noci and nonnoci neurons for any of the regions tested. 5. A comparison of the mean threshold currents for the five regions studied revealed that the lowest stimulation currents were obtained in NMC with NRM, CU, NGC, and PAG, each requiring progressively higher current intensities in order to produce inhibition. 6. These results indicate that stimulation in PAG and NRM not only inhibits the responses of noci neurons but also those of nonnoci neurons. Moreover, stimulation in reticular regions adjacent to these two regions is effective in inhibiting the responses of both noci and nonnoci neurons.     

Efferent projections of the periaqueductal gray in the rabbit.

The efferent projections of the periaqueductal gray in the rabbit have been described by anterograde tract-tracing techniques following deposits of tritiated leucine, or horseradish peroxidase, into circumscribed sites within dorsal, lateral or ventral periaqueductal gray. No attempts were made to place labels in the fourth, extremely narrow (medial), region immediately surrounding the aqueduct whose size and disposition did not lend itself to confined placements of label within it. These anatomically distinct regions, defined in Nissl-stained sections, corresponded to the same regions into which deposits of horseradish peroxidase were made in order for us to describe afferent projections to the periaqueductal gray. In this present study distinct ascending and descending fibre projections were found throughout the brain. Terminal labelling was detected in more than 80 sites, depending somewhat upon which of the three regions of the periaqueductal gray received the deposit. Therefore, differential projections with respect to both afferent and efferent connections of these three regions of the periaqueductal gray have now been established. Ventral deposits disclosed a more impressive system of ramifying, efferent fibres than did dorsal or lateral placements of labels. With ventral deposits, ascending fibres were found to follow two major pathways from periaqueductal gray. The periventricular bundle bifurcates at the level of the posterior commissure to form hypothalamic and thalamic components which distribute to the anterior pretectal region, lateral habenulae, and nuclei of the posterior commissure, the majority of the intralaminar and midline thalamic nuclei, and to almost all of the hypothalamus. The other major ascending pathway from the periaqueductal gray takes a ventrolateral course from the deposit site through the reticular formation or, alternatively, through the deep and middle layers of the superior colliculus, to accumulate just medial to the medial geniculate body. This contingent of fibres travels more rostrally above the cerebral peduncle, distributing terminals to the substantia nigra, ventral tegmental area and parabigeminal nucleus before fanning out and turning rostrally to contribute terminals to ventral thalamus, subthalamus and zona incerta, then continuing on to supply amygdala, substantia innominata, lateral preoptic nucleus, the diagonal band of Broca and the lateral septal nucleus. Caudally directed fibres were also observed to follow two major routes. They either leave the periaqueductal gray dorsally and pass through the gray matter in the floor of the fourth ventricle towards the abducens nucleus and ventral medulla, or are directed ventrally after passing through either the inferior colliculus or parabrachial nucleus. These ventrally directed fibres merge just dorsal to the pons on the ventral surface of the brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural study of vestibular and reticular projections to the abducens nucleus

Summary The origin of the synaptic boutons in the abducens nucleus was studied following lesions of the contralateral medial vestibular nucleus, the ipsilateral paramedian pontine reticular formation and the contralateral dorsomedial part of the reticular formation caudal to the abducens nucleus.Lesions in the rostral part of the contralateral medial vestibular nucleus resulted in degeneration of boutons located mainly on dendritic processes. On the other hand, lesions in both ipsilateral and contralateral reticular formations provoked degenerating terminals on the somata of the abducens neurones and on proximal dendrites in the abducens nucleus beneath the genu of the facial nerve.This work was supported by INSERM (U6), CNRS (GR 45), and grant DGRST 78.7.3017     

Frontal cortical projections to the periaqueductal gray in the rat: a retrograde and orthograde horseradish peroxidase study

Injection of horseradish peroxidase solution into the eye of some european salamander species labeled retrogradely pretectal neurons. These neurons lie near the commissura posterior and extend their dendrites into the visual pretectal neuropil. On the average, each animal contained 28 labeled cells. Their distribution is bilaterally with a slight preference to the contralateral pretectum. As in other vertebrates these retrogradely labeled pretectal cells may be considered as efferents to the salamander retina.     

Ultrastructural study of ascending projections to the lateral mammillary nucleus of the rat

Summary We examined the synaptic organization of ascending projections from the pars ventralis of the dorsal tegmental nucleus of Gudden (TDV) and the laterodorsal tegmental nucleus to the lateral mammillary nucleus (LM). The LM neuropil consists of terminals containing pleomorphic synaptic vesicles and forming symmetric synaptic contact, and terminals containing round synaptic vesicles and forming asymmetric synaptic contact. They make up 63% and 37%, respectively, of all axodendritic terminals. All axosomatic terminals contain pleomorphic vesicles and make symmetric contact. Following injection of WGA-HRP into the TDV, many anterogradely labeled terminals and retrogradely labeled cells are found in the LM. Labeled terminals contact mainly proximal (more than 2 m diameter) and intermediate (1–2 m diameter) dendrites. Serial ultrathin sections of the LM show that 55% of axosomatic terminals are labeled anterogradely. Following injection of WGA-HRP into the laterodorsal tegmental nucleus, many anterogradely labeled terminals are found in the LM, but no retrogradely labeled cells are present. Labeled terminals contact mainly distal (less than 1 m diameter) and intermediate dendrites as well as somata. In the LM neurons, 46% of axosomatic terminals are labeled anterogradely. All labeled terminals from these nuclei contain pleomorphic vesicles and make symmetric synaptic contact. These results indicate that almost all axosomatic terminals come from the TDV and the laterodorsal tegmental nucleus, which send inhibitory inputs to the lateral mammillary nucleus.