Neuroscience Letters, 140 (1992) 197-199 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
197
NSL 08691
Evidence that biocytin is taken up by axons G. Chevalier, J.M. Deniau and A. Menetrey D~partement des Neurosciences de la Vision, Institut des Neurosciences, U.A. 1199, Universit~ Pierre et Marie Curie, Paris (France)
(Received 27 January 1992; Accepted 25 March 1992) Key words:
Biocytin; Anterograde tracing; Axonal uptake
Unexpected anterograde labeling is systematically observed in the pontine nuclei following iontophoretic injection of biocytin in the substantia nigra pars reticulata. Control experiments using WGA-HRP have led to deny the existence of a nigropontine pathway. The possibility that biocytin is taken up by fibers of passage has been tested. Deposits of biocytin in the corpus callosum result in a massive axonal labeling of this fibrous system. This study, in contrast to previous reports, stresses that biocytin is easily taken up and transported by axons. Hence, this tracer has to be used with careful controls when injected in structures crossed by fibrous tracts.
Biocytin is a conjugate of biotin and lysine which was recently introduced as an anterograde tracer [5]. Due to its remarkable properties, biocytin rapidly attracted neuroanatomists. Besides its versatility, its ability to provide a detailed picture of the labeled axonal profiles, this tracer is also reputed as not being taken up by fibers en passage [4]. During the course of a study in which biocytin was iontophoretically injected in the substantia nigra pars reticulata, the presence of a profuse terminal labeling in the pontine nuclei attracted our attention. Since these nuclei are not classically recognized as a target for nigral efferents [2, 3, 7], this aberrant labeling suggested that biocytin might be taken up and transported through fibers of passage. The present study points up the major defect of this tracer. Male Sprague-Dawley rats (Charles River, France) weighing between 260 and 300 g were deeply anesthetised with sodium pentobarbital (50 mg/kg). Biocytin (Sigma) dissolved at 2~,% in buffered (Tris, pH 7.6; acetate, pH 4.5) or unbuffered solutions of 0.15, 0.25, 1 or 2 M sodium chloride was injected iontophoretically through glass micropipettes (tip diameter 10-20/tm) using anodal rectangular current pulses (1.2 s on and 1.8 s off) of 5/tA for 15-20 min. After a survival period of 30 h, rats were deeply anesthetized with Nembutal and perfused sequentially with physiological saline and a 4% paraformaldehyde/0.15% glutaraldehyde buffered solution. Following Correspondence: G. Chevalier, D6partement des Neurosciences de la Vision, Institut des Neurosciences, U.A. 1199, Universit6 Pierre et Marie Curie, 4 Place Jussieu, 75230 Paris Cedex 05, France.
a 12 h postfixation period at 4°C, tissue was cut using a vibrating or freezing microtome. Sections of 40/lm were collected and rinsed four times into potassium phosphate buffered saline (KPBS 0.02 M, pH 7.4). Finally, they were incubated in avidin-biotin-peroxidase complex (dilution l: 100 in KPBS) from 5 h to overnight. After several rinses in KPBS, sections were reacted with diaminobenzidine (0.05%) and hydrogen peroxide (0.01%). Reaction product was intensified using Adams' procedure [1]. The sections were mounted onto gelatin chrome-alum coated slides and dehydrated through alcohol to xylene for light microscopic examination. In addition some control experiments were performed using wheat germ agglutinin conjugated to horseradish peroxidase (WGAHRP). WGA-HRP (2% in 0.9% saline) was applied through micropipettes (10-20 Bm tip diameter) using 5 /2A anodal pulses (2 s on and 1 s off) for 15 min. After a survival period of 30-36 h, animals were perfused-fixed with 3% glutaraldehyde in 0.15 M phosphate buffer. Frozen sections of 50/2m were conventionally processed for H R P histochemistry using the tetramethyl benzidine method of Mesulam [6]. Biocytin was injected in the substantia nigra pars reticulata of 8 animals. As generally described in the literature, the deposit of this tracer resulted in small injection sites of 100-500pm diameter [4] (Fig. la). This latter was characterized by the presence of completely filled neurons intermingled with a dense local network of axonal profiles (Fig. lb). An important number of labeled axons were found to leave the injection site and could be followed in serial sections. Besides the efferent fiber systems
198
a
b
I
c , cl , Fig. 1. Pontine axonal labeling followinginjectionof biocytinin the substantia nigra pars reticulata, a: dark-fieldphotomicrographof the substantia nigra pars reticulata. Arrow points to a series of three discrete iontophoreticdeposits of biocytin (3% in 1 M sodium chloride), b: bright-fieldhigh magnificationview of two injection sites, c: dark-fieldphotomicrographof the basilar pontine nuclei localizingthe site of terminal axonal labeling (framed area), d: bright-fieldhigh magnificationview of the terminal axonal labeling. Bars = 500 ,urn (a,c), 100/lm (b) and 50/~m (d).
