Phaseolus Vulgaris Leucoagglutinin Injection Site Research Articles

Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity

In order to investigate converging projections originating from adjacent populations of cortical neurons, injections of two different anterograde tracers, biotinylated dextranamine (BDA) and Phaseolus vulgaris leucoagglutinin (PHA-L), were made in close proximity. When the two injection sites were separated by around 500 μm and the time between injections was 1–4 h, BDA-labeling of neuronal elements was found not only at the BDA injection site but also at the PHA-L injection site. This false-positive BDA labeling of neurons at the PHA-L injection site was so intense that labeled axons could be traced, both into the neighboring cortical gray matter and into white matter. Increasing the separation distance to 1000 μm resulted in much fewer falsely positive labeled neurons at the PHA-L injection site. Even more effective was extending the time interval between the two injections. Thus, if the BDA injection preceded the PHA-L injection by more than 12 h, virtually no false-positive labeling was associated with the PHA-L injection site. These procedures may be applied to other combinations of anterograde tracers, such as BDA with tetramethylrhodamine-conjugated dextran amine.

Medullary pathways mediating depressor responses from Na(+)-sensitive sites in nucleus of the solitary tract.

Two series of experiments were done in male Wistar rats to investigate the medullary pathways that mediate the depressor responses from sodium-sensitive sites in the nucleus of the solitary tract (NTS). In the first series, the anterograde tract tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophoresed unilaterally at sites in the NTS at which microinjections (20 nl) of a 154-175 mM NaCl solution elicited depressor responses. PHA-L injection sites were found to be localized within the medial subnucleus of the NTS (Sm). In the medulla, PHA-L-labeled fibers and presumptive terminal boutons were observed bilaterally, but with an ipsilateral predominance, throughout the rostrocaudal extent of the NTS the dorsal motor nucleus of the vagus, area postrema, the ventrolateral medulla (VLM), and nucleus ambiguus. The pontine region, containing the A5 catecholaminergic cell group and the parabrachial nucleus, also received projections from Sm. In the second series of experiments, the effect of blocking synaptic transmission in VLM with cobalt chloride (CoCl2; 5 mM, 100 nl) on the cardiovascular response elicited by microinjection (20 nl) of hypertonic saline (154-175 mM) into the ipsilateral Sm was investigated in the alpha-chloralose-anesthetized, paralyzed, and artificially ventilated rat. Microinjection of CoCl2 into VLM, at sites shown in the previous study to receive efferent projections from Sm, significantly attenuated the depressor (60%) and bradycardic (80%) responses to stimulation of Sm. These data indicate that the sodium-sensitive region of the caudal Sm innervates VLM neurons and suggest that these VLM neurons are involved in mediating the depressor and bradycardic responses elicited by changes in the extracellular concentration of sodium.

Efferent projections of the anterior perirhinal cortex in the rat

Because convulsive seizures develop very rapidly from kindling sites in the anterior perirhinal cortex, we studied perirhinal efferents by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PhAL). PhAL injections into the anterior perirhinal cortex labelled a prominent network of fibers within the frontal cortex that was most dense within layers I and II and layer VI. As individual PhAL injection sites within the perirhinal cortex were restricted to one or two adjacent laminae, we were able to determine that layer V was the main source of the perirhinofrontal projection. This was confirmed by frontal cortex injections of the retrograde tracer Fluorogold (FG). Other cortical areas with densely labelled fibers following perirhinal PhAL injections included the agranular insular, infralimbic, orbital, parietal, and entorhinal cortices. Moderate to mild fiber labelling was also noted in the posterior piriform, temporal and occipital cortices, and the claustrum. Subcortical labelling was seen in the nucleus accumbens; fundus striati; basal and lateral amygdala nuclei; the "acoustic thalamus"; and the central grey. Several of these cortical and subcortical projections were bilateral. The different laminar origin of these perirhinal efferents is discussed. These results confirmed our prediction of extensive direct projections from the anterior perirhinal cortex to the frontal cortex in the rat. The significance of this projection is discussed with special reference to the anatomical basis of convulsive limbic seizures.

Innervation of the amygdaloid complex by catecholaminergic cell groups of the ventrolateral medulla.

