Brain Research Bulletin,Vol. 26, pp. 813-816. 0 Pergamon Press plc, 1991. Printed in the U.S.A
0361-9230191 $3.00 + .OO
RAPID COMMUNICATION
Anterograde Transport of Lucifer Yellow-Dextran Conjugate H. T. CHANG Department of Anatomy and Neurobiology, The University of Tennessee, Memphis College of Medicine, 875 Monroe Avenue, Memphis, TN 38163
Received 27 December 1990 CHANG, H. T. Anrerograde transport of Lucifer Yellow-dextran conjugate. BRAIN RES BULL 26(5) 813-816, 1991. --Immunoperoxidase reaction was performed using a very sensitive antisera raised against Lucifer Yellow to demonstrate that dextran amines conjugated to Lucifer Yellow are anterogradely transported by axons. Anterograde
axonal transport
Anti-Lucifer
Yellow
Fluorescent
dextran amines
Subsequently, a conventional immunoperoxidase reaction was performed to demonstrate LY immunoreactivity using a rabbit antisera raised against LY conjugated to keyhole limpet hemocyanin (Chemicon International, Inc.). The sections were incubated sequentially in solutions containing the primary antisera (1: 1000 dilution), biotinylated donkey anti-rabbit IgG (Jackson, 1: loo), and avidin complexed with biotinylated horseradish peroxidase (Vector, 1:lOO). Detergent (0.3% Triton X-100) was added in the primary solution to enhance the penetration of antibodies into the tissue. The tissue bound peroxidase was visualized by reaction with diaminobenzidine (DAB) and hydrogen peroxidase. The sections were reexamined with epifluorescence, and both bright-field and dark-field illuminations. The specificity of the antisera to LY (anti-LY) was tested using a method modified from the spot-on-paper technique described by Larsson (6). Since nitrocellulose papers are normally used to bind protein samples, and LY and LY-D are not proteins, the papers may not bind to either LY or LY-D efficiently. We saturated the paper strips first with 10% normal serum derived from the host of the secondary antibodies (e.g., donkey or goat) for 2 hours, rinsed in PBS and air dried. Drops containing either pure LY or LY-D were then spotted on these nitrocellulose papers, and fixed with formaldehyde vapor in an oven at 80°C for l-2 hours. After several rinses with PBS to remove unbound LY and LY-D, the spotted papers were reacted for an immunoperoxidase reaction using the anti-LY. Spots made with 1 ~1 drops of 10 ng LY or LY-D could be detected easily using primary antisera at 1:lOOO dilution. Control immunocytochemical reactions were also performed in tissue sections containing processes which are labeled by extracellularly injected LY and fixed by the same fixative solution described above. Since the excitation and emission wavelength of LY are very different from those of Texas Red, immunofluorescence reactions were performed using Texas Red conjugated secondary antisera to label processes containing LY. Switching the
RECENT studies reported that dextran amines conjugated to tetramethylrhodamine (TRITC) (9) and fluorescein isothiocyanate (PITC) appear to be good anterograde tracers, whereas those conjugated to Lucifer Yellow (LY) appear to be not transported anterogradely (7). We report here that dextran amines conjugated to Lucifer Yellow (LY-D) are actually also anterogradely transported as efficiently as other fluorescent dextran amines and that the initial detection problems may be a result of the low quantum yield of the few LY molecules bound on the anterogradely transported dextran amine conjugate.