directed to the known target regions of the substantia nigra, i.e. thalamus, superior colliculus and peribrachial area [2, 3], an impressive number of labeled axons was regularly seen to run caudalwards within the longitudinal fasciculus pons before arborizing profusely within the basilar pontine nuclei (Fig. lc,d). This pontine labeling was observed irrespective of the biocytin solutions tested (biocytin concentration and type of medium). The extranigral origin of this unexpected labeling has been established by the failure to reveal the existence of any nigropontine pathway by means of W G A - H R E In full agreement with observations by others [7], application of W G A - H R P in several loci of the pontine nuclei results in retrograde labeling in all the midbrain areas known to project to the pons such as tectum, pretectum, zona incerta, but not in the pars reticulata of the substantia nigra. This negative result was strongly suggestive of a direct uptake of biocytin by fibers of passage. This has been tested by injecting biocytin into the corpus callosum
(Fig. 2a). A small deposit of tracer results in a stream of solidly stained axons which can be followed over long distances (Fig. 2b,c). By contrast, iontophoretic injections of W G A - H R P made in similar conditions (same electrode tip diameters and ejection current) never result in such an axonal labeling. Considering the cautions taken to minimize tissue damage (microiontophoresis using small tip diameter pipettes and injection current of low intensity) it appears likely that the considerable axonal labeling observed with biocytin results primarily from uptake through undamaged fibers. In conclusion, at variance with previous reports that biocytin is selectively taken up by perikarya and dendrites if injected iontophoretically [4, 5], the present data point out that this tracer is also easily taken up and transported through axons of passage. This potential source of misinterpretation has to be seriously considered when it is used for extracellular tract tracing.
199
This work was supported by Esprit Basic Research Action 3149 MUCOM.
a
b
C Fig. 2. a: arrow points to a biocytin injection in the corpus caUosum. In b and c are dark-field and bright-field views of the corpus callosum illustrating the stream of labeled axons which stems from the injection site. Bars = 500/.tin (a), 100/.tm (b) and 50/tm (c).
1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Deniau, J.M. and Chevalier, G., The lamellar organization of the rat substantia nigra pars reticulata. Distribution of projection neurons, Neuroscience, 46 (1992) 361-377. 3 Gerfen, C.R., Staines, W.A., Arbuthnott, G.W. and Fibiger, H.C., Crossed connections of the substantia nigra in the rat, J. Comp. Neurol., 207 (1982) 283-303. 4 Izzo, EN., A note on the use of biocytin in anterograde tracing studies in the central nervous system: application at both light and electron microscopic level, J. Neurosci. Methods, 36 (1991) 155-166. 5 King, M.A., Bruce, P.M.L., Hunter, B.E. and Walker, D.W., Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative, Brain Res., 497 (1989) 361-367. 6 Mesulam, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 7 Mihailoff, G.A., Kosinski, R.J., Azizi, S.A. and Border, B.G., Survey of noncortical afferent projections to the basilar pontine nuclei: a retrograde tracing study in the rat, J. Comp. Neurol., 282 (1989) 617-643.
