The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the labeling of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaloid body.

Groenewegen, H.J.,

Horst, G.J. ter,

Karst, H.,

Luiten, P.G.M.,

English

The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the labeling of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaloid body

ter Horst, G.J.

Groenewegen, H.J.

Karst, H.

Luiten, P.G.M.

University of Groningen

   University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison betweenautoradiographic and lectin tracing of neuronal efferentsHorst, G.J. ter; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Horst, G. J. T., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutininimmunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents.Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tr cing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover, the study of very small projections is limited by the inevitable background activity in the emulsion. Apart from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP), HRP conjugated with wheat germ agglutinin or free lectins 5have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate he an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable oss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4 h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest ermi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~.. ca l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~ .~ , .  ~.  ....... , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t;.: :~,~./ lh :;:~.-.,,t.~'4~fi %. . .  ~.~.. ."at)  ~ ~I~:. i~  ' , ' . .b l :  ..... ~ . . .~-> . \ 4"~.  ) ' ~  +--  ,_~li~:i,. C / I ~I I ':I:~.. am IImi:~:: t i '  :: ~ ....... : : " -~.7~! ::I: i,~iI 7 . : : t  '~"'~ '. ~ ' ' : . : \  .... .~  , -~ .  q: : \ . " .  "... ~-  ~ i~m: : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~ : - . . : /~ L . . .~ .~ ; ..- '~ .~ i~ .~". % . . .  . ~ /~. . . . - , - . : :~ .~ ...~:r~ ~.~i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~,  s ,  381 Fig. 1. Two series of identical transverse ctions from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH).  Abbreviations: abl, basolateral mygdaloid nucleus; abm, basomedial mygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; t, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed orsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI, others bend dor- sally towards tria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. DMH injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7,whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine t rminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph ofPHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of aPHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of the PHA-L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary, it may be concluded that the tech- nique of anterogradely transported PHA-L  is ba- sically as effective as the autoradiographic method for the study of neuronal efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of even small efferent projections. A disadvantage may be that the PHA-L  technique does not appear suitable for sur- vey studies of efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research FUNGO (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of Pure Research (ZWO). 1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous ystem. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde n u- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections ofthe ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus todorsal motor vagus and ambiguus nuclei n the rat, J. auton, nerv. Syst., in press. 

Phaseolus vulgaris leuco-agglutinin immunohistochemistry:A comparison between autoradiographic and lectin tracing of neuronal efferents

NARCIS 

Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronal efferents

   University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistryter Horst, G.J.; Groenewegen, H.J.; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):ter Horst, G. J., Groenewegen, H. J., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronalefferents. Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 25-04-2023Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tracing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover,  the study of very small projections is limited by the inevitable background activity in the emulsion. Apart  from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP),  HRP conjugated with wheat germ agglutinin or free lectins 5 have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate the an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable loss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4  h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest termi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~..  c a l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~  . ~ , .  ~ .  ... . . . .  , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t ; . : :~,~. / lh  :;:~.-.,,t.~'4~fi % . . .  ~ .~ . . . "a t )  ~ ~I~:. i ~  ' , ' . . b l :  ..... ~ . . . ~ - >  . \ 4 " ~ .  ) ' ~  + - -  ,_~li~:i,. C / I ~I ':I:~.. am IImi:~:: ~ t i '  :: ~ ....... :: " -~.7~! ::I: i,~iI 7 . : : t  '~" '~ '. ~ ' ' : . : \  .... . . ~  , - ~ .  q: : \ . " .  "... ~ -  ~ i ~ m : : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~  : - . . : /~  L . . . ~ . ~  ; ..- ' ~ . ~ i ~  .~". % . . .  . ~ / ~ . . . . - , - . : : ~ . ~  ...~:r~ ~ . ~ i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~ ,  s ,  381 Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH) .  Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed dorsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI,  others bend dor- sally towards stria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. D M H  injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7, whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of  the P H A - L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary,  it may be concluded that the tech- nique of  anterogradely transported P H A - L  is ba- sically as effective as the autoradiographic method for the study of  neuronal  efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of  even small efferent projections. A disadvantage may be that the P H A - L  technique does not appear  suitable for sur- vey studies of  efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research F U N G O  (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of  Pure Research (ZWO).  1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous system. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections of the ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

ARTS repository - University of Groningen

University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistryter Horst, G.J.; Groenewegen, H.J.; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):ter Horst, G. J., Groenewegen, H. J., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronalefferents. Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 24-12-2025Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tracing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover,  the study of very small projections is limited by the inevitable background activity in the emulsion. Apart  from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP),  HRP conjugated with wheat germ agglutinin or free lectins 5 have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate the an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable loss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4  h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest termi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~..  c a l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~  . ~ , .  ~ .  ... . . . .  , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t ; . : :~,~. / lh  :;:~.-.,,t.~'4~fi % . . .  ~ .~ . . . "a t )  ~ ~I~:. i ~  ' , ' . . b l :  ..... ~ . . . ~ - >  . \ 4 " ~ .  ) ' ~  + - -  ,_~li~:i,. C / I ~I ':I:~.. am IImi:~:: ~ t i '  :: ~ ....... :: " -~.7~! ::I: i,~iI 7 . : : t  '~" '~ '. ~ ' ' : . : \  .... . . ~  , - ~ .  q: : \ . " .  "... ~ -  ~ i ~ m : : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~  : - . . : /~  L . . . ~ . ~  ; ..- ' ~ . ~ i ~  .~". % . . .  . ~ / ~ . . . . - , - . : : ~ . ~  ...~:r~ ~ . ~ i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~ ,  s ,  381 Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH) .  Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed dorsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI,  others bend dor- sally towards stria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. D M H  injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7, whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of  the P H A - L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary,  it may be concluded that the tech- nique of  anterogradely transported P H A - L  is ba- sically as effective as the autoradiographic method for the study of  neuronal  efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of  even small efferent projections. A disadvantage may be that the P H A - L  technique does not appear  suitable for sur- vey studies of  efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research F U N G O  (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of  Pure Research (ZWO).  1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous system. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections of the ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

