Anterograde transsynaptic transport of WGA-HRP in rat olfactory pathways

The transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was studied in rat olfactory pathways. After applications of tracer to the vomeronasal organ, the olfactory epithelium or injections into the olfactory bulb, WGA-HRP reaction product was observed in second-order neuron terminal areas of each pathway, e.g. within posteromedial cortical amygdaloid nucleus, primary olfactory cortex and contralateral primary olfactory cortex, respectively. The results indicate that anterograde transsynaptic transport of WGA-HRP occurs in olfactory pathways, as has been shown in visual, somatosensory and limbic systems, and thus, anterograde transsynaptic transport may be a mechanism for neurons to exchange materials and/or messages.     

Anterograde transsynaptic transport of WGA-HRP in the limbic system of rat and monkey

The lectin tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), was injected into the entorhinal cortex in rat and monkey brains. Tracer labeling was followed in the entorhinal projection to dentate gyrus and hippocampus, i.e. along the perforant pathway. Besides labeling perforant pathway terminals in the outer two-thirds of stratum moleculare in the dentate gyrus, reaction product was also observed within stratum granulosum. We conclude that labeling of dentate granule cells was the result of anterograde transsynaptic transport of WGA-HRP. The evidence thus provides an example of anterograde transsynaptic transport: in the limbic system; and at an excitatory synapse.     

Neuronal projections to the guinea pig stellate ganglion investigated by retrograde tracing

Previous electrophysiological studies have revealed a peripheral sensory input to the stellate ganglion which does not originate from the dorsal root ganglia. The present retrograde tracing study aimed at evaluating whether the parent cell bodies are located in the periphery, i.e. in mediastinal ganglia. Following injection of Fast blue or wheat germ agglutinin-horseradish peroxidase into the right stellate ganglion of the guinea pig, retrogradely labelled cell bodies were observed in the intermediolateral and intercalated nuclei of the spinal cord as well as in dorsal root ganglia at segmental levels C8 to T6. In another case, the stellate ganglion was resected and replaced by a sponge soaked with 10 μl of Fast blue. Labelling of preganglionic and sensory neurons parallelled that obtained by tracer injections. In neither case, however, were retrogradely labelled neurons found within or around the thoracic viscera (thymus, trachea, bronchi, esophagus, heart, great vessels of upper mediastinum) when these were cut serially en bloc. Controls performed by injection of Fast blue into the inferior mesenteric ganglion and investigation of the distal colon showed that our experimental protocol was able to visualize a peripheral projection towards a sympathetic ganglion — in this case from myenteric ganglia to the inferior mesenteric ganglion. We conclude that, in contrast to the circuitry connecting prevertebral sympathetic ganglia with the gut, the neuronal cell bodies providing peripheral sensory input from thoracic viscera to the right stellate ganglion most likely are not located within the mediastinal ganglia. Instead, they may reside within the stellate ganglion itself.     

Three electrophysiological classes of guinea pig sympathetic postganglionic neurone have distinct morphologies

Sympathetic postganglionic neurones can be differentiated electrophysiologically into three classes (phasic, Ph; tonic, T; and long-afterhyperpolarising, LAH) based on their potassium channel expression and consequent differences in excitability. We tested whether neuronal morphology differs between these classes. Neurones in coeliac, inferior mesenteric, and lower lumbar paravertebral ganglia of guinea pigs were filled with biocytin during in vitro experiments in which electrical properties were recorded. The dimensions of somata and dendrites were measured in approximately equal numbers of stained neurones of each class. The three electrophysiological classes were distinct in terms of soma shape, soma size (Ph < T = LAH), total dendritic length (LAH < Ph < T) and average length of dendrites (LAH < Ph < T) (P < 0.0001, multivariate analysis of variance). The mean number of primary dendrites also differed (LAH 13, Ph 16, T 20). The majority of dendrites did not branch, the ratios of terminations to primary dendrites being 1.36 (LAH), 1.63 (Ph) and 1.81 (T). Overall, LAH neurones, with medium-sized somata but the smallest dendritic trees, were more distinct morphologically than Ph and T neurones. The morphological differences between classes were not dependent on differences in location. Further, there was no apparent relation between morphology and the pattern of synaptic input each class receives. The results indicate that three distinct groups of sympathetic postganglionic neurone exist in adult guinea pigs, although more than three functions are subserved by these neurones. © 1996 Wiley-Liss, Inc.     

