Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus.

Following microinjections of a colloidal gold complex into the nucleus paragigantocellularis (PGi) of the ventral medulla, and of latex microspheres into the nucleus abducens (Abd) of the same animal, retrogradely labeled neurons were identified in the region of the contralateral supraoculomotor nucleus (SOM) in the ventromedial periaqueductal gray (PAG). Neurons labeled from the Abd were found in the SOM in the ventromedial PAG throughout the midbrain, as well as scattered ventrally in the oculomotor nucleus. Neurons in the SOM area retrogradely labeled from the PGi were most numerous rostral to the dorsal raphe nucleus and extended throughout the level of the oculomotor nucleus. Direct comparison of the two labels revealed that the neurons that project to the Abd were located slightly more ventrally than PGi-projecting neurons. Almost no doubly labeled neurons were identified, although singly labeled neurons formed adjacent but separate populations. These results indicate that neurons in the SOM area projecting to the PGi are distinct from those projecting to the Abd, and that the PGi-projecting neurons are probably not pre-oculomotor neurons. Given their more dorsal location and the nature of their target neurons in the rostral ventrolateral medulla, PGi-projecting neurons may be related to visceromotor, as opposed to oculomotor functions.     

Retrograde labeling of rat dorsolateral septal nucleus neurons following intraseptal injections of WGA-HRP

Projection neurons in the rat dorsolateral septal nucleus (DLSN) were retrogradely labeled following intraseptal injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). Injections of WGA-HRP centered in the medial septum (MS) and parts of the intermediate and ventrolateral subdivisions of the lateral septum retrogradely labeled only a few centrally scattered multipolar-shaped neurons. In contrast, injections placed in the nucleus of the diagonal band of Broca (DBB) consistently resulted in labeling of DLSN neurons of all sizes and shapes. Large injections in rostral DBB appeared to retrogradely label every DLSN neuron, while similar injections in caudal DBB only labeled neurons in restricted regions of the nucleus. A collection of small cells forming the ventricular border of caudal DLSN and a group of larger cells situated in the dorsolateral tip of rostral DLSN were consistently labeled following each DBB injection. The pattern of retrogradely labeled neurons in the DLSN appeared in a complementary fashion to that seen in the other lateral septal nuclei. Our findings support the conclusion that the DLSN is a morphologically heterogeneous nucleus consisting almost entirely of projection neurons. The pattern of retrograde labeling in the lateral septum suggests that these projection neurons may be topographically organized since distinct subpopulations of cells were labeled following different injections in the MS/DBB complex. © 1996 Wiley-Liss, Inc.     

Reciprocal connections between the medial preoptic area and the midbrain periaqueductal gray in rat: a WGA-HRP and PHA-L study.

The midbrain periaqueductal gray (PAG) participates in diverse functions such as analgesia, autonomic regulation, sexual behavior, and defense/escape responses. Anatomical studies of the circuits involved in such functions have largely focused on the connections of PAG with the medulla. Projections to PAG from forebrain structures are extensive, but their organization has received little attention. Previous anatomic studies indicate that the medial preoptic area (MPO), involved in a variety of physiological and behavioral functions, is a major source of afferent input to the periaqueductal gray. Here, we have examined the topography of reciprocal connections between these two structures in the rat by using wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and Phaseolus vulgaris leucoagglutinin (PHA-L). Multiple WGA-HRP injections at several rostrocaudal levels of PAG retrogradely labeled large numbers of neurons in the medial preoptic area; labeled cells were primarily located in the medial preoptic nucleus, the median preoptic nucleus, and the region lateral to the medial preoptic nucleus. The distribution of labeled cells shifted medially to laterally along the rostral to caudal axis of the medial preoptic area. Rostrally, there was selective retrograde labeling in the central and lateral divisions of medial preoptic nucleus, whereas caudally, labeled cells were primarily located only in the lateral subdivision of medial preoptic nucleus. Tracer injections in PAG also produced strong anterograde labeling in MPO. WGA-HRP and PHA-L injections in the medial preoptic area resulted in dense anterograde labeling along the entire rostrocaudal axis of PAG. The terminal labeling in PAG from the medial preoptic area was not uniformly distributed throughout PAG, however. Instead, this projection formed one or two rostrocaudally oriented longitudinal columns that terminated in different subregions of PAG along the entire rostrocaudal axis of this structure. Rostrally, inputs from the medial preoptic area project heavily to dorsomedial PAG, and at mid-PAG levels, the projection becomes distinctly bipartite with two discrete longitudinal terminal columns in dorsomedial and lateral PAG; caudally, the heaviest labeling is in ventrolateral PAG. The projection also exhibited a central to peripheral (radial) gradient; labelled fibers and terminals were heaviest near the aqueduct and much lower in the peripheral parts of PAG. WGA-HRP injections in MPO also produced retrograde labeling of neurons at all rostrocaudal levels of PAG; more neurons were labeled in the rostral than the caudal half of PAG. The majority of labeled cells were located in dorsomedial and ventral/ventrolateral parts of PAG; only a few neurons in the dorsal raphe region appear to project to MPO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferent connections of the rostral medulla of the cat: A neural substrate for midbrain-medullary interactions in the modulation of pain

