Fluoro-green and fluoro-red: two new fluorescent retrograde tracers with a number of unique properties

BRAIN RESEARCH ELSEVIER

Brain Research 736 (1996) 61-67

Research report

Fluoro-Green and Fluoro-Red: two new fluorescent retrograde tracers with a number of unique properties Kai D o n g "' *, T i n g y u Q u a Farid A . K . M . A h m e d a Lixin Z h a n g a Koshi Y a m a d a a Noel G. Guison a Michael Miller a,b Takashi Yamadori a a First Department of Anatomy, Kobe University School of Medicine, 5-1, Kusunoki-cho, 7 Chome, Chuo-ku, Kobe 650, Japan b Unicersity Laboratory of Physiology, Oxford, UK

Accepted 28 May 1996

Abstract As a means of improving nerve tract-tracing in the peripheral and central nervous systems we experimented with two (retrograde) fluorescent emulsions, which we have tentatively named Fluoro-Green (FGr) and Fluoro-Red (FRe), and which we believe possess the following seven advantages: (1) they show little diffusion beyond the injection site; (2) their excitation/emission characteristics allow their use in double-tracing experiments; (3) they do not 'leak' from labeled cells; (4) their fluorescence is presented as large granules in the cytoplasm and its processes; (5) the fluorescence lasts for a sufficiently long time to permit repeated observation; (6) they may be used in combination with a wide variety of other neuroanatomical tracing methods; (7) they are economical, non-toxic and easy to utilize. Keywords: Fluoro-Green; Fluoro-Red; Retrograde fluorescent tracer; Double-labeling; Neuroanatomical study

1. Introduction Since the introduction of horseradish peroxidase (HRP), retrograde, axonal-tract, tracing technique ([2,3,11,16,17,19]; for review see [8]), a number of alternative axonal tracers have been developed to aid the neuroscientist in the study of neuronal connections in the central and peripheral nervous systems. Many of these new tracers are fluorescent [7,13-15] and their basic advantages are sensitivity and simplicity of use [12,13,15]. Furthermore, two or more fluorescent tracers of different emission characteristics (excitation and emission wavelengths) can be used to determine whether an individual, retrogradelabeled, neuron sends its axons and collaterals to different targets [4,6,12,13,15,22]. However, we believe there are four broad disadvantages at present to using such tracers: (1) a diffusion of tracer may take place at the injection site, particularly with a long survival period [1,6,12,13,21]; (2) with longer than optimal survival times, these tracers tend to diffuse out of the labeled neurons or axons. Adjacent neurons, glial cells or axons, may then take up the tracer and be labeled [1,6,12,13,21]; (3) it is sometimes difficult to distinguish labeled cells from those that are autofluores-

cent [20]; (4) the fluorescence of some tracers fades very rapidly. Such disadvantages as these have spurred efforts to develop new generations of fluorescent tracers, such as 4 - a c e t a m i d o , 4 ' - i s o t h i o c y a n o s t i l b e n - 2 , 2 ' - d i s u l f o n i c acid (SITS), Fluoro-Gold (FG) and Fluoro-Ruby (FR), and the usefulness of these new fluorescent tracers in retrograde axonal transport is now well established, as they do not diffuse from labeled cells despite wide variations in survival time [22,24,25]. However, they also have the dual disadvantage of fading rapidly plus a tendency to diffuse around the injection site. In the present experiment, we searched for improved tracers and found two emulsions of strong fluorescence that do not decay easily. Furthermore, in using these emulsions we obtained excellent results that are not available, we believe, with other markers of this type. The emulsions in question we have tentatively named: FluoroGreen (FGr) and Fluoro-Red (FRe).

