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.     

Anatomical evidence for convergence of olfactory, gustatory, and visceral afferent pathways in mouse cerebral cortex

Flavor perception requires the neural integration of olfactory, gustatory and, possibly, visceral afferent information. Presently, it is not known where, or how this integration takes place in the brain. Neuroanatomical data presented here suggest that pathways subserving these sensory modalities converge in mouse insular cortex after surprisingly few synaptic relays. Orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to label main olfactory bulb (MOB) efferents. A projection into layer I of insular cortex was present in every case. Bulb transections were made to provoke anterograde degeneration and EM analysis confirmed that the olfactory projection to insular cortex was a terminal pathway. WGA-HRP injections in the MOB-recipient zone of insular cortex resulted in ortho and retrograde labeling of ascending and descending gustatory-visceral afferent pathways. It is concluded that in the mouse, there is a remarkably direct convergence of olfactory and gustatory-visceral sensory pathways in insular cortex. Together with the descending connections from insular cortex to the amygdala and to brainstem autonomic structures, it is possible that the cortical integration of olfactory and gustatory-visceral information could modulate mechanisms involved in food selection and autonomic reactions relating to the chemical senses. Basic mechanisms subserving flavor perception might be usefully modelled in mouse insular cortex.     

Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane----endocytic structures----endosomes (presecondary lysosomes)----transfer vesicles----transmost Golgi saccule----vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport of molecules from nose to brain: Transneuronal anterograde and retrograde labeling in the rat olfactory system by wheat germ agglutinin-horseradish peroxidase applied to the nasal epithelium

Transneuronal anterograde labeling with the conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP) has been documented in the mammalian and immature avian visual system [6,14]. Transneuronal retrograde labeling was significant only in the chick [6]. The present study was performed to determine whether transneuronal labeling could be shown in the mammalian olfactory system, whether the phenomenon was robust in adults, and whether transneuronal retrograde transport could label several transmitter-specific centrifugal afferent projections to the olfactory bulb. In addition we wished to learn whether molecules that enter the nasal cavity can undergo transport to brain neurons. Gelfoam implants soaked with 1% WGA-HRP, surgically implanted into the nasal cavity, produced transneuronal labeling patterns that affirmed all of these questions. Transneuronal anterograde transport labeled the appropriate zones in the olfactory bulb and in all second order olfactory targets. In addition, there was transneuronal retrograde labeling of neurons in the olfactory bulb, anterior olfactory nucleus and in transmitter-specific projection neurons from the diagonal band (cholinergic), raphe (serotonergic) and locus coeruleus (noradrenergic). Transneuronal labeling was robust and consistent. The patterns of labeling indicated that transneuronal anterograde and retrograde transport occurred along known, specific circuits in the olfactory system. The present results suggest that nasal epithelial application of WGA-HRP may be a useful tool for assessing regeneration of primary olfactory neurons and the status of central circuitry following regeneration. The method should also facilitate the study of central olfactory connections after surgical or genetic lesions of the olfactory bulb. Finally, these experiments suggest the possibility that inhaled molecules including, possibly substances of abuse, may be transported to, and, possibly, influence the function of neurons in the brain, including some (diagonal band, raphe, locus coeruleus) which have extensive projections to wide areas of the CNS.     

Anterograde transsynaptic transport of WGA-HRP from spinal afferents to postganglionic sympathetic cells of the stellate ganglion of the guinea pig.

