Brain Research, 553 (1991) 135-148 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116749P

135

BRES 16749

Fluoro-Gold: composition, and mechanism of uptake Martin W. Wessendorf Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455 (U.S.A.)

(Accepted 29 January 1991) Key words: Hydroxystilbamidine; Stilbamidine; Retrograde neuronal tracing; Lysosome; Amidine; Fluorescence; Stilbene; Weak base

Determining the mechanism by which fluorescent retrograde neuronal tracers are taken up requires knowledge of their composition. It has been claimed that Fluoro-Gold, a retrogradely transported fluorescent neuronal tracer, is 2-hydroxy-4,4"-diamidinostilbene(hydroxystilbamidine), an amidine antibiotic. However, this appears questionable, since the fluorescence spectrograms reported for Fluoro-Gold differ markedly from the spectrograms previously reported for purified hydroxystilhamidine. To help clarify the mechanism by which Fluoro-Gold might be taken up, it was decided to examine its composition and determine whether hydroxystilbamidine was its active agent. Fluoro-Gold was found by mass spectrometry to contain a component with a molecular weight of 280 Da (identical to that of hydroxystilbamidine), and fluorescence spectroscopy demonstrated the existence of a substance with a fluorescence spectrum similar to that of purified hydroxystilbamidine. Although a major fluorescent impurity was also observed, chromatographic separation of different fluorescent components of Fluoro-Gold suggested that the fraction resembling hydroxystilhamidinewas responsible for its retrograde labeling of cells. It is concluded that hydroxystilhamidine is the active constituent of Fluoro-Gold. Chemically, hydroxystilbamidineis a weak base. In this respect it resembles True blue, DAPI, Granular blue, bis-benzimide, Nuclear yellow, and several other retrogradely transported molecules. It is suggested that these agents cross cell membranes in their uncharged form and are trapped in lysosomes and endosomes by a favorable pH gradient. Thus, the uptake of this type of retrograde tracer may be an example of a well-understood process occurring widely throughout biological systems: the trapping of weak bases in acidic cellular compartments.

INTRODUCTION Since its introduction in 198649, Fluoro-Gold has gained great popularity as a retrograde tracer. Its bright yellow fluorescence in cytoplasm and lysosomes, its good filling of dendritic processes, and its compatibility with immunocytochemistry have allowed its broad application. The question arises of why Fluoro-Gold labels cells. Understanding the uptake of Fluoro-Gold might shed light on the more general question of how fluorescent retrograde neuronal tracers work, as well as possibly facilitating the development of other agents with even better properties. However, progress in this matter has been slowed by the fact that the original description of Fluoro-Gold did not reveal its identity beyond suggesting that it was a stilbene compound 49. Recently, it has been claimed 'm that Fluoro-Gold is 2-hydroxy-4,4"-diamidino stilbene (hydroxystilbamidine, Chemical Abstracts #495-99-8; Fig. 1), an amidine antibiotic 17. If Fluoro-Gold were hydroxystilbamidine, it would suggest a mechanism by which Fluoro-Gold might be taken up. Hydroxystilbamidine, like chloroquin and many other fluorescent weak bases, is accumulated by

lysosomes2. Lysosomal uptake of weak bases in many types of cells appears to be by passive diffusion of the uncharged form of the dye, followed by trapping of the protonated form of the dye within the acidic interior of lysosomesa8'44. This mechanism appears feasible for the neuronal uptake of hydroxystilbamidine and, if accurate, would suggest that a variety of fluorescent weak bases could be useful for neuroanatomical tract-tracing. However, the purported identity of Fluoro-Gold appears to be open to question. The fluorescence spectrograms that were included in that study 48 are different from those previously published for purified hydroxystilbamidine 18. Furthermore, a previous study has reported the presence of fluorescent contaminants within commercially available hydroxystilbamidine 18. It therefore appears possible that the retrograde tracing obtained with Fluoro-Gold might be due to something other than hydroxystilbamidine. This possibility bears particular scrutiny in light of previous studies showing that contaminated reagents can influence results using retrograde tracers 42'48'51. Thus, to clarify the identity of Fluoro-Gold and thereby to elucidate the mechanism by which it is taken up, the composition of Fluoro-Gold was examined.

Correspondence: M. Wessendorf, Department of Cell Biology and Neuroanatomy, University of Minnesota, 321 Church St. S.E., Minneapolis, MN 55455, U.S.A.

136 MATERIALS AND METHODS

Materials Commercial preparations of hydroxystilbamidine diisethionate monohydrate and stilbamidine diisethionate used in these experiments were donated by Rhone-Poulenc (formely May and Baker), Dagenham, Essex, UK. These preparations will be abbreviated c-OHSA and c-SA respectively, to allow their characteristics to be distinguished from those of pure hydroxystilbamidine (OHSA) and stilbamidine (SA). The c-OHSA was described as product MB 1011A, batch LOP 4303; the c-SA was described as product MB 744, batch number L1. Fluoro-Gold (FG) was purchased from Fluorochrome, Inc., Englewood, Colorado.

Spectrophotometry Absorbance spectrograms were made using a Beckman 25 spectrophotometer. Spectrograms were made of solutions of either FG or c-OHSA at a concentration of 15 /~g/ml. Samples were prepared either in 0.05 M acetate buffer (pH 5.0) or 0.05 M glycinate buffer (pH 9.0). Spectrograms of c-SA were made at a concentration of 10/tg/ml in 0.05 M acetate buffer (pH 4.6). In all cases, the buffer used in the sample was also used as the reference solution.

Spectrofluorometry Fluorescence spectrograms were made using a Spex DM 3000 fluorescence spectrometer (Spex Industries, Inc., Edison, NJ). Spectrograms were corrected for variations in lamp intensity using a rhodamine screen 3~ and were uncorrected for variations in photodetector sensitivity. Artifactual peaks resulting from Raman scattering were identified by their movement of a constant wave number in response to changes in excitation wavelength3~. For the sake of clarity, such peaks were omitted from the spectrograms illustrated herein. The artifactual peaks observed at integral multiples of the excitation wavelength were also omitted.

tandem with a diode-array absorbance detector were used to monitor the appearance of fluorescent and/or UV absorbing fractions. The excitation wavelength was chosen based on the absorbance characteristics of the substances found in FG (see Fig. 5); the emission wavelength was chosen as a reasonable compromise between the emission spectra of FG and OHSA and the likely emission of any other fluorophore with absorbance characteristics similar to those of the components of FG. The diode array detector was used to monitor absorbance at 330 nm continuously; this wavelength was used because initial studies showed that FG, c-OHSA, SA and the two major contaminants all absorbed well at this wavelength. In addition to absorbance at 330 nm, absorbance spectrograms between 190 and 500 nm were captured and stored when peaks appeared. Absorbance between 500 and 600 nm was used as the reference value in all cases.