In order to study the organization of the rostral medulla of the cat and its contribution to pain control mechanisms, we have examined the afferent connections of the midline nucleus raphe magnus (NRM), the laterally located nucleus reticularis magnocellularis (Rmc), and the nucleus reticularis gigantocellularis (Rgc) located dorsal to Rmc. Iontophoretic injections of HRP were made into the three regions; the distribution of retrogradely labeled neurons in brainstem and spinal cord was then mapped. While significant differences characterize the source of afferents to Rgc and NRM/Rmc, there is little to distinguish that between NRM and Rmc. The predominant spinal projection is to Rgc; fewer labeled neurons were recorded after injections into Rmc. In contrast, no significant direct spinal projection to NRM was found. All three regions receive input from widespread areas within the medullary and pontine reticular formation. The most pronounced differences in the distribution of retrogradely labeled neurons were found in the midbrain. The major projection to both NRM and Rmc derives from the periaqueductal gray (PAG) and from the adjacent nucleus cuneiformis. Labeled cells are concentrated in the dorsal and lateral PAG; few are found in the ventrolateral PAG. In contrast, Rgc receives few afferents from the PAG; however, after Rgc injections, many cells were recorded in the deep layers of the contralateral tectum. None of the injection sites produced significant labeling of the catecholamine-rich dorsolateral pontine tegmentum or of the nucleus raphe dorsalis. The demonstration of significant PAG projections to NRM/Rmc provides anatomical evidence for the hypothesis that opiate and stimulation-produced analgesia involves connections from PAG to neurons of NRM and Rmc which, in turn, inhibit spinal nociceptors.     

Trigeminal and dorsal column nuclei projections to the anterior pretectal nucleus in the rat

The projections of the trigeminal (V) sensory nuclei (VSN) and the dorsal column nuclei (DCN) to the anterior pretectal nucleus (APT) of the rat were investigated by the use of anterograde and retrograde transport of wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into the APT retrogradely labeled neurons in the contralateral VSN and DCN. The labeled neurons in the VSN were most concentrated in the rostral V subnucleus interpolaris (Vi), but were also found in caudal V subnucleus oralis (Vo). No labeled neurons were seen in V subnucleus caudalis. In the DCN, retrogradely labeled neurons were observed in rostral portions of both the cuneate (Cu) and gracile (Gr) nuclei. Injections of WGA-HRP into the rostral Vi or caudal Vo resulted in dense anterograde terminal labeling in the ventral two-thirds of the APT; the labeling was maximal in the ventromedial part of the caudal half of the APT and did not extend into its most rostral portion. Labeling resulting from injections of tracer into Cu or Gr was located primarily in the ventral half of the APT, was maximal in the mid-levels of the nucleus and extended into its rostral portions. These results indicate the existence of prominent somatosensory projections to the APT and are consistent with recent findings suggesting a role for the APT in sensorimotor integration.     

