Wheat Germ Agglutinin-conjugated Horseradish Peroxidase Research Articles

Excitatory innervation of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis in the rat.

We examined the possible innervation of the caudal hypoglossal nucleus by the nucleus reticularis gigantocellularis of the medulla oblongata, based on single-neuron recording and retrograde tracing experiments in Sprague-Dawley rats. Under pentobarbital sodium (50 mg/kg, i.p.) anesthesia, electrical stimulation of the caudal portion of the nucleus reticularis gigantocellularis with repetitive 0.5-ms rectangular pulses increased (46 of 51 neurons) the basal discharge frequency of spontaneously active cells, or evoked spike activity in silent, hypoglossal neurons located at the level of the obex. This excitatory effect was related to the intensity (25-100 microA) and/or frequency (0.5-20 Hz) of the stimulating pulses to the nucleus reticularis gigantocellularis. Perikaryal activation of neurons by microinjection of L-glutamate (0.5 nmol, 25 nl) into the caudal portion of the nucleus reticularis gigantocellularis similarly produced an excitatory action on eight of 14 hypoglossal neurons. Retrogradely labeled neurons were found bilaterally within the confines of the nucleus reticularis gigantocellularis following unilateral microinjection of wheatgerm agglutinin-conjugated horseradish peroxidase or Fast Blue into the corresponding hypoglossal recording sites. Furthermore, the distribution of labeled neurons in the nucleus reticularis gigantocellularis substantially overlapped with the loci of electrical or chemical stimulation. These complementary electrophysiological and neuroanatomical results support the conclusion that an excitatory link exists between the nucleus reticularis gigantocellularis and at least the caudal portion of the hypoglossal nucleus in the rat.

Evidence for glutamate as neurotransmitter in trigemino-and spinothalamic tract terminals in the nucleus submedius of cats.

The nucleus submedius in the medial thalamus of cats is an important termination site for lamina I trigemino-and spinothalamic tract (TSTT) neurons, many of which are nociceptive-specific, and the nucleus submedius has been proposed to be a dedicated nociceptive substrate involved in the affective aspect of pain. In the present study, the distribution of glutamate was examined by immunocytochemical methods in order to evaluate the possible role of this amino acid as a neurotransmitter in TSTT terminals in the nucleus submedius. TSTT terminals were identified by anterograde transport of horseradish peroxidase and wheatgerm agglutinin-horseradish peroxidase conjugate from the spinal cord or the medullary dorsal horn. Quantitative analysis of immunogold labelling revealed that TSTT terminals contain about twice the tissue average of glutamate-like immunoreactivity. A strong positive correlation was found between the density of synaptic vesicles and the density of gold particles in these terminals, whereas no relationship was seen between these variables in GABAergic presynaptic dendrites. Enrichment of glutamate-like immunoreactivity (approximately 250% of the tissue average) was also observed in terminals of presumed cortical origin. Presynaptic dendrites and neuron cell bodies in the nucleus submedius were found to contain relatively low levels of glutamate-like immunoreactivity, at or below the tissue average. These observations provide evidence that glutamate is a neurotransmitter in lamina I TSTT terminals in the nucleus submedius. The findings also suggest glutamatergic neurotransmission between cortical afferents and nucleus submedius neurons. Glutamate is therefore likely to be an important mediator of nociceptive processing in the medial thalamus.

Differential mitotic phosphorylation of proteins of the nuclear pore complex.

During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.

Ultrastructure of spinal relay of hypoglossal afferents to the parabrachial nucleus.

Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injection into the hypoglossal nerve mainly resulted in retrograde labeling in the superior ganglia of the glossopharyngeal and vagal nerves ipsilaterally. Anterogradely labeled fibers were found in lamina I of the ipsilateral upper cervical spinal cord with a few distribution to laminae IV-V and VII-VIII. WGA-HRP injection into the PBN revealed intensive labeling of lamina I neurons of the upper cervical spinal cord ipsilaterally. These light microscopic observations appear to indicate the hypoglossal sensory inputs to the PBN through the spinal cord. In order to investigate the synaptic nature of this spinal relay, electron microscopic observations were carried out on lamina I of the first and second cervical spinal cord after cutting the hypoglossal nerve and WGA-HRP injection into the PBN in the same animal. The spinoparabrachial projection neurons were demonstrated to show a low cytoplasmic/nuclear ratio and have an oval or deeply indented nucleus with a centrally located nucleolus. Furthermore, dark and light type degenerating fibers were observed to make synaptic contacts with HRP-labeled somata and dendritic profiles.

