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The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei
Brain Research, 475 (1988) 259-271 Elsevier
259
BRE 14104
The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei Vincent A. Pieribone and Gary Aston-Jones Department of Biology, New York University, New York, NY IO003 (U.S.A.)
(Accepted 14 June 1988) Key words: Fluoro-Gold; Retrograde tracing; Fluorescent tracer; Locus coeruleus; Raphe; Substantia nigra; Nucleus basalis; Afferent pathway; Immunofluorescence; Rat
A method is described for identifying the afferents to confined areas within the central nervous system using iontophoretic application of the fluorescent tracer, Fluoro-Gold (FG). Unlike other fluorescent tracers, it is possible to make focal iontophoretic injections through small-tipped micropipettes, and electrophysiological recordings from the injection pipette can be used to define structures prior to injections. Retrograde labeling with FG appears to be as sensitive as wheatgerm agglutinin-conjugated horseradish peroxidase visualized with tetramethylbenzidine. Furthermore, iontophoretically applied FG does not appear to be taken up and trans-. ported retrogradely by fibers of passage. Finally, retrograde transport of FG can be combined with immunofluorescence without: appreciable loss of sensitivity in either label. INTRODUCTION Since their introduction in 1977 by Kuypers and coworkers 4-6'2°-22, fluorescent retrograde tracers have become important tools for determining neuroanatomical connectivity. The use of these tracers offers several advantages over retrograde transport techniques that utilize horseradish peroxidase (HRP) or its conjugates. Fluorescent tracers require minimal tissue processing, they can be combined to demonstrate collateralization of efferents 23'4°, and tissue containing the tracers can be processed for immunocytochemistry to identify neurotransmitters 7"15'16' 24,35.37,41
However, the accuracy of a retrograde tracing study examining the afferents to a small brain region is limited by the ability to retrogradely label only t h o s e neurons which have axons that terminate within the area of interest. It is difficult with most fluorescent tracers to accurately study afferents to small brain nuclei in the rat brain since: (1) iontophoretic injections are generally not possible and it may be im-
possible to make small pressure injections limited to the desired brain site; (2) pressure injections damage tissue; (3) fibers of passage through the injection site transport most fluorescent dyes; and (4) desired injection areas cannot be physiologically localized with electrical recordings from an injection pipette. Schmued and Fallon 36 recently introduced FluoroGold (FG) as a fluorescent retrograde tracer with several novel properties. The initial report did not include details concerning some potential applications of FG. We have now examined this compound in retrograde transport studies as well as in c o m b i n a t i o n with immunocytochemistry, and provide additional information on using this new tracer. In particular, below we present specific details for iontophoretic application of FG combined with electrophysiological recordings from FG-filled pipettes for the study of afferents to small nuclei in the rat brain. MATERIALS AND METHODS Male S p r a g u e - D a w l e y rats (n = 30) were anesthe-
Correspondence: V.A. Pieribone, Department of Biology, New York University, 1009 Main Building, Washington Square, New York, NY 10003, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
260 tized with chloral hydrate (400 mg/kg, intraperitoneal), and placed in a stereotaxic apparatus. A scalp incision was made, a hole was drilled in the skull overlying the injection target, and the dura was reflected. Injections were centered in one of four structures: locus coeruleus (LC), the head of the caudate nucleus (CA), frontal cortex (FCx), or fimbria. Five previously characterized projections were investigated: the nigral-striatal, coeruleocortical, dorsal raphe-cortical, and the paragigantocellularis- and prepositus hypoglossal-to-LC pathways 3. Injections into the CA, FCx, and fimbria were done with the skull horizontal and the following coordinates: 0.2 mm posterior to bregma, 3.5 mm lateral to midline and 5.0 mm from top of skull; 4.0 mm anterior to bregma, 2.0 mm lateral to midline and 3.0 mm from top of skull; and 1.3 mm posterior to bregma, 0.5 mm lateral to midline and 4.0 mm from top of skull, respectively. For LC injections the skull was placed at a 17° angle (nose tilted down) to avoid rupturing the overlying transverse sinus; coordinates were: 3.7 mm caudal to real lambda, 1.2 mm lateral to midline and 6.3 mm from skull. FG (Fluoro-chromes, Englewood, CO) was stored in desiccated light-tight containers at 4 °C until used. Glass capillary tubes (1.5 mm o.d., Omega-Dot) were cleaned by passing deionized water and 95% ethanol through the unpulled pipettes and then airdried. Capillary tubes were then heated, pulled, and the tips broken back to 10-60/~m diameter under microscopic control. These electrodes were filled with a 1% solution of FG in filtered (0.22/~m) 0.1 M sodium acetate buffer (pH 3.3) just prior to use. Injections into LC were made from micropipettes with 10-20/~m tips. Electrophysiological recordings from these pipettes aided in localizing LC by its distinctive discharge characteristics (see Results below) 39. Larger-tipped pipettes (50-60/~m) were used for injections into FCx and CA. Injection parameters for LC were +1.0/~A for 2-10 min, while for FCx and CA currents of +5.0/~A for 15-30 min were used. Iontophoretic injections employed pulsed current (4 s on and 4 s off) to avoid heating or clogging the tip (Finntronics constant-current source). It is also possible to make injections using a duty cycle durations of 7 s on and 7 s off. In some animals, pressure injections (100-200 ni) of the same FG solution were made into CA and FCx via a 50/~m-tipped glass pi-