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) or choleragenoid conjugated HRP (B-HRP) into lower cervical and upper thoracic dorsal root ganglia (DRG), HRP reaction product was observed in peripheral fibers of spinal afferents and in postganglionic cell bodies of the stellate ganglion (SG) in the guinea pig. After WGA-HRP injection into C8-T3 or T5 DRG, HRP-labelled cells were observed to cluster at the rami within the SG, with peak labelling observed 36 h after injection. SG cell labelling occurred with B-HRP as well, but not with native HRP after injection into thoracic DRG. Injection of this tracer in C8 DRG gave rise to a small number of labelled cells. In contrast to the labelling pattern following thoracic or C8 DRG injections, injection of WGA-HRP or native HRP into C6 DRG, led to random SG cell labelling. We conclude that the anterograde transsynaptic transport, following injection of WGA-HRP into thoracic DRG, provides a method to selectively label a population of postganglionic sympathetic neurons within the SG. A combination of transsynaptic and retrograde transport appears to be responsible for labelling after injection into C8 DRG, whereas labelling after C6 DRG injections seems to be due primarily to retrograde transport.     

Central Connections of the Ovine Olfactory Bulb Formation Identified Using Wheat Germ Agglutinin-Conjugated Horseradish Peroxidase

Pheromonal stimuli elicit rapid behavioral and reproductive endocrine changes in the ewe. The neural pathways responsible for these effects in sheep are unknown, in part, because the olfactory bulb projections have not been examined in this species. Using the anterograde and retrograde neuronal tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), we describe the afferent and efferent olfactory bulb connections of the Suffolk ewe. Injections of WGA-HRP limited to the main olfactory bulb resulted in retrograde labeling of cells in numerous telencephalic, diencephalic, and metencephalic regions. Terminal labeling was limited to layer Ia of ipsilateral cortical structures extending rostrally from the anterior olfactory nucleus (AON), piriform cortex, anterior-, and posterolateral-cortical amygdaloid nuclei to lateral entorhinal cortex caudally. Injections involving the accessory olfactory bulb and AON produced additional labeling of cells within the bed nucleus of the stria terminalis (BNST), medial nucleus of the amygdala, and a few cells in the posteromedial cortical nucleus of the amygdala. Terminal labeling included a small dorsomedial quadrant of BNST and also extended to the far lateral portions of the supraoptic nucleus. A clearly defined accessory olfactory tract and nucleus was not evident, perhaps due to limitations in the sensitivity of the method. With this possible exception, the afferent and efferent olfactory connections in the sheep appear similar to those reported for other species.     

Nuclear terminations of corticoreticular fiber systems in rats

Corticoreticular fiber systems were examined in adult albino and hooded rats using anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and anterograde degeneration. WGA-HRP injections were made stereotactically into the medial prefrontal cortex, the medial agranular cortex, the anterior cingulate cortex, the face motor cortex, the forelimb motor cortex, the trunk-hindlimb motor cortex, the face somatosensory cortex, the primary auditory cortex, the secondary visual cortex and the primary visual cortex. With exception of the cingulate cortex (which is relatively inaccessible to lesioning methods) and the primary visual cortex, electrocautery lesions were made into these same cortical areas. The precise locations of cortical injection/lesion sites were corroborated on the basis of cortical cytoarchitectonic criteria, patterns of retrograde and anterograde thalamic labeling, and patterns of anterograde labeling in non-reticular brainstem nuclei such as the red nucleus, trigeminal nuclei and dorsal column nuclei. The heaviest corticoreticular projections arise from the medial agranular cortex. The medial prefrontal cortex also gives rise to consistently strong corticoreticular projections. The anterior cingulate cortex sends robust corticoreticular projections to the upper brainstem but relatively weak projections to the lower brainstem. With respect to the primary motor cortex, the face area gives rise to the densest corticoreticular projections, rivaling those emanating from the medial agranular cortex. The trunk-hindlimb area gives rise to substantial corticoreticular projections, but those originating from the forelimb area are modest and directed chiefly to midbrain and medullary levels. The face area of the somatosensory cortex gives rise to rather weak corticoreticular projections, while those arising from the primary auditory cortex are fewer still. Descending projections from the secondary visual cortex are sparse, with labeled terminals occurring in a few pontine and medullary reticular nuclei. Only one brainstem reticular nucleus (nucleus cuneiformis) was found to receive projections from the primary visual cortex, and this input was extremely sparse. Corticoreticular projections to the upper brainstem terminate predominantly ipsilateral to the cortical injection site, whereas medullary corticoreticular projections distribute bilaterally. Corticoreticular fibers from the medial agranular, face motor and trunk-hindlimb motor cortex terminate heavily in somatomotor brainstem reticular nuclei such as the pontis oralis, the pontis caudalis and the gigantocellularis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization of projections from olfactory epithelium to olfactory bulb in the frog, Rana pipiens