Serotonin‐containing projections to the mesencephalic trigeminal nucleus of the cat

It is well known that the mesencephalic trigeminal nucleus (MTN) neurons transmit somatosensory information from proprioceptors in the oral-facial region. Several mechanisms of sensory transduction in these specialized receptors have been proposed, but the neurotransmitters which are responsible for mediating proprioceptive information are still unknown. The current study concentrates on the distribution of one putative neurotransmitter system, serotonin (SER), in the cat MTN. A second objective was to clarify the location and sources of serotoninergic projections on the MTN neurons. To determine whether SER was localized in the MTN, the peroxidase-antiperoxidase (PAP) immunocytochemical technique was applied at light and electron microscopic levels in colchicine-treated animals. The origin of SER-containing fibers in the MTN was studied using a double-labeling method combining retrograde transport with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and SER immunocytochemistry. There were no SER-containing neurons in the MTN. The cell bodies of immunonegative MTN neurons were closely surrounded by fine SER-positive fibers and terminals. The labeled fibers were in most cases very thin and sometimes varicose. Ultrastructurally, direct synaptic contacts between SER-containing terminals and perikarya of MTN neurons of all sizes could be seen. The majority of SER-labeled structures were synaptic terminals in which the immunoreactive material was located within the small round clear as well as the small granular vesicles (diameter 50-80 nm) and a few large dense-cored vesicles (up to 150 nm). Retrograde tracing demonstrated that most of cells in the nuclei raphe dorsalis, pontis and magnus were WGA-HRP-labeled. These results indicated that MTN neurons received serotoninergic projections from the raphe nuclei of the brainstem. In light of these morphological data, it is concluded that the MTN of the cat is under the influence of SER-containing axons and this serotoninergic input may modulate MTN neuronal activity at the first synaptic relay.

Cutaneous distribution of sympathetic postganglionic fibers from stellate ganglion: A retrograde axonal tracing study using wheat germ agglutinin conjugated with horseradish peroxidase.

Sympathetic postganglionic cells of the stellate ganglion (SG) projecting to the skin in young dogs were labeled retrogradely with wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). After application of WGA-HRP into the dermis and/or subcutaneous tissue of the upper extremity, numerous labeled cells were observed in the SG. The sympathetic postganglionic fibers from the SG were distributed in the skin area between the third cervical vertebra and the level of the thirteenth rib. These fibers decreased in number toward the rostral and caudal skin area with a peak on the upper extremity. Sympathetic postganglionic cells in the SG projecting to these skin areas were found ipsilaterally to the injection side. No labeled cells were observed in the SG after injecting WGA-HRP into the epidermis and/or subcutaneous tissue of the face, the level of the second cervical vertebra, and the lower extremity.Sympathetic postganglionic neurons of the SG which project in the cervical and thoracic sympathetic trunks were labeled retrogradely with unconjugated horseradish peroxidase (HRP) applied to the cut end of the sympathetic trunks proximal to the SG. The application was made in the cervical sympathetic trunk (CST) caudal to the superior cervical ganglion (SCG). In the thoracic sympathetic trunk (TST), the application was made between the fourth and fifth thoracic sympathetic ganglia (T4 and T5), and between T10 and T11. Labeled cell bodies were observed in SG bilaterally.

Subcortical contributions to head movements in macaques. II. Connections of a medial pontomedullary head-movement region.