The projections to the amygdaloid complex (AMG), originating in the catecholaminergic cell groups of the ventrolateral medulla (VLM), were studied in the rat by using either the retrograde tracer fluoro-gold (FG) or the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with tyrosine hydroxylase (TH) and/or phenylethanolamine-N-methyltransferase (PNMT) immunohistochemistry. In the first series of experiments, injections of FG were made into regions of the central nucleus of the amygdala (ACe) where dense TH and PNMT immunoreactivity was previously observed, and then sections of the brainstem were processed for TH and PNMT immunoreactivity. FG retrogradely labelled neuronal cell bodies were observed throughout the rostrocaudal extent of VLM, bilaterally, with a contralateral predominance. Approximately 44% of the FG labelled cell bodies in VLM were also immunoreactive to the catecholamine biosynthetic enzymes TH and/or PNMT. Most of these catecholaminergic neurons were part of the A1 noradrenergic cell group in the caudal VLM and to a lesser extent part of the C1 adrenergic cell group in the rostral VLM. In the second series of experiments, PHA-L was iontophoresed into VLM at different rostrocaudal levels where in the previous series of experiments FG retrogradely labelled cell bodies were observed. Transverse sections of the forebrain and brainstem were then processed for the demonstration of PHA-L and either TH or PNMT immunoreactivity in cell bodies, axons, and presumptive axon terminals. PHA-L injection sites within either the caudal or rostral VLM resulted in labelled axons and terminal bouton-like swellings primarily in the contralateral AMG and to a lesser extent in the ipsilateral AMG. The ACe was observed to receive the greatest innervation from either VLM site. Additionally, PHA-L labelled fibers and presumptive terminal boutons were observed within the intercalated, medial, basomedial, and basolateral nuclei of the AMG. Most of the PHA-L labelled fibers and presumptive terminal boutons in the AMG after a caudal VLM (A1 region) injection also displayed TH immunoreactivity, whereas after a PHA-L injection into the rostral VLM (C1 region) all of the labelled axons and axon terminals in the AMG also were immunoreactive to PNMT. These data demonstrate that catecholaminergic neurons in A1 and C1 regions of VLM innervate the AMG and suggest that these VLM neurons may be involved in relaying afferent information directly to the AMG which influences the activity of AMG neurons controlling autonomic, endocrine, and behavioural functions.

The striatopallidal projection displays a high degree of anatomical specificity in the primate

The striatopallidal projection in the squirrel monkey ( Saimiri sciureus) was studied with two highly sensitive anterograde tracers, the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) and biocytin. After small PHA-L injections into various sectors of the striatum, the striatopallidal projection was found to display a very precise topographical organization. Fibers from the head of the caudate nucleus emerge as several distinct fascicles that penetrate the dorsal portion of the pallidum at various points along its rostrocaudal extent. Each fascicle arborizes into the dorsal third of the pallidum as dense plexuses composed of numerous fibers that closely entwined the dendrites of pallidal neurons, hence forming typical ‘woolly’ fiber arrangements. In contrast, fibers from the postcommissural putamen emerge as a few compact bundles that reach the pallidum through its lateral surface. In the pallidum, thin fibers detach themselves from these compact bundles, sweep caudally, and arborize in the form of narrow and elongated bands aligned parallel to the medullary laminae. Each band appears composed of numerous, thin and weakly varicose fibers that make only en passant type of contact with pallidal cell bodies rostrally, but form a dense field of woolly fibers caudally. In cases in which two PHA-L injections were made at two different rostrocaudal levels in the putamen, two rostrocaudally distant fields of woolly fibers, separated one another by thin varicose fibers, occur in each band. Furthermore, each PHA-L injection site in the striatum gives rise to at least two bands in each pallidal segment, indicating that the primate striatum has a dual representation at pallidal level. Finally, injections of PHA-L and biocytin into two small and mediolaterally adjacent areas of the postcommissural putamen lead to the formation of two clearly distinguishable sets of bands in each pallidal segment. Even though they lie very close to one another these two types of bands never really overlap. This experiment shows that, in contrast to previous beliefs, axons of striatal neurons from two small adjacent populations do not converge upon the same pallidal neurons but instead project to several distinct subsets of pallidal neurons. The findings of the present study reveal that the striatopallidal projection system in primates is highly ordered and displays a high degree of specificity with respect to its target sites in the pallidum. Different anatomical strategies are used to maximally exploit the relatively small pallidal space and ensure that the finely tuned corticostriatal information is not blurred as it flows through the funnel-shaped pallidum.

Intrinsic circuitry in the cat superior colliculus: projections from the superficial layers.

The neuroanatomical tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was used to label the local projections of neurons whose cell bodies are located in the superficial layers of the cat superior colliculus. Small, localized groups of neurons in the superficial layers project to all regions of the ipsilateral colliculus, including the deep layers. Comparable distributions of labeled terminals are seen throughout the colliculus when the PHA-L injection site is located in the rostral, middle, or caudal one-third of the colliculus. In addition, there is some evidence of a topographic projection from superficial to deep layers. These results suggest that a complex anatomical substrate exists for communication between the superficial and deep layers of the colliculus. Connections such as these may underlie the transfer of visual information from neurons in the superficial layers to populations of neurons in the deep layers that respond prior to saccadic eye movements.

