Afferent projections to the self-stimulation regions of the dorsal pons, including the locus coeruleus, in the rat as demonstrated by the horseradish peroxidase technique

Afferent projections to the dorsal pons of the rat have been studied using the horseradish peroxidase (HRP) technique. HRP injections were made in each of the following regions: the vicinity of the locus coeruleus (LC); the periventncular gray, medial to the LC; the medial parabrachial region, lateral to the LC; the ventral cerebellum, dorsal to the LC; and the pontine reticular formation, ventral to the LC. Because intracranial self-stimulation (ICSS) has been obtained in these regions, the afferents have been discussed in terms of their possible contributions to that behavior. Previous ICSS studies of the dorsal pons have focussed on the LC as playing a central role. Presently identified inputs to the LC include: the dorsal raphe nucleus: the ventrolateral periaqueductal gray: the pontine reticular formation: the areas that contain the pontine and medullary noradrenergic and adrenergic cell groups: the lateral hypothalamic area: the contralateral LC: the deep cerebellar nuclei: the ventrolateral and parafascicular thalamic nuclei: and the parabrachial regions of the pons and midbrain.     

Descending modulation of visceral nociceptive transmission from the locus coeruleus/subcoeruleus in the rat

The purpose of the present investigation was to examine whether electrical stimulation in the locus coeruleus/subcoeruleus (LC/SC) could modulate visceral pain evoked by noxious colorectal distention (CRD). Experiments were performed on 40 pentobarbital anesthetized male Sprague–Dawley rats. Extracellular potentials of single L₆–S₂ spinal neuron were recorded with a carbon filament electrode. CRD (80 mmHg) was produced by inflating a balloon inside the descending colon and rectum. Electrical stimulation of the LC/SC (30, 50 and 70 μA, 100 Hz, 0.1 ms pulses) was delivered either ipsilaterally or contralaterally. Results showed that for 42/62 (68%) short-latency abrupt (SL-A) neurons, all of the short-latency sustained (SL-S) and long-latency (LL) neurons, LC/SC stimulation produced intensity-dependent attenuation of the CRD-evoked discharge. For 10/62 (16%) SL-A neurons, 6/8 (75%) inhibited (INHIB) neurons LC/SC stimulation increased the evoked discharge, for 10/62 (16%) SL-A neurons and 2/8 (25%) INHIB neurons, the evoked discharges were unaffected by the LC/SC stimulation. LC/SC stimulation also had different effects on the spontaneous activities of these neurons. The effects of LC/SC stimulation were the same both ipsilaterally and contralaterally either for the evoked discharges or for spontaneous activities. Following LC/SC lesions, LC/SC stimulation did not inhibit nociceptive responses, whereas inhibitory effects were observed by stimulation of the intact LC/SC contralateral to the recording site. These data suggest that the transmission of visceral pain was under the control of the centrifugal pathways from the LC/SC.     

Differential projections from locus coeruleus to olfactory bulb and olfactory tubercle: an HRP study

The microiontophoretic administration of horseradish peroxidase (HRP) to the olfactory bulb (OB) or olfactory tubercle (OT) in cats and rats yielded similar results in both species. After an OB HRP-injection ipsilateral and contralateral labelled neurons were seen in the piriform cortex, polymorphic layer of OT, magnocellular preoptic region, lateral hypothalamus, ventromedial hypothalamic nucleus and locus coeruleus (LC). In both species more labelled structures were found after an OT HRP-injection than after an OB HRP-injection. The substantia nigra in rats was more abundantly labelled after an OT injection than after an OB one. In cats the dorsal and the ventral raphe were also labelled. In either species, OT HRP-injections resulted in a higher frequency of LC labelled neurons than after OB injections. These results favor the hypothesis that the OT plays an important role as a relay station for efferent inflow from the brain stem en route to the OB.     

Raphe projections to the locus coeruleus in the rat

Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 μl of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus, lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among others, by most of the raphe nuclei. Thus, various activities of the locus coeruleus could be modulated or regulated by widespread projections from most raphe nuclei as well as several other regions of the brainstem.     