pette attached to a 1/~1 gas chromatography syringe (Precision Sampling, Baton Rouge, LA) 38. In all cases, electrodes were allowed to remain in place 5 min before and after injections. Animals that received injections into LC were allowed to survive 1-5 days. Animals that received injections into CA and FCx survived 5-40 days. Following the appropriate survival time, animals were deeply anesthetized with chloral hydrate and perfused transcardially with saline (100 ml for 45 s), followed by 450 ml of freshly prepared 4% paraformaldehyde (Merck) in 0.1 M phosphate buffer (pH 7.4) at 80 ml/min and then 450 ml of this same solution at 20 ml/min. All perfusion solutions were at room temperature. Brains were immersed in the same fixative at 4 °C for 90 min then transferred into phosphatebuffered 20% sucrose overnight, both at 4 °C. The following day, 40-/zm-thick frozen sections were collected into ice-cold isotonic 0.1 M phosphate-buffered saline (pH 7.4). Sections not subjected to immunohistochemistry were rinsed twice in buffer and mounted on gelatinized slides. Immunocytochemistry for neurochemical markers was done on free-floating sections. Sections were rinsed twice in Tris-buffered saline (TBS, pH 7.6) and placed in 3% normal goat serum containing 0.25% Triton for 30 min. Following 4-12 h incubation in a dilute solution of primary antisera in TBS with 1% goat serum added, sections were transferred to a solution of fluorescein isothiocyanate (FITC)- or rhodamine isothiocyanate (RITC)-conjugated, affinity purified, goat anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN) in 1% goat serum. Primary antisera were raised in rabbit against dopamine-fl-hydroxylase (DBH; Eugene Tech, N J) or serotonin conjugated to bovine serum albumin (Immuno Nuclear, Indianapolis, IN). Sections were rinsed between each step with 1% goat serum in TBS. Cutting and processing of sections was done under low ambient light conditions to avoid possible quenching of the FG. Slides were allowed to dry overnight in the dark and then quickly dehydrated through graded alcohols (70%, 95%, and 2 x 100%; 20 s each), cleared in xylenes and coverslipped using DPX (BDH, Poole, U.K.). Alternatively some sections were coverslipped in an aqueous media (glycerol/ phosphate buffer, 9:1) with added p-phenylenediamine 19'32 or polyvinyl alcohol (Lehrner, New Ha-
261 ven, CT). Coverslipped sections were kept frozen in the dark at - 2 0 °C until observation. Studies of LC afferents using W G A - H R P have been described 3 previously. Briefly, electrophysiological recordings from W G A - H R P injection pipettes were used to localize LC and iontophoretic injections were made in LC. Animals were allowed to survive one day and then perfused with a glutaraldehyde and paraformaldehyde mixture 27. Forty/~m frozen sections were processed by the technique of Mesulem et al. 27 using tetramethylbenzidine (TMB). A Nikon Microphot microscope equipped with epifluorescence was used for observation and photography of fluorescent material. The Nikon UV-10 (equivalent to the UV-1) fluorescent cube was used, producing an excitation peak at 330 nm and a lowend emission cut-off at 450 nm. On sections processed for FITC double-labeling, a notch filter (Omega Optical) eliminating transmission at 530 + 15 nm was placed in the light path between the emission filter and the ocular/camera to remove FITC fluorescence produced by UV excitation (see Discussion below). Color photographs were made with Kodak Ektachrome 200 or Kodachrome 200 film using an ASA-of 400. Black and white photographs were made on Kodak Panatomic film (ASA 32) or Tmax (ASA 100). RESULTS
Iontophoretic injection experiments Our previous experience indicates that in order to accurately place injections into the relatively small LC nucleus it is very helpful to record cellular activity through the injection pipette and localize LC neurons by their distinctive discharge characteristics 39 (Fig. 1 inset). Towards this end, we tried to dissolve FG (at concentrations of 0.5-4 % ) in the following solutions: 0.1 M phosphate/HC! buffer (pH 7.4), 0.1 M Tris/ HCI buffer (pH 7.6), 0.9% NaCI, Ringer's solution (with and without lactate36), deionized water, and 0.1 M sodium acetate/HCl buffer (pH 6.0 and 3.3). We also varied electrode tip diameters from 4 to 60 /.tm. All concentrations of FG were soluble in all of the above except for the Tris- and phosphate-buffered solutions in which FG precipitated even at low concentrations and after prolonged sonication. Four-
~t.