Fluorescent retrograde tracing Sprague-Dawley-derived rats (100-200 g; BioLab, St, Paul, MN) were anesthetized with ketamine (80 mg/kg i.m.) and xylazine (12.5 mg/kg i.m.) or pentobarbital (45 mg/kg i.p.), Samples of FG and c-OHSA (1% in distilled water) were injected into one side of the tongue as a series of small (0.5/zl) injections, with a total volume of 3 /~l. Samples of stilbamidine (1% in distilled water) were similarly injected, with a total volume of 5 #1. Samples of chromatographically purified c-OHSA were injected into a single site due to the small amount of substance available. Injections were made at a rate of 0.05 to 0.1/~1 per min. After 2 days, rats were given an overdose of chloral hydrate and perfused with a picric acid/formaldehyde fixative5s. Sections of brainstem were cut on a freezing microtome and mounted on slides. Photomicrographs were made using Kodak Ektar 1000 color print film (Eastman Kodak, Rochester, NY). RESULTS

Mass spectrometry

Spectrophotometry

Fast-atom bombardment (FAB) mass spectrograms were generated using a model VG 7070E-HF mass spectrometer (VG Analytical, Ltd., Manchester, UK). The accelerating voltage was 5 kV and the FAB gas was xenon. Meta-nitrobenzyl alcohol (m-NBA) was used as the matrix.

5.0 and p H 9.0 are illustrated in Fig. 2. A t p H 5.0, b o t h

A b s o r b a n c e s p e c t r o g r a m s o f F G and c - O H S A at p H substances a b s o r b e d m a x i m a l l y at 3 4 4 - 3 4 5 n m with a secondary peak

at a b o u t

310 nm.

At pH

9.0, b o t h

High pressure liquid chromatography

substances a b s o r b e d m a x i m a l l y at 318 n m with a s m a l l e r

Substances were separated chromatographically using a Vydak Cls column, 4.6 x 150 mm, 5/~m particle size (The Separations Group, Hesperia, CA) in a model 1090 Hewlett-Packard liquid chromatograph (Avondale, PA). The first two min of chromatograms were run isocratically using 50 mM acetate buffer (pH 4.6), followed by a gradient of from 0 to 30% acetonitrile in the same buffer over the course of 15 rain. Flow rate was 1 ml/min. A fluorescence detector (excitation = 330 nm; emission = 450 nm) in

s e c o n d a r y p e a k c e n t e r e d at 398-401 n m . c - S A a b s o r b e d m a x i m a l l y at 327 n m with n o s e c o n d a r y p e a k s (Fig. 3).

Spectrofluorometry E x c i t a t i o n and e m i s s i o n f l u o r e s c e n c e s p e c t r o g r a m s of F G at p H 5.0 are illustrated in Fig. 4 A . W h e n e x c i t e d at 280 n m , t h e e m i s s i o n s p e c t r u m o f F G f e a t u r e d a large p e a k at 407 n m and a small s e c o n d a r y p e a k close to 600

~

IH2

nm.

//C \~/~T~ OH / H

However,

when

the

excitation

wavelength

was

i n c r e a s e d to 390 n m , t h e s h a p e o f t h e e m i s s i o n s p e c t r u m was q u i t e different. T w o distinct p e a k s w e r e o b s e r v e d ,

NH

c e n t e r e d at 448 n m and 582 n m , with t h e l a t t e r b e i n g larger. T h e c h a n g e s in t h e e m i s s i o n s p e c t r u m as a

C~-~C

f u n c t i o n of the w a v e l e n g t h at which F G was e x c i t e d w e r e

H/ OHSA mw= 280

~ c ~ N

H I NH2

Fig. 1. The structure of hydroxystilbamidine (OHSA). Stilbamidine (SA), which has been reported to contaminate commercially obtained OHSA TM, lacks the hydroxyl group.

p a r a l l e l e d by c h a n g e s in its e x c i t a t i o n spectra as a f u n c t i o n of t h e w a v e l e n g t h at which e m i s s i o n was m o n i t o r e d . T h e e x c i t a t i o n s p e c t r u m for F G f e a t u r e d a p e a k at 312 n m if e m i s s i o n w e r e m e a s u r e d at 410 n m ; h o w e v e r , if e m i s s i o n w e r e m e a s u r e d at 600 n m intensity was g r e a t e s t w h e n excited by light 365 n m in w a v e l e n g t h .

137 The intensity of emission at 410 nm (exciting at 312 nm) was over 5 times greater than that at 600 nm (exciting at 365 nm). Differences as a function of wavelength among a sample's spectra suggest the presence of impurities in the sample 41. The present results suggested that there might be two major components in FG: a substance excited maximally at about 312 nm and emitting maximally at about 407 nm, and a second substance excited maximally at about 365 nm and emitting at 448 nm and 582 nm. However, differences in spectra can also result from the sample containing a single substance in two fluorescent forms, e.g. in an ionized form and an unionized form. To test this possibility, fluorescence spectra were obtained of FG in pH 9.0 glycinate buffer, at which pH the hydroxyl group of O H S A would be expected to be largely ionized TM. Some of the spectra at pH 9.0 were markedly different from those at p H 5.0 (compare Fig. 5, cm = 600 and ex = 390 to Fig. 4A, e m = 600 and ex = 390), suggesting that p H 9.0 was sufficient to ionize the hydroxyl group• However, large differences in spectra as a function of wavelength were still noted. Spectra of FG in 0.01 M citric acid (pH 2.0) were essentially the same as spectra at p H 5•0 and likewise differed with wave-

length (not shown).

>,'E

0.6

~ i 0.4 '~£ oo

250

300

H S.0

In addition to wavelength-dependent differences in spectra, a second type of evidence suggested the presence of two chemically distinct components in FG. When excited at 335 nm, the peak at 407 nm rapidly photobleached• However, the peak near 600 nm appeared to

u,,,v~,.t em-6oo

600 (410 nm) 40 (600 rim)"

a.o

I

300

1'0 t ~" 0.8

I

350

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-

400

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450

violet /

f

~

Blue

Green Orange Yellow

I

ern~O0

Red

1IX) (280 nm) 10 (390 nm)

ex-280 /

i

FG

0.4

450

described under Materials and Methods• The difference between this spectrogram and the spectogram of peak 3 of Fig. 10 argues that SA is not the active component of OHSA.