METHOD
Surgeries were performed on male adult hooded rats anesthetized with Nembutal (50 mglkg). Glass micropipette (tip diameter 20 km) containing 5% LY-D (Molecular Probe No. D-1825, Lot 9A-2) dissolved in saline was positioned stereotaxically into the ventral striatum. The fluorescent dextran amine conjugate was iontophoretically injected into the brain with negative current pulses ( - 5 PA, 2 second pulse, 50% duty cycle) for 15 minutes. After a survival period of 5 to 7 days, the animals were anesthetized deeply with Nembutal and perfused through the heart with 200 ml of saline and followed with 500 ml of 0.1 M phosphate buffered (pH 7.4) fixative solution (4% paraformaldehyde, 0.05% glutaraldehyde, and 15% saturated picric acid). The brains were postfixed overnight in the same fixative solution and cut into serial parasagittal sections (50 km thick) on a Vibratome. Sections were mounted under glass coverslips in phosphate buffered saline (PBS) containing 50% glycerine and 2.5% DABCO (1,4 diazo-bicycle[2,2,2]octane, Sigma) (5) and examined with an Olympus BH-2 microscope equipped with an epifluorescence attachment. The fluorescent injection sites were identified and recorded on Kodak T-Max-400 film. The sections were then removed from the slides and rinsed in several changes of PBS. 813
FIG. 1. (A) Fluorescence micrograph showing the LY-D injection site in ventral striatum. Note that many neurons and processes at the injection site are fluorescent whereas the ventral pallidum under the anterior commissure (ac) are devoid of detectable anterogradely labeled processes. Arrowheads point to some of the brightly fluorescent perivascular macrophages. (B) In the same section as in A, most of the neurons and processes in the injection site are no longer fluorescent after the immunoperoxidase reaction to label LY + processes. Many of the perivascular macrophages (arrowheads), however, remain fluorescent. (C) Bright-field illumination of the same section as in B reveals that the injection site contains many LY + neurons and processes and the expected terminal field in the ventral pallidum under the anterior commissure (ac) is also filled with LY + labeled processes. (D) The trajectory of anterogradely labeled LY + fibers from the injection site in ventral striatum and through the ventral pallidum is now visible even at low magnification bright-field micrograph. The boxed area corresponds to the region shown in A, B and C. (E) The labeled LY + fibers can be better appreciated with higher signal-to-noise ratio using dark-field illumination. (F) Anterogradely labeled LY + fibers and termimals in the substantia nigra pars reticulata (SNr) can be found easily in this dark-field micrograph. Figures A, B, C and F are at the same magnification, D and E are at the same magnification.
ANTEROGRADE AXONAL TRANSPORT OF LUCIFER YELLOW
fluorescence filter sets unequivocally identified the immunolabeled processes (Texas Red) as only those also labeled with LY. Preadsorption of the primary antisera solution (1: 1000 dilution) with 10 p_g/mlpure LY abolished or reduced drastically the immunolabeling of the LY containing processes. Nevertheless, as the possible cross-reaction of this primary antisera with other antigens has not been exhaustively tested, immunolabeled processes in this study are referred to as those contain~g LY-like immuno~activity, and are designated as LY + processes. RESULTS
As shown in Fig. IA, the injection site of LY-D in the ventral striatum was easily visible. Some brightly and some weakly fluorescent neurons together with some brightly labeled perivascular macrophages or pericytes were found at the injection site. The expected terminal field in the neighboring ventral pallidum, however, was not filled with detectable fluorescent processes. This observation was consistent with that reported previously (7). On the other hand, after i~uno~roxida~ reaction using the antiLY, the expected terminal fields in both the ventral pallidum (Fig. lC, D, E) and the substantia nigra pars reticulata (Fig. 1F) were filled with densely labeled LY + axonal processes. The level of anterograde labeling appears to be comparable to those made with other more popular tracers, including the lectin PHA-L (3). Interestingly, many of the perivascular macrophages at the injection site remained fluorescent and unstained with the i~uno~~xidase reaction products (Fig. 1B arrowheads). DISCUSSION
Anti-Licker Yellow as a Tool for Ne~roanatom~