~
,,J.
Fig. 1. Schematic diagram of the electrophysiologicalrecording and injection paradigm. Single-cell recordings are made through a micropipette filled with FG. Once LC has been identified by its electrical discharge properties, the electrode is connected to a constant current source and iontophoretic current is applied to eject FG. Inset: oscilloscopephotograph of extracellular recording (500 Hz-10 kHz bandpass) from an individual LC neuron made through a FG injection pipette (10/xm tip). Bar = 5 ms, 0.2 mV.
/~m-tipped electrodes filled with the other solutions yielded acceptable single cell recordings. However, we found that only the acetate-buffered solution of FG at pH 3.3 could be used to reliably iontophorese tracer into the brain. Electrodes filled with many of the other solutions extruded FG into a saline solution in vitro but once inserted into the brain, failed to pass current for more than a minute. Although the initial impedance of such electrodes was between 5 and 30 Mr2, the impedance increased to over 200 MQ after applying current for 30-60 s. When such electrodes were subsequently examined, the tips contained air bubbles and solid deposits of FG. Injection sites that resulted from these electrodes were very small (10-50/zm 2) and inconsistent in size. Larger electrode tips and lower concentrations of FG allowed for slightly longer injection times before tips became blocked. Electrodes that were cleaned with water and ethanol prior to use tended to pass current for a longer period of time and produced larger injection sites than uncleaned electrodes, using equal currents. We also found that low current magnitudes were necessary (+ 1.0 # A ) and that current be delivered in pulses rather than continuously. With continuous
262 current the electrodes became clogged and did not p r o d u c e injection sites. Pulses of 4 or 7 s in duration were found to work well and it was concluded that pulse durations on the o r d e r of a few seconds were best to avoid heating or clogging the tip. Overall, we found that a 1% solution of F G in 0.1 M sodium acetate buffer (pH 3.3) iontophoresed through precleaned micropipettes with 10-20 ktm tip diameters using pulsed + 1/~A current gave the most satisfactory and consistent results. Electrodes with 10 lCm tips exhibited impedances between 20 and 25 M Q and yielded clear single-cell recordings from LC neurons (Fig. l inset), allowing identification of the site for accurate and reliable injections. In some areas containing larger neurons (e.g. Purkinje neurons in the cerebellum or sensory neurons in the mesencephalic nucleus of the fifth cra-
nial nerve), excellent single-cell recordings were obtained using 20-~m tipped electrodes, while in other locations excellent multiunit activity was recorded. W e could consistently m a k e injections lasting at least 20 min using the above parameters; longer periods of iontophoresis may be possible but were not explored. Using these procedures all injections were centered in the desired structure and the same injection p a r a m e t e r s p r o d u c e d injection sites of similar volume from animal to animal. A typical injection into LC is depicted in Fig. 2a. This injection lasted 4 min and apparently filled the LC nucleus, producing an injection site covering an a p p r o x i m a t e volume of 0.13 mm 3 (equivalent to a cube 500/~m in each dimension). No necrosis was detected at the injection site from iontophoretic injections of a 1% solution of FG. Neg-
Fig. 2. a: fluorescent photomicrograph of a coronal section containing a typical injection of FG in LC. Photograph is made under episcopic ultraviolet illumination, b: same section as in a except using blue episcopic illumination to show FITC immunofluorescence for dopamine-fl-hydroxylase. Note that the presence of FG does not interfere with FITC immunofluorescence nor does the deposit of FG produce a lesion in LC. Medial (IV ventricle) is to the right and dorsal is at top. Bar = 1001Lm.
263 ative retaining currents were not used since: (1) little or no leakage of tracer was apparent along the injection tract; and (2) negative currents were also found to eject F G appreciably. Pressure injections of this solution (0.1-0.2 /~1) produced similar results but with injection sites of substantially greater volume
FG. Such n o n - a q u e o u s m o u n t i n g did not appear to have any adverse effects on the brightness or persistence of either FITC or R I T C immunofluorescence. P e r m o u n t (Fisher) fluoresces u n d e r ultraviolet illumination and was not used. In some cases mercaptoethanol was added to the D P X as a free radical scavenger 12 in efforts to reduce the quenching rate,
and often with necrotic cores. F G sections coverslipped in aqueous m o u n t a n t s tended to quench rapidly when subjected to highpower microscopic observation. Sections that were dehydrated, cleared with xylenes and coverslipped in D P X quenched at a much slower rate and allowed
fluorescent intensity. W h e n Histoclear (National Diagnostics, N J) was substituted for xylenes, the F G
better optical resolution of the structures containing
cleared material fluoresced brightly and contained
J
~at
~'~
but this was found to increase fading and suppress F G
emission was suppressed and appeared yellowish. Retrogradely labeled F G n e u r o n s in xylene-
W
~
Fig. 3. a: epifluorescent photomicrograph of a coronal section through dorsal raphe containing neurons retrogradely labeled with FG 35 days after an injection in FCx. Note the extensive dendritic fillingand sharp cellular morphology. Midline is in the center and dorsal (third ventricle) is at the top. b: retrogradely labeled neurons in nucleus basalis from the same animal as in a. Medial is to the right and dorsal at the top. c: epifluorescent photomicrograph of a coronal section though the ipsilateral LC from the same animal as in a and b. Note the large number of retrogradely labeled neuronal profiles in LC but the relative paucity of processes extending beyond the bounds of the nucleus proper except ventromedially. Medial (IV ventricle) is to the right and dorsal is at the top. Bar = 100/~m.