-

•

400

Fig. 3. Absorbance spectrogram of c-SA at pH 4,6, obtained as

50 (600 nm)" A

A

350 Wavelength (nm)

300(410nm)

1°t

o•a

0.8

,\

.x.2So c-OHSA

200 (280 nrn) 10 (390 nm)

~J~

500

.--

B

200

c-OHSA

0.60

.

0.4-

.

0.2-

I

I

I

I

f

300

350

400

450

500

Wavelength (nm)

Fig. 2. Absorbance spectrograms of FG and c-OHSA at pH 5 and pH 9, obtained as described under Materials and Methods. Note the similarity between the spectrograms of FG and OHSA.

300

400 5~ Wavelength (nm)

600

700

Fig. 4. Fluorescence spectrograms of F G and c-OHSA in 0•01 M acetate buffer, pH 5.0. Excitation spectra are to the left; emission spectra are to the right. Intensity is given in counts per second x 10-3; scales on the left refer to excitation spectra and scales on the right refer to emission spectra• A: spectrograms of FG. Excitation spectra are those from monitoring emission at 410 nm and 600 nm; note the difference between the two spectra. Emission spectra are those resulting from excitation at 280 nm and 390 nm. Again, note the difference between the two spectra• B: excitation and emission spectra for c-OHSA, obtained as in A. Arrows point to a shoulder on the excitaton spectrum for FG obtained when monitoring emission at 600 nm. The lack of that shoulder in the spectrum for c-OHSA suggests the presence of a contaminant in FG emitting at 600 nm• Note also the relatively greater intensity of F G emission at about 600 nm compared to c-OHSA (ex = 390 nm).

138 150 (410 nrr 40 (600 mn)-

A O. O v

~=410A ~/,A

/~

pFG.0 ex= 390 ex--280 /f'\\///

_75 (280 nm) 40 (390 nm)

>, ,t-, I= .= ¢:

err

ill

it, 200

300

400

500

600

700

Wavelength(nm) Fig. 5. Fluorescence spectra of FG in 0.01 M glycinate buffer, pH 9.0. Arrangement of spectra and labeling of axes are as in Fig. 4. If the two fluorescent components observed in Fig. 4A were due to equilibrium between the ionized and unionized forms of the 2-hydroxyl group on OHSA, it would be expected that changing the pH to favor one of the forms would abolish the spectra of the other component. Note the differences between pH 5.0 (Fig. 4A) and pH 9.0, in terms of the excitation spectrum resulting from monitoring emission at 600 nm and the emission spectrum produced by excitation at 390 nm. These differences suggest that pH 9.0 was sufficient to ionize the hydroxyl group of OHSA. However, despite these differences there still appear to be two components present. Measuring emission at 410 nm, FG is maximally excited at 312 nm. Measuring emission at 600 nm, the excitation spectrum is bimodal, with maxima at 324 nm and 397 nm. Exciting at 280 nm, emission is maximal at 427 nm; exciting at 390 nm, emission is maximal at 599 am.

emilmion

FG I

I

I

I

|

I

450

500

550

600

650

700

Wavelength(nm) Fig. 7. Emission of a solution of 10 #g/ml FG in 0.01 M acetate buffer, pH 5.0, upon excitation at 365 nm. 365 nm is the U.V. excitation wavelength predominantly used in fluorescence microscopy. The emission appears to be largely that of OHSA (compare to Festy and Daune18).

c o m p o n e n t s w o u l d be visible u n d e r t h o s e c o n d i t i o n s , a solution of F G was e x c i t e d at 365 n m ; the e m i s s i o n s p e c t r u m is s h o w n in Fig. 7. T h e e m i s s i o n s p e c t r u m r e s e m b l e d that of F G e x c i t e d at 390 nm. H o w e v e r , the shorter-wavelength

p e a k was l o c a t e d at 420 n m , the

l o n g e r - w a v e l e n g t h p e a k o c c u r r e d at 599 n m , a n d the r e l a t i v e sizes of t h e two p e a k s w e r e r e v e r s e d . T h e f l u o r e s e n c e spectra of c - O H S A w e r e similar to

be u n a f f e c t e d (Fig. 6). A l t h o u g h it a p p e a r e d possible that F G c o n t a i n e d m o r e

t h o s e of F G , a l t h o u g h n o t i d e n t i c a l (Fig. 4B). E x c i t a t i o n

t h a n o n e f l u o r e s c e n t s u b s t a n c e , it was u n c l e a r if all of

of c - O H S A at 280 n m r e s u l t e d in a large p e a k at 409 n m

t h e s e substances w o u l d b e visible u n d e r the illumination

with a small s e c o n d a r y p e a k at a r o u n d 600 n m ; e x c i t a t i o n

u s e d for f l u o r e s e n c e m i c r o s c o p y . F l u o r e s c e n c e micros-

at 390 n m r e s u l t e d in e m i s s i o n at 446 n m and 584 nm.

c o p y of s u b s t a n c e s e x c i t e d by U V light is m o s t o f t e n

Similarly, w h e n e m i s s i o n was m o n i t o r e d

p e r f o r m e d using a m e r c u r y arc l a m p , f r o m which the 365

m a x i m u m e x c i t a t i o n was n o t e d at 311 n m . H o w e v e r ,

nm

w h e n e m i s s i o n was m o n i t o r e d at 600 n m , t h e w a v e l e n g t h

mercury

'line'

is isolated.

To

determine

which

at 410 nm,

m o s t strongly exciting c - O H S A was 354 n m r a t h e r t h a n 365 n m , as with F G . T h i s d i f f e r e n c e was r e f l e c t e d in the

70-

/--

// ~ P.

r 91

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II/_\\\

-. "

'

/

am, =2nd

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~ I

-- - '. " = 3,~

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:/

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III

500

600

c

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..-, 700 It.-

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~ 400

r- . . . . . . . . . . . . . . . . ~ . . , ~ . . ~ 500

6()0

I

._>

7()0

Wavelength(nm) Fig. 6. Photobleaching of FG solutions. Intensity is given in counts per s x 10 -3. The emission spectra shown resulted from excitation of FG (10 gg/ml in 0.01 M acetate buffer, pH 5.0) at 335 nm. Four successive spectra are shown; each spectrum required illumination of the FG solution for about 70 s. Note that the peak at about 407 nm photobleaehed rapidly, whereas the peak at about 600 nm appeared unaffected. Inset: the portions of the spectra between 500 and 700 nm, drawn at a higher gain. Again, note that the peak at about 600 nm appeared not to photobleach.

