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Two new fluorescent retrograde neuronal tracers which are transported over long distances
Neuroscience Letters, 18 (1980) 25-30
25
© Elsevier/North-Holland Scientific Publishers Ltd.
TWO NEW F L U O R E S C E N T R E T R O G R A D E N E U R O N A L T R A C E R S W H I C H ARE T R A N S P O R T E D OVER LONG D I S T A N C E S
M. BENTIVOGLIO, H.G.J.M. KUYPERS, C.E. CATSMAN-BERREVOETS, H. LOEWE and O. DANN
Department of Anatomy, Erasmus University Medical School, Rotterdam (The Netherlands); (H.L.) Hoechst Company, Frankfurt am Main (F.R.G.) and
(0. D.) Institute for Pharmacy and Foodchemistry, Friedrich-A leyander University, Erlangen (F.R.G.) (Received January 7th, 1980) (Revised version received February 18th, 1980) (Accepted February 18th, 1980)
SUMMARY
Two new fluorescent retrograde neuronal tracers are reported: Nuclear Yellow (Hoechst S 769121), which mainly labels the neuronal nucleus; and Fast Blue (diamidino compound 253/50), which mainly labels the neuronal cytoplasm. Both tracers appear to be transported effectively over long distances in rat and cat.
Several fluorescent substances, i.e. Evans Blue, DAPI-Primuline, propidium iodide, bisbenzimide (Bb), Granular Blue (GB) and True Blue (TB) are transported retrogradely through axons to their parent cell bodies and produce fluorescent labeling of different features of the cell [1, 4, 6]. Two other fluorescent retrograde tracers have been found: (1) Hoechst S 769121, which is related to Bb. It mainly labels the neuronal nucleus and therefore will be referred to as Nuclear Yellow (NY)*; (2) the diamidino compound 253/50, synthesized by Dann and his collaborators. This substance is related to TB, but is transported more rapidly and effectively over long distances in cat and therefore will be referred to as Fast Blue (FB)*. These two tracers have been compared with Bb, TB and GB [1, 4]. Hoechst S 769121: 2-(4-sulfamylphenyl)-6-(6-(4-methylpiperazino)-2-benzimidazolyl)benzimidazole trishydrochloride, i.e. Nuclear Yellow (NY), is difficult to dissolve in water and has been used in 10% suspension; however, in later * See footnote p. 26
26 experiments 107owas preferred [2]. Fast Blue (FB) (diamidino compound 253/50)* is soluble in water and has been used in concentrations of 3% and 5°7o. True Blue (TB) is almost water-insoluble [1] and has been used in suspensions of 2°7o and 5°70; Granular Blue (GB) is slightly water-soluble [1] and has been used in a suspension of 5%. The tracers were injected in brain and spinal cord of rats and cats by means of either a Hamilton syringe with a 22-gauge needle or a micropipette. Each needle and micropipette was only used for one tracer in order to avoid contamination. The animals were perfused as described earlier [1, 4, 6]. Brain and spinal cord were kept in cacodylate buffered (pH 7.2) 30O7o sucrose for up to 24 h. The injected spinal segments were kept separate from the other tissue slices since the tracer remaining at the injection site might dissolve in the sucrose solution and from there penetrate cut surfaces of the other slices. The material was cut transversely in frozen sections 30 ~m thick. Initially they were collected in distilled water and then were either first stored at 4°C for up to 12 h or were immediately mounted and air dried at room temperature, but were not coverslipped. However, later in cat a different procedure was adopted in order to prevent migration of NY out of retrogradely labeled neurons to surrounding cells [2]. The material was studied with a Leitz Ploemopack fluorescence microscope, equipped with filter-mirror systems A, D and N2, which provide excitation light at 360 nm, 390 nm and 550 nm wavelengths.