2. Materials and methods 2.1. A n i m a l a n d t r a c e r i n j e c t i o n

* Corresponding author. Fax: [email protected]

+ 81 (78) 360-1968; e-maih

F o r t y - t w o male albino rats were used (Wistar s t r a i n / J a p a n C l e a / b o d y weight approx. 300 g). As shown

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K. Dong et al./Brain Research 736 (1996) 61 67

in Table 1, they were divided into nine groups. After anesthesia with an intraperitoneal injection of 10% chloral hydrate (300 m g / k g ) , each animal was placed in a stereotaxic headholder. An incision was made in the scalp, and two holes were drilled in the skull to approach the superior colliculus (SC) a n d / o r dorsal part of the lateral geniculate nucleus (dLGN) on the left side, using the atlas of Paxinos and Watson [18] for reference. Glass micropipettes were made with internal diameters of about 40 ~m, and then loaded via suction with an emulsion of 0.02 to 0.05 txl FGr, or the same amount of FRe. The FGr- or FRe- filled micropipette was stereotaxically lowered into the SC or dLGN on the same side. About 0.02 ixl of FGr or FRe was pressure-injected through a thin plastic tube connected to a 1 g l Hamilton syringe for more than 5 min. Then, the incision was sutured and the rat given penicillin. Similarly, rats in the control group were injected with the same volume of fluorescent tracers, Fluoro-Gold and Fluoro-Ruby, into the dLGN a n d / o r SC (Table 1). 2.2. Tracers

Fluoro-Green (excitation peak: 450 nm, emission peak: 505 nm, pH 4.5) and Ftuoro-Red (excitation peak: 540 nm, emission peak: 585 nm, pH 4.0) which are normally used in fluorescent pencils, are emulsions of fluorescent dyes prepared by the Tombow Pencil Co., Tokyo, Japan. 2.2.1. Fluoro-Green (FGr)

FGr is a mixed green-yellow emulsion of fluorescent dyes in fluid form including: (A) Solution of 1% basic yellow 40 {1H-Benzimidazolium, 2-[7-(diethylamino)-2-oxo-2H- 1-benzopyran-3yl]-l,3,dimethyl-, methyl sulfate} prepared by Nippon Keiko Chemical Co., Japan. (B) dilution solution: (1) distilled water (70%); (2) ethylene glycol (8%); (3) glycerin (8%); (4) D-sorbital (12%); (5) thiabendazade (2%). The proportion of fluid to diluent is 5 0 / 5 0 % .

Table 1 Grouping of paired or unpaired injection sites of dLGN and SC with different fluorescent tracers Groups n Injectionsites FluoroF l u o r o - F l u o r o - FluoroGreen Red Gold Ruby l

5

2 3 4 5 6 7 8 9

6 5 5 6 6 3 3 3

SC dLGN dLGN SC

SC dLGN SC dLGN dLGN dLGN

FRe is also a mixed pink emulsion of fluorescent dyes in fluid from including: (A) The solution of 0.5% basic-red 1 {3,6-bis(ethyl amino)-9-[2-(ethoxycarbonyl)phenyl]-2,7-dimethyl, chloride (9CI)} and 0 . 5 % basic-violet 11 {3,6bis(diethylamino)-9-[2-(methoxycarbomyl)phenyl]-(T- 1) tetrachlore} prepared by Nippon Keiko Chemical Co. (B) dilution solution: (1) distilled water (74%); (2) ethylene glycol (8%); (3) glycerin (8%); (4) D-sorbital (8%); (5) thiabendazade (2%). The proportion of fluid to diluent is 3 9 / 6 1 % . Both dyes are usually stored at room temperature under normal lighting conditions. Both emulsions are easy to use to suction and inject and both are and available from the original user. 2.3. Further procedures

Following a survival period of 2 - 1 4 days, the rats were anesthetized as before, and perfused. Perfusates included saline, followed by 10% formalin in 0.2 M phosphate buffer (pH 6.4-7.2). The eyes were surgically isolated following a brief saline rinse during perfusion and the retina separated from the pigmented epithelium before being mounted on a non-fluorescent glass slide. Each retina was then dried in an incubator (37°C) and coverslipped with HSR solution (International Geagents Co., Kobe, Japan). The retinas were observed under a fluorescent microscope (Olympus AX80, Japan) equipped with B(blue, excitation wavelength: 4 5 0 - 4 8 0 nm, emission wavelength: 515 nm) and G-systems (green, excitation wavelength: 5 1 0 - 5 5 0 nm, emission wavelength: 580 nm). The distribution of the ganglion cells retrogradely labeled with FGr a n d / o r FRe were then confirmed. The brain was removed following perfusion and placed in air at room temperature for 24 h. Frozen sections were cut at 40 Ixm in the frontal plane, and then mounted in serial order into gelatinized glass slides. The injection site as well as the extent of tracer diffusion within the SC and dLGN was confirmed, and the distributions of the cells retrogradely labeled with FGr a n d / o r FRe were also observed by fluorescent microscope. Finally, the sections were lightly counterstained with 1% neutral red for reconfirmation of injection site. In addition, two sets of randomly selected sections were exposed to ultraviolet irradiation for 5 h and stored at room temperature in daylight for 6 months, respectively. The random-sections procedure was designed to investigate the longevity of the fluorescence and its storage properties.