264 many white/gold particles. Labeling was particulate in the cytoplasm and the nucleolus often appeared brightly labeled. The particulate nature of FG labeling was very useful in double-labeling studies, providing good contrast with the diffuse immunocytochemical labeling obtained with FITC- or RITC-conjugated antibodies. FITC has an excitation spectra that includes the peak excitation of the UV-10 filter cube (365 nm) resulting in significant excitation or 'bleed-through' with UV excitation. Thus, it was difficult to photograph a neuron with intense FITC labeling and very weak FG labeling. We found that a 530 + 15 nm notch filter removed a substantial portion of the green FITC emission without markedly affecting the FG emission. On the other hand, R I T C immunohistochemistry was also compatible for use with FG transport, and offered the advantage that R I T C did
not fluoresce appreciably during illumination with the UV-10 cube for FG. Additionally, neurons that were heavily labeled with FG were not visible under R I T C illumination, while some bleed-through of FG was apparent under FITC illumination. FG never appeared to 'leak' from retrogradely labeled neurons unless tissue was not well fixed, in which case the retrograde labeling was markedly reduced. In fixed tissue, cellular morphology was sharp and unambiguous. As reported earlier 36, retrograde labeling with FG became more intense as the survival times were extended. With small iontophoretic injections, however, there seemed to be a narrower survival period for optimal labeling than with larger, pressure injections. Staining intensities from iontophoretic deposits seemed to reach a peak after one week and retrograde labeling from animals surviving more than one
Fig. 4. Epifluorescent photomicrograph of a coronal section through the ipsilateral SN in an animal that received a 150 nl injection of FG into CA 35 days previously. Note retrogradely labeled somata in SN pars compacta (upper left) and SN pars reticulata (lower right) whose processes arborize in SN pars reticulata. Medial is to the left and dorsal is at the top. Bar = 100~m.
265 week gradually decreased in intensity. Animals allowed to survive over one month after iontophoretic injections of FG yielded weak or no labeling in areas known to be afferent to the injection site. Conversely, with large, pressure injections the intensity of more distantly labeled neurons became greater as survival times were extended (up to 35 days). However, after survival times of over 40 days the number and intensity of retrogradely labeled cells reached a peak and began to decline. With survival times of one week or greater, the dendritic processes of retrogradely labeled neurons became clearly visible such that secondary and tertiary bifurcations could be seen extending up to 150/tm from retrogradely labeled neurons (Figs. 3, 4).
Pressure injection experiments Pressure injections made into CA (3 rats) pro-
duced robust retrograde neuronal labeling in substantia nigra (SN) (Fig. 4) and injections into FCx (11 rats) produced labeling in nucleus basalis (Fig. 3b), dorsal raphe (Fig. 3a), and LC (Fig. 3c) among other expected sites. In these cases, animals were allowed to survive 35 days, the optimal time for large injections. Note the number and extent of FG-filled dendritic processes of retrogradely labeled neurons in SN pars compacta extending into SN pars reticularis in Fig. 4. For comparison, Fig. 3c shows neurons in LC of the same animal as in Fig. 3a and b that received an injection in FCx. Note that although nucleus basalis and raphe neurons exhibited dendrites extending considerable distances from the somata, the dendritic arborization of labeled LC neurons appears to be predominantly limited to the boundaries of the nucleus proper, with processes extending somewhat further in the ventromedial LC area as
Fig. 5. a: UV illuminated photomicrograph of rostral LC from an animal injected in FCx with FG. b: photomicrograph of the same field as in a but illuminated to reveal FITC immunofluorescence for DBH. Note that all neurons containing FG in LC also contain DBH immunoreactivity. Medial is to the right and dorsal is at the top. Bar = 50/~m.
266
267 previously reported 13. A large fraction (approximately 80%) of the neurons in the LC ipsilateral to the injected FCx were retrogradely labeled. In this same animal, retrogradely labeled neurons in the region of dorsolateral parabrachial nucleus contain processes that extend hundreds of microns from the cell soma into the surrounding parabrachial area (not shown).