Results with Nuclear Yellow (Hoechst S 769121). The retrograde transport of many compounds related to Bb has been tested earlier (D. van der Kooy and M. Bentivoglio, unpublished observations). Hoechst S 769121 (NY) will be described, since it is very suitable for double-labeling experiments to be reported separately [5]. In 6 rats 0.2/~1 10% NY was injected in caudate-putamen followed by survival times ranging from 1 to 26 days. The NY injection produced a fluorescent injection area similar to that of Bb [4], and resulted in NY retrograde labeling of neurons in substantia nigra (SN), center median-parafascicular complex ( C M - P f ) , and dorsal raphe (DR). When viewed with filter-mirror system A, the neurons showed a bright golden-yellow fluorescent nuclear labeling which closely resembled the labeling obtained with Bb [4]. However, especially when buffered formaline was used [3], they showed little cytoplasmic fluorescence, except for fine golden-yellow dust. Long survival times did not produce an increase in fluorescence of the retrogradely labeled neurons. In 3 rats 1 ~tl 10% NY was injected at C 2 - C 3 , followed by 5 days survival. In these rats neurons of sensorimotor cortex and descending brain stem pathways
* For research purposes small samples of Nuclear Yellow can be obtained from Dr. H. Loewe, Hoechst Aktiengesellschaft, Post Box 800320, 6230 Frankfurt am Main 80, F.R.G. and small samples of Fast Blue can be obtained from Prof. Dr. O. Dann, lnstitut f~3rPharmazie und Lebensmittelchemie der FriedrichAlexander Universit~t, Post Box 2044, D 8520 Erlangen, F.R.G.
27 showed the same labeling (Fig. 1A, B) as in SN, C M - C f and DR after caudateputamen injections. This same retrograde neuronal labeling was also obtained in cortex and brain stem o f a cat in which _ 30 #l 10% NY was injected at L 1 - L 2 . This demonstrated that NY, as Bb [4], is effectively transported over long distances both in rat and cat. In all cases NY-labeled neurons, as Bb-labeled ones [4], were surrounded by golden-yellow fluorescent glial nuclei. After spinal injections fluorescent glial nuclei were also present in pyramidal tract and cerebral peduncle. This indicates that NY, as Bb [4], when transported retrogradely through axons, may migrate to adjoining glial cells. In the rats with NY injections in caudate-putamen, brilliantly fluorescent glial nuclei were present throughout the substantia nigra, pars reticulata (SNR) where many striatonigral fibers terminate. After spinal injections fluorescent glial nuclei were present in the inferior cerebellar peduncle as well as in the spinoreticular bundles and their distribution area in medullary reticular formation. In the rats with caudate-putamen injections, which interrupted fiber bundles of the internal capsule, fluorescent glial nuclei were also present in cerebral peduncle and the corresponding bundles in pons. Therefore NY, as Bb [4], when injected into cell groups or fiber bundles, also proceeds anterogradely through the axons and from there may migrate into adjoining glial cells. Injections o f NY as well as of Bb may produce fluorescence o f pial and ependymal nuclei, probably due to effusion of the tracers into the CSF. When this fluorescence is very pronounced, some of the underlying glial and neuronal nuclei may show a dull fluorescence. In order to avoid confusion these cases were discarded. Results with Fast Blue (FB). The retrograde transport of Fast Blue (diamidino compound 253/50) was tested in the following experiments. In 6 rats 0.3 #l 5°7o FB was injected in caudate-putamen on one side, followed by 12, 18 or 24 h and 5, 10 or 20 days survival. In the first two animals 0.3 #l 5o7o Granular Blue (GB) was injected in caudate-putamen on the other side. The FB fluorescent injection area closely resembled the sharply delineated injection areas of TB and GB [1] and did not display any overt changes in size following different survival times. In these cases, many neurons in C M - P f , SN and DR were retrogradely labeled and labeling already occurred after 12 h survival. When viewed with filter-mirror system A they displayed a blue fluorescent cell body and dendrites with fine silver fluorescent granules, while the nucleus showed little fluorescence (Fig 1C, D). Lengthening the survival time increased the fluorescence and, in addition, produced large orange fluorescent granules in the cytoplasm. After survival for 5 days or more, the tissue around the FB fluorescent neurons gradually became fluorescent with some fluorescent glial nuclei. The axons through which FB was transported also showed a blue fluorescence, but were not surrounded by fluorescent glial nuclei. Thus, the FB
28
Fig. 1. Photomicrographs of NY- and FB-labeled neurons viewed with filter-mirror system A providing excitation light of 360 nm wavelength and with oil immersion optics. A and B: NY-labeled neurons in red nucleus of rat following NY injections into the spinal cord. Note brilliant fluorescence of nucleus including brilliant fluorescent ring around nucleolus (arrows) and duller fluorescence of cytoplasm. Note also fluorescent glial nuclei around retrogradely labeled neurons. C and D: FB-labeled neurons in rat medullary reticular formation after FB injection into spinal cord. Note labeling of cell body and proximal dendrites, containing also fine fluorescent granules. Note also that nucleus (arrow) shows little fluorescence.