Projections from the periaqueductal gray to the rostromedial pericoerulear region and nucleus locus coeruleus: anatomic and physiologic studies.

Previous studies showed that the nucleus locus coeruleus (LC) receives two major afferent inputs from 1) nucleus paragigantocellularis and 2) nucleus prepositus hypoglossi, both in the rostral medulla. Recent reports suggested that the midbrain periaqueductal gray (PAG) projects to the rostromedial pericoerulear area and LC. Since the PAG is a major site for control of central antinociception, and since descending noradrenergic fibers have been implicated in pain modulation, we have investigated in detail the functional anatomy of projections from PAG to the dorsolateral pontine tegmentum. A combined anatomical and electrophysiological approach was used to assess the organization and synaptic influence of PAG on neurons in the rostromedial pericoerulear region and in LC proper. Injections of the tracer wheatgerm agglutinin conjugated to horseradish peroxidase encompassing LC proper and the rostromedial pericoerulear area retrogradely labeled neurons in PAG located lateral and ventrolateral to the cerebral aqueduct; injections restricted to LC proper did not consistently label PAG neurons. Deposits of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into this same region of PAG labeled axons that robustly innervated the zone rostral and medial to LC. Only sparse fibers were observed in LC proper. Consistent with these results, focal electrical stimulation of LC antidromically activated only a few PAG neurons (6 of 100); all of these driven cells were located lateral and ventrolateral to the cerebral aqueduct. The majority of neurons in the rostromedial pericoerulear area were robustly activated by single pulse stimulation of PAG. In contrast, single pulse electrical stimulation of lateral PAG produced weak to moderate synaptic activation of some LC neurons; stimulation of ventrolateral PAG produced predominant inhibition of LC discharge, perhaps through recurrent collaterals subsequent to antidromic activation of neighboring LC cells. Taken together, these results indicate that PAG strongly innervates the region rostral and medial to LC, including Barrington's nucleus, but only weakly innervates LC proper. Although recent studies indicate that the dendrites of LC neurons ramify heavily and selectively in the rostromedial pericoerulear region, the results of the present physiological studies suggest that PAG preferentially targets rostromedial pericoerulear neurons rather than LC dendrites.     

Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat

We studied afferents to the parabrachial nucleus (PB) from the spinal cord and the spinal trigeminal nucleus pars caudalis (SNVc) in the rat by using the anterograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into medial PB retrogradely labeled neurons in the promontorium and in lamina I of the dorsal rostral SNVc, while injections into lateral PB and the K?lliker-Fuse nucleus retrogradely labeled neurons in these areas as well as in lamina I throughout the caudal SNVc and spinal dorsal horn. Injections of WGA-HRP into the caudal SNVc and dorsal horn of the spinal cord resulted in terminal labeling in the dorsal, central, and external lateral subnuclei of PB and the K?lliker-Fuse nucleus, all of which are known to receive cardiovascular and respiratory afferent information. Injections of WGA-HRP into the promontorium and dorsal rostral SNVc resulted in terminal labeling in the same PB subnuclei, as well as in the medial and the ventral lateral PB subnuclei, which are sites of relay for gustatory information ascending from the medulla to the forebrain. The spinal and trigeminal projection to PB may mediate the convergence of pain, chemosensory, and temperature sensibilities with gustatory and cardiorespiratory systems in PB.     