1. In the companion article, a variety of head movements were elicited by stimulation in, and adjacent to, the gigantocellular reticular nucleus (Cowie and Robinson 1994). We refer to this area, caudal to the abducens nucleus, as the gigantocellular head movement region. In the present paper, the anatomical connections of this region, as determined by injections of wheat-germ agglutinin conjugated horseradish peroxidase (WGA-HRP), are reported. The majority of efferent and afferent connections were with areas related to head movements. 2. Efferent fibers from the region projected via two paths to the caudal medulla and upper cervical spinal cord. Labeled fibers descended in the anterolateral funiculus of the ipsilateral spinal cord to terminate in lateral parts of the ventral horn. A second pathway descended bilaterally in the medial longitudinal fasciculus to the anterior funiculi and medial portions of the ventral gray. These efferents paralleled the head-movement topography demonstrated physiologically. Other projections included efferents to the interstitial nucleus of Cajal, caudal field H of Forel, paramedian pontine reticular formation, and caudal vestibular nuclei. Other efferent fibers projected to the trigeminal, facial, and hypoglossal nuclei, as well as to the parvocellular reticular field, which contains interneurons for these motor groups. However, no efferent or afferent labeling involved the ocular motor nuclei. 3. Afferents to the gigantocellular head movement region arose mainly from head-movement areas. In all animals, labeled cells were found in the intermediate and deep layers of the caudal superior colliculus. Labeled neurons also were found in the caudal field H of Forel, interstitial nucleus of Cajal, pontine medial tegmentum including the pontine paramedian reticular formation, nucleus subcoeruleus, and vestibular nuclear complex. Caudally, filled cells were located in the parvocellular, magnocellular, dorsal, and ventral reticular nuclei, the supraspinal nucleus, and the upper cervical ventral horn. 4. In one animal, the ipsilateral frontal cortex contained retrogradely labeled neurons. These cells were found in layer V of cortical areas 4 and 6. Other afferent cells were found consistently in the periventricular and periaqueductal gray matter. 5. A control injection into the caudal vestibular nuclear complex showed projections to the gigantocellular reticular formation and labeled cells in the vestibular and parvocellular reticular nuclei. These observations show that the connections of the gigantocellular region are not typical of all head movement sites. 6. These data indicate that the gigantocellular head-movement region has the requisite efferent and afferent connections to function in the subcortical control of head, but not eye, movements.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAergic projection from the basal forebrain to the visual sector of the thalamic reticular nucleus in the cat

We examined the projection from the basal forebrain to thalamic and cortical regions of the visual system in cats, with particular reference to the visual sector of the thalamic reticular nucleus, the lateral geniculate nucleus, and the striate cortex. First, we made injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the visual sector of the thalamic reticular nucleus and found cells labeled by retrograde transport in the lateral nucleus basalis magnocellularis. Injection of biocytin into the basal forebrain resulted in the anterograde labeling of a dense band of fibers and terminals within the entire thalamic reticular nucleus; this labeling extended through the visual sector including the perigeniculate nucleus. No orthograde labeling was found in the lateral geniculate nucleus. Next, we addressed the issue of putative neurotransmitters used by this pathway using a variety of immunocytochemical and histochemical markers. In this fashion, we identified two populations of cells in the nucleus basalis magnocellularis of the cat; large cholinergic cells that contain choline acetyltransferase, NADPH-diaphorase, and calbindin and that project to striate cortex and smaller cells that contain gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, and parvalbumin and that project to the visual sector of the thalamic reticular nucleus. We also examined at the electron microscopic level terminals in the visual sector of the thalamic reticular nucleus that were labeled from a biocytin injection in the basal forebrain. Most of these terminals form symmetric contacts onto dendrites and were revealed by postembedding immunocytochemical staining to be positive for GABA.

The periaqueductal gray in the cat projects to lamina VIII and the medial part of lamina VII throughout the length of the spinal cord.

The periaqueductal gray (PAG) plays an important role in analgesia as well as in motor activities, such as vocalization, cardiovascular changes, and movements of the neck, back, and hind limbs. Although the anatomical pathways for vocalization and cardiovascular control are rather well understood, this is not the case for the pathways controlling the neck, back, and hind limb movements. This led us to study the direct projections from the PAG to the spinal cord in the cat. In a retrograde tracing study horseradish peroxidase (HRP) was injected into different spinal levels, which resulted in large HRP-labeled neurons in the lateral and ventrolateral PAG and the adjacent mesencephalic tegmentum. Even after an injection in the S2 spinal segment a few of these large neurons were found in the PAG. Wheat germ agglutinin-conjugated HRP injections in the ventrolateral and lateral PAG resulted in anterogradely labeled fibers descending through the ventromedial, ventral, and lateral funiculi. These fibers terminated in lamina VIII and the medial part of lamina VII of the caudal cervical, thoracic, lumbar, and sacral spinal cord. Interneurons in these laminae have been demonstrated to project to axial and proximal muscle motoneurons. The strongest PAG-spinal projections were to the upper cervical cord, where the fibers terminated in the lateral parts of the intermediate zone (laminae V, VII, and the dorsal part of lamina VIII). These laminae contain the premotor interneurons of the neck muscles. This distribution pattern suggests that the PAG-spinal pathway is involved in the control of neck and back movements. Comparing the location of the PAG-spinal neurons with the results of stimulation experiments leads to the supposition that the PAG-spinal neurons play a role in the control of the axial musculature during threat display.