One hypothesis for the coding of olfactory quality is that regions of the olfactory epithelium are differentially sensitive to particular odor qualities and that this regional sensitivity is conveyed to the olfactory bulb in a topographic manner by the olfactory nerve. A corollary to this hypothesis is that there is a sufficiently orderly connection between the epithelium and the olfactory bulb to convey this topographical coding. Thus we examined topography in the projection from epithelium to bulb in the frog, which has been the subject of numerous electrophysiological studies but has not yet been examined using modern neuroanatomical techniques. The tracer WGA-HRP was applied to the ventral or to the dorsal olfactory epithelium, or both. Anterograde transport of label to the olfactory bulb was seen after as few as 2 days; label was still present in the bulb as long as 21 days postinjection. In cases where WGA-HRP was applied to the entire epithelium, there was dense anterograde labelling of the ipsilateral olfactory bulb. In addition, a small medial portion of the contralateral bulb was labelled. Injections limited to either the ventral or dorsal epithelium produced patterns of anterograde labelling in the glomerular layer of the olfactory bulb, which varied with the size and location of the injection. With very large injections in either the dorsal or ventral epithelium, label appeared to be evenly distributed in the glomerular layer. With smaller injections in the ventral epithelium, there was heavier labelling in the lateral than in the medial portions of the glomerular layer, although light labelling was found in all regions of the glomerular layer. In contrast, injection sites restricted to the dorsal epithelium produced more anterograde labelling in the medial than lateral portions of the glomerular layer. These patterns extended throughout the dorsal-ventral extent of the bulb. Within the limits of the anterograde tracing technique used, we were unable to detect any systematic relationship between the pattern of labelling in the glomerular layer and the medial-lateral or rostral-caudal location of the injection site in either the ventral or dorsal epithelium. We conclude that in the frog, as in other amphibia, there is only a limited degree of topographic order between the epithelium and the olfactory bulb.     

Fixation of transsynaptically transported WGA-HRP and fluorescent dyes used in combination

The transsynaptic transport of WGA-HRP is a powerful tool for the identification of last order interneurones in the spinal cord, but differentiation of primarily labelled motoneurones from transsynaptically labelled interneurones can be difficult. We therefore combined the transsynaptic labelling of interneurones with WGA-HRP and the labelling of motoneurones with fluorescent dyes (fluorogold, red beads, fast blue, DAPI) by injecting a mixture of the tracers into the nervi mediani of rats. The influence of different fixatives on the simultaneous preservation of WGA-HRP and fluorescence is evaluated. It is shown that, of several perfusion protocols, only perfusion with paraformaldehyde in addition to 1.4% lysine and 0.23% periodate allowed simultaneous visualisation of transsynaptic WGA-HRP and fluorescent dyes.     

Reciprocal connections between medial prefrontal cortex and lateral posterior nucleus in rats

The connections of the posterior part of the medial prefrontal cortex with the thalamic lateral posterior nucleus in rats were studied using anterograde and retrograde axonal transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and tritiated leucine. After injections of WGA-HRP into the medial prefrontal cortex, an area confirmed to receive direct projections from the visual cortex, retrogradely labeled neurons were observed ipsilaterally in the lateral posterior nucleus of the thalamus, as well as in the mediodorsal, anteromedial, ventromedial, ventrolateral, laterodorsal, centrolateral, paracentral, rhomboid, parafascicular and posterior nuclei. In the lateral posterior nucleus, the labeled cells were located mainly in the lateroventral portion of its anterior half. In contrast, the posterior half of this nucleus was free of label. Axons labeled by the anterograde transport of tritiated leucine were dispersed over the same region which contained retrogradely labeled cells. The functional significance of these connections is discussed with special reference to their possible role in visuomotor integration in rats.     