Descending projections of the locus coeruleus and subcoeruleus/medial parabrachial nuclei in monkey: Axonal transport studies and dopamine-β-hydroxylase immunocytochemistry

Spinal projections originating in the dorsolateral pons in the ventral locus coeruleus and in the subcoeruleus and medial parabrachial nuclei were identified in monkeys (Macaco fascicularis) using the retrograde horseradish peroxidase tracing technique. Anterograde autoradiographic tracing studies were then carried out to determine the brain stem and spinal cord terminations of the neurons. Finally, results obtained with the axonal transport tracing methods were compared with the pattern of staining for noradrenergic cells and terminals revealed immunocytochemically with an antibody to dopamine-β-hydroxylase (DβH), the synthesizing enzyme for norepinephrine. The major findings of these studies are that two presumed noradrenergic cell groups of the dorsolateral pons, one corresponding to the nucleus locus coeruleus, the second to the subcoeruleus/medial parabrachial nuclei, give rise to descending project-ions. They differ significantly in their patterns of termination in the lower brain stem and spinal cord. Among the major terminations of the locus coeruleus pathway are projections to parasympathetic neurons of the dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, and the sacral spinal cord. The terminations of the descending subcoeruleus/medial parabrachial pathway, in contrast, include project-ions to sympathetic preganglionic neurons of the intermediolateral cell column of the thoracic cord and heavier projections to somatic cranial nerve nuclei. Both pathways have additional widespread and bilateral terminations in various nuclei of the reticular formation, in the spinal dorsal horn (including the marginal zone), in the region around the central canal and in the ventral spinal gray matter. Since the origins and terminations of both these pathways correspond closely to the locations and patterns of terminations of noradrenaline-containing neurons, demonstrated here with DβH immunocytochemistry, norepinephrine (or epinephrine) is suggested to be the transmitter in both these descending systems.     

The afferent projections to the centrum medianum of the cat as demonstrated by retrograde transport of horseradish peroxidase

The afferent projections of nucleus centrum medianum (CM) of the thalamus were studied, in the cat, by means of retrograde transport of electrophoretically ejected horseradish peroxidase. Several variations of method — survival time, fixatives, substrates, etc. — were tried to improve the amount of visible reaction product.Labeled neurons were localized primarily in two categories of nuclei in the brain. The first consisted of structures making up or closely related to the basal ganglia: the entopeduncular nucleus, the pars reticulata of the substantia nigra, and motor cortex. The second category was made up of nuclei closely related to postural and orienting functions: the deep layers of the superior colliculus ipsilaterally, and the medial and lateral vestibular nuclei bilaterally. Other nuclei containing retrogradely labeled neurons were the periaqueductal gray and locus coeruleus. Brain stem reticular projections were sparse and widely scattered. These results identify CM as an important element in the loop system linking medial thalamus and neostriatum; the probable attention and orientation related functions of this system are discussed.     

Cerebellar afferents from motor nuclei of cranial nerves, the nucleus of the solitary tract, and nuclei coeruleus and parabrachialis in sheep, demonstrated with retrograde transport of horseradish peroxidase

Following HRP injections in the cerebellar cortex of the sheep (except the ventral part of the anterior lobe, the flocculus and ventral paraflocculus), labeled cells were evident in motor nuclei of cranial nerves (XII, VII, VI, III, visceromotor nucleus of X and nucleus ambiguus), in the solitary tract nucleus, the nucleus coeruleus and the parabrachial nucleus.     

Nonidet P40 and the retrograde transport of horseradish peroxidase in undamaged visceral nerves

The cell bodies of origin of peripheral nerves, in particular visceral nerves, are often difficult to identify using standard horseradish peroxidase (HRP) methods. The non-ionic surfactant Nonidet-P40, when applied to intact peripheral nerve along with HRP, allows the investigator to examine the neurons of origin of the nerve without cutting the fibers or injecting label into its peripheral terminal field.     

Cerebellar inhibition and ICSS from stimulation in the area of the nucleus locus coeruleus

The purpose of this study was to determine the relationship between intracranial self-stimulation (ICSS) and the long-lasting inhibition (LLI) of cerebellar Purkinje cells which are produced by stimulation around the dorsal pontine nucleus locus coeruleus (LC). No strong correlation was found between the dorsal pontine sites which produced LLI and those sites which yielded ICSS. Moreover, ICSS sites were no more effective than non-ICSS sites in producing LLI. LLI of Purkinje cells was produced most effectively by stimulation of an area dorsolateral to the LC where axons arising from the LC collect to ascend to the cerebellum. The LLI produced by stimulation of this dorsolateral region was less often associated with short latency excitations, compared to the LLI produced by stimulation of the cerebellar white matter. This characteristic may be useful as an indication of LC-produced LLI. Sites yielding ICSS were scattered around the LC but were most consistent ventrolateral to the LC. These results indicate that ICSS and LLI of Purkinje cells appear to be independent phenomena which depend on different mechanisms.     