29 fluorescence resembles the GB fluorescence [1]. However, in the animals with FB and GB injections in caudate-putamen on either side the FB fluorescence was much more pronounced than the GB fluorescence at the same survival time. Findings in rats and cats showed, further, that FB is transported effectively over long distances. Thus, in two rats in which 2 ~1 5% FB was injected in lumbo-sacral cord, followed by 48 h survival time, neurons of the descending brain stem pathway and neurons in sensorimotor cortex were retrogradely labeled (Fig. 1C, D). Moreover, in 2 cats in which 11 ~1 3% FB was injected at T5 and T7 respectively, followed by 6 or 7 days survival, and in 2 other cats in which 15 ~1 3% FB was injected at T1, followed by 9 or 22 days survival, neurons of the descending brain stem pathways as well as neurons in sensorimotor cortex were labeled. However, after 22 days the neuropil around retrogradely labeled neurons in brain stem showed a pronounced fluorescence. Further, in other experiments the impression was gained that FB gave better retrograde labeling when injected into the termination area of axons than in the axon bundles themselves. For long distance transport studies in adult cats FB is now being used instead of TB in our laboratory, since long distance transport of TB, which is quite effective in rats [1] was found to be much less effective in adult cats and required rather long survival times. Thus, in 4 cats with 15 ~1 5% TB injections at C 2 - C 3 , followed by 5, 9, 21 or 35 days survival, relatively few neurons of the descending brain stem pathways were labeled and only very few faintly labeled neurons were found in cortex. However, in kittens TB is more effectively transported over long distances than in adult cats, since in three 30-day-old kittens with 7 ~1 TB injections at C 2 - C 3 followed by 8, 24 or 28 days survival, good fluorescent labeling of neurons in brain stem and sensorimotor cortex occurred, even after 8 days survival. Moreover, in adult cats TB gives good retrograde labeling when transport over relatively short distances is combined with relatively long survival times, e.g. from sensorimotor cortex to thalamus and basal forebrain area (4 cats) with 14-40 days survival and from ventral thalamus and mesencephalic tegmentum to deep cerebellar nuclei (3 cats) with 20 or 40 days survival, respectively. The present findings show that Nuclear Yellow and Fast Blue are both transported over long distances even in cats. NY preferentially labels the nucleus while FB preferentially labels the cytoplasm o f cell body and dendrites. Because of these characteristics the two tracers can be successfully combined in double labeling experiments to be reported separately [5]. NY, as Bb [4], also labels glial nuclei around retrogradely labeled neurons and along axons through which it is transported, even after relatively short survival times. FB, as TB [1], also gives fluorescence of glial nuclei and neuropil around retrogradely labeled neurons, but only after relatively long survival times.
30
ACKNOWLEDGEMENTS The authors Oudenalder
thank
Mr.
E. D a l m ,
Miss P. van Alphen
and Mr. W. van den
f o r t h e i r t e c h n i c a l a s s i s t a n c e , D r . R. L e m o n f o r r e a d i n g t h e m a n u s c r i p t
a n d M i s s G . v a n G e l d e r f o r t y p i n g it. T h i s s t u d y w a s in p a r t s u p p o r t e d b y G r a n t 1346-15 o f t h e F U N G O / Z W O
(Dutch
Organization
for Fundamental
R e s e a r c h in
Medicine). REFERENCES 1
2
3 4
5
6
Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thyminerich DNA, Neurosci. Lett., 12 (1979) 235-240. Bentivoglio, M., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E., Retrograde neuronal labeling by means of bisbenzimide and Nuclear Yellow (Hoechst S 769121). Measures to control diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18 (1980) 19 24. Hilwig, J. and Gropp, A., pH-Dependent fluorescence of DNA and RNA in cytologic staining with '33258 Hoechst', Exp. Cell Res., 91 (1979) 457-460. Kuypers, H.G.J.M., Bentivoglio, M., Van der Kooy, D. and Catsman-Berrevoets, C.E., Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies, Neurosci. Lett., 12 (1979) 1 7. Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, T.B., Double retrograde neuronal labeling through divergent axon collaterals using two fluorescent tracers with same excitation wavelength which label different features of the cell, Exp. Brain Res., in preparation. Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padt, R.E., Retrograde axonal transport of fluorescent substances in rat's forebrain, Neurosci. Lett., 6 (1977) 127-135.