   University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistryter Horst, G.J.; Groenewegen, H.J.; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):ter Horst, G. J., Groenewegen, H. J., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronalefferents. Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tracing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover,  the study of very small projections is limited by the inevitable background activity in the emulsion. Apart  from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP),  HRP conjugated with wheat germ agglutinin or free lectins 5 have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate the an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable loss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4  h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest termi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~..  c a l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~  . ~ , .  ~ .  ... . . . .  , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t ; . : :~,~. / lh  :;:~.-.,,t.~'4~fi % . . .  ~ .~ . . . "a t )  ~ ~I~:. i ~  ' , ' . . b l :  ..... ~ . . . ~ - >  . \ 4 " ~ .  ) ' ~  + - -  ,_~li~:i,. C / I ~I ':I:~.. am IImi:~:: ~ t i '  :: ~ ....... :: " -~.7~! ::I: i,~iI 7 . : : t  '~" '~ '. ~ ' ' : . : \  .... . . ~  , - ~ .  q: : \ . " .  "... ~ -  ~ i ~ m : : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~  : - . . : /~  L . . . ~ . ~  ; ..- ' ~ . ~ i ~  .~". % . . .  . ~ / ~ . . . . - , - . : : ~ . ~  ...~:r~ ~ . ~ i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~ ,  s ,  381 Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH) .  Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed dorsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI,  others bend dor- sally towards stria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. D M H  injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7, whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of  the P H A - L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary,  it may be concluded that the tech- nique of  anterogradely transported P H A - L  is ba- sically as effective as the autoradiographic method for the study of  neuronal  efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of  even small efferent projections. A disadvantage may be that the P H A - L  technique does not appear  suitable for sur- vey studies of  efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research F U N G O  (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of  Pure Research (ZWO).  1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous system. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections of the ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistryter Horst, G.J.; Groenewegen, H.J.; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):ter Horst, G. J., Groenewegen, H. J., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronalefferents. Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tracing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover,  the study of very small projections is limited by the inevitable background activity in the emulsion. Apart  from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP),  HRP conjugated with wheat germ agglutinin or free lectins 5 have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate the an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable loss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4  h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest termi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~..  c a l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~  . ~ , .  ~ .  ... . . . .  , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t ; . : :~,~. / lh  :;:~.-.,,t.~'4~fi % . . .  ~ .~ . . . "a t )  ~ ~I~:. i ~  ' , ' . . b l :  ..... ~ . . . ~ - >  . \ 4 " ~ .  ) ' ~  + - -  ,_~li~:i,. C / I ~I ':I:~.. am IImi:~:: ~ t i '  :: ~ ....... :: " -~.7~! ::I: i,~iI 7 . : : t  '~" '~ '. ~ ' ' : . : \  .... . . ~  , - ~ .  q: : \ . " .  "... ~ -  ~ i ~ m : : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~  : - . . : /~  L . . . ~ . ~  ; ..- ' ~ . ~ i ~  .~". % . . .  . ~ / ~ . . . . - , - . : : ~ . ~  ...~:r~ ~ . ~ i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~ ,  s ,  381 Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH) .  Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed dorsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI,  others bend dor- sally towards stria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. D M H  injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7, whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of  the P H A - L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary,  it may be concluded that the tech- nique of  anterogradely transported P H A - L  is ba- sically as effective as the autoradiographic method for the study of  neuronal  efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of  even small efferent projections. A disadvantage may be that the P H A - L  technique does not appear  suitable for sur- vey studies of  efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research F U N G O  (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of  Pure Research (ZWO).  1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous system. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections of the ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