31)0

350

4~o

4~o

s6o

s~o

66o

Wavelength(nm) Fig. 8. Fluorescence spectra of c-SA. Excitation spectrum obtained by monitoring emission at 450 nm is on the left. Emission spectrum obtained by exciting at 280 nm is on the right. Note that the wavelength at which c-SA emitted maximally was 402 nm, close to the maximum of one of the components of FG. However, the wavelength at which c-SA was maximally excited was 335 nm, different from that of the FG component.

139 lack of a 'shoulder' on the excitation spectrum for c - O H S A that was present on the spectrum for F G (Fig. 4, arrows). c-SA was maximally excited by light 335 nm in wavelength and emitted light with greatest intensity at a wavelength of 405 nm (Fig. 8). However, unlike F G and c - O H S A its excitation and emission spectra varied as a function of wavelength only to a minimal extent.

75t 50

o ill

0 t

a

t ~

0

Mass spectrometry F A B mass spectrograms of a sample of F G showed major cationic peaks at mass-to-charge values of 281, 289, and 307 (Fig. 9). The latter two peaks are characteristic for m - N B A , the matrix used in these experiments. The peak at 281 was the largest of the three. Assuming that it represents the adduct ion of a proton with a component of F G 62, it would suggest the presence of a substance in F G with a mol.wt, of 280 Da, the same as hydroxystilbamidine. No peak above baseline was observed at a mass-to-charge value of 265, the value expected for stilbarnidine.

High pressure liquid chromatography W h e n 20/zg F G were chromatographically separated by H P L C , 3 peaks were observed that absorbed at 330 nm (Fig. 10A and B). All 3 peaks had retention times between 8 and 10 min, corresponding to concentrations of acetonitrile between 12 and 16%. Peak 3 was by far the largest, and the absorbance spectrum strongly resembling that shown in Fig. 2 for c - O H S A (Fig. 10, bottom). Both peaks 1 and 3 emitted fluorescence at 450 nm. In contrast, peak 2 did not (Fig. 10C). H P L C separation of 2 0 / z g c - O H S A resulted in an identical pattern of peaks (Fig. l l A , B ) . The absorbance spectra of the peaks strongly resembled those from separation of FG; similarly, only peaks 1 and 3 were fluorescent (not shown). To determine whether or not the

A 1

1

0

,

,

;




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rr

...........d,h,.,,,..I,hh,,,.,.,,,,dllh,,,,,,,,,ll l..h..,.,I II,,,I ....I,l,,..,.Jb

I 200

I 220

f 240

I 260

280

C

...............................

I

I

I

300

320

340

Mass/Charge Fig. 9. Mass spectrogram of FG. Peak size reflects relative abundance. Peaks at 289 and 307 result from m-nitrobenzyi alcohol, the matrix used in these experiments. The peak at 281 represents FG. Assuming that the peak represents the adduct ion of a proton with FG, it would imply that FG contains a component having a mol.wt, of 280, the same as OHSA.

I

8

.

.

.

.

I

9

'

'

'

'

I

10

'

'

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1 1 min

Fig. 11. Comparison of chromatograms of FG (A), c-OHSA (B), and of a mixture of equal masses of FG and c-OHSA (C). Note the single large peak in C, suggesting that FG and c-OHSA have identical retention times.

140 (Fig. 11C).

was the case in the p r e s e n t study using H P L C since the

Previous studies r e p o r t e d that c - O H S A was c o n t a m i n a t e d with S A TM. It was n o t possible to test w h e t h e r this

r e t e n t i o n time for S A was identical to that of the third peak (not shown). H o w e v e r , it a p p e a r e d that the third

Fig. 12. Retrograde labeling of hypoglossai motoneurons by FG, OHSA, and SA. A: retrograde labeling obtained following an injection of 3/~l of 1% FG into the tongue. B: retrograde labeling obtained following a similar injection of c-OHSA into the tongue. Note the similarity to the labeling shown in A. C: retrograde labeling obtained following injection of a small amount of HPLC-purified c-OHSA into the tongue. Note the similarity to the labeling shown in A and B. D: retrograde labeling obtained following an injection of 5/~l 1% c-SA into the tongue. Note the qualitative difference in color of labeling between D and FG labeling, suggesting that SA is not the active component of FG. E: retrograde labeling resulting from injection of the 'blue fraction' obtained by column chromatography of FG. This fraction appeared to be identical to the contaminant in FG that was maximally excited at 312 nm and that emitted maximally at 407 nm (see Fig. 13). Note lack of labeling. F: retrograde labeling resulting from injection of the 'pink fraction' obtained by column chromatography of FG. Note labeling indistinguishable from that of FG. Bar = 200/~m.

141 peak was predominantly not SA, since the absorbance spectra of the two were different (compare Fig. 3 to Fig. 5, bottom).

Retrograde transport Injection of FG (1% in distilled water, 3/~1) into the tongue of rats resulted in labeling of neurons in the hypoglossal nucleus (Fig. 12A). Similar labeling resulted from injection of c-OHSA into the tongue (Fig. 12B). To test whether peak 3 was responsible for the retrograde labeling observed, as opposed to the fluorescent contaminant represented by HPLC peak 1, c-OHSA was chromatographically purified. Seventy/~g c-OHSA were applied to the column and the fraction representing the trailing portion of peak 3 was collected. This fraction was dried in vacuo and reconstituted using 10/A distilled water. A large amount of white precipitate was noted; it was presumed that this represented undissolved sodium acetate remaining from the buffer. The reconstituted material was injected into the tongue of a rat. Strong yellow-gold labeling indistinguishable from that of FG was noted (Fig. 12C). Since SA co-migrated with peak 3 in the chromatographic system that was employed, it was possible that OHSA was contaminated with SA. To test whether SA might be responsible for the retrograde neuronal labeling observed with FG, SA (5/zl, 1% in distilled water) was injected into the tongue of rats and the animals allowed to survive 3 days. Labeled somata were observed in the hypoglossal nucleus after this procedure. However, the fluorescence of SA appeared weaker than that from c-OHSA and had a blue-white color rather than yellowgold (Fig. 12D). The spectrofluorometric studies suggested that in addition to OHSA, FG and c-OHSA contained a substance emitting maximally at 407-409 nm and maximally excited at 311-312 nm. However, that substance

a e-

@

_> 0

re

A

,.