25
© Elsevier/North-Holland Scientific Publishers Ltd.
TWO NEW F L U O R E S C E N T R E T R O G R A D E N E U R O N A L T R A C E R S W H I C H ARE T R A N S P O R T E D OVER LONG D I S T A N C E S
M. BENTIVOGLIO, H.G.J.M. KUYPERS, C.E. CATSMAN-BERREVOETS, H. LOEWE and O. DANN
Department of Anatomy, Erasmus University Medical School, Rotterdam (The Netherlands); (H.L.) Hoechst Company, Frankfurt am Main (F.R.G.) and
(0. D.) Institute for Pharmacy and Foodchemistry, Friedrich-A leyander University, Erlangen (F.R.G.) (Received January 7th, 1980) (Revised version received February 18th, 1980) (Accepted February 18th, 1980)
SUMMARY
Two new fluorescent retrograde neuronal tracers are reported: Nuclear Yellow (Hoechst S 769121), which mainly labels the neuronal nucleus; and Fast Blue (diamidino compound 253/50), which mainly labels the neuronal cytoplasm. Both tracers appear to be transported effectively over long distances in rat and cat.
Several fluorescent substances, i.e. Evans Blue, DAPI-Primuline, propidium iodide, bisbenzimide (Bb), Granular Blue (GB) and True Blue (TB) are transported retrogradely through axons to their parent cell bodies and produce fluorescent labeling of different features of the cell [1, 4, 6]. Two other fluorescent retrograde tracers have been found: (1) Hoechst S 769121, which is related to Bb. It mainly labels the neuronal nucleus and therefore will be referred to as Nuclear Yellow (NY)*; (2) the diamidino compound 253/50, synthesized by Dann and his collaborators. This substance is related to TB, but is transported more rapidly and effectively over long distances in cat and therefore will be referred to as Fast Blue (FB)*. These two tracers have been compared with Bb, TB and GB [1, 4]. Hoechst S 769121: 2-(4-sulfamylphenyl)-6-(6-(4-methylpiperazino)-2-benzimidazolyl)benzimidazole trishydrochloride, i.e. Nuclear Yellow (NY), is difficult to dissolve in water and has been used in 10% suspension; however, in later * See footnote p. 26
26 experiments 107owas preferred [2]. Fast Blue (FB) (diamidino compound 253/50)* is soluble in water and has been used in concentrations of 3% and 5°7o. True Blue (TB) is almost water-insoluble [1] and has been used in suspensions of 2°7o and 5°70; Granular Blue (GB) is slightly water-soluble [1] and has been used in a suspension of 5%. The tracers were injected in brain and spinal cord of rats and cats by means of either a Hamilton syringe with a 22-gauge needle or a micropipette. Each needle and micropipette was only used for one tracer in order to avoid contamination. The animals were perfused as described earlier [1, 4, 6]. Brain and spinal cord were kept in cacodylate buffered (pH 7.2) 30O7o sucrose for up to 24 h. The injected spinal segments were kept separate from the other tissue slices since the tracer remaining at the injection site might dissolve in the sucrose solution and from there penetrate cut surfaces of the other slices. The material was cut transversely in frozen sections 30 ~m thick. Initially they were collected in distilled water and then were either first stored at 4°C for up to 12 h or were immediately mounted and air dried at room temperature, but were not coverslipped. However, later in cat a different procedure was adopted in order to prevent migration of NY out of retrogradely labeled neurons to surrounding cells [2]. The material was studied with a Leitz Ploemopack fluorescence microscope, equipped with filter-mirror systems A, D and N2, which provide excitation light at 360 nm, 390 nm and 550 nm wavelengths.