Neural projections from the frontal cortex to the oculomotor nucleus: an anatomical study using retrograde axonal, anterograde axonal and transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase in cats.

Neural projections from the frontal cerebral cortex to the oculomotor nucleus (3N) were investigated in 1- to 2-year-old cats by retrograde and anterograde axonal and transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the 3N area and its surrounding tissues, retrogradely labeled cells were observed in the anterior sigmoid gyrus, ventral bank of the cruciate sulcus, medial and lateral walls and base of the presylvian sulcus, gyrus rectus and gyrus proreus. Following injection of WGA-HRP into these frontal cortical areas, anterogradely labeled nerve terminals were observed in the mesencephalic periaqueductal gray matter (PAG) just overlying the 3N. Only a few terminals were observed within the 3N. Following injection of WGA-HRP into the extraocular muscles of 1-month-old kittens, transneuronally labeled small cells were observed in the PAG just overlying the 3N and in the mesencephalic reticular formation, ventrolateral to it. These small cells may represent intercalated neurons of the cortico-oculomotor projections in the cat.

Trigeminal sensory innervation on perforators of the circle of willis in rabbits by wheat germ agglutinin-conjugated horseradish peroxidase anterograde tracing

Distribution patterns of sensory innervation from the trigeminal ganglion to the perforators of the circle of Willis in rabbits were investigated by wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) anterograde tracing. Twenty Japanese white rabbits were anesthetized by inhaling 1% halothane. Using a microsurgical technique, 4 μl of 2% WGA-HRP in 1 M KCl solution, colored with brilliant blue, was micro-injected into the medial part of the left trigeminal ganglion in 14 animals with a pressure injection system. Another six served as controls to exclude the possibility of labeling non-trigeminal axons. Forty-eight hours later, the perforators in the cisternal and intracerebral segments along with their parent arteries were dissected from the brain according to Dacey's dissecting technique after transcardial perfusion, reacted with the 3,3′,5,5′-tetramethyl benzidine method of Mesulam. The results revealed that sensory nerves on the perforators of the circle of Willis were less densely innervated than those on their parent arteries due to the difference in diameter. The posteromedial perforating arteries arising from the Pl segment of the posterior cerebral artery to the tegmentum, posteroventral thalamus and posterior hypothalamus were more prominently and consistently innervated than other perforators. The sensory fibers were seen on the cisternal segment of the perforating arteries. A parallel or twisted pattern was found in the perforators less than 100 μm in diameter, while a meshwork pattern was visualized in the proximal part of some bigger ones. Fine sensory fibers could be traced on the perforators as small as 40 μm in diameter. The present study demonstrates for the first time the detailed distribution patterns of sensory nerves from the trigeminal ganglion to perforators of the circle of Willis in rabbits. Pathophysiological implications of the findings are discussed in relation to cerebral vasospasm following subarachnoid hemorrhage.

Nadph-diaphorase in the spinal trigeminal nucleus oralis and rostral solitary tract nucleus of rats

NADPH-diaphorase histochemical staining demonstrated a distinct neural group that might synthesize nitric oxide in the lower brainstem of rats. The NADPH-diaphorase stain revealed a Golgi-like network in the dorsomedial spinal trigeminal nucleus oralis and rostrolateral solitary tract nucleus, whereas this network was more dense in the latter nucleus. The distribution of NADPH-diaphorase-positive neurons in these areas overlapped with parts of central terminations from the chorda tympani nerve, as demonstrated with transganglionic transport of wheatgerm agglutinin conjugated horseradish peroxidase. The number of NADPH-diaphorase-positive neurons changed after chorda tympani nerve lesion relative to the contralateral side. The control value (%) was 106.0 ± 4.9 (mean ± S.E.M.). One hour after the nerve lesion, the value increased to115.2 ± 9.1 ( P > 0.05). It then decreased to83.9 ± 5.2 two days after the lesion ( P < 0.05), and remained at this reduced level for one or two weeks,83.2 ± 3.0 ( P <0.01) and83.7 ± 2.3 ( P <0.01), respectively. This statistically significant reduction recovered to control level103.4 ± 2.9 four weeks after the lesion. These results show that NADPH-diaphorase-positive neurons in the lower brainstem could be regulated trans-synaptically by primary afferents, possibly gustatory inputs.