--+jill"

.x::o

I

I

!

I

300

400

500

600

Wavelength (nm) Fig. 13. Excitation (left) and emission (right) spectra of the 'blue fraction' obtained by column chromatography of FG. Unlike crude FG, the spectra of this fraction change little as a function of wavelength. This suggests that the fraction contains only one fluorescent component.

did not appear to be separated by HPLC and thus is was unclear what its contribution was to FG's retrograde labeling. To test whether that substance could be transported retrogradely, it was isolated by column chromatography using a variation of the method of Festy and Daune TM. Five mg of FG were layered onto a 53 crn column of Sephadex G-10 (column volume = approx. 45 ml). Elution with 0.01 M HCI quickly resulted in separation of FG into at least two fractions as detected by a hand-held near-UV light. The faster-migrating fraction fluoresced blue, whereas the slower-migrating fraction fluoresced bright pink. Where the two fractions were adjacent, a white-fluorescing region could be observed but it was unclear whether this represented a separate fraction or mixing of the blue and pink components. What appeared to be the majority of the blue fraction was eluted in a total volume of approx. 150 ml. Four samples of the blue fraction, each about 5 ml, were dried in vacuo and reconstituted in 5 /~1 distilled water; the samples emitted maximally at 402-404 nm, were maximally excited at 312 nm, and appeared to be nearly pure by spectrofluorometric analysis (Fig. 13). Injection of the substance into the tongues of four rats resulted in no visible labeling in the hypoglossal nucleus (Fig. 12E). In contrast, injection of a similar sample of the pink fraction resulted in labeling indistinguishable from that of FG (Fig. 12F). DISCUSSION Understanding the uptake of fluorescent retrograde tracers would be facilitated by understanding their chemical properties. The latter is complicated when authors omit the identity of a tracer from its description 7' 28.49 Thus it would be beneficial if the developers of fluorescent probes would at least identify tracers by chemical name and give the source and lot number. However, even if a substance has been identified in this manner, knowledge of a product's chemical composition requires analytical studies since commercial preparations of fluorescent substances may contain significant impurities 18,42. One powerful technique for assessing the purity of fluorescent substances is fluorescence spectroscopy. Fluorescence spectra of pure substances are independent of wavelength, e.g. the emission spectrum of a pure substance will have the same shape no matter at what wavelength it is excited41. Thus if the spectra of a sample are found to vary as a function of wavelength it suggests the presence of more than one fluorescent substance in the sample. The relative size of the fluorescence peaks can be used to estimate to what extent the fluorescence of a contamitant will be visible. It follows that fluores-

142 cence spectroscopy can be superior to methods of analysis by weight or by molar ratio, since the latter allow no estimate of the intensity of fluorescence from an unidentified contaminant to be made. Based on fluorescence spectroscopic and photobleaching data, it appears that FG contains at least two major fluorescent components. One of the components was excited maximally at 365 nm (correcting for the presence of a relatively minor contamitant it would appear to be excited maximally at 354 nm - - Fig. 4, arrows) and emitted with maxima at 448 and 582 nm. This component resembles purified O H S A TM. A second, unidentified component was maximally excited at about 312 nm and emitted maximally at 407 nm. The emission of the unknown component of FG was 5 times more intense than the emission of the OHSA-Iike component and thus this unidentified compound is by far the most strongly fluorescent constituent of FG. Studies by Murgatroyd 35 and Schmued 48 reported that c-OHSA and FG had emission maxima of 410 nm and 409 nm, respectively, and excitation maxima of 280 nm and 324 nm, respectively. These values differ greatly from those of purified O H S A TMbut are similar to the values noted for the major unidentified component in the present study.Thus, the spectra of Schmued 48 and of Murgatroyd 35 may be those of contaminants similar to that noted in the present study. Festy and Daune reported that c-OHSA was contaminated with SA18; SA also emits maximally in the same range as the unidentified component of FG (402 nm - Fig. 8). However, the unidentified component appears not to be SA since SA was maximally excited at 335 nm rather than 312 nm, and since mass spectrometry showed no evidence that FG contained a substance with the molecular weight of SA. A previous study 49 stated that FG appeared to be pure by thin-layer chromatography. No chromatograms were illustrated and the conditions were not specified; thus it is unclear whether the conditions that were employed were sufficient to separate the components of FG. However, the present HPLC experiments illustrate that it may be difficult to separate the major components of FG by some types of chromatography. The same study reported that FG appeared to be greater than 99% pure by an unspecified form of 'qualitative analysis'. Although the results of the present study would tend to be inconsistent with those values, it should be noted that a high degree of purity is not necessarily incompatible with there being a major fluorescent contaminant. The intensity of fluorescence of a substance is a function of many factors, of which its concentration is only one 31'41. The differences in spectra noted for FG in the present study could conceivably be the result of different forms of a