Results with Nuclear Yellow (Hoechst S 769121). The retrograde transport of many compounds related to Bb has been tested earlier (D. van der Kooy and M. Bentivoglio, unpublished observations). Hoechst S 769121 (NY) will be described, since it is very suitable for double-labeling experiments to be reported separately [5]. In 6 rats 0.2/~1 10% NY was injected in caudate-putamen followed by survival times ranging from 1 to 26 days. The NY injection produced a fluorescent injection area similar to that of Bb [4], and resulted in NY retrograde labeling of neurons in substantia nigra (SN), center median-parafascicular complex ( C M - P f ) , and dorsal raphe (DR). When viewed with filter-mirror system A, the neurons showed a bright golden-yellow fluorescent nuclear labeling which closely resembled the labeling obtained with Bb [4]. However, especially when buffered formaline was used [3], they showed little cytoplasmic fluorescence, except for fine golden-yellow dust. Long survival times did not produce an increase in fluorescence of the retrogradely labeled neurons. In 3 rats 1 ~tl 10% NY was injected at C 2 - C 3 , followed by 5 days survival. In these rats neurons of sensorimotor cortex and descending brain stem pathways
* For research purposes small samples of Nuclear Yellow can be obtained from Dr. H. Loewe, Hoechst Aktiengesellschaft, Post Box 800320, 6230 Frankfurt am Main 80, F.R.G. and small samples of Fast Blue can be obtained from Prof. Dr. O. Dann, lnstitut f~3rPharmazie und Lebensmittelchemie der FriedrichAlexander Universit~t, Post Box 2044, D 8520 Erlangen, F.R.G.
27 showed the same labeling (Fig. 1A, B) as in SN, C M - C f and DR after caudateputamen injections. This same retrograde neuronal labeling was also obtained in cortex and brain stem o f a cat in which _ 30 #l 10% NY was injected at L 1 - L 2 . This demonstrated that NY, as Bb [4], is effectively transported over long distances both in rat and cat. In all cases NY-labeled neurons, as Bb-labeled ones [4], were surrounded by golden-yellow fluorescent glial nuclei. After spinal injections fluorescent glial nuclei were also present in pyramidal tract and cerebral peduncle. This indicates that NY, as Bb [4], when transported retrogradely through axons, may migrate to adjoining glial cells. In the rats with NY injections in caudate-putamen, brilliantly fluorescent glial nuclei were present throughout the substantia nigra, pars reticulata (SNR) where many striatonigral fibers terminate. After spinal injections fluorescent glial nuclei were present in the inferior cerebellar peduncle as well as in the spinoreticular bundles and their distribution area in medullary reticular formation. In the rats with caudate-putamen injections, which interrupted fiber bundles of the internal capsule, fluorescent glial nuclei were also present in cerebral peduncle and the corresponding bundles in pons. Therefore NY, as Bb [4], when injected into cell groups or fiber bundles, also proceeds anterogradely through the axons and from there may migrate into adjoining glial cells. Injections o f NY as well as of Bb may produce fluorescence o f pial and ependymal nuclei, probably due to effusion of the tracers into the CSF. When this fluorescence is very pronounced, some of the underlying glial and neuronal nuclei may show a dull fluorescence. In order to avoid confusion these cases were discarded. Results with Fast Blue (FB). The retrograde transport of Fast Blue (diamidino compound 253/50) was tested in the following experiments. In 6 rats 0.3 #l 5°7o FB was injected in caudate-putamen on one side, followed by 12, 18 or 24 h and 5, 10 or 20 days survival. In the first two animals 0.3 #l 5o7o Granular Blue (GB) was injected in caudate-putamen on the other side. The FB fluorescent injection area closely resembled the sharply delineated injection areas of TB and GB [1] and did not display any overt changes in size following different survival times. In these cases, many neurons in C M - P f , SN and DR were retrogradely labeled and labeling already occurred after 12 h survival. When viewed with filter-mirror system A they displayed a blue fluorescent cell body and dendrites with fine silver fluorescent granules, while the nucleus showed little fluorescence (Fig 1C, D). Lengthening the survival time increased the fluorescence and, in addition, produced large orange fluorescent granules in the cytoplasm. After survival for 5 days or more, the tissue around the FB fluorescent neurons gradually became fluorescent with some fluorescent glial nuclei. The axons through which FB was transported also showed a blue fluorescence, but were not surrounded by fluorescent glial nuclei. Thus, the FB
28
Fig. 1. Photomicrographs of NY- and FB-labeled neurons viewed with filter-mirror system A providing excitation light of 360 nm wavelength and with oil immersion optics. A and B: NY-labeled neurons in red nucleus of rat following NY injections into the spinal cord. Note brilliant fluorescence of nucleus including brilliant fluorescent ring around nucleolus (arrows) and duller fluorescence of cytoplasm. Note also fluorescent glial nuclei around retrogradely labeled neurons. C and D: FB-labeled neurons in rat medullary reticular formation after FB injection into spinal cord. Note labeling of cell body and proximal dendrites, containing also fine fluorescent granules. Note also that nucleus (arrow) shows little fluorescence.