Interhemispheric connections in neonatal owl monkeys ( Aotus trivirgatus) and galagos ( Galago crassicaudatus)

Interhemispheric connections were studied by injecting a mixture of horseradish peroxidase (HRP) and wheatgerm agglutinin conjugated with horseradish peroxidase (WGA-HRP) into multiple sites in dorsolateral occipital and parietal cortex of one cerebral hemisphere of three galagos ( Galago crassicaudatus) and two owl monkeys ( Aotus trivirgatus) within seven days of birth. Cortex was either separated from the rest of the brain, flattened and cut parallel to the surface to aid reconstructing surface-view patterns of labeled neurons and processes, or cut in standard coronal or parasagittal planes to better reveal laminar patterns of connections. In both primate species, the surface-view pattern of callosal connections in infants was remarkably adult-like. In infant owl monkeys, callosal connections were concentrated along the margin of area 18 with area 17, and only a few labeled cells were found within area 17. Other visual areas including the second visual area, V-II, and the middle temporal visual area, MT, had patchy distributions of labeled neurons that extended over large parts of the visual field representations. Primary motor, auditory, and somatosensory fields also had patchy distributions of labeled neurons, with regions of areas 3b and adjoining somatosensory fields having few callosal connections in portions that appeared to correspond with representations of the hand and foot. Results were very similar in galagos, except that newborn galagos, as in adults, had a patchy distribution of callosally projecting neurons that extended well within area 17. Furthermore, the labeled neurons were concentrated in patches that aligned with the cytochrome oxidase blobs of area 17. Finally, callosal connections were concentrated in cytochrome oxidase poor regions of area 3b.

GABAergic projection from the intercalated cell masses of the amygdala to the basal forebrain in cats.

The intercalated cell masses (ICMs) are dense clusters of small GABAergic cells interposed between the basolateral and centromedial nuclear groups of the amygdala. Until now, the ICMs have been largely ignored in anatomical studies of the amygdaloid complex. Thus, this study was undertaken to identify some of their targets by means of tract-tracing methods combined with immunohistochemical techniques. Wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into numerous cortical areas and dorsal thalamic nuclei, in the anterior commissure and/or stria terminalis nuclei, and in the caudate nucleus, as well as into lateral and preoptic hypothalamic areas. Very few retrogradely labeled cells were seen in the ICMs following these injections. In contrast, massive retrograde labeling was found in the rostral groups of ICMs after WGA-HRP injections involving the substantia innominata and horizontal limb of the diagonal band. Furthermore, these retrogradely labeled intercalated cells were also GABA-immunoreactive. Results of iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the rostral ICMs confirmed that they contribute a massive projection to the entire extent of the substantia innominata and horizontal limb of the diagonal band. Electron microscopic observations of ultrathin sections prepared for postembedding GABA or glutamate immunocytochemistry revealed that the ICM terminals labeled with PHA-L displayed GABA, but not glutamate immunoreactivity, and formed symmetric synapses with dendritic profiles. The present findings constitute the first direct demonstration of an amygdalofugal GABAergic projection to the basal forebrain. Considering that the basal forebrain contains a group of cholinergic and GABAergic neurons collectively projecting to the entire cortical mantle, this GABAergic projection of the ICMs could allow the amygdaloid complex to influence the activity of widespread cortical regions to which it is not directly connected, at least in the cat.

Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord.

Exposure of the lumbar spinal cord of rats to X-rays 3 days after birth results in changes in the composition of central glia. Shortly after irradiation, there is both retardation of central myelin formation and a loss of integrity of the astrocyte-derived glia limitans on the dorsal surface of the cord. Subsequently, Schwann cells invade, undergo division and myelinate axons in the dorsal funiculi in the irradiated region of the cord, creating there an environment similar to that of peripheral nerve. The present study was undertaken to compare the ability of lesioned dorsal root axons to grow back into the altered glial environments that exist within the spinal cord after irradiation. This regrowth was assessed by injecting Fluoro-Gold into the spinal cord and subsequently examining neurons in the dorsal root ganglia (DRG) for the presence of this label. Numbers of retrogradely labeled neurons were counted in the DRG in both injured and contralateral non-injured sides. Non-irradiated control rats had almost no labeled DRG neurons on the injured side, whereas Fluoro-Gold labeled neurons were observed in substantial numbers in the DRG on the injured side of irradiated rats. There was a definite trend in the data, indicating that the longer the interval between irradiation and root injury, the greater the number of labeled neurons. Since the Fluoro-Gold labeling technique does not allow for visualization of the labeled axons within the spinal cord, a few animals were used to assess anterograde labeling with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP/HRP) from the dorsal root into the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential projections to the raphe nuclei from the medial parabrachial-Kölliker-Fuse (NPBM-KF) nuclear complex and the retrofacial nucleus in cats: retrograde WGA-HRP tracing