Distribution and connections of inspiratory premotor neurons in the brainstem of the pigeon (Columba livia)

We have recorded extracellular, inspiratory-related (IR) unit activity in the medulla at locations corresponding to those of neurons retrogradely labeled by injections of retrograde tracers in the lower brachial and upper thoracic spinal cord, injections that covered cell bodies and dendrites of motoneurons innervating inspiratory muscles. Bulbospinal neurons were distributed throughout the dorsomedial and ventrolateral medulla, from the spinomedullary junction through about 0.8 mm rostral to the obex. Almost all of the 104 IR units recorded were located in corresponding parts of the ventrolateral medulla, rostral to nucleus retroambigualis, where expiratory related units are found. Injections of biotinylated dextran amine at the recording sites labeled projections both to the spinal cord and to the brainstem. In the lower brachial and upper thoracic spinal cord, bulbospinal axons traveled predominantly in the contralateral dorsolateral funiculus and terminated in close relation to the dendrites of inspiratory motoneurons retrogradely labeled with cholera toxin B-chain. In the brainstem, there were predominantly ipsilateral projections to the nucleus retroambigualis, tracheosyringeal motor nucleus (XIIts), ventrolateral nucleus of the rostral medulla, infraolivary superior nucleus, ventrolateral parabrachial nucleus, and dorsomedial nucleus of the intercollicular complex. In all these nuclei, except XIIts, retrogradely labeled neurons were also found, indicating reciprocity of the connections. These results suggest the possibility of monosynaptic connections between inspiratory premotor neurons and inspiratory motoneurons, which, together with connections of IR neurons with other brainstem respiratory-vocal nuclei, seem likely to mediate the close coordination that exists in birds between the vocal and respiratory systems. The distribution of IR neurons in birds is similar to that of the rostral ventral respiratory group (rVRG) in mammals. J. Comp. Neurol. 379:347–362, 1997. © 1997 Wiley-Liss, Inc.     

Presynaptic and postsynaptic relations of mu-opioid receptors to gamma-aminobutyric acid-immunoreactive and medullary-projecting periaqueductal gray neurons

The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the mu-opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., gamma-aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary-projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual-labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR-immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual-immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA-immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary-projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG.     

Mesodiencephalic projections to the vestibular complex in the cat

The distribution of cells in the rostral medial mesencephalon and caudal diencephalon which project to the vestibular complex was mapped in the cat by using retrograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Subsequent experiments using anterograde transport of WGA-HRP clarified the position of the terminations of the mesodiencephalic-derived afferents in the vestibular complex. After large injections which involved the entire vestibular complex, retrogradely labeled cells were seen in both the ipsilateral and contralateral interstitial nucleus of Cajal (INC) and were more numerous in its rostral pole. Labeled cells also occurred in the perifascicular region, both immediately adjacent to the fasciculus retroflexus and rostroventral to it. Fusiform midline cells of the Edinger-Westphal nucleus were also labeled, as well as a number of cells in the adjacent somatic portion of the oculomotor complex (OMC). Another group of labeled cells was observed within the contralateral medial terminal nucleus of the accessory optic tract (MTN) and in the posterior hypothalamic nucleus. Injections limited to subregions of the vestibular complex resulted in similar but slightly varying distributions and numbers of retrogradely labeled cells. After injections covering the caudal half of the medial vestibular nucleus (MVN) and descending vestibular nucleus (DVN), labeled cells in the INC and tegmentum dorsal to it were especially prominent, but none was seen in the MTN or OMC. Injections placed in the rostral MVN, lateral vestibular nucleus, y group, and superior vestibular nucleus resulted in a distribution of labeled cells similar to that seen following global vestibular injections, but these cells were fewer in number. After an injection confined to the y group, a small number of retrogradely labeled cells were seen in the rostral pole of the INC and immediately ventral to the fasciculus retroflexus. Projections from the rostral medial mesencephalon and caudal diencephalon to the MVN, DVN, and y group were confirmed by using anterograde transport of WGA-HRP. Direct projections from the INC-perifascicular regions and somatic neurons of the OMC to the caudal vestibular complex could play a role in eye-head coordination. Those projections from the rostral INC and MTN to the rostral vestibular complex may play a role in vertical eye movements and responses to visual stimuli which move in the vertical plane.     

Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat.