3. R e s u l t s

SC dLGN

2.2.2. Fluoro-Red (FRe)

SC SC

As shown in Fig. 1 (A, by FGr; B, by FRe), the injected dyes yielded very small areas in the SC (diameter 0.2 mm)

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K. Dong et al. / Brain Research 736 (1996) 6 1 - 6 7

1

dLGN L

M

sc A

B

Fig. 1. Photographsof typical injection sites of FGr (volume: 0.05 pA; survival time: 4 days) in the SC (A) and FRe (volume: 0.05 ~1; survival time: 4 days) in the dLGN (B). dLGN, dorsal lateral geniculate nucleus; L, lateral; M, medial; SC, superior colliculus. Scale bar = 0.1 mm.

and dLGN (diameter 0.17 mm) without noticeable diffusion into the surrounding structures. In contrast, the average diameters of injection sites with the same volume of Fluoro-Gold into the SC or dLGN were about 1.4 mm. Fig. 2 demonstrates the appearance of labeled retinal ganglion cells with FGr (Fig. 2A, C, E) a n d / o r FRe (Fig. 2B, D, F). The FGr-labeled cells had intense, white/green-colored granules fluorescing within the cytoplasm and processes under filter system B (blue), while the FRe-labeled cells had bright, orange-colored granules in the cytoplasm as observed under filter system G (green). Since the fluorescence of FGr and FRe had different excitation wavelengths, the labeled cells could be visualized by changing filters (B- and G-systems) alternately. Double-labeled cells can be clearly shown using this technique. We did not find any labeling of glial cells, suggesting that both dyes did not leak from the labeled ganglion cells in spite of the prolonged survival periods (up to 14 days). Nor did we find any labeling of fibers in the sections of brain tissue, indicating that both dyes were transported as retrograde markers. Our purpose was to show double-labeled ganglion cells projecting bilaterally to two different structures via axonal bifurcation. Therefore, in group 5 (Table 1), we injected FGr and FRe into the left SC and dLGN, respectively. In group 6, the tracers injected into the same areas were reversed. We also injected FG a n d / o r FR into the same portions of the SC and dLGN as controls (groups 7, 8, 9). As shown in Fig. 2G, H and I, the double labeled cells exhibited granules of FGr and FRe with two different colors in the cytoplasm and processes of the single ganglion cell. Microscopic examination of the injection sites with the FGr or FRe revealed small spherical areas of bright fluorescence. When injected with these tracers, no tissue necrosis in the central region of the injection was detectable, suggesting that both tracers are non-toxic. Since the dyes were originally intended for use in coloring pencils their non-toxicity might be expected. The observation of the specimens exposed to ultraviolet irradiation for 5 h demonstrated that the fluorescence of the labeling did

not decay basically (Fig. 3). Furthermore, the fluorescence remained in the specimens for 6 months while they were stored under normal conditions of lighting of lighting and temperature.