Abstract After injection of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP, 2 × 30 nl) into the nucleus raphe magnus (NRM) of 6 cats, a number of retrogradely labelled neurons were observed in the rostral pons, mainly in the pontine pneumotaxic area, i.e., the medial parabrachial and Kolliker-Fuse (NPBM-KF) nuclear complex, and the tegmental field. In addition, a cluster of labelled cells was observed in the retrofacial nucleus (RFN) and its adjacent areas in the medulla. Control injections of the same volume of WGA-HRP into the medullary magnocellular tegmental field (2 mm lateral to the NRM) resulted in a much lower number of labelled neurons in the areas described above, and the labelled cells in the tegmental fields were predominant ipsilateral to the injected side. Injections into the nucleus raphe pallidus caudal to the NRM resulted in a diffuse distribution of labelled neurons, mainly in the tegmental fields of the pons and medulla. This study demonstrates that the NRM receives specific convergent projections from the NPBM-KF complex and the RFN in the medulla. It is suggested that these pathways are involved in the control of respiration.

The trajectory of the sympathetic nerve fibers to the rat cochlea as revealed by anterograde and retrograde WGA-HRP tracing

Wheat germ agglutinin-horseradish peroxidase conjugate was injected in the unilateral superior cervical ganglion (SCG), and the projection pathways of postganglionic sympathetic nerve fibers innervating the cochlea were traced in the rat. The labeled axons advanced along the internal carotid artery (ICA), and a few advanced caudally in the major petrosal nerve (MPN) and entered the facial nerve, while the majority ran rostral to the pterygopalatine ganglion at the point where they crossed the MPN in the carotid canal. The rest of the labeled fibers remained on the surface of the ICA and advanced to the cranial cavity. Most of the labeled fibers along the facial nerve joined the cochlear nerve and finally reached the osseous spiral lamina through the spiral ganglion. Some of the labeled fibers ran along the anterior inferior cerebellar artery from the basilar artery which was previously thought to have been the only pathway. We could not find any labeled fiber on the modiolar artery from anterior inferior cerebellar artery in the cochlea. These observations are consistent with our hypothesis that the sympathetic fibers innervating the neural tissues or related structures follow nerve fibers and meninges as matrices of projection pathways rather than arteries.

Organization of cortical afferents to the rostral, limbic sector of the rat thalamic reticular nucleus.

The organization of limbic cortical afferents to the thalamic reticular nucleus (TRN) is described. Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), biocytin, neurobiotin, or fluorescent dextrans was delivered into the rat cingulate, retrosplenial, and, for comparison, somatosensory cortices. In other species a slab-like arrangement of cortical terminals has been described for sensory TRN sectors. Here this is seen in the rat somatosensory sector. Terminals from limbic cortices did not cluster into slabs but were found to fill the entire thickness of distinct rostral TRN regions. The cingulate and retrosplenial recipient TRN regions overlap, as do the projections from these cortical areas to anterior thalamic nuclei. Retrosplenial fibres contacted the dorsal and rostral TRN, which is known to be connected to the retrosplenial-recipient anteroventral, anterodorsal, and laterodorsal thalamic nuclei. Cingulate terminals occupied more ventral regions of the rostral TRN. This area is connected to thalamic nuclei also innervated by the cingulate cortex: the mediodorsal and anteromedial nuclei. A loose, but clear, topography could be defined for the cingulate-reticular pathway: rostrocaudal and mediolateral directions in the cortex are represented by ventrodorsal and rostrocaudal directions in the TRN, respectively. This organization of limbic corticoreticular pathway corresponds to the arrangement of limbic corticothalamic connections. The ultrastructure of the limbic cortical axon terminals was similar to that of the cortical boutons (D-type) described previously. The labelled terminals formed asymmetrical synapses onto dendritic profiles of reticular neurons. These findings, together with data in the literature, show significant morphological and connectional differences within the TRN that imply functional heterogeneities.