Horst, G.J. ter

University of Groningen Research Database

  
 University of Groningen
Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between
autoradiographic and lectin tracing of neuronal efferents
ter Horst, Gerrit; Karst, H.; Luiten, P.G.M.
Published in:
Brain Research
DOI:
10.1016/0006-8993(84)90500-6
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1984
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Horst, G. J. T., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin
immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents.
Brain Research, 307(1), 379-383. DOI: 10.1016/0006-8993(84)90500-6
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 10-02-2018
Brain Research, 307 (1984) 379-383 379 
Elsevier 
BRE 20303 
Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison 
between autoradiographic and lectin tracing of neuronal efferents 
G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 
1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, 
Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) 
(Accepted March 27th, 1984) 
Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  
immunohistochemistry - -  neuroanatomical tr cing 
The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- 
ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical 
methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. 
In recent years the study of neural connections un- 
derwent a tremendous development by the introduc- 
tion of methods that employ exogenous compounds 
which can be transported by the living neuron. For 
the study of efferent connections, the autoradio- 
graphic technique that is based on the anterograde 
transport of tritiated amino acids has generally been 
accepted as the most reliable method 1. A disadvan- 
tage of the widely applied autoradiographic tech- 
nique for neuronal tracing, however, is the indirect 
identification of labeling in the overlying emulsion 
that does not allow the positive identification of the 
actual structures. Secondly, the spread of tracer at 
the injection site obscures the determination of the 
effective tracer uptake area. Moreover, the study of 
very small projections is limited by the inevitable 
background activity in the emulsion. Apart from ma- 
jor projections, it is also impossible to discriminate 
between labeling of fibers and terminals in autoradi- 
ographic material. Besides, a time-consuming proce- 
dure and radioactive waste materials offer additional 
problems to this method. 
Although several other exogenous compounds like 
horseradish peroxidase (HRP), HRP conjugated 
with wheat germ agglutinin or free lectins 5have been 
shown to be transported in the anterograde direc- 
tion, most of these substances also appear to be car- 
ded in the opposite direction. Recently, Gerfen and 
Sawchenko 2 have indicated that the plant lectin Pha- 
seolus vulgaris leuco-agglutinin (PHA-L) may be 
used as a selective anterograde tracer for light micro- 
scopy. Before this technique can be accepted as a 
routine method it is essential to demonstrate he an- 
terograde transport properties of PHA-L by compa- 
rison with such transport of tritiated amino acids 
studied in the same system. In this paper we report 
our observations on anterograde transport after ion- 
tophoretically delivered PHA-L and tritiated leucine 
([3H]Leu) in the ventromedial hypothalamic nude- 
us (VMH). [3H]Leu was injected and processed 
according to the procedures of Ricardo 4. The brain 
sections were coated with Kodak NTB-2 nuclear 
track emulsion and exposed for 12-16 weeks at 4 °C. 
The slides were developed in Kodak D-19b at 16 °C, 
counterstained, coverslipped and examined with 
dark-field illumination. For the PHA-L experiments 
basically the procedure as described by Gerfen and 
Sawchenko 2 was followed. During the course of the 
experiments a number of parameters such as survival 
times, fixation and immunohistochemical reactions 
Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, 
The Netherlands. 
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 
380 
were varied in order to obtain optimal results. The 
experiments were performed in male albino Wistar 
rats weighing approximately 300 g. The following 
procedure proved to give the best results in our 
hands. 
Five mg Phaseolus vulgaris leuco-agglutinin ob- 
tained from Vector Laboratories, Burlingame, 
U.S.A., was dissolved in 0.05 M Tris buffered saline 
(pH = 7.4) to a 2.5% solution. This solution was di- 
vided into 10 ktl portions and stored at -20 °C. Prior 
to an experiment one portion was stored at 4 °C and 
could be used for 2 weeks without appreciable oss of 
reactivity, provided it is kept refrigerated between 
experiments. Small PHA-L deposits into the rat hy- 
pothalamus and several other structures like periaq- 
ueductal gray, reticular formation, ventral pallidum 
and amygdala were obtained by means of iontopho- 
resis. Bevelled glass micropipettes (10-20 #m in di- 
ameter) were filled with the PHA-L solution and po- 
sitioned in the brain stereotactically. A positive 
pulsed DC current of 4-7/~A was applied to the pi- 
pette using a Midgard CS-3 constant current source 
for 30 min. Following iontophoresis the pipette was 
left in situ for 10 min to avoid loss of tracer in the pi- 
pette track. Animals were allowed to survive for 
7-10 days. Satisfactory fixation was obtained with a 
transcardial prerinse of 50 ml phosphate buffered 
(0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. 
heparin/ml, which was followed by 500 ml of a solu- 
tion of 4% paraformaldehyde in 0.05 M phosphate 
buffer (pH = 7.4) or a prerinse of 50 ml of a phos- 
phate buffered solution containing 0.8% NaC1, 0.8% 
sucrose and 0.4% D-glucose (37 °C), followed by 500 
ml fixative made up of 0.5% paraformaldehyde, 
2.5% glutaraldehyde and 4% sucrose in phosphate 
buffer. The advantage of the latter fixative is a lower 
background precipitate in the subsequent immuno- 
chemical staining procedure. 
Following 2-4 h post-fixation brains were stored 
overnight at 4 °C in 20-30% phosphate buffered su- 
crose and cut in 40/tm transverse sections on a freez- 
ing microtome. Sections were collected in chilled Tris 
buffered saline (TBS) and rinsed overnight prior to 
incubation. 
Immunohistochemical procedure 
Every third section was transferred to a solution 
made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M 
NaCI and 0.5% Triton X-100 (TBS-T). This solution 
contained the primary antibody goat-anti-Phaseolus 
(Vector) diluted to a concentration of 1:2000. The 
tissue container placed on a rocker was stored for 36 
h at 4 °C or 18 h at room temperature with the same 
effect on the reaction. Then the sections were rinsed 
3 x 15 min TBS-T and incubated with rabbit-anti- 
goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 
20 °C. Sections were washed thoroughly again in 
TBS-T and transferred to a goat peroxidase-antiper- 
oxidase (PAP) (Nordic, Immunochemicals) in TBS- 
T solution (1:400) for at least 4 h. The prolonged 
PAP incubation strongly intensified the density of the 
reaction product. After rinsing in several changes of 
TBS (pH = 7.4) the sections were incubated with 40 
mg diaminobenzidine (DAB) in 100 ml Tris buffer 