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

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

Transneuronal transport of WGA-HRP in immature rat visual pathways

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to study transneuronal transport in the developing rat visual pathways. Intraocular injections of WGA-HRP were made in neonatal albino rat pups at different ages from the day of birth, postnatal day 0 (P0), to one month of age. Transneuronal labeling in geniculostriate fibers and in tectoparabigeminal terminals was observed as early as P1 and showed little change with eye-opening. However, at early ages, consistent transneuronal labeling was found to require injection of up to 4 times the amount of tracer (0.18 mg WGA-HRP) as adults (0.04 mg WGA-HRP), delivered in two injections. Control injections of HRP alone produced heavy anterograde labeling at all ages, without requiring increased injections. The results suggest that transneuronal transport precedes synaptic transmission, and may illustrate a mechanism for exchanging molecules between neurons. One explanation for the requirement of increased tracer is that axonal and/or transneuronal transport of WGA-HRP may be selectively limited to certain cells in the postnatal retina.     

Enhancement of horseradish peroxidase histochemistry and/or uptake with radiocontrast media.

The effects of dissolving horseradish peroxidase (HRP) or HRP conjugated to wheatgerm agglutinin (WGA-HRP) in radiocontrast media (MD76) on the intraaxonal transport and enzymatic activity of these tracers were evaluated both in vivo and in vitro. The in vivo studies showed that more reaction product was visible in the L4 dorsal horn following soaking of the sciatic or peroneal nerve, or following cutaneous injection of WGA-HRP, when WGA-HRP was dissolved in MD76, as compared to dissolving WGA-HRP in distilled water. Additionally, there appeared to be an enhancement of anterograde transport and a reduction of retrograde transport of WGA-HRP when this tracer was dissolved in MD76, as compared to dissolving it in distilled water. The in vitro studies indicated that radiocontrast media increased the enzymatic activity of both WGA-HRP and HRP as compared to their enzymatic activity in distilled water when assayed spectrophotometrically. Data are presented that indicate some binding (about 4%) of HRP with MD76. This binding of HRP with organically bound iodine may account for the enhancement of anterograde transport and/or increased enzymatic activity.     

The use of wheat germ agglutinin--horseradish peroxidase conjugates for studies of anterograde axonal transport

Injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), into the hemisected spinal cord of the rat, cat and monkey consistently resulted in the intense anterograde labeling of ascending spinal projections such as the spinothalamic tract and spinocerebellar tracts and their terminal fields. Injections of WGA-HRP in the dorsal column nuclei resulted in the anterograde labeling of the medial lemniscus and its terminal fields in the thalamus. Injection of similar amounts of horseradish peroxidase alone (HRP) in hemisected animals or the dorsal column nuclei resulted in little anterograde labeling. The rate of the anterograde transport of WGA-HRP in cut axons appears to be greater than 200 mm/day. Small amounts of transneuronal labeling appeared to occur after injection of WGA-HRP in both cut axons and undamaged cell bodies. These results suggest that the amount of anterograde labeling observed after injection of WGA-HRP into both cut axons and undamaged cell bodies is significantly greater than the anterograde labeling observed after injections of HRP alone. Therefore, in the central nervous system WGA-HRP appears to be a far more effective anterograde tracer than HRP alone.     