Distribution of postganglionic parasympathetic fibers originating in the pterygopalatine ganglion in the maxillary and ophthalmic nerve branches of the trigeminal nerve; HRP and WGA-HRP study in the guinea pig.

The distribution of the postganglionic parasympathetic fibers originating in the pterygopalatine ganglion (PTPG) has been traced in the guinea pig by means of the HRP and WGA-HRP methods. The greatest number of labeled cells were observed when WGA-HRP was injected in the lacrimal gland. After applying HRP to all the ramifications of the maxillary and ophthalmic divisions of the trigeminal nerve, labeled neurons were found in the PTPG. Numerous PTPG fibers were detected in the ethmoidal and sphenopalatine nerves. The presence of PTPG fibers in the supraorbital, infratrochlear, zygomaticotemporal, zygomaticofacial-inferior palpebral, sphenopalatine and infraorbital-superior alveolar nerves has not hitherto been reported in mammals.     

Sympathetic neurons in the cat spinal cord projecting to the stellate ganglion.

Horseradish peroxidase (HRP) was injected into the stellate ganglia of anesthetized cats. After a 2-day survival time the spinal cord was processed for the HRP reaction to study the distribution of the preganglionic neurons which project to the stellate ganglia. HRP-labeled neurons in the cord were located exclusively on the side ipsilateral to the injected stellate ganglia and were concentrated in four distinct areas: (1) intermediolateral cell column (78.2%), (2) lateral funiculus (18.5%), (3) intercalated area and (4) central autonomic area (3.3%) for the latter 2 areas). Their distribution pattern in the very rostral pole of the thoracic sympathetic preganglionic cell column was different from the typical thoracic pattern in that a greater proportion of the cells were located in the lateral funiculus, and a few labeled cells were observed in the ventral horn. Longitudinally, a wide range (C8-T8) of spinal cord levels projected to the stellate ganglia, with a peak at the T2 level. These observations infer that a large population of axonal processes of sympathetic preganglionic neurons traveled several segments of the spinal cord through an intraspinal pathway before exiting a particular ventral root.     

Calbindin-immunoreactive nerve terminals in the guinea pig coeliac ganglion originate from colonic nerve cells

Previous work has shown that calbindin-immunoreactive (calbindin-IR) nerve terminals are numerous in guinea pig prevertebral ganglia. A high proportion of those colonic nerve cells that project to the inferior mesenteric ganglia are calbindin-IR, but none of the neurons that project from the small intestine to the coeliac ganglion are immunoreactive for calbindin. The present work was designed to determine the source of the calbindin-IR fibres and the pathways by which they reach the coeliac ganglion. Sections through the major nerve trunks that connect with the coeliac ganglion revealed numerous calbindin-IR fibres in the inferior coeliac nerves and in the intermesenteric nerves, while there were very few fibres in the splanchnic or superior coeliac nerves. When all peripheral nerve connections to a lobe of the coeliac ganglion were cut, all calbindin-IR terminals degenerated. Cutting the ileo-caeco-colic nerves caused a substantial reduction in the density of nerve fibres in the coeliac ganglion, whereas no significant reduction could be detected when the intermesenteric nerves were cut. However, lesion of both the ileo-caeco-colic and intermesenteric nerves caused all the calbindin-IR nerve fibres in the coeliac ganglion to degenerate. It is concluded that most or all of the calbindin-reactive nerve terminals in the coeliac ganglion originate from the large intestine and that most reach the ganglion via the ileo-caeco-colic nerves. Thus many colonic intestinofugal neurons, supplying both the coeliac and inferior mesenteric ganglia, are immunoreactive for calbindin, whereas small intestinal intestinofugal neurons are not immunoreactive for this protein.     

Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.     