(pH = 7.4) to which 0.9 ml H202 1.5% was added. 
The DAB incubation should be extended to at least 1 
h for optimal density of the precipitate. Then the sec- 
tions were washed thoroughly, mounted, counter- 
stained and coverslipped. The PHA injection sites 
appeared as clusters of labeled cell bodies (Fig. 2C). 
The amount of cells and cluster size can be correlated 
to the pipette diameter and current density. There 
also appeared to be a relation between the amount of 
labeled somata and the number of marked fibers, 
which suggests that the effective injection site can be 
defined by the amount of labeled cell bodies. In the 
case of VMH injections as exemplified in Fig. 1 ef- 
ferent pathways ascend ventrally and dorsally from 
this hypothalamic nucleus. The ventral bundle tra- 
verses the lateral hypothalamic area (LH), courses 
dorsal to the tractus opticus (TO) (Fig. 2E, F) and 
enters the amygdala. Within this nuclear complex the 
bundle divides in lateral and medial directed fibers. 
In experiments in which [3H]Leu was injected into the 
VMH local increases of silver grains can be observed 
in several amygdaloid nuclei, which suggest ermi- 
nation areas. In the PHA-L material these supposed 
termination fields coincide with fine branching of fi- 
bers concomitaiat with the appearance of large num- 
bers of varicosities (Fig. 2A, B). These morphologic- 
al criteria we used for the identification of terminal 
fields in PHA-L material (see also ref. 2). 
The lateral coursing fibers in the amygdala termi- 
nate within the lateral nucleus (AL) after travelling 
dorsal to the central nucleus (AC). The fibers cours- 
ing medially terminate within the medial nucleus 
3H-LEU PHA-L 
L i L irn, 
" t  ....... ~'~ . .  • . '~.. ca l  ..... . . .  ...... ...... 
: .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... 
A .....i ......... ...... 
• .. . . .~ .~ , .  ~.  ....... , 
I ~  : :  ..... :<ha e ' / ~  
I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t;.: :~,~./ lh :;:~.-.,,t.~'4~fi 
%. . .  ~.~.. ."at)  ~ ~I~:. i~  ' , ' . .b l :  
..... ~ . . .~-> . \ 4"~.  ) ' ~  +--  ,_~li~:i,. 
C / I ~I I ':I:~.. am IImi:~:: t i '  :: ~ ....... : : " -~.7~! ::I: i,~iI 7 
. : : t  '~"'~ '. ~ ' ' : . : \  .... .~  , -~ .  q: : 
\ . " .  "... ~-  ~ i~m: : . -  ?.. . . . .  • ,-x.~*//~ 
k .... " .~ : - . . : /~ L . . .~ .~ ; ..- '~ .~ i~ .~". 
% . . .  . ~ /~. . . . - , - . : :~ .~ ...~:r~ ~.~i . . .  
. . . . .  ~ '-' ~ \ ~ ,  ~,  s ,  
381 
Fig. 1. Two series of identical transverse ctions from anterior (A) to posterior (C) representing the efferent projections to the amyg- 
dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- 
pothalamic nucleus (MH).  Abbreviations: abl, basolateral mygdaloid nucleus; abm, basomedial mygdaloid nucleus; ac, central 
amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- 
terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, 
lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; t, stria terminalis; to, tractus 
opticus; zi, zona incerta. 
(AM), basolateral nucleus (ABL) and periamygda- 
loid cortex (PAC). Fibers directed orsally from the 
VMH injection site traverse the dorsomedial nucleus 
(DMH) and divide in lateral and dorsal coursing bun- 
dies. This dorsal bundle terminates partly within the 
nucleus reuniens (RE) and the rest ascends to the ha- 
benular complex. Lateral coursing fibers travel un- 
der zona incerta (ZI) towards the capsula interna 
(CAI). Some fibers cross this CAI, others bend dor- 
sally towards tria terminalis (ST). 
The PHA-L results show a remarkable similarity 
with this and previous autoradiographic studies on 
VMH projections3, 6. The [3H]Leu injections, how- 
ever, appear as rather diffuse deposits, which do not 
allow the precise delineation of the tracer uptake 
area. Moreover, the tracer diffusion prohibits the 
identification of short connections. DMH injections 
of PHA-L (not illustrated) show a strong short pro- 
jection to the VMH which could not be identified af- 
ter similar injections with [3H]Leu. Furthermore, 
after the same DMH injections of PHA-L long de- 
scending projections of individual fibers could be ob- 
served to the lower medulla, previously demon- 
strated with retrograde transport methods 7,whereas 
these minor projections can hardly be discerned in 
the autoradiographic material. Another advantage 
382 
Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of 
[3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- 
tomicrograph showing tritiated leucine t rminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- 
micrograph ofPHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of aPHA-L 
iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- 
minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- 
ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 
383 
of the PHA-L  technique over the autoradiographic 
method is that in several projection areas it is possi- 
ble to identify detailed contacts between the efferent 
terminals and the receiving somata (Fig. 2D). In 
these cases large numbers of labeled boutons can be 
seen embracing the target perikarya. 
In summary, it may be concluded that the tech- 
nique of anterogradely transported PHA-L  is ba- 
sically as effective as the autoradiographic method 
for the study of neuronal efferents, but offers several 
additional advantages. Its superior sensitivity en- 
ables the user to study the fine structure of even small 
efferent projections. A disadvantage may be that the 
PHA-L  technique does not appear suitable for sur- 
vey studies of efferents from large brain areas. 
The authors wish to express their appreciation to 
Mrs. Joke Poelstra for typing the manuscript. This 
work was supported in part by the Foundation for 
Medical Research FUNGO (Grant 13-46-36) which 
is subsidized by the Dutch Organization for the Ad- 
vancement of Pure Research (ZWO). 
1 Edwards, S. B. and Hendrickson, A., The autoradiographic 
tracing of axonal connections in the central nervous ystem. 
In L. Heimer and M. J. Robards (Eds.), Neuroanatomical 
Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 
171-205. 
2 Geffen, C. R. and Sawchenko, P. E., An anterograde n u- 
roanatomical tracing method that shows the detailed mor- 
phology of neurons, their axons and terminals: immunohis- 
toehemical localization of an axonally transported plant lec- 
tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- 
search, 290 (1984) 219-238. 
3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- 
radiographic study of the efferent connections of the ventro- 
medial nucleus of the hypothalamus, J. comp. Neurol., 183 
(1979) 785-816. 
4 Ricardo, J. A., Efferent connections of the subthalamic re- 
gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 
43-60. 
5 Ruda, M. and Coulter, J. D., Axonal and transneuronal 
transport of Wheat Germ Agglutinin demonstrated by im- 
munocytochemistry, Brain Research, 249 (1982) 237-246. 
6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- 
ent connections ofthe ventromediai hypothalamus of the rat, 
J. comp. Neurol., 169 (1976) 409-422. 
7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- 
ing pathways from hypothalamus todorsal motor vagus and 
ambiguus nuclei n the rat, J. auton, nerv. Syst., in press. 


Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents

University of Groningen Digital Archive

Brain Research, 307 (1984) 379-383  379 
Elsevier 
BRE 20303 
Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison 
between autoradiographic and lectin tracing of neuronal efferents 
G. J. TER HORST  1, H. J. GROENEWEGEN  2, H. KARST  1  and P. G. M. LUITEN  1 
1Department of  Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of  Anatomy, 
Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) 
(Accepted March 27th, 1984) 
Key words: Phaseolus vulgaris leuco-agglutinin  -- autoradiography -- anterograde transport -- 
immunohistochemistry  -- neuroanatomical tracing 
The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- 
ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical 
methods. This  comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. 
In recent years the study of neural connections un- 
derwent a tremendous development by the introduc- 
tion of methods that employ exogenous compounds 
which can be transported by the living neuron.  For 
the  study  of  efferent  connections,  the  autoradio- 
graphic technique  that is based on the  anterograde 
transport of tritiated amino acids has generally been 
accepted as the most reliable method 1.  A  disadvan- 
tage  of  the  widely  applied  autoradiographic  tech- 
nique  for neuronal tracing,  however, is the indirect 
identification  of labeling  in  the  overlying emulsion 
that does not allow the positive identification of the 
actual  structures.  Secondly,  the  spread of tracer at 
the injection  site obscures the determination of the 
effective tracer uptake area. Moreover, the study of 
very small  projections  is  limited  by  the  inevitable 
background activity in the emulsion. Apart from ma- 
jor projections,  it is also impossible to discriminate 
between labeling of fibers and terminals in autoradi- 
ographic material. Besides, a time-consuming proce- 
dure and radioactive waste materials offer additional 
problems to this method. 
Although several other exogenous compounds like 
horseradish  peroxidase  (HRP),  HRP  conjugated 
with wheat germ agglutinin or free lectins  5 have been 
shown  to  be transported  in  the  anterograde  direc- 
tion, most of these substances also appear to be car- 
ded in the opposite direction. Recently, Gerfen and 
Sawchenko  2  have indicated that the plant lectin Pha- 
seolus  vulgaris  leuco-agglutinin  (PHA-L)  may  be 
used as a selective anterograde tracer for light micro- 
scopy.  Before  this  technique  can  be accepted  as  a 
routine method it is essential to demonstrate the an- 
terograde transport properties of PHA-L by compa- 
rison  with  such  transport  of  tritiated  amino  acids 
studied in the same system. In this paper we report 
our observations on anterograde transport after ion- 
tophoretically delivered PHA-L and tritiated leucine 
([3H]Leu)  in the ventromedial hypothalamic nude- 
us  (VMH).  [3H]Leu  was  injected  and  processed 
according to the procedures of Ricardo  4.  The brain 
sections  were  coated  with  Kodak  NTB-2  nuclear 
track emulsion and exposed for 12-16 weeks at 4 °C. 
The slides were developed in Kodak D-19b at 16 °C, 
counterstained,  coverslipped  and  examined  with 
dark-field illumination.  For the PHA-L experiments 
basically the procedure as described by Gerfen and 
Sawchenko  2 was followed. During the course of the 
experiments a number of parameters such as survival 
times,  fixation  and  immunohistochemical  reactions 
Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, 
The Netherlands. 
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 
were varied in order to obtain optimal results.  The 
experiments were performed in male albino Wistar 
rats  weighing  approximately 300  g.  The  following 
procedure  proved  to  give  the  best  results  in  our 
hands. 
Five  mg  Phaseolus  vulgaris  leuco-agglutinin  ob- 
tained  from  Vector  Laboratories,  Burlingame, 
U.S.A., was dissolved in 0.05 M Tris buffered saline 
(pH =  7.4) to a 2.5% solution. This solution was di- 
vided into 10 ktl portions and stored at -20 °C. Prior 
to an experiment one portion was stored at 4 °C and 
could be used for 2 weeks without appreciable loss of 
reactivity, provided it is  kept refrigerated between 
experiments. Small PHA-L deposits into the rat hy- 
pothalamus and several other structures like periaq- 
ueductal gray, reticular formation, ventral pallidum 
and amygdala were obtained by means of iontopho- 
resis. Bevelled glass micropipettes (10-20 #m in di- 
ameter) were filled with the PHA-L solution and po- 
sitioned  in  the  brain  stereotactically.  A  positive 
pulsed DC current of 4-7/~A was applied to the pi- 
pette using a Midgard CS-3 constant current source 
for 30 min.  Following iontophoresis the pipette was 
left in situ for 10 min to avoid loss of tracer in the pi- 
pette  track.  Animals  were  allowed  to  survive  for 
7-10 days. Satisfactory fixation was obtained with a 
transcardial  prerinse  of  50  ml  phosphate  buffered 
(0.05 M; pH =  7.4) saline at 37 °C containing 10 I.U. 
heparin/ml, which was followed by 500 ml of a solu- 
tion of 4% paraformaldehyde in 0.05 M  phosphate 
buffer (pH =  7.4) or a prerinse of 50 ml of a phos- 
phate buffered solution containing 0.8% NaC1, 0.8% 
sucrose and 0.4% D-glucose (37 °C), followed by 500 
ml  fixative  made  up  of  0.5%  paraformaldehyde, 
2.5%  glutaraldehyde and 4%  sucrose in phosphate 
buffer. The advantage of the latter fixative is a lower 
background precipitate in the subsequent  immuno- 
chemical staining procedure. 
Following 2-4 h  post-fixation brains were stored 
overnight at 4 °C in 20-30% phosphate buffered su- 
crose and cut in 40/tm transverse sections on a freez- 
ing microtome. Sections were collected in chilled Tris 
buffered saline (TBS) and rinsed overnight prior to 
incubation. 
Immunohistochemical procedure 
Every third section was transferred to a  solution 
made up of 0.05  M  Tris buffer (pH  =  8.6),  0.5  M 
NaCI and 0.5% Triton X-100 (TBS-T). This solution 
contained the primary antibody goat-anti-Phaseolus 
(Vector) diluted to a  concentration of 1:2000.  The 
tissue container placed on a rocker was stored for 36 
h at 4 °C or 18 h at room temperature with the same 
effect on the reaction. Then the sections were rinsed 
3  x  15 min TBS-T and incubated with  rabbit-anti- 
goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 
20 °C.  Sections  were  washed  thoroughly  again  in 
TBS-T and transferred to a goat peroxidase-antiper- 
oxidase (PAP) (Nordic, Immunochemicals) in TBS- 
T  solution  (1:400)  for at  least  4  h.  The prolonged 
PAP incubation strongly intensified the density of the 
reaction product. After rinsing in several changes of 
TBS (pH =  7.4) the sections were incubated with 40 
mg diaminobenzidine (DAB)  in  100 ml Tris buffer 
(pH =  7.4) to which 0.9 ml H202 1.5% was added. 
The DAB incubation should be extended to at least 1 
h for optimal density of the precipitate. Then the sec- 
tions  were  washed  thoroughly,  mounted,  counter- 