Reciprocal parabrachial-cortical connections in the rat

The projection from the parabrachial nucleus (PB) to the cerbral cortex in the rat was studied in detail using the autoradiographic method for tracing anterograde axonal transport and the wheat germ agglutinin-horseradish peroxidase (WGA-HRP) method for both anterograde and retrograde tracing. PB innervates layers I, V and VI of a continuous sheet of cortex extending from the posterior insular cortex caudally, through the dorsal agranular and the granular anterior insular cortex and on rostrally into the lateral prefrontal cortex. Within the prefrontal area, PB fibers innervate primarily layer V of the ventrolateral cortex caudally, but more rostrally the innervated region includes progressively more dorsal portions of the prefrontal area, until by the frontal pole the entire lateral half of the hemisphere is innervated. This projection originates for the most part in a cluster of neurons in the caudal ventral part of the medial PB subdivision, although a few neurons in the adjacent parts of the PB, the Kolliker-Fuse nucleus and the subcoeruleus region also participate.After injection of WGA-HRP into the PB region, retrogradely labeled neurons were found in layer V of the same cortical areas which receive PB inputs. The importance of this monosynaptic reciprocal brainstem-cortical projection as a possible anatomical substrate for the regulation of cortical arousal is discussed.     

Enucleation-induced transsynaptic labeling with WGA-HRP in the developing rat visual system

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) is a neuroanatomical tracer which is transported transneuronally. In order to investigate whether transport of WGA-HRP is across synapses, labeling was studied in the developing retinotectal pathway where it is known that enucleation results in increased ipsilateral synaptic connections from the remaining eye. While little or no transneuronal labeling was evident in controls, after enucleation transneuronal labeling was consistently observed. Furthermore, the critical period for enucleation-induced transneuronal labeling coincides with the known critical period for enucleation-induced neuronal survival and synaptic formation. The results suggest that transneuronal exchange of WGA-HRP depends on the presence of synapses, and is therefore transsynaptic.     

Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats: reciprocal connections with host thalamus demonstrated with WGA-HRP

Fetal frontal cortex was transplanted into lesion cavities formed in host motor/sensory cortex of adult rats. Eight to twenty-eight weeks later wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into host thalamus and the brain was sectioned and reacted using a sensitive TMB procedure. A large amount of fine granular WGA-HRP was detected in most transplants. This could represent anterograde transport demonstrating that injured adult host thalamic neurons sprouted axons into fetal cortical transplants. Conversely, none or very few retrogradely labeled pyramidal neurons were present in the transplants. This indicates that pyramidal neurons in transplants either did not sprout into adult host brain or sprouted such short distances that they did not pick up the WGA-HRP. These results are compatible with the hypothesis that high trophic/growth factor levels in newborn or fetal brain and low levels in adults determine the more extensive connections seen in newborn hosts compared with those in adult transplanted hosts. The data are also consistent with the proposal that adult host brains impair axonal growth. Functionally, the data suggest that although corticofugal effects of fetal cortical transplants in adult host brains are likely to be limited, transplants could exert beneficial trophic effects on adult host thalamic neurons.     

Quantitative analysis of the olfactory pathway for drug delivery to the brain

Following intranasal administration to rats, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) concentrated in the olfactory nerve and glomerular layers of the olfactory bulb resulting in a mean olfactory bulb concentration of 140 nM. A negligible amount of label was detected in the olfactory bulb following intravenous administration of WGA-HRP or intranasal administration of unconjugated HRP. This is the first quantitative assessment of intraneuronal transport of a protein into the brain using the olfactory route.     

Intracortical termination of the retino-geniculo-striate pathway studied with transsynaptic tracer (wheat germ agglutinin-horseradish peroxidase) and cytochrome oxidase staining in the macaque monkey

The tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), was used as a transneuronal marker in the macaque monkey to study retino-geniculo-striate pathway terminals in area 17. Concomitantly, we stained matching sections for metabolic capacity using the cytochrome oxidase staining technique²⁶. Terminal labeling by WGA-HRP and cytochrome oxidase activity staining revealed duplicate patterns in layers II and III, IVA, IVCα, IVCβ, and VI. The absence of WGA-HRP-labeled neuronal cell bodies in area 17 supports the conclusion that WGA-HRP is a transsynaptic marker in the macaque visual pathways.