29 fluorescence resembles the GB fluorescence [1]. However, in the animals with FB and GB injections in caudate-putamen on either side the FB fluorescence was much more pronounced than the GB fluorescence at the same survival time. Findings in rats and cats showed, further, that FB is transported effectively over long distances. Thus, in two rats in which 2 ~1 5% FB was injected in lumbo-sacral cord, followed by 48 h survival time, neurons of the descending brain stem pathway and neurons in sensorimotor cortex were retrogradely labeled (Fig. 1C, D). Moreover, in 2 cats in which 11 ~1 3% FB was injected at T5 and T7 respectively, followed by 6 or 7 days survival, and in 2 other cats in which 15 ~1 3% FB was injected at T1, followed by 9 or 22 days survival, neurons of the descending brain stem pathways as well as neurons in sensorimotor cortex were labeled. However, after 22 days the neuropil around retrogradely labeled neurons in brain stem showed a pronounced fluorescence. Further, in other experiments the impression was gained that FB gave better retrograde labeling when injected into the termination area of axons than in the axon bundles themselves. For long distance transport studies in adult cats FB is now being used instead of TB in our laboratory, since long distance transport of TB, which is quite effective in rats [1] was found to be much less effective in adult cats and required rather long survival times. Thus, in 4 cats with 15 ~1 5% TB injections at C 2 - C 3 , followed by 5, 9, 21 or 35 days survival, relatively few neurons of the descending brain stem pathways were labeled and only very few faintly labeled neurons were found in cortex. However, in kittens TB is more effectively transported over long distances than in adult cats, since in three 30-day-old kittens with 7 ~1 TB injections at C 2 - C 3 followed by 8, 24 or 28 days survival, good fluorescent labeling of neurons in brain stem and sensorimotor cortex occurred, even after 8 days survival. Moreover, in adult cats TB gives good retrograde labeling when transport over relatively short distances is combined with relatively long survival times, e.g. from sensorimotor cortex to thalamus and basal forebrain area (4 cats) with 14-40 days survival and from ventral thalamus and mesencephalic tegmentum to deep cerebellar nuclei (3 cats) with 20 or 40 days survival, respectively. The present findings show that Nuclear Yellow and Fast Blue are both transported over long distances even in cats. NY preferentially labels the nucleus while FB preferentially labels the cytoplasm o f cell body and dendrites. Because of these characteristics the two tracers can be successfully combined in double labeling experiments to be reported separately [5]. NY, as Bb [4], also labels glial nuclei around retrogradely labeled neurons and along axons through which it is transported, even after relatively short survival times. FB, as TB [1], also gives fluorescence of glial nuclei and neuropil around retrogradely labeled neurons, but only after relatively long survival times.
30
ACKNOWLEDGEMENTS The authors Oudenalder
thank
Mr.
E. D a l m ,
Miss P. van Alphen
and Mr. W. van den
f o r t h e i r t e c h n i c a l a s s i s t a n c e , D r . R. L e m o n f o r r e a d i n g t h e m a n u s c r i p t
a n d M i s s G . v a n G e l d e r f o r t y p i n g it. T h i s s t u d y w a s in p a r t s u p p o r t e d b y G r a n t 1346-15 o f t h e F U N G O / Z W O
(Dutch
Organization
for Fundamental
R e s e a r c h in
Medicine). REFERENCES 1
2
3 4
5
6
Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thyminerich DNA, Neurosci. Lett., 12 (1979) 235-240. Bentivoglio, M., Kuypers, H.G.J.M. and Catsman-Berrevoets, C.E., Retrograde neuronal labeling by means of bisbenzimide and Nuclear Yellow (Hoechst S 769121). Measures to control diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18 (1980) 19 24. Hilwig, J. and Gropp, A., pH-Dependent fluorescence of DNA and RNA in cytologic staining with '33258 Hoechst', Exp. Cell Res., 91 (1979) 457-460. Kuypers, H.G.J.M., Bentivoglio, M., Van der Kooy, D. and Catsman-Berrevoets, C.E., Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies, Neurosci. Lett., 12 (1979) 1 7. Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, T.B., Double retrograde neuronal labeling through divergent axon collaterals using two fluorescent tracers with same excitation wavelength which label different features of the cell, Exp. Brain Res., in preparation. Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padt, R.E., Retrograde axonal transport of fluorescent substances in rat's forebrain, Neurosci. Lett., 6 (1977) 127-135.