Distribution and origin of serotoninergic afferents to guinea pig cochlear nucleus

The distribution of serotoninergic fibers in the guinea pig cochlear nucleus was studied with serotonin immunohistochemistry. In addition, the origin of the serotoninergic fibers was determined by combining the retrograde transport of wheat germ agglutinin-apohorseradish peroxidase (gold conjugated) with serotonin immunohistochemistry. Immunoreactivity was present in varicose and nonvaricose fibers that were unevenly distributed throughout the cochlear nucleus. The fibers were most prominent in the superficial layers of the dorsal cochlear nucleus and the anterior spherical cell area of the anteroventral cochlear nucleus. Although less prominent, serotonin-positive fibers were also present in the remaining part of the anteroventral cochlear nucleus and the posteroventral cochlear nucleus. A few positive fibers were present in the auditory nerve root and the dorsal and intermediate acoustic stiae. Double-labeled cells were found throughout the rostral- caudal extent of the serotoninergic system from the caudal linear nucleus to the nucleus raphe pallidus. However, most were confined to the dorsal (52%) and median (18%) raphe nuclei. Some serotoninergic cell groups contained retrogradely labeled cells that were not serotonin immunoreactive, indicating nonauditory afferents to cochlear nucleus containing other neurotransmitter substances. Serotonin may tonically modulate auditory processing within the cochlear nucleus as well as influence certain ascending auditory pathways. Most of the serotonin in the cochlear nucleus comes from superior raphe nuclei that also project to basal ganglia motor systems and limbic strctures. Therefore, the effect of serotonin on the cochlear nucleus may be related to level of arousal or behavioral state. © 1995 Willy-Liss, Inc.     

Nitric oxide synthase in trigeminal ganglion cells projecting to the cochlea of rat and guinea pig

Nitric oxide (NO) influences electrophysiological and morphological parameters of the mammalian cochlea. Recently, the isoform of the NO-producing enzyme neuronal NO synthase (nNOS) has been demonstrated in spiral ganglion cells and olivocochlear neurons. The cochlea is also innervated by fibers stemming from the trigeminal ganglion (TG) and superior cervical ganglion (SCG). Whether these ganglion cells contain nNOS is not known yet. We therefore identified TG and SCG cells upon injection of Fluoro-Gold (FG) into the cochlea and retrograde neuronal transport of FG in rat and guinea pig. These ganglion cells were investigated for neuronal NOS immunohistochemically. Perikarya labeled by FG were found in the ipsilateral TG and SCG. In both species investigated, a considerable number of FG-labeled TG cells were also nNOS-immunoreactive whereas SCG cells were not. These data, demonstrating the existence of nNOS-containing TG cells that project to the cochlea, provide evidence that these neurons are further sources of nitric oxide in the cochlea.     

Mast cells in the guinea pig superior cervical ganglion: a functional and histological assessment.

We have previously found that antigenic stimulation of mast cells in the guinea pig superior cervical ganglion leads to membrane depolarization of principal neurons and a long-term increase in the efficacy of ganglionic transmission. In this study experiments were conducted to discern the histological, immunological and pharmacological characteristics of the mast cells within the superior cervical ganglion. Mast cells within the superior cervical ganglion could be stained with toluidine blue or berberine sulfate, the latter indicating that heparin-like molecules were present in the granules. Stainable mast cells were distributed throughout the ganglion with no gross evidence of regional localization. The number of mast cells stained with toluidine blue was reduced significantly (P less than 0.01) in contralateral ganglia that had been exposed to the sensitizing antigen (ovalbumin), indicating antigen-induced degranulation. The superior cervical ganglion contained 208 +/- 6 picomole of histamine (mean +/- SEM, n = 66). Ovalbumin evoked the release of histamine from the superior cervical ganglion in a concentration-dependent fashion. At maximally effective concentrations, ovalbumin released 33 +/- 2% of the total histamine stores (mean +/- SEM, n = 61). Similar values were obtained with antigen-challenged stellate ganglia. A temperature of 37 degrees C and an extracellular calcium concentration of 1 mM was required to elicit optimal antigen-induced responses. In addition to releasing histamine, antigenic stimulation of the ganglion resulted in a 3- to 5-fold increase in the synthesis and release of arachidonic acid metabolites including peptidoleukotriene, thromboxane B2, prostaglandins (PG) E2, F2 alpha, D2, the PGD2 metabolite 9 alpha 11 beta-PGF2, and the prostacyclin metabolite 6-keto PGF1 alpha. Various putative mast cell secretagogues were examined for their ability to activate the superior cervical ganglion mast cell, as indicated by evoked histamine release. In contrast to rat peritoneal mast cells, high concentrations of substance P, compound 48/80, and nerve growth factor failed to stimulate the ganglion mast cells. Preganglionic nerve stimulation, electrical field stimulation of axons and cell bodies, or depolarizing concentrations of potassium chloride also failed to activate the superior cervical ganglion mast cells. These results suggest that substances released by membrane depolarization do not influence the function of the resident mast cells. The results demonstrate that the mast cells within sympathetic ganglia can be actively sensitized to respond to specific antigen. These mast cells are similar to lung parenchymal mast cells with respect to histological, immunological and pharmacological characteristics...     