The periaqueductal or midbrain central gray matter (CG) in the rat contains a dense network of adrenergic and noradrenergic fibers. We examined the origin of this innervation by using retrograde and anterograde axonal tracers combined with immunohistochemistry for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). Following injections of the fluorescent tracers Fast Blue or Fluorogold into the CG, double-labeled neurons in the medulla were identified mainly in the noradrenergic A1 group in the caudal ventrolateral medulla (VLM) and A2 group in the medial part of the nucleus of the solitary tract (NTS); and in the adrenergic C1 group in the rostral ventrolateral medulla and C3 group in the rostral dorsomedial medulla. Injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into these cell groups resulted in a distinct pattern of axonal labeling in various subdivisions of the CG. Anterogradely labeled fibers originating in the medial NTS were predominantly found in the lateral portion of the dorsal raphe nucleus and in the adjacent part of the lateroventral CG (CGlv). Following PHA-L injections into the C3 region the anterogradely labeled fibers were diffusely distributed in the CGlv and the dorsal raphe nucleus at caudal levels, but rostrally tended to be located laterally in the CGlv. In contrast, ascending fibers from the caudal and rostral VLM terminated in the rostral dorsal part of the CGlv and in the dorsal nucleus of the CG, whereas ventral parts of the CG, including the dorsal raphe nucleus, contained few afferent fibers. Double-label studies with antisera against DBH and PNMT confirmed that noradrenergic neurons in the A1 and A2 groups and adrenergic neurons in the C1 and C3 groups contributed to these innervation patterns in the CGlv. Noradrenergic and adrenergic projections from the medulla to the CG may play an important role in a variety of autonomic, sensory and behavioral processes.     

Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: retrograde and anterograde tracing studies

The nucleus paragigantocellularis in the ventrolateral medulla has been implicated in cardiovascular, pain, and analgesic functions; and it has also been found to be a major afferent to the pontine nucleus locus coeruleus. In the present study, afferents to the nucleus paragigantocellularis were identified in the rat by means of the retrograde tracers wheat germ agglutinin-conjugated horseradish peroxidase or Fluoro-Gold. Projections to the nucleus paragigantocellularis arise from a wide variety of nuclei with autonomic, visceral, and sensory-related functions. Major afferents with consistent and robust retrograde labeling include most laminae of the spinal cord, the caudal lateral medulla, the contralateral paragigantocellularis, the nucleus of the solitary tract, the A1 area, the lateral parabrachialis, the K?lliker-Fuse nucleus, the periaqueductal gray, and a preoculomotor nucleus in the ventral central gray, the supraoculomotor nucleus. Other notable afferents, seen only after large caudal injections into the nucleus paragigantocellularis, include the lateral hypothalamus, the paraventricular nucleus of the hypothalamus, and the medial prefrontal cortex. Minor afferents include the gigantocellular nucleus, the area postrema, the caudal raphe groups, the inferior colliculus, the A5 area, and the locus coeruleus. The projection from the supraoculomotor nucleus, not previously reported as an afferent to the ventrolateral medulla, was confirmed with anterograde tracing by means of Phaseolus vulgaris-leucoagglutinin. Iontophoretic deposits of Phaseolus vulgaris-leucoagglutinin into the nucleus of the solitary tract (commissuralis level) or into the periaqueductal gray also yielded terminal fiber labeling in the nucleus paragigantocellularis. Fibers from the supraoculomotor nucleus and the nucleus of the solitary tract were densest in the lateral aspect of the nucleus paragigantocellularis (corresponding to the rostroventrolateral reticular nucleus), while fibers from the periaqueductal gray were more medially located. Previous studies have defined inputs to the rostral ventrolateral medulla from the cochlear nucleus as well as from the colliculi. In the present study, deposits of wheat germ agglutinin-conjugated horseradish peroxidase or Phaseolus vulgaris-leucoagglutinin into the cochlear nucleus or the superior colliculus yielded only sparse anterograde labeling in the nucleus paragigantocellularis, but heavily labeled adjacent areas. The inferior collicular injections yielded strong but restricted anterograde labeling in the rostromedial paragigantocellularis, medial to the facial nucleus. These results indicate that the paragigantocellularis area receives inputs from diverse brain structures. Neurons in the nucleus paragigantocellularis afferent to the locus coeruleus, being distributed throughout this region, may provide a channel where several types of information are integrated and transmitted to the extensive locus coeruleus noradrenergic efferent network...     

Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla

Projections from the nucleus tractus solitarii (NTS) to autonomic control regions of the ventrolateral medulla, particularly the nucleus reticularis rostroventrolateralis (RVL), which serves as a tonic vasomotor center, were analyzed in rat by anterograde, retrograde, and combined axonal transport techniques. Autonomic portions of the NTS, including its commissural, dorsal, intermediate, interstitial, ventral, and ventrolateral subnuclei directly project to RVL as well as to other regions of the ventrolateral medulla. The projections are organized topographically. Rostrally, a small cluster of neurons in the intermediate third of NTS, the subnucleus centralis, and neurons in proximity to the solitary tract selectively innervate neurons in the retrofacial nucleus and nucleus ambiguus. Neurons generally located in more caudal and lateral sites in the NTS innervate the caudal ventrolateral medulla (CVL). The RVL, CVL, and nucleus retroambiguus are interconnected. A combined retrograde and anterograde transport technique was developed so as to prove that projections from the NTS to the ventrolateral medulla specifically innervate the region of RVL containing neurons projecting to the thoracic spinal cord or the region of the nucleus containing vagal preganglionic neurons. When the retrograde tracer, fast blue, was injected into the thoracic spinal cord, and wheat germ agglutinin-conjugate horseradish peroxidase (HRP) was injected into the NTS, anterogradely labeled terminals from the NTS surrounded the retrogradely labeled neurons in the RVL and in the nucleus retroambiguus in the caudal medulla. Among the bulbospinal neurons in the RVL innervated by the NTS were adrenaline-synthesizing neurons of the C1 group. When fast blue was applied to the cervical vagus, and HRP was injected into the NTS, anterogradely labeled terminals from the NTS surrounded retrogradely labeled neurons in the rostral dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, the retrofacial nucleus, and the dorsal portion of the RVL, a region previously shown to contain cardiac vagal preganglionic neurons. This combined anterograde and retrograde transport technique provides a useful method for tracing disynaptic connections in the brain. These data suggest that the RVL is part of a complex of visceral output regions in the ventrolateral medulla, all of which receive afferent projections from autonomic portions of the NTS. Bulbospinal neurons in the RVL, in particular the C1 adrenaline neurons, may provide a portion of the anatomic substrate of the baroreceptor and other visceral reflexes.     

Localization of neurons afferent to the optic tectum in longnose gars

Afferent pathways to the optic tectum in the longnose gar were determined by unilateral tectal injections of HRP. Retrogradely labeled cells were observed in the ipsilateral caudal portion of the rostral entopeduncular nucleus and bilaterally in the rostral half of the lateral zone of area dorsalis of the telencephalon. The following diencephalic cell groups were also labeled following tectal injections: the ipsilateral anterior, ventrolateral, and ventromedial thalamic nuclei, the periventricular pretectal nucleus, and the central pretectal nucleus (bilaterally); the ventromedial thalamic and central pretectal nuclei revealed the largest number of labeled cells. At midbrain levels, retrogradely labeled cells were seen in the ipsilateral torus longitudinalis, nucleus isthmi, and accessory optic nucleus; cells were labeled bilaterally in the torus semicircularis and a rostral tegmental nucleus. Only a few cells were labeled in the contralateral optic tectum, suggesting that few of the fibers of the intertectal commissure are actually commissural to the tectum. At hindbrain levels, retrogradely labeled cells were seen bilaterally in the locus coeruleus, the superior, medial, and inferior reticular formations, the eurydendroid cells of the cerebellum, and the nucleus of the descending trigeminal tract; the contralateral dorsal funicular nucleus also exhibited labeling. Clearly, the tectum in gars receives a substantial number of nonvisual afferents from all major brain areas, most of which have been reported in other vertebrates. The functional significance of these afferent sources and their probable homologues in other vertebrate groups are discussed.     