Double labeling As documented above, injections of FG into FCx" produced robust retrograde neuronal labeling in LC. When sections were subsequently processed for immunocytochemistry, LC neurons could be readily identified as DBH-positive under blue (FITC) or green (RITC) illumination (Fig. 5b), while neurons containing FG could be unambiguously identified in the same section using U V illumination (Fig. 5a). Even neurons in the injection site containing a dense deposit of FG could be processed for immunohistochemistry without appreciable loss of immunofluorescence (Fig. 2). As expected 13, nearly all of the neurons (approximately 95%) in LC that contained FG also exhibited immunoreactivity for D B H . Similar results were seen for dorsal raphe neurons retrogradely from FCx and subsequently processed for serotonin immunofluorescence. These and other results lead us to conclude that the presence of FG in a neuron does not interfere with the presence or visualization of FITC- or RITC-linked antibodies, and vice-versa.
Sensitivity of the FG method The sensitivity of FG retrograde labeling was compared to that of W G A - H R P revealed by tetramethylbenzidine (TMB), a technique previously shown to be highly sensitive 27-29. We examined afferents to L C 3°'31 as: (i) it is a small subcortical nucleus that requires dense focal deposits of tracer to effectively label afferents; (ii) a sensitive tracer is required to
demonstrate the afferents arising from such small injections; and (iii) our recent retrograde transport study of afferents to LC using W G A - H R P and TMB histochemistry 3 provides a good basis for comparison. Injections of either tracer were judged to be restricted to LC if they did not substantially encroach on neighboring structures (i.e. parabrachial and central grey) and if the injections did not produce retrograde labeling in areas (e.g. nucleus tractus solitarius and vestibular nuclei) afferent to these neighboring structures but that do not project to the LC 3. Injections of FG and W G A - H R P that produced injection sites of apparently similar volume (Fig. 2a and Fig. 6b insert, respectively) resulted in a similar pattern of labeling and similar numbers of retrogradely labeled neurons in the two major sources of afferents to LC, nucleus paragigantoceilularis (Fig. 6) and nuclei prepositus hypoglossi (not shown3).
Fibers of passage Iontophoretic injections placed in the fimbria (+ 1 /~A, 5 min, 2 animals) resulted in no retrograde labeling in septai nuclei and only 1 - 2 labeled neurons per section in hippocampus. Also, an injection into the corpus callosum in one subject resulted in no retrogradely labeled neurons in cerebral cortex. The absence of FG-labeled neurons in areas sending axons through either fimbria or corpus callosum indicates that FG, when applied iontophoretically onto undamaged axons, is not taken up and retrogradely transported to a significant degree. This has been reported previously for pressure injection of FG and SITS (a similar molecule) into various fiber tracts 36. DISCUSSION To study the neurochemical identity of afferents to small deep brain nuclei we sought a double-labeling method that would meet specific criteria: (1) the retrograde tracer must be compatible with immuno-
Fig. 6. a: epifluorescent photomicrograph of a coronal section through PGi showing neurons retrogradely labeled with FG. This animal received an iontophoretic injection of FG in LC (similar to Fig. 2) and survived 5 days. b: a dark-field photomicrograph of PGi containing retrogradely labeled neurons arising from an injection of WGA-HRP in LC. The animal was allowed to survive 24 h and sections of the brain were processed for HRP histochemistry using TMB. Note the similar distribution pattern and numbers of retrogradely labeled neurons. Medial is to the right and dorsal is at the top. Inset: a bright-field photomicrograph of a typical iontophoretic injection of WGA-HRP in LC. Bar = 100/~m (for inset, bar = 130~m).
268 c y t o c h e m i s t r y ; (2) it m u s t a c c u r a t e l y reveal the n e u -
d e c r e a s e d sensitivity for b o t h labels. (ii) T h e r e is n o
r o n s a f f e r e n t to a small i n j e c t i o n site; a n d (3) it can-
sufficiently sensitive s u b s t r a t e f o r H R P t h a t c a n w i t h -
n o t label fibers t h a t pass t h r o u g h but d o not t e r m i -
stand subsequent immunocytochemistry. The sub-
n a t e in t h e i n j e c t i o n site.