Regional topography within noradrenergic locus coeruleus as revealed by retrograde transport of horseradish peroxidase.

A hitherto unsuspected degree of regional topographic organization in the noradrenergic nucleus, locus coeruleus, was revealed by the use of retrograde transport of horseradish peroxidase (HRP) from terminal areas receiving noradrenergic innervation. HRP was injected into hippocampus, hypothalamus, thalamus, caudate-putamen, septum, amygdala-piriform cortex, cerebellum and cortex. Successful transport was obtained from all areas, including the caudate-putamen and cerebral cortex. The pattern of HRP positive cells in the ipsilateral locus coeruleus was markedly different depending on the location of the HRP injection. Thus, hippocampal injections labeled cells in the dorsal locus coeruleus but not at all in the ventral tip. Injections of HRP into caudate-putamen or cerebellum labeled the ventral tip along with the rest of the dorsal portion. HRP injections into the septum labeled cells only in the dorsal half of the dorsal locus coeruleus. There thus exists a three tier division of locus coeruleus into the ventral one third, dorsal one third and intermediate one third. A further division was seen in the anterior-posterior plane with HRP injections into the thalamus labeling the posterior pole of locus very intensely but with little transport to more anterior levels; conversely HRP injection into the hypothalamus resulted in intense labeling only in the anterior pole of locus coeruleus. Amygdala-piriform cortex HRP injections revealed a further pattern with very intensely reactive cells scattered sparsely throughout the nucleus. Cortical HRP injections yielded weaker labeling also in occasional, scattered cells. All HRP transport to locus coeruleus was shown to be noradrenergic by degeneration with 6-hydroxydopamine and due to terminal, rather than fiber of passage, uptake by control injection into the dorsal NA bundle. It is concluded that the locus coeruleus is not an homogenous nucleus with respect to the origin of the noradrenergic projections to sundry forebrain, spinal and cerebellar areas but is comprised of distinct subdivisions of noradrenergic neurons.     

Galanin-containing neurons in the solitary nucleus and locus coeruleus of spontaneously hypertensive rats are associated with genetic hypertension

Spontaneously hypertensive (SHR) rats contained more galanin (GAL) content and GAL mRNA in locus coeruleus (LC) at the prehypertensive, but not at the well-established hypertensive stage, than did age-matched Wistar-Kyoto (WKY) rats. However, there was also more GAL content, but not GAL mRNA, in the nucleus tractus solitarii (NTS) of SHR rats than WKY rats at both stages. This study suggests that galaninergic neurons in the LC and NTS may participate in the pathogenesis of genetic hypertension.     

Spinal projections of the locus coeruleus and the nucleus subcoeruleus in the Harlan and the Sasco Sprague-Dawley rat

The descending projections of the locus coeruleus (LC) and the nucleus subcoeruleus (SC) to the lumbar spinal cord were examined in rats from two vendors using retrograde transport of fluorescent latex beads. There was a vendor difference observed which agrees with previous findings. The differential dorsal horn and ventral horn projections of the Harlan and the Sasco Sprague-Dawley rats, reported by Fritschy and Grzanna, and Clark and Proudfit were confirmed. In the Harlan rat more cells were labeled in the LC following injections in the dorsal horn. In contrast, in the Sasco rat, more cells were labeled in the LC from injections in the ventral horn. Although, in all studies, the LC in rats from these vendors projected to some extent to both the dorsal and the ventral horn. A difference in labeling was noted also for the depth of placement of the tracer in the dorsal horn. When the site of injection was in the nucleus proprius, a predominately contralateral projection of the LC was noted. In contrast, when horseradish peroxidase (HRP) gel implants were placed to include the superficial laminae, the cells in the LC were labeled predominately ipsilaterally. The SC has a major projection to the dorsal horn in the Harlan rats while cells in the SC were predominately labeled following ventral horn injections in the Sasco rats. These cells send mostly ipsilateral projections to the dorsal and ventral horn of the spinal cord. Double labeled studies confirmed that 91% of LC and 86% of SC neurons projecting to the spinal cord were noradrenergic. The present results confirmed a difference in the descending catecholamine projections of rats purchased from different vendors. These strain differences may prove useful in studies of motor and sensory systems.     