Efferent projections of the subthalamic nucleus in the squirrel monkey as studied by the PHA‐L anterograde tracing method

The organization of the efferent connections of the subthalamic nucleus was studied in the squirrel monkey (Saimiri sciureus) by using the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) as an anterograde tracer. At the level of the basal forebrain, anterogradely labeled fibers and axon terminals were mostly found in the striatopallidal complex and the substantia innominata. In cases in which the PHA-L injection sites were placed in the central or the lateral third of the subthalamic nucleus, numerous anterogradely labeled fibers were seen to arise from the injection loci and innervate massively the globus pallidus. At pallidal levels the fibers formed bands lying parallel and adjacent to the medullary laminae. The number and the complexity of the topographical organization of these bands varied with the size and the location of the PHA-L injection site. When examined at a higher magnification, the bands of subthalamopallidal fibers appeared as rich plexuses of short axon collaterals with small bulbous enlargements that closely surrounded the cell bodies and primary dendrites of pallidal cells. In contrast, PHA-L injection involving the medial tip of the subthalamic nucleus did not produce bandlike fiber patterns in the globus pallidus. Instead, the labeled fibers formed a diffuse plexus occupying the ventral part of the rostral pole of the globus pallidus as well as the subcommissural pallidal region. The substantia innominata contained a moderate number of labeled fibers and axon terminals following injection of PHA-L in the medial tip of the subthalamic nucleus. A small to moderate number of anterogradely labeled fibers were seen in the putamen after all PHA-L injections. These subthalamostriatal fibers were long, linear, and branched infrequently. At midbrain level the substantia nigra contained a significant number of anterogradely labeled fibers and axon terminals following PHA-L injection in the subthalamic nucleus. The subthalamonigral fibers descended along the ventromedial part of the cerebral peduncle and swept laterally to reach their target. Most of these fibers formed small plexuses along the base of the pars reticulata, whereas a few others ascended along the cell columns of the pars compacta that impinged deeply within the pars reticulata. More caudally in the brainstem, a small number of fibers occurred in the area of the pedunculopontine nucleus and in the periaqueductal gray. These findings indicate that besides its well-known connection with the pallidum, the subthalamic nucleus gives rise to widespread projections to other components of the basal ganglia in primates.

Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raph� nuclei and sympathetic preganglionic neurons in the rat spinal cord

Electrophysiological and anatomical studies have suggested the existence of a pathway between the caudal raphé nuclei and regions of the spinal cord containing the sympathetic preganglionic neurons. However synaptic connections between cells in the raphé nuclei and identified sympathetic preganglionic neurons have not yet been shown. We have used a combination of anterograde tracing using Phaseolus vulgaris leucoagglutinin (PHA-L), retrograde tracing using a conjugate of cholera B chain and HRP and electron microscopy to look for such a pathway in rats. When PHA-L had been injected into the regions mainly restricted to the raphé pallidus and raphé magnus, synaptic contacts were found between PHA-L containing terminals and preganglionic neurons retrogradely labelled from the adrenal medulla. Out of the 43 synaptic contacts analysed, 26 were onto somata and 14 onto dendrites. 75% of the total appeared to have symmetric membrane specialisations, 20% asymmetric and the remainder could not be classified. Synaptic contacts were not seen in an animal in which the PHA-L injection site involved cells in the ventral raphé obscurus and surrounding gigantocellular reticular formation. These findings provide evidence of the existence of a direct monosynaptic pathway between cells in the raphé pallidus and/or caudal raphé magnus, and identified sympathetic preganglionic neurons and give further support for a role for the caudal raphé nuclei in sympathetic autonomic regulation.

Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transported Phaseolus vulgaris leucoagglutinin

The present paper deals with a detailed analysis of cortical projections from the magnocellular basal nucleus (MBN) and horizontal limb of the diagonal band of Broca (HDB) in the rat. The MBN and HDB were injected iontophoretically with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). After immunocytochemical visualization of labeled efferents, the distribution of projections over the cortical mantle, olfactory regions and amygdala were studied by light microscopy. Based on differences in cortical projection patterns, the MBN was subdivided in anterior, intermediate and posterior portions (MBNa, MBNi and MBNp). All subdivisions maintain neocortical projections and are subject to an anterior to posterior topographic arrangement. In the overall pattern, however, the frontal cortex is the chief target. Furthermore, all MBN parts project to various regions of meso- and allocortex, which are progressively more dense when the tracer injection is more anteriorly plaed. The most conspicuous finding, however, was a ventrolateral to dorsomedial cortical projection pattern as the PHA-L injection site moved from posterior to anterior. Thus, the posterior MBN projects predominantly to lateral neo- and mesocortex while the anterior MBN sends more fibers to the medial cortical regions. Furthermore, the MBN is a source of considerable afferent input to the olfactory nuclei and as such should be regarded as a transition to the HDB. The HDB, apart from projecting densely to olfactory bulb and related nuclei, maintains a substantial output to the medial prefrontal cortical regions and entorhinal cortex, as well. Comparison of young vs aged cases indicate that aging does not appear to have a profound influence on cortical innervation patterns, at least as studied with the PHA-L method.

An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L)

A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50–200 μm in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specialization, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be visualized in the same tissue sections as PHA-L localized with immunofluorescence techniques.