s t r a t e T M B o f f e r s t h e g r e a t e s t sensitivity for h i s t o chemical
D o u b l e - l a b e l i n g t e c h n i q u e s e m p l o y i n g H R P histo-
localization o f r e t r o g r a d e l y
HRP 27-29, but
c h e m i s t r y c o m b i n e d w i t h i m m u n o c y t o c h e m i s t r y ~'25"
transported
t h e T M B r e a c t i o n p r o d u c t is n o t s t a b l e
27,33,34,42 h a v e t w o m a j o r d r a w b a c k s . (i) I m m u n o c y t o -
at t h e n e u t r a l p H r e q u i r e d for i m m u n o c y t o c h e m -
c h e m i s t r y f o r d e t e c t i o n o f p r o t e i n s is g e n e r a l l y p e r -
istry. W e h a v e f o u n d , as p r e v i o u s l y r e p o r t e d 3a, t h a t
f o r m e d o n tissue fixed with 4 % p a r a f o r m a l d e h y d e
diaminobenzidine (DAB)-induced
a n d little o r n o a d d e d g l u t a r a l d e h y d e , since t h e l a t t e r
the TMB reaction product decreases the m e t h o d ' s
stabilization
of
can m a r k e d l y r e d u c e t h e a n t i g e n i c i t y o f p r o t e i n s 11'2~.
sensitivity as a r e t r o g r a d e label. In o u r h a n d s , i o n t o -
In c o n t r a s t , the o p t i m a l f i x a t i o n for p r e s e r v a t i o n o f
phoretic injections of WGA-HRP
retrogradely transported
r o b u s t l a b e l i n g in P G i a n d P r H w h e n e x a m i n e d w i t h
contains
substantial
HRP
e n z y m a t i c activity
glutaraldehyde
and
T M B h i s t o c h e m i s t r y a l o n e 3. H o w e v e r , w h e n t h e s e
little p a r a f o r m a l d e h y d e ( 1 % ) , as t h e l a t t e r r e d u c e s
same sections were subsequently subjected to D A B -
H R P e n z y m a t i c activity a n d t h e r e b y d e c r e a s e s t h e
i n d u c e d s t a b i l i z a t i o n t h e r e was a m a r k e d ( a p p r o x i -
m e t h o d ' s sensitivity 27. T h u s ,
(1.25%)
into LC p r o d u c e d
for double
labeling
m a t e l y 4 0 % ) d e c r e a s e in t h e n u m b e r o f r e t r o g r a d e l y
using H R P - b a s e d r e t r o g r a d e t r a n s p o r t , a c o m p r o -
l a b e l e d n e u r o n s . In t h e original p a p e r 34 c o n c e r n i n g
mise fixation p r o c e d u r e is g e n e r a l l y u s e d , r e s u l t i n g in
D A B - i n d u c e d s t a b i l i z a t i o n t h e a u t h o r s r e p o r t an av-
TABLE I
Summary o f retrograde tracer properties Information from chart comes from work in our laboratory and refs. 1,2,4-7,9,10A5-18,20-22,24,40,41. NA = no information available.
Retrograde tracer
Exc. max. (nm)
Era. max. (nm)
No cellular leakage ~
Equivalent sensitivity to HRP
Injection by iontophoresis b
Compatible with immuno.'
Injection site necrosis
Transport by all systems d
No fiber tract uptake e
Fluoro-Gold WGA-HRP Fast blue True blue Diamino yellow Nuclear yellow Prop. iodine Bisbenzimide Granular blue DAPI Primuline Evans blue
330 385 365 385 370 340 360 355 360 400 540
530 460 420 510 520 600 500 425 490 465 735
+ + + + + + + + -
+ + + + - * -
+ + NA NA + NA NA NA NA
+
+ + + + + + + -
+ ** + ** +** + ** NA NA NA NA NA NA -
+ + -
+ + -
+ (-) -
+ + + -
-*** - *** NA - *** - *** NA NA
" Many fluorescent tracers (minus signs) leak from retrogradely labeled neurons resulting in uptake by neighboring cells and producing erroneous retrograde labeling. b Pluses indicate those tracers that can be repeatable iontophoreses (see refs. l, 9). c Acceptable tracers (plus signs) are those that do not yield a substantial reduction in the number of retrogradely labeled neurons when combined with immunohistochemistry and whose labeling does not becomes obscured as a result of the immunohistochemical process• a Certain tracers (minus signs) are not avidly transported transport in some systems when compared to other systems or retrograde tracers 15. c Pluses indicate tracers which do not did result in retrograde labeling when injected into fiber tracts. * Shown not to be as sensitive as HRP in the peripheral nervous system TM. ** These compounds appear to be transported equally in all systems tested. *** Although direct evidence for uptake from undamaged fibers of passage does not exist, these tracers produce considerable necrosis at the injection site-' when administered as recommended and uptake and transport occurs in damaged fibers.