Ever since its initial discovery by Stewart in 1978 (lo), LY has been used by neurophysiologists as an intracellular dye useful for revealing the morphology of neurons recorded intracellularly, and to probe membrane coupling between adjacent cells. More recently, neuroanatomists have injected LY intracelluiarly in lightly fixed brain slices or whole-mounts as a means to label individual neurons. Frequently, a photo-catalysis reaction (8) is performed to convert LY to polymerized DAB. The more stable DAB reaction products also enable further analysis at the electron microscopic level. Photo-catalysis is limited, however, by the fact that only processes directly under the small field of illumination (as determined by the objective lens used for focusing the stimulating light) may be labeled with DAB. Moreover, fine processes labeled with LY (like those anterogradely fibers in this study) often may be bleached by the light before any photo-catalysis reaction has occurred. The availability of anti-LY has overcome most of these problems. We have shown here that anti-LY can be used to mark the LY-D-labeled fibers with convention immuno~roxidase reaction products within the entire tissue section. In alternative paradigms, anti-LY may be used to amplify the original weak LY fluorescence signal with an immunofluorescence reaction. Labeling of Perivascu~ar Cells Near the injection Site
As reported in previous studies, numerous perivascular cells with presumably phagocytic fluorescent granules are found near the LY-D injection site. It remains unclear why many of them are not labeled with the immunoperoxidase reaction products after the anti-LY imrnun~~oche~~~ reaction. In separate studies we have found similar cells, near injection sites of the fluorescent retrograde tracer Pluoro-Gold, which are also relatively resistant to immunocytochemical labeling by antisera raised against Fluoro-
Gold (1). One possible reason is that these cells or their phagosomes are enclosed by membranes less sensitive to the detergent treatment used in this study, and thus the irnrnun~yt~he~c~ reagents (e.g., primary or secondary antisera molecules) cannot readily gain access to the fluorescent or the nonfluorescent antigens within these granules. This would explain the persistence of their fluorescent granules in this study and the failure to notice their presence in studies employing nonfluorescent tracers (e.g., PHA-L). The functional roles of these p&vascular macrophages in the brain remain unclear. Nevertheless, since these cells can be visualized and identified by their phagocytized fluorescent tracers, it would be possible to perform thin-section immunocytochemistry at either light or electron microscopic level to investigate their anatomical properties. Fluorescent Dextran Amine Conjugates as Anterograde Tracers
The problem of low signal-to-noise ratio of the various fluorescent dextran amine conjugates has been noted by previous authors (2, 4, 7). According to the supplier (Molecule Probe), the dextran amine conjugates usually have 1 to 2 fluorescent dyes per dextran amine. Since these fluorescent dyes are very small (molwt. about 500) in contrast to the parent dextran amines (molwt. lO,OOO),and virtually all of the conjugates are similarly charged (anionic), it is likely that comparable amount of dextran amines are ~~s~~ed irrespective of the fluorescent dyes that they are conjugated to. The fluorescence signal of transported dextran amine conjugates, therefore, probably depends on the quantum yield, the integrity, and the number of the original fluorescent dyes on the conjugate. Since LY has only one-fifth of the quantum yield as FITC (lo), even if all of the LY moieties on the LY-D remain fluorescent after the uptake by neurons and the subsequent histological treatments, LY-D in the terminal field would be only L/sas bright as those conjugated to FITC. This low quantum yield of LY may thus be the major reason for the failure of detecting anterogradely transported LY-D by conventional fluorescence microscopy in previous studies. Evidence sup~~ing this view is provided in this study in which we have used a very sensitive polyclonal anti-LY to detect the presence of LY-D within the tissue sections. Fibers labeled by the immunoperoxidase reaction products are found densely in the expected terminal fields in the target nuclei. The often-overlooked fact illustrated by this result is that the immunocyt~he~cal procedure may be used to amplify the signalto-noise ratio several orders of magnitudes. This amplification by the technique of immunocytochemistry is not surprising as the polyclonal primary antisera can bind to multiple epitopes on the original antigen, and subsequently multiple epitopes on the immunoglobulins of the polyclonal primary antisera can be detected and bound by the ~lyclonal secondary antisera. A similar finding to this study can be expected if an immunoperoxidase reaction was performed using a primary antiserum raised against dextran. As discussed in previous studies (2, 4, 7, 9), a great advantage of anterograde labeling with fluorescent dextran amines is that ante~gradely labeled fibers are visible with conventional fIuorescence microscopy without the need to perform histochemistry or immunocytochemistry as required by the popular lectin tracers (e.g., PHA-L) (3). On the other hand, the result from this study indicates that the fluorescence signal from the original dyes on the dextran amine conjugates may be insuffricient to reveal the full extent of the anterograde labeling. False negative results may be obtained if only conventional fluorescence microscopy was used to visualize fibers containing anterogradely transported dextran amine conjugates.