197
NSL 08691
Evidence that biocytin is taken up by axons G. Chevalier, J.M. Deniau and A. Menetrey D~partement des Neurosciences de la Vision, Institut des Neurosciences, U.A. 1199, Universit~ Pierre et Marie Curie, Paris (France)
(Received 27 January 1992; Accepted 25 March 1992) Key words:
Biocytin; Anterograde tracing; Axonal uptake
Unexpected anterograde labeling is systematically observed in the pontine nuclei following iontophoretic injection of biocytin in the substantia nigra pars reticulata. Control experiments using WGA-HRP have led to deny the existence of a nigropontine pathway. The possibility that biocytin is taken up by fibers of passage has been tested. Deposits of biocytin in the corpus callosum result in a massive axonal labeling of this fibrous system. This study, in contrast to previous reports, stresses that biocytin is easily taken up and transported by axons. Hence, this tracer has to be used with careful controls when injected in structures crossed by fibrous tracts.
Biocytin is a conjugate of biotin and lysine which was recently introduced as an anterograde tracer [5]. Due to its remarkable properties, biocytin rapidly attracted neuroanatomists. Besides its versatility, its ability to provide a detailed picture of the labeled axonal profiles, this tracer is also reputed as not being taken up by fibers en passage [4]. During the course of a study in which biocytin was iontophoretically injected in the substantia nigra pars reticulata, the presence of a profuse terminal labeling in the pontine nuclei attracted our attention. Since these nuclei are not classically recognized as a target for nigral efferents [2, 3, 7], this aberrant labeling suggested that biocytin might be taken up and transported through fibers of passage. The present study points up the major defect of this tracer. Male Sprague-Dawley rats (Charles River, France) weighing between 260 and 300 g were deeply anesthetised with sodium pentobarbital (50 mg/kg). Biocytin (Sigma) dissolved at 2~,% in buffered (Tris, pH 7.6; acetate, pH 4.5) or unbuffered solutions of 0.15, 0.25, 1 or 2 M sodium chloride was injected iontophoretically through glass micropipettes (tip diameter 10-20/tm) using anodal rectangular current pulses (1.2 s on and 1.8 s off) of 5/tA for 15-20 min. After a survival period of 30 h, rats were deeply anesthetized with Nembutal and perfused sequentially with physiological saline and a 4% paraformaldehyde/0.15% glutaraldehyde buffered solution. Following Correspondence: G. Chevalier, D6partement des Neurosciences de la Vision, Institut des Neurosciences, U.A. 1199, Universit6 Pierre et Marie Curie, 4 Place Jussieu, 75230 Paris Cedex 05, France.
a 12 h postfixation period at 4°C, tissue was cut using a vibrating or freezing microtome. Sections of 40/lm were collected and rinsed four times into potassium phosphate buffered saline (KPBS 0.02 M, pH 7.4). Finally, they were incubated in avidin-biotin-peroxidase complex (dilution l: 100 in KPBS) from 5 h to overnight. After several rinses in KPBS, sections were reacted with diaminobenzidine (0.05%) and hydrogen peroxide (0.01%). Reaction product was intensified using Adams' procedure [1]. The sections were mounted onto gelatin chrome-alum coated slides and dehydrated through alcohol to xylene for light microscopic examination. In addition some control experiments were performed using wheat germ agglutinin conjugated to horseradish peroxidase (WGAHRP). WGA-HRP (2% in 0.9% saline) was applied through micropipettes (10-20 Bm tip diameter) using 5 /2A anodal pulses (2 s on and 1 s off) for 15 min. After a survival period of 30-36 h, animals were perfused-fixed with 3% glutaraldehyde in 0.15 M phosphate buffer. Frozen sections of 50/2m were conventionally processed for H R P histochemistry using the tetramethyl benzidine method of Mesulam [6]. Biocytin was injected in the substantia nigra pars reticulata of 8 animals. As generally described in the literature, the deposit of this tracer resulted in small injection sites of 100-500pm diameter [4] (Fig. la). This latter was characterized by the presence of completely filled neurons intermingled with a dense local network of axonal profiles (Fig. lb). An important number of labeled axons were found to leave the injection site and could be followed in serial sections. Besides the efferent fiber systems
198
a
b
I
c , cl , Fig. 1. Pontine axonal labeling followinginjectionof biocytinin the substantia nigra pars reticulata, a: dark-fieldphotomicrographof the substantia nigra pars reticulata. Arrow points to a series of three discrete iontophoreticdeposits of biocytin (3% in 1 M sodium chloride), b: bright-fieldhigh magnificationview of two injection sites, c: dark-fieldphotomicrographof the basilar pontine nuclei localizingthe site of terminal axonal labeling (framed area), d: bright-fieldhigh magnificationview of the terminal axonal labeling. Bars = 500 ,urn (a,c), 100/lm (b) and 50/~m (d).