269 erage 25% reduction in the number of retrogradely labeled neurons seen before and after the stabilization of TMB. When this stabilization method is further coupled with the non-optimal fixation (higher than 2% paraformaldehyde) required for the combination of HRP transport and immunocytochemistry, the total number of labeled neurons was further reduced, making this technique unsuitable for our purposes. Additionally, we have found it difficult to unambiguously differentiate all singly and doubly labeled neurons based upon the variable difference in color between the black cobalt/DAB (or cobalt/ DAB/TMB) reaction product and the brown DAB reaction product, as is generally employed with these techniquesS.27,33.34.42. We also examined double-labeling methods employing retrograde transport of various fluorescent retrograde dyes combined with immunocytochemistry. From our experience and after reviewing the literature (see Table I), many of these tracers present problems for determining afferents to small brain nuclei: (i) Many fluorescent tracers are less sensitive than HRP visualized with TMB 2 and therefore presumably much less sensitive than W G A - H R P transport z7. (ii) Most fluorescent tracers are retrogradely transported by fibers of passage either by direct uptake into undamaged fibers or by uptake into fibers damaged as a result of their cytotoxic properties 2' 18,20-22.35. One advantage of conjugated HRP over free HRP stems from the observation that the former is not taken up by undamaged fibers of passage 1°'14. (iii) Many fluorescent tracers 'leak' from retrogradely labeled neurons during the immunocytochemical process 2'4-6-15A8,20,21,35,37, resulting in a loss of retrogradely labeled neurons and possible erroneous labeling of adjacent cells 2. (iv) With the exception of REFERENCES 1 Alheid, G.F. and Carlsen, J., Small injections of fluorescent tracers by iontophoresis or chronic implantation of micropipettes, Brain Research, 235 (1974) 174-178. 2 Aschoff, A. and Hollander, H., Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual systemof the albino rat, J. Neurosci. Methods., 6 (1982) 179-197. 3 Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T., The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network, Science, 234 (1986) 734-737. 4 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berre-
propidium iodine, none of the fluorescent dyes has been successfully injected iontophoreticallyt'9; application by pressure can damage tissue and limits the minimum size of injection. (v) Dyes ejected via drawn glass pipettes typically require a minimum tip diameter of about 50/~m because of the viscous nature of the dye emulsions 24. Electrodes with this size tip diameter do not yield cellular recordings useful in localizing the desired injection site. We found that FG offers decided advantages in many of these areas. It can be iont0Phoresed from small-tipped pipettes which can be used to record physiological activity and identify the nuclei to be injected. It appears to be as sensitive as W G A - H R P at detecting afferents from small injection sites. FG is apparently not taken up by fibers of passage and does not have significant cytotoxic effects when applied as outlined. Retrograde transport of FG is compatible with immunocytochemistry (and a wide variety of other histochemical techniques36). It has the additional advantage of producing extensive dendritic filling of retrogradely labeled neurons. ACKNOWLEDGEMENTS The authors are grateful to Dr. Michael Shipley for his helpful advice, Matthew Ennis and Elisabeth Van Bockstaele for their assistance in the laboratory, and Stephanie Aston-Jones for her artwork. Supported by PHS Grants AA06607 and NS24698, BRSG Grant RR07062, ONR Contract N00014-86-K-0493, Air Force Office of Sponsored Research, Spencer Foundation, Alzheimer Disease and Related Disorders Assoc., American Federation for Aging Research, NYU Scientific Equipment and Curricular Development Funds. voets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rat by means of substances binding specifically to adeninethymine rich DNA, Neurosci. Lett., 12 (1979) 235-240. 5 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30. 6 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 prevent diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18 (1980) 19-24. 7 Bj6rklund, A. and Skagerberg, G., Simultaneous use of