single compound, e.g. ionized and unionized forms of O H S A that are both fluorescent 41. However, it appears unlikely that this is the case, since the two components were still visible when the pH was changed (Fig. 5). Despite the presence of a strongly fluorescent contaminant, it appears that bonafide O H S A is a major component of FG. FG contained a substance with a molecular weight of 280 Da, the same as the molecular weight of bonafide OHSA. In addition, the absorbance spectra of FG were similar to those previously published for c-OHSA, and as stated above, one of the fluorescence components of FG - - that excited selectively by emission at 390 nm - - had a fluorescence emission spectrum similar to that published for purified O H S A TM. The question remains of whether O H S A is the only component of FG, or even the major component of FG, to retrogradely label neurons. Several points argue that bonafide O H S A is responsible for the retrograde labeling observed with FG. First, the fuorescent emission of retrogradely transported FG can be observed microscopically through sets of filters passing light 410-490 nm in wavelength, sets passing 520-560 nm, and sets passing 590 nm and longer (Wessendorf, unpublished observations). This is consistent with the emission spectrum reported for purified O H S A TM. Second, O H S A is an aryl amidine. In this way it is similar to other fluorescent retrograde tracers, including SA (present study), DAPI 3°, True Blue, and Granular Blue 6. Third, emission elicited by excitation at 365 nm (the wavelength of the 'line' present in the mercury arc lamps most commonly used for near-ultraviolet microscopic excitation) is largely that of OHSA. Fourth, none of the other fluorescent fractions observed in FG appeared to be responsible for its labeling of neurons. (A) Retrograde labeling indistinguishable from that of FG can be obtained using HPLC-purified c-OHSA, in which the fluorescent contaminant represented by peak 1 in Fig. 10 has been removed. (B) The contaminant emitting at 600 nm and producing the 'shoulder' on the excitation curve for FG (arrow, Fig. 4) is not responsible for FG's retrograde labeling, c-OHSA, which does not contain that contaminant, retrogradely labels neurons in a fashion identical to that of FG. (C) The substance emitting maximally at 407 nm and maximally excited at 312 nm is not responsible for the retrograde labeling obtained with FG. A chromatographic fraction containing this substance and that appeared spectroscopically not to contain O H S A produced no visible labeling when injected into the tongue of a rat. Thus, by elimination, it appears that the labeling obtained with FG is due to OHSA. However, confirmation of this hypothesis awaits testing the transport of pure OHSA.

143

Pharmacology c-OHSA is a member of the class of antibiotics known as diamidines, the most commonly used agent of this group being pentamidine. (Pentamidine is used to treat Pneumocystis carinii infections in AIDS patients46.) c-OHSA was developed nearly 50 years ago by the British firm of May and Baker, Ltd, apparently as part of a systematic search for aromatic diamidines with antitrypanosomal activity5'21'22. c-OHSA found use against North American blastomycosis6° and was employed until recently for that condition 26'61. The drug has also been reported to have beneficial effects in multiple myeloma 53 and experimental leukemia 34. However, c-OHSA appears to be no longer distributed in the United States as a pharmaceutical agent. For a review of some aspects of the pharamacology of c-OHSA, see Festy 17.

Fluorescence and cell labeling Although one study reported that c-OHSA did not fluoresce25, the strong fluorescence of c-OHSA was early used as a means of assaying the drug in bodily fluids 32'47. In addition, c-OHSA has been reported to fluorescently label many cellular components in a histochemically useful fashion, including lysosomes2'5°, elastic fibers, mucosubstances, fungal hyphae 35'36, and DNA and RNA 35'55'56. The usefulness of fluorescence microscopy in visualizing cellular uptake of c-OHSA was recognized by the early 1950'S 39'54-56. Mammalian cells labeled by c-OHSA were reported to have a striking appearance; to 'stand out like firey beacons '57. Although the original description of Fluoro-Gold was published in 198649, that work may not be the first description of retrograde labeling of neurons by OHSA. Therapeutically administered OHSA that had been given systemically would be expected to retrogradely label cells projecting outside the blood-brain barrier, such as cells of cranial nerve motor nuclei. Thus it is interesting to note that in 1950, Snapper et al. 55 reported that, 'in the brain of cancer mice treated with this substance (i.e. c-OHSA) large numbers of ganglion cells exhibit fluorescent nuclei'. Although labeling would be expected to be in the cytoplasm and nucleolus rather than the nucleus 49, it is possible that Snapper et al. 55 is the first description of retrograde labeling of neurons by cOHSA. It was recognized at an early date that the fluorescence of OHSA is dependent upon its molecular environment 56, 57, i.e. that it may appear as different colors under different conditions. Descriptions of the fluorescence of OHSA or c-OHSA under UV excitation have ranged from blue to yellow to orange to red 18'35'36'39'4°'49'56. These differences in color appear to be due in part to differences in pH 18'49. In the present study it was found

that the emission spectrum of the OHSA-like component of FG at pH 5.0 contained a blue component that was not present at pH 9.0. This observation suggests that FGlabeled neurons might appear orange or red at pH 9.0 rather than yellow. Factors other than pH also appear capable of affecting the fluorescence of OHSA, including the viscosity of its solvent and temperature TM. Interestingly, it has also been reported that the interaction of c-OHSA with DNA or RNA can change its fluorescence. Snapper et al. 57 noted that addition of either nucleic acid to solutions of c-OHSA changed the color of fluorescence from reddish to yellow and increased its intensity; this effect appeared to be independent of pH. Similarly, Festy and Daune TM examined spectrofluorograms of OHSA in the presence and absence of polynucleotides and observed a large increase in the blue component of the fluorescent emission. These and other reports (see above) suggest that OHSA is capable of binding to DNA molecules. However, it does not appear to be an intercalating agent TM. Rather than inserting itself between base pairs, OHSA appears to bind DNA by aligning across the minor groove of the helix adjacent to A-T rich regions 19.

Toxicology The toxicity of FG is a matter of legitimate concern to those who use it but unfortunately little has been published on this subject. However, there are many studies relating to the toxicity of c-OHSA. The acute toxicity of c-OHSA appears to be relatively low and when it was used therapeutically in humans relatively large amounts were given. For instance, in treatment of North American blastomycosis, 225 mg were administered i.v. daily and a total of 8-16 g or more were given in the course of treatment of an infection26'6x. However, it should be noted that c-OHSA and OHSA interact with D N A 15'1s'19'57'59 and that OHSA has been reported to be a mutagen in yeast (Mounolou and Festy, cited in Delain et al.15). Therefore, although no mutagenicity was noted in tests utilizing salmonella bacteria 35, the possibility exists that c-OHSA is a carcinogen and thus FG should be treated with caution. This is especially important given that c-OHSA can be sequestered in tissue for periods of years 54. A study of the subsequent medical histories of patients treated with c-OHSA would be helpful in assessing the possible risks associated with this substance. In addition to carcinogenesis, another possible hazard associated with FG is neurotoxicity. A previous study has reported that c-OHSA was contaminated with SA, the unhydroxylated form of OHSA TM. c-SA has been reported to be neurotoxic9,1°,37,64. Administration to humans results in a trigeminal neuropathy that initially causes numbness of the nose and face and progresses to