Afferent and efferent connections of the olfactory bulbs in the lizard Podarcis hispanica.

The connections of the olfactory bulbs of Podarcis hispanica were studied by tract-tracing of injected horseradish peroxidase. Restricted injections into the main olfactory bulb (MOB) resulted in bilateral terminallike labeling in the medial part of the anterior olfactory nucleus (AON) and in the rostral septum, lateral cortex, nucleus of the lateral olfactory tract, and ventrolateral amygdaloid nucleus. Bilateral retrograde labeling was found in the rostral lateral cortex and in the medial and dorsolateral AON. Ipsilaterally the dorsal cortex, nucleus of the diagonal band, lateral preoptic area, and dorsolateral amygdala showed labeled cell bodies. Retrogradely labeled cells were also found in the midbrain raphe nucleus. Results from injections into the rostral lateral cortex and lateral olfactory tract indicate that the mitral cells are the origin of the centripetal projections of the MOB. Injections in the accessory olfactory bulb (AOB) produced ipsilateral terminallike labeling of the ventral AON, bed nucleus of the accessory olfactory tract, central and ventromedial amygdaloid nuclei, medial part of the bed nucleus of the stria terminalis, and nucleus sphericus. Retrograde labeling of neurons was observed ipsilaterally in the bed nucleus of the accessory olfactory tract and stria terminalis, in the central amygdaloid nucleus, dorsal cortex, and nucleus of the diagonal band. Bilateral labeling of somata was found in the ventral AON, the nucleus sphericus (hilus), and in the mesencephalic raphe nucleus and locus coeruleus. Injections into the dorsal amygdala showed that the mitral neurons are the cells of origin of the AOB centripetal projections. Reciprocal connections are present between AOB and MOB. To our knowledge, this is the first study to address the afferent connections of the olfactory bulbs in a reptile. On the basis of the available data, a discussion is provided of the similarities and differences between the reptilian and mammalian olfactory systems, as well as of the possible functional role of the main olfactory connections in reptiles.     

A combined immunocytochemical and retrograde tracing study of noradrenergic connections between the caudal medulla and bed nuclei of the stria terminalis

Region-specific noradrenergic inputs to the bed nuclei of the stria terminalis (BST) from the caudal medulla were studied using combined Fast Blue injections and tyrosine hydroxylase immunoreactivity (TH-ir). Injections into the rostral, dorsal, ventral and lateral BST resulted in predominantly ipsilateral retrograde labelling restricted to the mediodorsal and ventrolateral caudal medulla. Mediodorsal projecting neurones comprised the A2 TH-ir and a second non-aminergic group medial to A2. All ventrolateral retrogradely labelled neurones showed TH-ir and corresponded to A1. Injections into the caudal BST did not label the A2 and very few A1 neurones, indicating a paucity of noradrenergic inputs from this area of the medulla.     

Light and electron microscopic evidence for topographic and monosynaptic projections from neurons in the ventral medulla to noradrenergic dendrites in the rat locus coeruleus

Physiological studies have shown that afferents from the nucleus paragigantocellularis (PGi) in the rostral ventral medulla underlie the modulation of locus coeruleus (LC) activity by a variety of stimuli. However, there have been no anatomical demonstrations of a monosynaptic projection from neurons in the PGi to the LC. Thus, biotinylated dextran amine (BDA) was iontophoretically injected into the ventral medulla and single-tissue sections were processed for peroxidase localization of BDA and gold–silver labeling of tyrosine hydroxylase (TH). Discrete microinjections of BDA were placed into either the medial or lateral aspects of the ventral medulla. For medially placed injections, a medio-dorsal pathway to the LC was observed. This trajectory resulted in a predominant innervation of the ventral LC. Lateral injection placements yielded a fiber pathway that coursed more laterally within the medullo-pontine reticular formation and primarily innervated the dorsolateral LC. These light microscopic data suggested that neurons in the PGi use distinct pathways to innervate the LC and are topographically organized within this structure. Electron microscopic analyses of the LC region indicated that axon terminals originating from either subregion were equally likely to contact noradrenergic neurons in the LC. Approximately 57% and 62% of BDA-labeled terminals originating from the medial (n=150) or lateral (n=150) aspects of the ventral medulla, respectively, formed heterogeneous synaptic contacts (i.e., inhibitory- and excitatory-type) with dendrites containing TH. It is well known that the PGi is a functionally diverse region that is involved in sensory integration, autonomic regulation and pain modulation. It is also known that LC efferents are spatially organized with respect to their postsynaptic targets. Taken together, our findings that subdivisions of the ventral medulla topographically and monosynaptically innervate the LC suggest that regionally specific PGi neurons target subsets of LC neurons with efferent targets that may possess analogous functional correlates.     