816
ACKNOWLEDGEMENTS I thank H. Kuo and Q. Tian for their skillful technical assistance. This study was supported by USPHS Grant AGO5944, Biomedical Research Support Grant RR05423, and a grant from the Alzheimer’s Disease and Related Disorders Association.
REFERENCES 1. Chang, H. T.; Kuo, H.; Whittaker, J. A.; Cooper, N. G. F. Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold. J. Neurosci. Methods 3531-37; 1990. 2. Fritzsch, B.; Wilm, C. Dextran amines in neuronal tracing. Trends Neurosci. 13:14; 1990. 3. Gerfen, C. R.; Sawchenko, P. E. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant Iectin, Phaseolus vulgaris-leucoagglutinin. Brain Res. 290:219-238; 1984. 4. Glover, J. C.; Petursdottir, G.; Jansen, J. K. Fluorescent dextranamines used as axonal tracers in the nervous system of the chicken embryo. J. Neurosci. Methods 18(3):243-54; 1986. 5. Johnson, G. D.; Davidson, R. S.; McNamee, K. C.; Russell, G.: Goodwin, D.; Holborow, E. J. Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Im-
munol. Methods 55:231-242; 1982. 6. Larsson, L.-I. A novel immunocytochemical model system for specificity and sensitivity screening of antisera aganist multiple antigens. J. Histochem. Cytochem. 29408-410; 1981. 7. Nance, D. M.; Burns, J. Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: Applications and pitfalls. Brain Res. Bull. 25:139-145; 1990. 8. Sandell, J. H.; Masland, R. H. Photoconversion of some fluorescent markers to a diaminobenzidine product. J. Histochem. Cytochem. 36:555-559; 1988. 9. Schmued, L. C.; Kyriakidis, K.; Heimer, L. In vivo anterograde and retrograde axonal transport of the fluorescent rhodamlne-dextranamine, Fluoro-Ruby, within the CNS. Brain Res. 526:127-134; 1990. 10. Stewart, W. W. Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14: 741-759; 1978.
0361-9230191 $3.00 + .OO
RAPID COMMUNICATION
Anterograde Transport of Lucifer Yellow-Dextran Conjugate H. T. CHANG Department of Anatomy and Neurobiology, The University of Tennessee, Memphis College of Medicine, 875 Monroe Avenue, Memphis, TN 38163
Received 27 December 1990 CHANG, H. T. Anrerograde transport of Lucifer Yellow-dextran conjugate. BRAIN RES BULL 26(5) 813-816, 1991. --Immunoperoxidase reaction was performed using a very sensitive antisera raised against Lucifer Yellow to demonstrate that dextran amines conjugated to Lucifer Yellow are anterogradely transported by axons. Anterograde
axonal transport
Anti-Lucifer
Yellow
Fluorescent
dextran amines
Subsequently, a conventional immunoperoxidase reaction was performed to demonstrate LY immunoreactivity using a rabbit antisera raised against LY conjugated to keyhole limpet hemocyanin (Chemicon International, Inc.). The sections were incubated sequentially in solutions containing the primary antisera (1: 1000 dilution), biotinylated donkey anti-rabbit IgG (Jackson, 1: loo), and avidin complexed with biotinylated horseradish peroxidase (Vector, 1:lOO). Detergent (0.3% Triton X-100) was added in the primary solution to enhance the penetration of antibodies into the tissue. The tissue bound peroxidase was visualized by reaction with diaminobenzidine (DAB) and hydrogen peroxidase. The sections were reexamined with epifluorescence, and both bright-field and dark-field illuminations. The specificity of the antisera to LY (anti-LY) was tested using a method modified from the spot-on-paper technique described by Larsson (6). Since nitrocellulose papers are normally used to bind protein samples, and LY and LY-D are not proteins, the papers may not bind to either LY or LY-D efficiently. We saturated the paper strips first with 10% normal serum derived from the host of the secondary antibodies (e.g., donkey or goat) for 2 hours, rinsed in PBS and air dried. Drops containing either pure LY or LY-D were then spotted on these nitrocellulose papers, and fixed with formaldehyde vapor in an oven at 80°C for l-2 hours. After several rinses with PBS to remove unbound LY and LY-D, the spotted papers were reacted for an immunoperoxidase reaction using the anti-LY. Spots made with 1 ~1 drops of 10 ng LY or LY-D could be detected easily using primary antisera at 1:lOOO dilution. Control immunocytochemical reactions were also performed in tissue sections containing processes which are labeled by extracellularly injected LY and fixed by the same fixative solution described above. Since the excitation and emission wavelength of LY are very different from those of Texas Red, immunofluorescence reactions were performed using Texas Red conjugated secondary antisera to label processes containing LY. Switching the
RECENT studies reported that dextran amines conjugated to tetramethylrhodamine (TRITC) (9) and fluorescein isothiocyanate (PITC) appear to be good anterograde tracers, whereas those conjugated to Lucifer Yellow (LY) appear to be not transported anterogradely (7). We report here that dextran amines conjugated to Lucifer Yellow (LY-D) are actually also anterogradely transported as efficiently as other fluorescent dextran amines and that the initial detection problems may be a result of the low quantum yield of the few LY molecules bound on the anterogradely transported dextran amine conjugate.