144 facial parethesias and pain 9'1°'64. Since the present study was unable to confirm that either c-OHSA or FG are contaminated with SA, it is unclear whether possible contamination with SA poses a significant health hazard to users of FG. In addition, the neurotoxicity that was noted after c-SA administration appears to have occurred in patients who had received in excess of 1 gram of c-SA 9'1°'64, doses higher than would likely be encountered accidentally in the course of neuroanatomical studies. However, given the tendency of c-SA to accumulate in tissue 54, it appears possible that prolonged exposure to smaller doses might result in similar pathology. Thus, although the present study found no evidence for contamination of FG with SA, it would be prudent to handle FG with caution. Besides the actions described above, c-OHSA has been reported to interfere with enzyme function and normal metabolic activity in several systems 3'4'13A6"2°' 23,24,33,63. At least one of these actions appears worth mentioning for a toxicological reason. Bone resorption, 2-deoxyglucose incorporation, and thymidine incorporation in cultured rat femur TM have been reported to be decreased by c-OHSA. Since the effects on bone resorption were elicited by nanomolar concentrations of cO H S A it appears possible that experimental animals to which FG has been administered could be vulnerable to bone pathology. Moreover, since c-OHSA accumulates within tissue 54, it is conceivable that bone pathology might also occur in investigators frequently exposed to FG. Given the similarity between FG and c-OHSA, the toxicity of FG would be expected to be similar to c-OHSA. However, FG appears to contain at least one component that is not present in c-OHSA (Fig. 4 - arrows). Thus it is possible that there are hazards associated with FG that would not have been recognized using c-OHSA.

Possible mechanism of retrograde neuronal labeling by OHSA For a substance to function as a retrograde neuronal tracer, a number of conditions need to be fulfilled. These include, in part, (1) that the substance be accumulated by nerve fibers and (2) that it be sequestered in the cell body. With respect to the accumulation of OHSA, a previous study suggested that the substance probably did not diffuse across plasma membranes and that, 'certain reactive groups facilitate active endocytotic vesicular uptake of the tracer at terminals '49. Such a mechanism may exist for some tracers; for instance, the uptake of different anthracine antibiotic fluorescent tracers has been reported to be related to the sugar moieties present

on the different molecules 8. It has been reported that c-OHSA is actively taken up by some protozoal cells 12, suggesting that active transport might be involved in its entrance into neurones; it would be of interest to determine whether the uptake of O H S A can be competitively blocked by related drugs such as pentamidine. However, uptake of O H S A into cells may be due at least in part to simple diffusion, c-OHSA, chloroquin, and a number of other weak bases have been shown to be taken up by lysosomes2'38. It has been proposed that the uncharged form of weakly basic compounds is in equilibrium across cell membranes, and that the charged form does not cross membranes readily TM. As a result, it would be expected that if a weak base became protonated intracellularly (e.g. in an acidic compartment such as a lysosome or endosome) it would accumulate in that compartment 14'38. O H S A is a weak base having two amidine groups available for protonation and thus would be expected to be accumulated into neuronal lysosomes or endosomes by this means. That FG can be accumulated in neuronal lysosomes has previously been reported 5°. The 3 factors proposed to influence uptake of weak bases into lysosomes have been outlined in detail by de Duve14; for the present purpose these need only be described briefly. First is the difference in pH between the extracellular fluid and the lysosome. The ratio of hydrogen ion concentration within the lysosome to that in the extracellular fluid provides the maximum extent to which any substance can be concentrated by this mechanism. Assuming an extracellular pH of 7.4 and an intralysosomal pH of 4.744, the maximum extent of concentration possible by this mechanism would be about 500-fold. Second is the permeability of the uncharged and charged forms of the molecule in the relevant cellular membranes. High permeability of the uncharged form facilitates diffusion of the substance through the cellular membranes; low permeability of the charged form slows the substance's diffusion from the lysosome. The exact permeabilities of the charged and uncharged forms of O H S A appear never to have been determined, but membrane permeability would be expected to decrease with protonation. Third is the pK of the weak base. If its pK is one unit or more below the pH of the extracellular fluid, the substance will be maximally accumulated within the lysosome. Substances with pK's between lysosomai pH and extracellular pH would be accumulated to a lesser extent. Substances with pK's below lysosomal pH would not be accumulated. The pK's of the amidine groups on O H S A are presumably close to the pK of the amidine group of amidinobenzene, or about 11.643. Thus the latter 3 factors are consistent with O H S A being taken up into lysosomes by pH-trapping.

145 It appears possible, but less likely, that O H S A is significantly accumulated in organelles other than lysosomes and endosomes. Secretion granules are acidic in many cell types (for review, see ref. 45) and it is possible that O H S A is accumulated therein. In addition, since O H S A is a weak acid (due to its phenolic hydroxyl group) as well as a weak base (due to its amidine groups), it appears possible that it might be accumulated in basic compartments such as mitochondria ~4. However, as mentioned above, the maximum extent to which a c o m p o u n d can be accumulated into any compartment is equal to the ratio of hydrogen ion concentrations inside and outside the compartment TM. The p H gradient across mitochondria is two orders of magnitude less than that across lysosomes (compare Addanki et al. 1 to Pool and Ohkuma44). The p H gradient across secretory vesicles appears to be a function of the particular granules being studied, but generally has been reported to be an order of magnitude less than that in lysosomes 45. It therefore seems likely that accumulation of O H S A into these organelles would be less important than lysosomal accumulation. This conjecture is supported with respect to mitochondria by the report that chromomycin, olivomycin, and mithramycin, 3 fluorescent antibiotics that are weak acids but not weak bases 52, do not retrogradely label neurons 6. Assuming that O H S A is taken up by diffusion, the uptake of O H S A from undamaged axons of passage would appear possible, since the uncharged form of the tracer would appear capable of diffusing into axons and

being accumulated by any lysosomes present. Although it was initially claimed that F G was not transported retrogradely by undamaged axons of passage 49, recent studies indicate that this can occur 11. The amount of uptake from undamaged fibers of passage appears to be different for different systems of neurons 11. It could be speculated that uptake of O H S A would be slower from more heavily myelinated fibers, due to slower diffusion across the myelin sheath. Necrosis is frequently noted at F G injection sites 1L49. It is possible that one of the contaminants in F G is neurotoxic and is responsible for this effect. However, cell death may simply be due to osmotic effects. Although c - O H S A has been reported to stabilize lysosomes against release of their contents 63, it is likely that high concentrations of O H S A within lysosomes could cause them to burst from osmotic pressure and kill the cell. If this were the case, the presence of necrosis would be expected to depend upon the concentration of F G in the injection site, as has been reported 49. The fact that accumulation of FG appears to cause pathological changes in lysosomes 5° supports this hypothesis. With respect to sequestration of O H S A within the cell soma, it is possible that the fluorophore becomes covalently attached to some substrate within the cell. However, covalent modification of O H S A is not the only means by which it might be sequestered in the cell. For example, the p H gradient within lysosomes that favors accumulation of O H S A in the nerve terminal would also tend to hold O H S A within the cell body. In addition, the