stained  and  coverslipped.  The PHA  injection sites 
appeared as clusters of labeled cell bodies (Fig. 2C). 
The amount of cells and cluster size can be correlated 
to the pipette diameter and current density.  There 
also appeared to be a relation between the amount of 
labeled  somata  and  the  number  of marked  fibers, 
which suggests that the effective injection site can be 
defined by the amount of labeled cell bodies. In the 
case of VMH injections as exemplified in Fig.  1 ef- 
ferent pathways ascend ventrally and dorsally from 
this hypothalamic nucleus.  The ventral bundle  tra- 
verses the lateral  hypothalamic area (LH), courses 
dorsal to the tractus opticus (TO) (Fig. 2E,  F) and 
enters the amygdala. Within this nuclear complex the 
bundle divides in lateral and medial directed fibers. 
In experiments in which [3H]Leu was injected into the 
VMH local increases of silver grains can be observed 
in several  amygdaloid nuclei, which  suggest  termi- 
nation areas. In the PHA-L material these supposed 
termination fields coincide with fine branching of fi- 
bers concomitaiat with the appearance of large num- 
bers of varicosities (Fig. 2A, B). These morphologic- 
al criteria we used for the identification of terminal 
fields in PHA-L material (see also ref. 2). 
The lateral coursing fibers in the amygdala termi- 
nate within the lateral nucleus (AL) after travelling 
dorsal to the central nucleus (AC). The fibers cours- 
ing  medially  terminate  within  the  medial  nucleus 3H-LEU  PHA-L 
L  i  L  irn, 
"t  .......  ~'~ ..  •  .  '~..  cal  .....  ...  ......  ...... 
:  .:,>,~...~ ....... :  .~.~.  .::  ....  ....~_~._..  ..... 
A  .....i  .........  ...... 
•  ..  ...~  .~,.  ~.  .......  , 
I~  ::  .....  :<ha  e'/~ 
It.....  .....:~-  ......:...;.....,.li~-~t;.::~,~./lh  :;:~.-.,,t.~'4~fi 
%...  ~.~..."at)  ~  ~I~:.  i~  '  ,'..bl: 
.....  ~  ...~->  .  \  4"~.  )'~  +-- ,_~li~:i,. 
C  /  I  ~I  I  ':I:~..  am IImi:~::  ~ti'  ::  ~  .......  :  : "  -~.7~! ::I:  i,~iI 7 
.  :  :t  '~"'~  '.  ~'':.:\  ....  .  .~  ,-~.  q:  : 
\.".  "... ~-  ~i~m::.-  ?..  ....  •  ,-x.~*//~ 
k  ....  ".~  :  -  ..:/~  L...~.~  ;  ..-  '~.~i~  .~". 
%...  .  ~/~....-,-.::~.~  ...~:r~ ~.~i... 
.....  ~  '-'  ~  \~,  ~, s, 
381 
Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- 
dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- 
pothalamic nucleus (MH). Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central 
amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- 
terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, 
lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus 
opticus; zi, zona incerta. 
(AM),  basolateral nucleus  (ABL) and periamygda- 
loid cortex (PAC). Fibers directed dorsally from the 
VMH injection site traverse the dorsomedial nucleus 
(DMH) and divide in lateral and dorsal coursing bun- 
dies. This dorsal bundle terminates partly within the 
nucleus reuniens (RE) and the rest ascends to the ha- 
benular complex.  Lateral coursing fibers travel un- 
der  zona  incerta  (ZI)  towards  the  capsula  interna 
(CAI). Some fibers cross this CAI, others bend dor- 
sally towards stria terminalis (ST). 
The PHA-L results show a  remarkable similarity 
with  this  and  previous  autoradiographic  studies  on 
VMH  projections3,  6.  The  [3H]Leu  injections,  how- 
ever, appear as rather diffuse deposits, which do not 
allow  the  precise  delineation  of the  tracer  uptake 
area.  Moreover,  the  tracer  diffusion  prohibits  the 
identification of short connections.  DMH injections 
of PHA-L (not illustrated) show a strong short pro- 
jection to the VMH which could not be identified af- 
ter  similar  injections  with  [3H]Leu.  Furthermore, 
after the same DMH injections of PHA-L long de- 
scending projections of individual fibers could be ob- 
served  to  the  lower  medulla,  previously  demon- 
strated with retrograde transport methods  7, whereas 
these  minor projections can hardly be discerned  in 
the  autoradiographic  material.  Another  advantage 382 
Fig. 2. A-F: photomicrographs  illustrating labeling obtained after autoradiographic  and PAP immunohistochemical processing :of 
[3H]leucine  and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- 
tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- 
micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L 
iontophoretic injection  into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- 
minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- 
ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 
of the  PHA-L technique  over the  autoradiographic 
method is that in several projection areas it is possi- 
ble to identify detailed contacts between the efferent 
terminals  and  the  receiving  somata  (Fig.  2D).  In 
these cases large numbers of labeled boutons can be 
seen embracing the target perikarya. 
In summary,  it  may be  concluded  that  the  tech- 
nique  of  anterogradely  transported  PHA-L  is  ba- 
sically  as  effective  as  the  autoradiographic  method 
for the study of neuronal efferents, but offers several 
additional  advantages.  Its  superior  sensitivity  en- 
ables the user to study the fine structure of even small 
efferent projections. A  disadvantage may be that the 
PHA-L technique  does not appear suitable  for sur- 
vey studies of efferents from large brain areas. 
The authors wish to express  their appreciation  to 
Mrs.  Joke  Poelstra  for typing the  manuscript.  This 
work was  supported  in  part  by the  Foundation  for 
Medical  Research  FUNGO  (Grant  13-46-36)  which 
is subsidized by the Dutch Organization for the Ad- 
vancement of Pure Research (ZWO). 
1 Edwards, S. B. and Hendrickson,  A., The autoradiographic 
tracing of axonal connections in the central nervous system. 
In L. Heimer and M. J.  Robards (Eds.), Neuroanatomical 
Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 
171-205. 
2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- 
roanatomical  tracing  method that shows the detailed  mor- 
phology of neurons,  their axons and terminals:  immunohis- 
toehemical localization of an axonally transported plant lec- 
tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- 
search, 290 (1984) 219-238. 
3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- 
radiographic study of the efferent connections of the ventro- 
medial nucleus of the hypothalamus,  J.  comp. Neurol.,  183 
(1979) 785-816. 
4 Ricardo, J. A., Efferent  connections of the subthalamic re- 
gion in the rat: I. Zona incerta,  Brain Research,  214 (1981) 
43-60. 
5 Ruda,  M.  and Coulter,  J.  D.,  Axonal and transneuronal 
transport  of Wheat  Germ Agglutinin  demonstrated  by im- 
munocytochemistry, Brain Research,  249 (1982) 237-246. 
6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- 
ent connections of the ventromediai hypothalamus of the rat, 
J. comp. Neurol., 169 (1976) 409-422. 
7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- 
ing pathways from hypothalamus to dorsal motor vagus and 
ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