METHOD
Surgeries were performed on male adult hooded rats anesthetized with Nembutal (50 mglkg). Glass micropipette (tip diameter 20 km) containing 5% LY-D (Molecular Probe No. D-1825, Lot 9A-2) dissolved in saline was positioned stereotaxically into the ventral striatum. The fluorescent dextran amine conjugate was iontophoretically injected into the brain with negative current pulses ( - 5 PA, 2 second pulse, 50% duty cycle) for 15 minutes. After a survival period of 5 to 7 days, the animals were anesthetized deeply with Nembutal and perfused through the heart with 200 ml of saline and followed with 500 ml of 0.1 M phosphate buffered (pH 7.4) fixative solution (4% paraformaldehyde, 0.05% glutaraldehyde, and 15% saturated picric acid). The brains were postfixed overnight in the same fixative solution and cut into serial parasagittal sections (50 km thick) on a Vibratome. Sections were mounted under glass coverslips in phosphate buffered saline (PBS) containing 50% glycerine and 2.5% DABCO (1,4 diazo-bicycle[2,2,2]octane, Sigma) (5) and examined with an Olympus BH-2 microscope equipped with an epifluorescence attachment. The fluorescent injection sites were identified and recorded on Kodak T-Max-400 film. The sections were then removed from the slides and rinsed in several changes of PBS. 813
FIG. 1. (A) Fluorescence micrograph showing the LY-D injection site in ventral striatum. Note that many neurons and processes at the injection site are fluorescent whereas the ventral pallidum under the anterior commissure (ac) are devoid of detectable anterogradely labeled processes. Arrowheads point to some of the brightly fluorescent perivascular macrophages. (B) In the same section as in A, most of the neurons and processes in the injection site are no longer fluorescent after the immunoperoxidase reaction to label LY + processes. Many of the perivascular macrophages (arrowheads), however, remain fluorescent. (C) Bright-field illumination of the same section as in B reveals that the injection site contains many LY + neurons and processes and the expected terminal field in the ventral pallidum under the anterior commissure (ac) is also filled with LY + labeled processes. (D) The trajectory of anterogradely labeled LY + fibers from the injection site in ventral striatum and through the ventral pallidum is now visible even at low magnification bright-field micrograph. The boxed area corresponds to the region shown in A, B and C. (E) The labeled LY + fibers can be better appreciated with higher signal-to-noise ratio using dark-field illumination. (F) Anterogradely labeled LY + fibers and termimals in the substantia nigra pars reticulata (SNr) can be found easily in this dark-field micrograph. Figures A, B, C and F are at the same magnification, D and E are at the same magnification.