4. Discussion As neuroanatomists continue to refine studies on the topography and connections of the brain, the need for small injections of tracers becomes increasingly important. In previous studies, an iontophoretic technique has been developed to yield a small injection site [23]. But in our experience, increasing the current and time produces a larger injection site than expected, while reducing the current and time results in a failure of injection. Furthermore, iontophoretic injection of both FGr and FRe into brain tissue failed in our experience to produce satisfactory results although we tried a number of times under varying conditions. In contrast with the injection sites of dyes normally introduced under pressure or by iontophoresis, the size of injection sites with FGr or FRe is limited and shows little diffusion (less than 1 / 7 of injection sites with the same volume of Fluoro-Gold). This we believe is due to the FGr and FRe dyes themselves (Fig. 1). Fluorescent tracers have been extensively used in neuroanatomical studies because of their sensitivity and relative ease of use, plus their potential for carrying out multiple-labeling studies [4-6,9,10,12,13,15,22]. However, previous studies have noted that some fluorescent dyes may leak from labeled cells or axons to surrounding tissue and may then be taken up by other neurons, glial cells, or axons, perhaps leading to unreliable findings [ 1,6,7,9,10,12-14,21 ]. In the present experiment using FGr and FRe as fluorescent tracers, no evidence of dye leakage from labeled cells or axons was found, and we believe that this may be ascribed to distinct mechanisms of tracer uptake by axonal terminals. That is, the tracer is actively taken up in an endocytotic vesicular pattern and then

K. Dong et al./Brain Research 736 (1996) 6 1 - 6 7

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1

l

K. Dong et al. / Brain Research 736 (1996) 6 1 - 6 7

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Fig. 3. Photomicrographs(black and white) of FGr-labeledcells taken at the same field and sensitivitysetting, begining from 0 to 5 h (with intervalsof 1 h), following the persistent irradiation with B-system. The shutter speed is automaticallyadjusted by computer (12 s for 0 h, 76 s for after l h, 96 s for after 2 h, 114 s for after 3 h, 131 s for after 4 h, 143 s for after 5 h). h, hour{s).

retrogradely transported to the cell body where it appears to undergo little degradation. Furthermore, following the injections of FGr or FRe into the dLGN, well accepted afferent structures, including layer 6 of ipsilateral visual cortex ( O c l : a small number of labeled cells; Oc2: a large number of labeled cells, see Fig. 5A), ipsilateral parabigeminal nucleus (PBg), olivary pretectal nucleus (OPT), pretectal nucleus (PPTg), parvocellular division of ventral lateral geniculate nucleus (vLGPC, see Fig. 5B) contralat-

eral intergeniculate leaflet (IGL) and bilateral optic tract nucleus (OT) were confirmed. Following injections of FGr or FRe into the SC (see Fig. 4), labeled cells in the layer 5 of ipsilateral Oc2 (Fig. 4A, B), ipsilateral zona incerta (ZI), principal sensory trigeminal nucleus (Pr5), anterior pretectal area (APT); in the contralateral SC (Fig. 4D), prepositus hypoglossal nucleus (PrH), and bilateral IGL, PBg, PPT, OT (Fig. 4C), lateral posterior thalamic nucleus (LP), inferior colliculus (IC) were also observed, suggest-

Fig. 2. Photomicrographs of retrogradely labeled ganglion cells in the retina following injections of FGr and/or FRe into the dLGN and/or SC, respectively. A, C, E: FGr-labeledcells (× 325, x 325, X 650, respectively)demonstrated by the B-system. B, D, F: FRe-labeledcells (× 325, × 325, × 150, respectively) demonstrated by the G-system. G, H, I: double-labeled cells (arrows, X325). G: FGr-labeled cells; H: FRe-labeled cells; I: double-labeledcells with FGr and FRe demonstrated by double exposure, burn-timefor C and D: 2 h.

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K. Dong et al. / Brain Research 736 (1996) 61 67

&

• ,F

D

Fig. 4. Photographs of labeled cells in various regions of the brain following injection of FGr or FRe into the SC. A: cells labeled with FGr in the ipsilateral occipital cortex (area 2) (X 150). B: enlarged photograph of cells labeled with FGr in the ipsilateral occipital cortex (area 2) ( × 400), C: cells labeled with FGr in the ipsilateral optic tract nucleus ( × 150). D: cells labeled with FRe in the controlateral intermediate gray layer (X 400).

ing that the retrograde labeling of both tracers works equally well in regions other than the retina. However, the specific substance taken up by axonal terminals is unknown. Although we tried to purify these emulsions and used single constituent components of them for injection, the results were not satisfying. Perhaps the mixture of constituents in the emulsion produces the unique characteristics of the dye. The basic disadvantage of fluorescent dyes as labeling material is that they tend to fade or degrade very rapidly with time or exposure to light. Therefore, it is impossible to preserve specimens for later confirmatory examination