   University of GroningenPhaseolus vulgaris leuco-agglutinin immunohistochemistryter Horst, G.J.; Groenewegen, H.J.; Karst, H.; Luiten, P.G.M.Published in:Brain ResearchDOI:10.1016/0006-8993(84)90500-6IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1984Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):ter Horst, G. J., Groenewegen, H. J., Karst, H., & Luiten, P. G. M. (1984). Phaseolus vulgaris leuco-agglutinin immunohistochemistry: A comparison between autoradiographic and lectin tracing of neuronalefferents. Brain Research, 307(1), 379-383. https://doi.org/10.1016/0006-8993(84)90500-6CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023Brain Research, 307 (1984) 379-383 379 Elsevier BRE 20303 Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents G. J. TER HORST 1, H. J. GROENEWEGEN 2, H. KARST 1 and P. G. M. LUITEN 1 1Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren and 2Department of Anatomy, Free University, Van der Boechorststraat 9, 1081 BT Amsterdam (The Netherlands) (Accepted March 27th, 1984) Key words: Phaseolus vulgaris leuco-agglutinin - -  autoradiography - -  anterograde transport - -  immunohistochemistry - -  neuroanatomical tracing The autoradiographic pattern of anterograde labeling as a result from injections with tritiated amino acids is compared to the label- ing of efferents with Phaseolus vulgaris leuco-agglutinin after lectin injections in the same nucleus visualized by immunohistochemical methods. This comparison is made for efferents from the ventromedial hypothalamic nucleus to the amygdaioid body. In recent years the study of neural connections un- derwent a tremendous development by the introduc- tion of methods that employ exogenous compounds which can be transported by the living neuron. For the study of efferent connections, the autoradio- graphic technique that is based on the anterograde transport of tritiated amino acids has generally been accepted as the most reliable method 1. A disadvan- tage of the widely applied autoradiographic tech- nique for neuronal tracing, however, is the indirect identification of labeling in the overlying emulsion that does not allow the positive identification of the actual structures. Secondly, the spread of tracer at the injection site obscures the determination of the effective tracer uptake area. Moreover,  the study of very small projections is limited by the inevitable background activity in the emulsion. Apart  from ma- jor projections, it is also impossible to discriminate between labeling of fibers and terminals in autoradi- ographic material. Besides, a time-consuming proce- dure and radioactive waste materials offer additional problems to this method. Although several other exogenous compounds like horseradish peroxidase (HRP),  HRP conjugated with wheat germ agglutinin or free lectins 5 have been shown to be transported in the anterograde direc- tion, most of these substances also appear to be car- ded in the opposite direction. Recently, Gerfen and Sawchenko 2 have indicated that the plant lectin Pha- seolus vulgaris leuco-agglutinin (PHA-L) may be used as a selective anterograde tracer for light micro- scopy. Before this technique can be accepted as a routine method it is essential to demonstrate the an- terograde transport properties of PHA-L by compa- rison with such transport of tritiated amino acids studied in the same system. In this paper we report our observations on anterograde transport after ion- tophoretically delivered PHA-L and tritiated leucine ([3H]Leu) in the ventromedial hypothalamic nude- us (VMH). [3H]Leu was injected and processed according to the procedures of Ricardo 4. The brain sections were coated with Kodak NTB-2 nuclear track emulsion and exposed for 12-16 weeks at 4 °C. The slides were developed in Kodak D-19b at 16 °C, counterstained, coverslipped and examined with dark-field illumination. For the PHA-L experiments basically the procedure as described by Gerfen and Sawchenko 2 was followed. During the course of the experiments a number of parameters such as survival times, fixation and immunohistochemical reactions Correspondence: G. J. ter Horst, Department of Animal Physiology, State University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V. 380 were varied in order to obtain optimal results. The experiments were performed in male albino Wistar rats weighing approximately 300 g. The following procedure proved to give the best results in our hands. Five mg Phaseolus vulgaris leuco-agglutinin ob- tained from Vector Laboratories, Burlingame, U.S.A., was dissolved in 0.05 M Tris buffered saline (pH = 7.4) to a 2.5% solution. This solution was di- vided into 10 ktl portions and stored at -20 °C. Prior to an experiment one portion was stored at 4 °C and could be used for 2 weeks without appreciable loss of reactivity, provided it is kept refrigerated between experiments. Small PHA-L deposits into the rat hy- pothalamus and several other structures like periaq- ueductal gray, reticular formation, ventral pallidum and amygdala were obtained by means of iontopho- resis. Bevelled glass micropipettes (10-20 #m in di- ameter) were filled with the PHA-L solution and po- sitioned in the brain stereotactically. A positive pulsed DC current of 4-7/~A was applied to the pi- pette using a Midgard CS-3 constant current source for 30 min. Following iontophoresis the pipette was left in situ for 10 min to avoid loss of tracer in the pi- pette track. Animals were allowed to survive for 7-10 days. Satisfactory fixation was obtained with a transcardial prerinse of 50 ml phosphate buffered (0.05 M; pH = 7.4) saline at 37 °C containing 10 I.U. heparin/ml, which was followed by 500 ml of a solu- tion of 4% paraformaldehyde in 0.05 M phosphate buffer (pH = 7.4) or a prerinse of 50 ml of a phos- phate buffered solution containing 0.8% NaC1, 0.8% sucrose and 0.4% D-glucose (37 °C), followed by 500 ml fixative made up of 0.5% paraformaldehyde, 2.5% glutaraldehyde and 4% sucrose in phosphate buffer. The advantage of the latter fixative is a lower background precipitate in the subsequent immuno- chemical staining procedure. Following 2-4  h post-fixation brains were stored overnight at 4 °C in 20-30% phosphate buffered su- crose and cut in 40/tm transverse sections on a freez- ing microtome. Sections were collected in chilled Tris buffered saline (TBS) and rinsed overnight prior to incubation. Immunohistochemical procedure Every third section was transferred to a solution made up of 0.05 M Tris buffer (pH = 8.6), 0.5 M NaCI and 0.5% Triton X-100 (TBS-T). This solution contained the primary antibody goat-anti-Phaseolus (Vector) diluted to a concentration of 1:2000. The tissue container placed on a rocker was stored for 36 h at 4 °C or 18 h at room temperature with the same effect on the reaction. Then the sections were rinsed 3 x 15 min TBS-T and incubated with rabbit-anti- goat whole serum (Sigma) in TBS-T (1:400) for 2 h at 20 °C. Sections were washed thoroughly again in TBS-T and transferred to a goat peroxidase-antiper- oxidase (PAP) (Nordic, Immunochemicals) in TBS- T solution (1:400) for at least 4 h. The prolonged PAP incubation strongly intensified the density of the reaction product. After rinsing in several changes of TBS (pH = 7.4) the sections were incubated with 40 mg diaminobenzidine (DAB) in 100 ml Tris buffer (pH = 7.4) to which 0.9 ml H202 1.5% was added. The DAB incubation should be extended to at least 1 h for optimal density of the precipitate. Then the sec- tions were washed thoroughly, mounted, counter- stained and coverslipped. The PHA injection sites appeared as clusters of labeled cell bodies (Fig. 2C). The amount of cells and cluster size can be correlated to the