Laminar origins of spinothalamic projections in the cat as determined by the retrograde transport of horseradish peroxidase

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the locations of cells of origin of the spinothalamic tract in the cat. Injections of from 0.2–3.0 μl of 30% HRP were made unilaterally in various regions of the somatosensory thalamus. Massive injections of the caudal thalamus in several cats showed the spinothalamic cells of origin to be located mainly in laminae I, VII and VIII in the lumbar enlargement, and in laminae I, V and VII–VIII in the cervical enlargement. Small injections of HRP were made into the three major spinothalamic terminal zones in the thalamus, to determine the laminar origin(s) of the spinal projections to each zone. Neurons in lamina I in both cervical and lumbar enlargements were found to project almost exclusively to the rostral VB-caudal VL border zone. A small number of neurons in laminae VII and VIII also project there but a larger number project to the intralaminar region. Neurons projecting to the PO regions were located mainly in laminae IV and V. This anatomical segregation of thalamic afferents probably reflects a functional segregation of input, since the functional properties of spinal neurons vary according to their laminar location. Comparison of these data with the differential projection spinothalamic neurons in the rat and monkey indicate that it is unlikely that the proposed “paleo-” and “neospinothalamic” systems would arise from anatomically separate groups of spinal neurons.     

Afferent projections to the thalamic mediodorsal nucleus in the cat studied by retrograde and anterograde axonal transport of horseradish peroxidase

The afferent pathways to the thalamic mediodorsal nucleus (MD) in the cat were studied using the methods of anterograde and retrograde axonal transport of horseradish peroxidase (HRP) and wheat germ agglutinin conjugated to HRP (WGA-HRP). The MD receives fibers from the prefrontal cortex in a topically organized manner in accordance with the thalamocortical projections. The medial or ventral portion of the MD receives afferents from the islands of Calleja of the olfactory tubercle, the nucleus of the diagonal band, the amygdala and the claustrum. The lateral hypothalamic nucleus sends a moderate number of fibers to the medial MD, but other hypothalamic nuclei send only a few fibers to the MD. The lateral or dorsal portion of the MD receives fibers from the nucleus of the diagonal band, the ventral pallidum and the entopeduncular nucleus, but only few from the olfactory tubercle and the amygdala. The thalamic reticular nucleus sends many fibers to the MD without showing any topography. The MD, particularly its lateral part, receives afferents from brainstem structures, such as the substantia nigra, superior colliculus, reticular formation, raphe nuclei and nucleus loci coerulei. Only the interpeduncular nucleus sends fibers mainly to the medial part of the MD. The cerebellar nuclei send only a few fibers to the lateral part of the MD at posterior levels.     

The identification of brainstem neurones projecting to thoracic respiratory motoneurones in the cat as demonstrated by retrograde transport of HRP

Brainstem neurones which project to the immediate vicinity of the spinal motoneurones which supply the intercostal and abdominal respiratory muscles were identified by means of the retrograde transport of horseradish peroxidase (HRP). A combined electrophysiological and histological technique was used in which recording of phasic inspiratory or expiratory motoneurone activity within upper (T3-T4) or lower (T8-T9) thoracic segments was followed by the ion-tophoretic injection of HRP at these recording sites. HRP labelled cells were concentrated in those brainstem regions known to contain phasic respiratory neurones, namely the ventrolateral nucleus of the solitary tract (vl-NTS) or dorsal respiratory group (DRG), the ambiguus complex or ventral respiratory group (VRG) and the parabrachial pontine (PB) nuclei. In 18 cats, 248 cells were labelled in these three respiratory regions of the brainstem while 668 were much more diffusely distributed in other regions of the medulla and pons. The ipsilateral and contralateral contributions within the respiratory regions were respectively; 23%:77% (DRG), 33%:67% (VRG), 95%:5% (PB). These results are considered in the general context of previous electrophysiological and histological findings, but also with particular reference to a related study of the projections from brainstem neurones to the phrenic nucleus [32].