directed to the known target regions of the substantia nigra, i.e. thalamus, superior colliculus and peribrachial area [2, 3], an impressive number of labeled axons was regularly seen to run caudalwards within the longitudinal fasciculus pons before arborizing profusely within the basilar pontine nuclei (Fig. lc,d). This pontine labeling was observed irrespective of the biocytin solutions tested (biocytin concentration and type of medium). The extranigral origin of this unexpected labeling has been established by the failure to reveal the existence of any nigropontine pathway by means of W G A - H R E In full agreement with observations by others [7], application of W G A - H R P in several loci of the pontine nuclei results in retrograde labeling in all the midbrain areas known to project to the pons such as tectum, pretectum, zona incerta, but not in the pars reticulata of the substantia nigra. This negative result was strongly suggestive of a direct uptake of biocytin by fibers of passage. This has been tested by injecting biocytin into the corpus callosum
(Fig. 2a). A small deposit of tracer results in a stream of solidly stained axons which can be followed over long distances (Fig. 2b,c). By contrast, iontophoretic injections of W G A - H R P made in similar conditions (same electrode tip diameters and ejection current) never result in such an axonal labeling. Considering the cautions taken to minimize tissue damage (microiontophoresis using small tip diameter pipettes and injection current of low intensity) it appears likely that the considerable axonal labeling observed with biocytin results primarily from uptake through undamaged fibers. In conclusion, at variance with previous reports that biocytin is selectively taken up by perikarya and dendrites if injected iontophoretically [4, 5], the present data point out that this tracer is also easily taken up and transported through axons of passage. This potential source of misinterpretation has to be seriously considered when it is used for extracellular tract tracing.
199
This work was supported by Esprit Basic Research Action 3149 MUCOM.
a
b
C Fig. 2. a: arrow points to a biocytin injection in the corpus caUosum. In b and c are dark-field and bright-field views of the corpus callosum illustrating the stream of labeled axons which stems from the injection site. Bars = 500/.tin (a), 100/.tm (b) and 50/tm (c).
1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Deniau, J.M. and Chevalier, G., The lamellar organization of the rat substantia nigra pars reticulata. Distribution of projection neurons, Neuroscience, 46 (1992) 361-377. 3 Gerfen, C.R., Staines, W.A., Arbuthnott, G.W. and Fibiger, H.C., Crossed connections of the substantia nigra in the rat, J. Comp. Neurol., 207 (1982) 283-303. 4 Izzo, EN., A note on the use of biocytin in anterograde tracing studies in the central nervous system: application at both light and electron microscopic level, J. Neurosci. Methods, 36 (1991) 155-166. 5 King, M.A., Bruce, P.M.L., Hunter, B.E. and Walker, D.W., Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative, Brain Res., 497 (1989) 361-367. 6 Mesulam, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 7 Mihailoff, G.A., Kosinski, R.J., Azizi, S.A. and Border, B.G., Survey of noncortical afferent projections to the basilar pontine nuclei: a retrograde tracing study in the rat, J. Comp. Neurol., 282 (1989) 617-643.