Compartmentation of glutamate and glutamine in the lateral cervical nucleus: further evidence for glutamate as a spinocervical tract neurotransmitter.

Previous observations indicate that spinocervical tract terminals contain relatively high levels of glutamate. To examine whether these high glutamate levels are likely to represent a neurotransmitter pool or an elevated metabolic pool, the distributions of glutamate- and glutamine-like immunoreactivities were examined in adjacent immunogold-labeled sections of the lateral cervical nucleus. Spinocervical tract terminals were identified by anterograde transport of horseradish peroxidase and wheat germ agglutinin-horseradish peroxidase conjugate from the spinal cord. Spinocervical tract terminals were found to contain significantly higher levels of glutamate-like immunoreactivity than other examined tissue compartments (large neuronal cell bodies, terminals with pleomorphic vesicles, astrocytes, and average tissue level). In contrast, the highest levels of glutamine-like immunoreactivity were detected in astrocytes. The different analyzed tissue elements formed three groups with respect to glutamate:glutamine ratios: one high ratio group including spinocervical tract terminals, a second group with intermediate ratios consisting of neuronal cell bodies and terminals containing pleomorphic synaptic vesicles, and a third low ratio group including astrocytes. Our findings indicate the presence of a compartmentation of glutamate and glutamine in the lateral cervical nucleus, similar to that postulated in biochemical studies of the central nervous system. The results also show that spinocervical tract terminals have high glutamate: glutamine ratios, similar to those previously observed in putative glutamatergic terminals in the cerebellar cortex. Thus, spinocervical tract terminals display biochemical characteristics that would be expected of glutamatergic terminals and the present findings therefore provide further evidence for glutamate as a spinocervical tract neurotransmitter.

Direct projections from the ventrolateral medulla oblongata to the limbic forebrain: anterograde and retrograde tract-tracing studies in the rat.

Neurons in the ventrolateral medulla oblongata, a brain region implicated in central vasomotor regulation, have previously been reported to project to some forebrain limbic structures. The aim of the present study was (1) to describe the termination pattern of ventral medullary afferents in forebrain limbic areas using anterograde tract tracing, and (2) to determine the location and some morphological characteristics of the projection neurons using retrograde tract tracing from selected forebrain sites. Following ionophoretic microinjections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the rostral ventrolateral medulla, labelled afferents were observed in the hippocampus, entorhinal and retrosplenial cortices, dorsal septum, nucleus accumbens, and the medial prefrontal cortex. Anterogradely labelled axons, ascending from the caudal ventrolateral medulla, could be traced only to the rostral aspects of the investigated forebrain limbic structures. Here, the main target of the ascending projection was in the ventral septum. However, labelled terminals were also present in the nucleus accumbens, the dorsolateral septum, and in the infralimbic cortex. The density of the ventrolateral medullary projections into all examined forebrain areas was low. The location of the cells in the ventral medulla oblongata which give rise to direct forebrain projections was examined using retrograde tract tracing with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). Following WGA-HRP injections into the septo-accumbens region, retrogradely labelled cells were present in both the rostral and caudal ventrolateral medulla. When the tract tracer injection was restricted to the ventral region of the septal complex, the labelled cells were concentrated in the caudal aspects of the ventrolateral medulla (and the nucleus of the solitary tract). Following tracer injections into the anterior cingulate cortex or the hippocampus or the entorhinal cortex, retrogradely labelled cells in the medulla oblongata were predominantly in the rostral ventrolateral medulla. As a first attempt to reveal the chemical nature of the projection cells, the contribution of tyrosine hydroxylase-immunoreactive cells to the innervation of the septo-accumbens area was also investigated: tyrosine hydroxylase-immunoreactive cells of both the caudal ventrolateral medulla and the nucleus of the solitary tract were found to contribute to the innervation of the septo-accumbens area. The distribution of retrogradely labelled cells as well as the termination pattern of the anterogradely labelled terminals indicated that the innervation of the various forebrain limbic areas arises from cells, diffusely distributed in the rostral and/or the caudal ventrolateral medulla oblongata.(ABSTRACT TRUNCATED AT 400 WORDS)