ANTEROGRADE AXONAL TRANSPORT OF LUCIFER YELLOW
fluorescence filter sets unequivocally identified the immunolabeled processes (Texas Red) as only those also labeled with LY. Preadsorption of the primary antisera solution (1: 1000 dilution) with 10 p_g/mlpure LY abolished or reduced drastically the immunolabeling of the LY containing processes. Nevertheless, as the possible cross-reaction of this primary antisera with other antigens has not been exhaustively tested, immunolabeled processes in this study are referred to as those contain~g LY-like immuno~activity, and are designated as LY + processes. RESULTS
As shown in Fig. IA, the injection site of LY-D in the ventral striatum was easily visible. Some brightly and some weakly fluorescent neurons together with some brightly labeled perivascular macrophages or pericytes were found at the injection site. The expected terminal field in the neighboring ventral pallidum, however, was not filled with detectable fluorescent processes. This observation was consistent with that reported previously (7). On the other hand, after i~uno~roxida~ reaction using the antiLY, the expected terminal fields in both the ventral pallidum (Fig. lC, D, E) and the substantia nigra pars reticulata (Fig. 1F) were filled with densely labeled LY + axonal processes. The level of anterograde labeling appears to be comparable to those made with other more popular tracers, including the lectin PHA-L (3). Interestingly, many of the perivascular macrophages at the injection site remained fluorescent and unstained with the i~uno~~xidase reaction products (Fig. 1B arrowheads). DISCUSSION
Anti-Licker Yellow as a Tool for Ne~roanatom~
Ever since its initial discovery by Stewart in 1978 (lo), LY has been used by neurophysiologists as an intracellular dye useful for revealing the morphology of neurons recorded intracellularly, and to probe membrane coupling between adjacent cells. More recently, neuroanatomists have injected LY intracelluiarly in lightly fixed brain slices or whole-mounts as a means to label individual neurons. Frequently, a photo-catalysis reaction (8) is performed to convert LY to polymerized DAB. The more stable DAB reaction products also enable further analysis at the electron microscopic level. Photo-catalysis is limited, however, by the fact that only processes directly under the small field of illumination (as determined by the objective lens used for focusing the stimulating light) may be labeled with DAB. Moreover, fine processes labeled with LY (like those anterogradely fibers in this study) often may be bleached by the light before any photo-catalysis reaction has occurred. The availability of anti-LY has overcome most of these problems. We have shown here that anti-LY can be used to mark the LY-D-labeled fibers with convention immuno~roxidase reaction products within the entire tissue section. In alternative paradigms, anti-LY may be used to amplify the original weak LY fluorescence signal with an immunofluorescence reaction. Labeling of Perivascu~ar Cells Near the injection Site
As reported in previous studies, numerous perivascular cells with presumably phagocytic fluorescent granules are found near the LY-D injection site. It remains unclear why many of them are not labeled with the immunoperoxidase reaction products after the anti-LY imrnun~~oche~~~ reaction. In separate studies we have found similar cells, near injection sites of the fluorescent retrograde tracer Pluoro-Gold, which are also relatively resistant to immunocytochemical labeling by antisera raised against Fluoro-
Gold (1). One possible reason is that these cells or their phagosomes are enclosed by membranes less sensitive to the detergent treatment used in this study, and thus the irnrnun~yt~he~c~ reagents (e.g., primary or secondary antisera molecules) cannot readily gain access to the fluorescent or the nonfluorescent antigens within these granules. This would explain the persistence of their fluorescent granules in this study and the failure to notice their presence in studies employing nonfluorescent tracers (e.g., PHA-L). The functional roles of these p&vascular macrophages in the brain remain unclear. Nevertheless, since these cells can be visualized and identified by their phagocytized fluorescent tracers, it would be possible to perform thin-section immunocytochemistry at either light or electron microscopic level to investigate their anatomical properties. Fluorescent Dextran Amine Conjugates as Anterograde Tracers