A spinal cord pathway connecting primary afferents to the segmental sympathetic outflow system

The sympathetic innervation of lumbar dorsal root ganglia (DRGs) and the possible presence of spinal cord circuits connecting primary sensory afferents to the sympathetic outflow to DRGs were investigated. We used simultaneous tracing of the sympathetic input to and sensory output from DRGs. Adult male rats received unilateral microinjections of the Bartha strain of pseudorabies virus into four lumbar DRGs. At 24 h post-inoculation, productive infection was detected in both DRG neurons and sympathetic postganglionic neurons. Infection of spinal cord neurons was first observed in sympathetic preganglionic neurons of the intermediolateral column. Subsequently, the infection spread to the contralateral intermediolateral column, the area around the central canal and the superficial dorsal horn layers. To investigate the relationship between infected spinal cord neurons and primary afferents from the corresponding DRGs, we injected pseudorabies virus for retrograde tracing together with cholera toxin B for anterograde tracing. We found that infected LIV/LV and LX neurons were in close apposition to cholera toxin B labeled afferents. Importantly, immunohistochemical detection of bassoon, a pre-synaptic zone protein, identified such contacts as synapses. Together, this suggests synaptic contacts between primary sensory afferents and neurons regulating sympathetic outflow to corresponding DRGs.     

SIF cells, cyclic AMP responses, and catecholamines of the guinea pig superior cervical ganglion.

The superior cervical ganglion (SCG) of the guinea pig has been investigated by a multidisciplinary approach. Dopamine (50 micron) produced no increase in cyclic AMP levels above control values of 27.9 pmole/mg protein, but 50 micron isoproterenol produced cyclic AMP levels of 210 pmole/mg protein, indicating the existence of a beta-adrenergic receptor-adenylate cyclase complex. The SIF cells were studied by fluorescence histochemistry, which indicated that two morphological types were present. A few Type I cells of the guinea pig SCG were solitary, but most were present in clusters containing many Type II cells. Immunohistochemical localization of antibodies to dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) demonstrated that types of SIF cell localize antibodies to DBH but not PNMT, providing strong evidence that norepinephrine is the neurotransmitter for all the SIF cells of the guinea pig SCG. Determination of the ratio of norepinephrine to dopamine confirmed that no other dopamine pools exist in the guinea pig SCG.     

Synaptic innervation of sympathetic ganglion cells in the bullfrog.

The synaptic innervation of the ganglion cells in the ninth and tenth paravertebral sympathetic ganglia of the bullfrog was investigated by histochemical and electron microscopic techniques and by intracellular recording. The neurons were unipolar and most ganglion cells were innervated by a single preganglionic axon. The preganglionic fiber stained for acetylcholinesterase and was observed to spiral around the axon hillock of the ganglion cell before arborizing and making synaptic contact with the neuron. Most synapses were located on the soma near the axon hillock region, with features typical for cholinergic junctions. The axosomatic location of the synapses was manifested physiologically by a decrease in membrane resistance (increased conductance) at the peak of the fast EPSP (excitatory postsynaptic potential) and by a demonstrable reversal potential for the fast EPSP.     

Effect of nifedipine on neurons of guinea pig celiac ganglion.