Nucleus

Lysosomu

Lysosomefs

@ Terminal

;.s,

CellSoma

OHS+A

Fig. 14. Schematic representation of the mechanisms proposed to account for the uptake and sequestration of the active component of FG (i.e. OHSA). Relative size of the type suggests the relative concentration of OHSA species in each compartment; 'OHSA +' indicates forms of OHSA in which one or both of the amidine groups are protonated. It is proposed that pools of the unprotonated form of OHSA are in equilibrium across cell membranes but that protonated forms of OHSA are trapped. Thus in the case of nerve terminals at the injection site, unprotonated OHSA would be in equilibrium between extraceilular, cytosolic and lysosomal compartments. However, because of the relatively low pH of the interior of the lysosome, the concentration of OHSA + therein would tend to be relatively higher compared to other compartments. The low pH of some secretory granules might allow significant accumulation in that compartment as well. In the cell soma, uncharged OHSA would again tend to equilibrate across the lysosomal, cytosolic, nuclear, and extracellular compartments. However, because of the low pH of the lysosome most OHSA there would be trapped in the form of OHSA +. In addition, the ability of OHSA to bind nucleic acids (such as in the RNA in the nucleolus) could further slow its diffusion from the cell. It is proposed that these mechanisms might describe the uptake and sequestration of other fluorescent retrograde tracers as well.

146 diffusion of O H S A from the cell body might also be slowed by the binding of O H S A to nucleic acids 1s'57. In particular, O H S A appears to be sequestered by R N A in the nucleolus 49. However, given the diffuse cytoplasmic labeling sometimes observed in retrogradely labeled cells 49, it would appear possible that O H S A can bind to cytoplasmic R N A as well. c-OHSA appears to bind to cellular macromolecules other than nucleic acids 35'36 and this phenomenon may contribute to its sequestration within neurons as well. Fig. 14 shows a schematic representation of the mechanisms proposed to underlie the accumulation and sequestration of OHSA. Many fluorescent retrograde tracers other than O H S A are weak bases 6'29'3° and would thus be expected to be accumulated in lysosomes. In addition, many of these compounds bind nucleic acids 6. Thus, the mechanisms that have been proposed herein to account for the efficacy of O H S A may also account for the efficacy of other fluorescent weak bases that are retrogradely transported to neurons, such as Granular Blue, DAPI, DIPI, bis-benzimide, Nuclear Yellow, and True Blue. It would also appear likely that fluorescent amines and amidines as a group, especially those that bind nucleic acids, will retrogradely label neurones. The latter conclusion is supported by the observation that stilbamidine, a fluorescent amidine that binds nucleic acids 57, is transported retrogradely (Fig. 12D). There probably are fluorescent substances that are not taken up by the proposed mechanism. For instance, the lipophilic dyes Di I and Di O appear to be transported by membrane diffusion 27. Furthermore, it is possible that propidium iodide 29 is not taken up by means of pHtrapping. Propidium iodide has two quaternary amine moieties, which would be charged at any pH and which would be expected to minimize the rate at which propidium iodide would diffuse across lipid membranes. In addition, the aromatic primary amines that would provide the only basic character to propidium iodide would be expected to have pK's close to that of aniline, or 4.643. This value for the pK's would be close enough to the pH of lysosomes44 that little if any pH-trapping would be expected 14. The same argument with respect to pK's could be made regarding Evans blue and primulin 3°. Thus there may be as-yet undescribed means by which small fluorescent molecules enter cells. Based upon the mechanism that has been proposed, several predictions can be made regarding the behavior of weak-base retrograde tracers. First, it would be REFERENCES 1 Addanki, S., Cahill, F.D. and Sotos, J., Determination of intramitochondrial pH and intramitochondrial-extramitochondrial pH gradient of isolated heart mitochondria by the use of

expected that fluorescent weak bases that have little or no affinity for nucleic acids would label lysosomes and other acidic compartments within the cell, but would not produce nuclear or nucleolar labeling. Second, the uptake of a tracer would be expected to be strongly related to the membrane permeability of the tracer in uncharged form. All else being equal, a fluorescent weak base that was highly permeable in its unprotonated form would be expected to be a better retrograde tracer than a fluorescent weak base that was only marginally permeable. Third, the leakage of a tracer from a cell would be expected to be strongly related to the membrane permeability of the tracer in its protonated form. Thus it would be expected that tracers such as Nuclear yellow or bis-benzimide, which have reported to leak from cells quickly 7'29, would have higher membrane permeabilities in their protonated forms than O H S A , which leaks only slowly. Fourth, lysosomal lysis would be expected to occur at a sufficient concentration of any weak base. Thus it would be expected that all weak-base retrograde tracers would be capable of provoking necrotic injection sites. Fifth, no differences would be expected in the actual rate of transport among tracers employing the proposed mechanism. However, there may be differences among tracers in the rate at which they are taken up into cells and the rate at which they diffuse out of cells. This may account for the differences reported in the rates at which different tracers accumulate within the cell body 7. Sixth, any weak-base retrograde tracer would be expected to be taken up by undamaged axons of passage and this phenomenon would be expected to be more prominent with lightly myelinated fibers. Seventh, it would be expected that the nuclear staining observed with Nuclear yellow 7 reflects a relatively higher affinity for D N A than for RNA. Nucleolar staining, rather than nuclear staining, would be expected if a weak-base retrograde tracer had a higher affinity for RNA than for DNA. Finally, it would be expected that the uptake of weak-base retrograde tracers would be decreased or blocked if lysosomal p H were increased with ammonium chloride 44. Acknowledgements. The author wishes to thank Dr. Alan Schwartz for the suggestion that pH gradients might influence the uptake of OHSA, Dr. Robert Wohlhueter for assistance with the HPLC studies, Mr. Todd Brelje for suggesting the use of mass spectrometry in this study, Mr. Stephen Schneli for excellent technical assistance, and Dr. Robert Elde and Dr. Glenn Giesler for critical reading of the manuscript. These studies were supported by PHS Grant DA 05466 from ADAMHA.

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