The problem of low signal-to-noise ratio of the various fluorescent dextran amine conjugates has been noted by previous authors (2, 4, 7). According to the supplier (Molecule Probe), the dextran amine conjugates usually have 1 to 2 fluorescent dyes per dextran amine. Since these fluorescent dyes are very small (molwt. about 500) in contrast to the parent dextran amines (molwt. lO,OOO),and virtually all of the conjugates are similarly charged (anionic), it is likely that comparable amount of dextran amines are ~~s~~ed irrespective of the fluorescent dyes that they are conjugated to. The fluorescence signal of transported dextran amine conjugates, therefore, probably depends on the quantum yield, the integrity, and the number of the original fluorescent dyes on the conjugate. Since LY has only one-fifth of the quantum yield as FITC (lo), even if all of the LY moieties on the LY-D remain fluorescent after the uptake by neurons and the subsequent histological treatments, LY-D in the terminal field would be only L/sas bright as those conjugated to FITC. This low quantum yield of LY may thus be the major reason for the failure of detecting anterogradely transported LY-D by conventional fluorescence microscopy in previous studies. Evidence sup~~ing this view is provided in this study in which we have used a very sensitive polyclonal anti-LY to detect the presence of LY-D within the tissue sections. Fibers labeled by the immunoperoxidase reaction products are found densely in the expected terminal fields in the target nuclei. The often-overlooked fact illustrated by this result is that the immunocyt~he~cal procedure may be used to amplify the signalto-noise ratio several orders of magnitudes. This amplification by the technique of immunocytochemistry is not surprising as the polyclonal primary antisera can bind to multiple epitopes on the original antigen, and subsequently multiple epitopes on the immunoglobulins of the polyclonal primary antisera can be detected and bound by the ~lyclonal secondary antisera. A similar finding to this study can be expected if an immunoperoxidase reaction was performed using a primary antiserum raised against dextran. As discussed in previous studies (2, 4, 7, 9), a great advantage of anterograde labeling with fluorescent dextran amines is that ante~gradely labeled fibers are visible with conventional fIuorescence microscopy without the need to perform histochemistry or immunocytochemistry as required by the popular lectin tracers (e.g., PHA-L) (3). On the other hand, the result from this study indicates that the fluorescence signal from the original dyes on the dextran amine conjugates may be insuffricient to reveal the full extent of the anterograde labeling. False negative results may be obtained if only conventional fluorescence microscopy was used to visualize fibers containing anterogradely transported dextran amine conjugates.
816
ACKNOWLEDGEMENTS I thank H. Kuo and Q. Tian for their skillful technical assistance. This study was supported by USPHS Grant AGO5944, Biomedical Research Support Grant RR05423, and a grant from the Alzheimer’s Disease and Related Disorders Association.
REFERENCES 1. Chang, H. T.; Kuo, H.; Whittaker, J. A.; Cooper, N. G. F. Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold. J. Neurosci. Methods 3531-37; 1990. 2. Fritzsch, B.; Wilm, C. Dextran amines in neuronal tracing. Trends Neurosci. 13:14; 1990. 3. Gerfen, C. R.; Sawchenko, P. E. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant Iectin, Phaseolus vulgaris-leucoagglutinin. Brain Res. 290:219-238; 1984. 4. Glover, J. C.; Petursdottir, G.; Jansen, J. K. Fluorescent dextranamines used as axonal tracers in the nervous system of the chicken embryo. J. Neurosci. Methods 18(3):243-54; 1986. 5. Johnson, G. D.; Davidson, R. S.; McNamee, K. C.; Russell, G.: Goodwin, D.; Holborow, E. J. Fading of immunofluorescence during microscopy: A study of the phenomenon and its remedy. J. Im-
munol. Methods 55:231-242; 1982. 6. Larsson, L.-I. A novel immunocytochemical model system for specificity and sensitivity screening of antisera aganist multiple antigens. J. Histochem. Cytochem. 29408-410; 1981. 7. Nance, D. M.; Burns, J. Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: Applications and pitfalls. Brain Res. Bull. 25:139-145; 1990. 8. Sandell, J. H.; Masland, R. H. Photoconversion of some fluorescent markers to a diaminobenzidine product. J. Histochem. Cytochem. 36:555-559; 1988. 9. Schmued, L. C.; Kyriakidis, K.; Heimer, L. In vivo anterograde and retrograde axonal transport of the fluorescent rhodamlne-dextranamine, Fluoro-Ruby, within the CNS. Brain Res. 526:127-134; 1990. 10. Stewart, W. W. Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14: 741-759; 1978.