The effect of nifedipine on electrophysiological membrane properties and nicotinic neurotransmission of guinea pig celiac ganglion neurons was studied using intracellular recordings in vitro. Nifedipine in concentrations of 0.1-10 microM did not affect membrane potential, membrane input resistance or the amplitude and duration of action potentials induced by intracellular current injection. Higher doses of nifedipine (0.1-1 mM) significantly reduced the amplitude and extended the duration of action potentials induced by intracellular current injection. Superfusion of the ganglia with nifedipine in concentrations of 0.1-10 microM significantly inhibited nicotinic fast excitatory postsynaptic potentials (f-EPSPs) and orthodromic action potentials evoked by nerve stimulation. This depressant effect of nifedipine on synaptic transmission was eliminated with high Ca2+ (12.5 mM). Nifedipine (10 microM) did not affect the postsynaptic effect of exogenous acetylcholine (ACh), but significantly reduced the quantal content but not the quantal size of evoked f-EPSPs in a low Ca2+ (0.5 mM), high Mg2+ (5.5 mM) Krebs solution. Nifedipine in concentration of 10 microM did not affect afterspike hyperpolarization (AH) and post-tetanic hyperpolarization (PTH), which have been recognized to be generated mainly by an increase of calcium-dependent potassium conductance. Higher doses of nifedipine (0.1-1 mM) significantly depressed AH and PTH. These experimental results suggest that nifedipine in concentrations of 0.1-10 microM exerts an inhibitory effect on nicotinic neurotransmission without affecting the membrane properties of the guinea pig celiac ganglion neurons. This inhibitory effect of nifedipine on synaptic transmission may result from blocking L-type calcium channels and reducing the quantal release of ACh from the presynaptic nerve terminals.     

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells: roles of neurotrophin receptors

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.     

Contralateral projections of trigeminal mandibular primary afferents in the guinea pig as seen by transganglionic transport of horseradish peroxidase

Transganglionic transport of horseradish peroxidase (HRP) was used to investigate contralateral projections of trigeminal mandibular fibers in the guinea pig. After application of HRP to the buccal, lingual, auriculotemporal, mylohyoid, mental and inferior alveolar nerves, crossing fibers and contralateral endings were found in the caudal region of the nucleus of the solitary tract (most of these belonging to the buccal and lingual nerves), the dorsomedial region of the subnucleus caudalis of the trigeminal sensory nuclear complex (TSNC), and the dorsal horns of the first 5 cervical spinal cord segments (C1-C5). The greatest numbers of crossing fibers in the medullary and cervical dorsal horn segments belonged to the mental and mylohyoid nerves, though these nerves did not project contralaterally to C4-C5. Contralateral buccal and lingual endings were scattered sparsely from the subnucleus caudalis to C5, and only very few contralateral auriculotemporal terminals were observed. Though laminae I-V of the dorsomedial region of the medullary and cervical dorsal horns all exhibited contralateral endings of the mental and mylohyoid nerves, most such endings were found in laminae IIi-III, followed by lamina IV, which suggests their involvement in the reception of mechanical stimuli and in the sensory motor reflexes of the orofacial region. The contralateral buccal and lingual terminals were distributed somatotopically in the first 5 cervical cord segments, with the lingual endings rostral to the buccal terminals within each segment. In C4 and C5 lingual endings appeared exclusively in laminae I and IIo, suggesting that like the ipsilateral lingual projections at this level, which also terminate in these laminae, they may be involved in pain and temperature sensation.     

Anterograde tracing of retinal afferents to the tree shrew hypothalamus and raphe

The anterograde neuronal transport of Cholera toxin B subunit (CTB) was used in this study to label the termination of retinal afferents in the hypothalamus of the tree shrew Tupaia belangeri. Upon pressure-injection of the substance into the vitrous body of one eye, a major projection of the retinohypothalamic tract (RHT) was found to the hypothalamic suprachiasmatic nuclei (SCN). Although the innervation pattern was bilateral, the ipsilateral SCN received a somewhat stronger projection. Labeling was also found in the supraoptic nucleus and its perinuclear zone, respectively, mainly ipsilaterally as well as in the bilateral para- and periventricular hypothalamic regions without lateral predominance. In the raphe region, scattered fibers and terminals were seen in the dorsal and median raphe nuclei. CTB-immunoreactive structures were observed neither in the locus ceruleus nor in vagal nuclei. Our results, partly in contradiction to earlier studies using different tracing techniques in another tree shrew species (Tupaia glis), reveal that hypothalamic nuclei, in particular the SCN, are contacted by retino-afferent fibers which are thought to mediate the effects of light to the endogenous ‘clock’ and to parts of the neuroendocrine system.