Two physiologically distinct populations of neurons in the ventrolateral medulla innervate the locus coeruleus

Recent anatomic studies indicate that the nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla, strongly innervates the locus coeruleus (LC) while no such input derives from the more caudally located lateral reticular nucleus (LRN). In the present study, focal electrical stimulation of the LC was used to antidromically activate neurons in the ventrolateral medulla. A substantial number of PGi neurons were antidromically driven from the ipsilateral LC, while antidromic activation was virtually absent in LRN. Furthermore, several physiologic properties of antidromically driven cells in PGi define two populations within this group of neurons afferent to LC. These findings provide physiologic confirmation of an anatomically identified input to LC.     

Sonic motor nucleus and its connections with octaval and lateral line nuclei of the medulla in a rockfish, Sebastiscus marmoratus

The sonic motor nucleus and its fiber connections were examined in a rockfish, Sebastiscus marmoratus by means of tracer methods using horseradish peroxidase (HRP), biocytin, and carbocyanine dye (DiI). Sebastiscus has a swimbladder and a pair of extrinsic sonic/drumming muscles. The sonic muscle is ipsilaterally innervated by the occipital nerve which is composed of two ventral roots arising from the sonic motor nucleus. The sonic motor neurons are distributed in the most ventral part of the ventral column from the caudal medulla to the rostral spinal cord, and form a ventrally located columnar nucleus. Each neuron in this nucleus possesses a long thick dendrite and several short dendrites. The long dendrite extends dorsolaterally and branches in the lateral funiculus, whereas the short dendrites branch around their cell bodies. After biocytin injections into the sonic motor nucleus, two groups of premotor neurons were retrogradely labeled bilaterally, one in the dorsomedial portion of the descending octaval nucleus (DO) and the other in the medial zone of the reticular formation (RF) in the medulla. The DO premotor neurons were multipolar with several dendrites branching near the cell bodies, and the RF premotor neurons were bipolar. One of the two dendrites of the RF premotor neurons extends laterally into the ventral portion of the DO, and the other dendrite extends into the ventromedial area in the medulla. In the ventromedial dendritic field of the RF premotor neurons, descending fibers arising from the optic tectum (TO) and torus semicircularis (TS) traverse in the tractus tectobulbaris and terminate bilaterally. After DiI insertion into the ventromedial dendritic field, retrogradely labeled neurons were found bilaterally in the TS and TO. The majority of tectal neurons were located in the stratum griseum centrale. These neurons had two short basal dendrites branching in the cell layer and a long apical dendrite extending to the stratum fibrosum et griseum superficiale and stratum opticum. The toral neurons were bipolar and were distributed throughout the TS. Furthermore, biocytin injections into the medial nucleus of the lateral line system revealed that the nucleus projects bilaterally to the RF premotor neurons. These results show that premotor neurons for the sonic motor nucleus are located in the dorsomedial portion of the DO and the medial zone of the RF in the medulla. It is suggested that the sonic motor nucleus receives auditory input via the DO premotor neurons and input from RF premotor neurons which receive lateral line input via the medial nucleus, vestibular input through the lateral dendrite extending into the ventral portion of the DO, and information from the TO and TS via the tractus tectobulbaris.