pipette diameter and current density. There also appeared to be a relation between the amount of labeled somata and the number of marked fibers, which suggests that the effective injection site can be defined by the amount of labeled cell bodies. In the case of VMH injections as exemplified in Fig. 1 ef- ferent pathways ascend ventrally and dorsally from this hypothalamic nucleus. The ventral bundle tra- verses the lateral hypothalamic area (LH), courses dorsal to the tractus opticus (TO) (Fig. 2E, F) and enters the amygdala. Within this nuclear complex the bundle divides in lateral and medial directed fibers. In experiments in which [3H]Leu was injected into the VMH local increases of silver grains can be observed in several amygdaloid nuclei, which suggest termi- nation areas. In the PHA-L material these supposed termination fields coincide with fine branching of fi- bers concomitaiat with the appearance of large num- bers of varicosities (Fig. 2A, B). These morphologic- al criteria we used for the identification of terminal fields in PHA-L material (see also ref. 2). The lateral coursing fibers in the amygdala termi- nate within the lateral nucleus (AL) after travelling dorsal to the central nucleus (AC). The fibers cours- ing medially terminate within the medial nucleus 3H-LEU PHA-L L i L irn, " t  ....... ~'~ . .  • . '~..  c a l  ..... . . .  ...... ...... : .:,>,~...~ . . . . . . .  : .~.~. .:: . . . .  ....~_~._.. ..... A .....i ......... ...... • .. . . .~  . ~ , .  ~ .  ... . . . .  , I ~  : :  ..... :<ha e ' / ~  I t . . . . .  .....:~- . . . . . . : . . . ; . . . . . , . l i~-~t ; . : :~,~. / lh  :;:~.-.,,t.~'4~fi % . . .  ~ .~ . . . "a t )  ~ ~I~:. i ~  ' , ' . . b l :  ..... ~ . . . ~ - >  . \ 4 " ~ .  ) ' ~  + - -  ,_~li~:i,. C / I ~I ':I:~.. am IImi:~:: ~ t i '  :: ~ ....... :: " -~.7~! ::I: i,~iI 7 . : : t  '~" '~ '. ~ ' ' : . : \  .... . . ~  , - ~ .  q: : \ . " .  "... ~ -  ~ i ~ m : : . -  ?.. . . . .  • ,-x.~*//~ k .... " .~  : - . . : /~  L . . . ~ . ~  ; ..- ' ~ . ~ i ~  .~". % . . .  . ~ / ~ . . . . - , - . : : ~ . ~  ...~:r~ ~ . ~ i . . .  . . . . .  ~ '-' ~ \ ~ ,  ~ ,  s ,  381 Fig. 1. Two series of identical transverse sections from anterior (A) to posterior (C) representing the efferent projections to the amyg- dala after iontophoretic Phaseolus vulgaris leuco-agglutinin (PHA-L) and [3H]leucine (3H-LEU) injections into the ventromedial hy- pothalamic nucleus (MH) .  Abbreviations: abl, basolateral amygdaloid nucleus; abm, basomedial amygdaloid nucleus; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; al, lateral amygdaloid nucleus; am, medial amygdaloid nucleus; cai, capsula in- terna; el, elaustrum; cpf, piriform cortex; dmh, dorsomedial hypothalamic nucleus; f, fornix; fmt, fasiculus mammillothalamicus; lh, lateral hypothalamic area; Ira, lemniscus medialis; pac, peri-amygdaloid cortex; re, nucleus reuniens; st, stria terminalis; to, tractus opticus; zi, zona incerta. (AM), basolateral nucleus (ABL) and periamygda- loid cortex (PAC). Fibers directed dorsally from the VMH injection site traverse the dorsomedial nucleus (DMH) and divide in lateral and dorsal coursing bun- dies. This dorsal bundle terminates partly within the nucleus reuniens (RE) and the rest ascends to the ha- benular complex. Lateral coursing fibers travel un- der zona incerta (ZI) towards the capsula interna (CAI). Some fibers cross this CAI,  others bend dor- sally towards stria terminalis (ST). The PHA-L results show a remarkable similarity with this and previous autoradiographic studies on VMH projections3, 6. The [3H]Leu injections, how- ever, appear as rather diffuse deposits, which do not allow the precise delineation of the tracer uptake area. Moreover, the tracer diffusion prohibits the identification of short connections. D M H  injections of PHA-L (not illustrated) show a strong short pro- jection to the VMH which could not be identified af- ter similar injections with [3H]Leu. Furthermore, after the same DMH injections of PHA-L long de- scending projections of individual fibers could be ob- served to the lower medulla, previously demon- strated with retrograde transport methods 7, whereas these minor projections can hardly be discerned in the autoradiographic material. Another advantage 382 Fig. 2. A-F: photomicrographs illustrating labeling obtained after autoradiographic and PAP immunohistochemical processing :of [3H]leucine and Phaseolus vulgaris leuco-agglutinin injections into the ventromedial hypothalamic nucleus (VMH). A: dark-field pho- tomicrograph showing tritiated leucine terminal labeling within the periamygdaloid cortex (see also Fig. 1A, B). B: bright,field photo- micrograph of PHA-L terminal labeling within the periamygdaloid cortex in the area illustrated in A. C: photomicrograph of a PHA-L iontophoretic injection into the ventromedial hypothalamic nucleus (VMH). D: bright-field photomicrograph illustrating PHA-L ter- minal labeling embracing a receiving neuron (arrow). E, F: photomicrographs showing the labeling in the amygdalofugal pathway af- ter PHA-L (E) and [3H]Leu (F) injections into VMH, respectively. 383 of  the P H A - L  technique over the autoradiographic method is that in several projection areas it is possi- ble to identify detailed contacts between the efferent terminals and the receiving somata (Fig. 2D). In these cases large numbers of labeled boutons can be seen embracing the target perikarya. In summary,  it may be concluded that the tech- nique of  anterogradely transported P H A - L  is ba- sically as effective as the autoradiographic method for the study of  neuronal  efferents, but offers several additional advantages. Its superior sensitivity en- ables the user to study the fine structure of  even small efferent projections. A disadvantage may be that the P H A - L  technique does not appear  suitable for sur- vey studies of  efferents from large brain areas. The authors wish to express their appreciation to Mrs. Joke Poelstra for typing the manuscript. This work was supported in part by the Foundation for Medical Research F U N G O  (Grant 13-46-36) which is subsidized by the Dutch Organization for the Ad- vancement of  Pure Research (ZWO).  1 Edwards, S. B. and Hendrickson, A., The autoradiographic tracing of axonal connections in the central nervous system. In L. Heimer and M. J. Robards (Eds.), Neuroanatomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 171-205. 2 Geffen, C. R. and Sawchenko, P. E., An anterograde neu- roanatomical tracing method that shows the detailed mor- phology of neurons, their axons and terminals: immunohis- toehemical localization of an axonally transported plant lec- tin, Phaseolus vulgaris, leucoagglutinin (PHA-L), Brain Re- search, 290 (1984) 219-238. 3 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An auto- radiographic study of the efferent connections of the ventro- medial nucleus of the hypothalamus, J. comp. Neurol., 183 (1979) 785-816. 4 Ricardo, J. A., Efferent connections of the subthalamic re- gion in the rat: I. Zona incerta, Brain Research, 214 (1981) 43-60. 5 Ruda, M. and Coulter, J. D., Axonal and transneuronal transport of Wheat Germ Agglutinin demonstrated by im- munocytochemistry, Brain Research, 249 (1982) 237-246. 6 Saper, C. B., Swanson, L. W. and Cowan, W. N., The effer- ent connections of the ventromediai hypothalamus of the rat, J. comp. Neurol., 169 (1976) 409-422. 7 Ter Horst, G. J., Luiten, P. G. M. and Kuipers, F., Descend- ing pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J. auton, nerv. Syst., in press. 

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Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents

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