[22]. For example, the intense fluorescence of FG labeled cells can be kept for only about 10 s to 1 min under ultraviolet irradiation, and for 2 - 5 days only at a temperature of -20°C. That of Evans Blue (EB)-labeling can be preserved for 1 week. As for FR, its labeling is not sensitive and not clear in brain sections. Furthermore, the existence of this dye in brain tissue, but not in neurons, which may reflect its diffusion via blood vessels, has been observed in our laboratory (unpublished observation). The disadvantage of SITS is the instability of its fluorescence, which cannot be maintained despite storage in a deep freezer at temperature of - 2 0 ° C . However, when the

B

Fig. 5. Photographs of labeled cells in two regions of the brain following injection of FRe into the dLGN. A: a number of labeled cells in layer 6 of ipsilateral visual cortex (Oc2) ( × 75). B: some cells in the contralateral parvocellular division of ventral lateral geniculate nucleus (vLGPC) ( x 100).

K. Dong et a l . / B r a i n Research 736 (1996) 6 1 - 6 7

specimens labeled with FGr and FRe were coverslipped w i t h H S R s o l u t i o n a n d left u n d e r the i r r a d i a t i o n o f B - a n d G - s y s t e m s , r e s p e c t i v e l y , f o r 5 h, o r p r e s e r v e d for 6 m o n t h s at r o o m t e m p e r a t u r e a n d in d a y l i g h t , the f l u o r e s c e n c e o f t h e t w o d y e s w i t h i n the l a b e l e d cells d e c r e a s e d v e r y little, s u g g e s t i n g t h a t t h e s e s p e c i m e n s m i g h t b e p r e s e r v e d for m u c h l o n g e r p e r i o d s t h a n t h o s e o f o t h e r tracers in c u r r e n t use. T h e p h o t o g r a p h s o f l a b e l e d cells f o l l o w i n g 2 h o f i r r a d i a t i o n w i t h B - a n d G - s y s t e m s are s h o w n in Fig. 2C, D a n d t h o s e o f l a b e l e d cells f o l l o w i n g 0 - 5 h o f i r r a d i a t i o n w i t h B - s y s t e m in Fig. 3. T o c o n c l u d e , the a d v a n t a g e s o f t h e s e t w o tracers, w e b e l i e v e , are: 1. T h e size o f i n j e c t i o n site c a n b e c o n t r o l l e d easily b y volume of infused tracer without any diffusion during o p t i m a l s u r v i v a l t i m e s ( 2 - 1 4 days); 2. n e i t h e r t r a c e r l e a k s o u t o f t h e l a b e l e d cells a n d a x o n s ; 3. cells l a b e l e d w i t h e i t h e r t r a c e r c a n b e e a s i l y v i s u a l i z e d via i n t e n s e f l u o r e s c e n c e o f the large, b r i g h t , d y e g r a n ules w i t h i n the cell b o d y (see Fig. 2E); 4. b o t h t r a c e r s c a n b e c o m b i n e d w i t h e a c h o t h e r for d o u b l e - l a b e l i n g a n d m a y b e u s e d in c o m b i n a t i o n w i t h o t h e r t r a c i n g m e t h o d s for m u l t i p l e - l a b e l i n g studies; 5. the i n t e n s e f l u o r e s c e n c e o f l a b e l i n g w i t h t h e s e tracers f a d e s v e r y little w i t h time, or w i t h u l t r a v i o l e t irradiation, a n d the s p e c i m e n s c a n b e s t o r e d in a n o r d i n a r y r o o m e n v i r o n m e n t for l o n g p e r i o d s , p e r m i t t i n g r e p e a t e d observation; 6. b o t h d y e s are e c o n o m i c a l , n o n - t o x i c a n d e a s y to utilize. Therefore, we believe that both FGr and FRe may have w i d e a p p l i c a t i o n s in n e u r o a n a t o m i c a l t r a c t - t r a c i n g studies.

Acknowledgements T h e a u t h o r s e x p r e s s t h e i r t h a n k s to Mr. T o m i y o s h i S e t s u for his e x p e r t t e c n i c a l a s s i s t a n c e . T h e first t w o a u t h o r s c o n t r i b u t e d to this w o r k e q u a l l y .

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