270 retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the CNS, J. Neurosci. Methods, 1 (1979) 261-277. 8 Bowker, R.M., Steinbusch, H.W.M. and Coulter, J.D., Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method, Brain Research, 211 (1981) 412-417. 9 Brann, M.R. and Emson, P.C., Microiontophoretic injection of fluorescent tracer combined with simultaneous immunofluorescent histochemistry for the demonstration of efferents from the caudate-putamen projecting to the globus pallidus, Neurosci. Lett., 16 (1980) 61-65. 10 Brodal, P., Dietrichs, E., Bjaalie, J.G., Nordby, T. and Walberg, F., Is lectin-coupled horseradish peroxidase taken up and transported by undamaged as well as damaged fibers in the central nervous system, Brain Research, 278 (1983) 1-9. 11 Clements, J.R. and Beitz, A.J., The effects of different pretreatment conditions and fixation regimes of serotonin immunoreactive, J. Histochem. Cytochem., 33 (1985) 778-784. 12 Franklin, A.L. and Filion, G., A new technique for retarding fading of fluorescence: DPX-BME, Stain Technol., 60 (1985) 125-135. 13 Grzanna, R. and Molliver, M.E., The locus coeruleus in the rat: an immunohistochemical delineation, Neuroscience, 5 (1980) 21-40. 14 Herkenham, M. and Nauta, W.J.H., Afferent connections of the habenular nuclei in the rat a horseradish peroxidase study, with a note on the fiber-of-passage problem, J. Comp. Neurol., 173 (1977) 123-146. 15 Hokfelt, T., Skagerberg, G., Skirboll, L. and Bjorklund, A., Combination of retrograde tracing with neurotransmitter histochemistry, In Hokfelt, T. and Bjorklund, A. (Ed.), Handbook o f Chemical Neuroanatomy, Vol. 1, Methods in Chemical Neuroanatomy, Elsevier, New York, 1983, pp. 228-285. 16 Hokfelt, T., Terenius, L., Kuypers, H.G.J.M. and Dann, O., Evidence for enkephalin-immunoreactive neurons in the medulla oblongata projecting to the spinal cord, Neurosci. Lett., 14 (1979) 55-60. 17 Huisman, A.M., Kuypers, H.G.J.M. and Ververs, B., Retrograde neuronal labeling of cells of origin of descending brainstem pathways in rat using SITS as a retrograde tracer, Brain Research, 289 (1983) 305-310. 18 Illert, M., Fritz, N., Aschoff, A. and Hollander, H., Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat, J. Neurosci. Methods, 6 (1982) 199-218. 19 Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J., Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy, J. lmmunol. Methods, 55 (1982) 231-242. 20 Keizer, K., Kuypers, H.G.J.M., Huisman, A.M. and Dann, O., Diamino yellow dihydrochloride (DY.2HCI); a new fluorescent retrograde tracer, which migrates only very slowly out of the cell, Exp. Brain Res., 51 (1983) 179-191. 21 Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padts, R.E., Retrograde axonal transport of fluorescent sub-
stances in the rat's forebrain, Neurosci. Lett., 6 (1977) 1'27-135. 22 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. 23 Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, A.T., Double retrograde neuronal labeling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cell, Exp. Brain Res., 40 (1980) 383-392. 24 Kuypers, H.G.J.M. and Huisman, A.M., Fluorescent neuronal tracers, Adv. Cell Neurobiol., 5 (1984) 307-370. 25 Ljungdahl, A., Hokfelt, T., Goldstein, M. and Park, D., Retrograde peroxidase tracing of neurons combined with transmitter histochemistry, Brain Research, 84 (1975) 313-319. 26 McLeane, I.W. and Nakane, P.K., Periodate-lysine-paraformaldehyde fixative a new fixative for both light and immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. 27 Mesulam, M,M. (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley, New York, 1982. 28 Morrell, J.l., Greenberger, L.M. and Pfaff, D.W., Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics, J. Histochem. Cytochem., 29 (1981) 903-916. 29 Olsson, Y., Arvidson, B., Hartman, M,, Pettersson, A. and Tengvar, C., Horseradish peroxidase histochemistry. A comparison between various methods used for identifying neurons labeled by retrograde axonal transport, J. Neurosci. Methods, 7 (1983) 49-59. 30 Pieribone, V.A., Aston-Jones, G., Shipley, M.T. and Enhis, M., Neurochemical identity of afferents to the locus coeruleus: retrograde transport and alkaline phosphatase immunohistochemistry, Soc. Neurosci. Abstr., 12 (1986) 38.6. 31 Pieribone, V.A., Aston-Jones, G. and Bohn, M.C., Adrenergic and non-adrenergic neurons in the C1 and C3 areas project to the locus coeruleus: a fluorescent double labeling study, Neurosci. Lett., 85 (1988) 297-303. 32 Platt, J.L. and Michael, A.F., Retardation of fading and enhancement of intensity of immunofluorescence by pphenylenediamine, J. Histochem. Cytochem., 31 (1983) 840-842. 33 Priestley, J.V., Somogyi, P. and CueUo, A.C., Neurotransmitter-specific projection neurons revealed by combining PAP immunohistochemistry with retrograde transport of HRP, Brain Research, 220 (1981) 231-240. 34 Rye, D.B., Saper, C.B. and Wainer, B.H., Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry, J. Histochem. Cytochem., 32 (1984) 1145-1145. 35 Sawchenko, P.E. and Swanson, L.W., A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemistry techniques, Brain Research, 210 (1981) 31-51. 36 Schmued, L.C. and Fallon, J.H., Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Research, 377 (1986) 147-154. 37 Skirboll, L., Hokfelt, T., Norell, G., Phillipson, O., Kuy-
271 pers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E., Visser, T.J., Steinbusch, H., Verhofstad, A., Cuello, A.C., Goldstein, M. and Brownstein, M., A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing, Brain Res. Rev., 8 (1984) 99-127. 38 Shipley, M.T., A simple, low cost hydraulic pressure device for making microinjections in the brain, Brain Res. Bull., 8 (1982) 237-239. 39 Valentino, R.J., Foote, S.L. and Aston-Jones, G., Corticotropin-releasing factor activates noradrenergic neurons of
the locus coeruleus neurons, Brain Research, 270 (1983) 363-367. 40 Van der Kooy, D. and Kuypers, H.G.J.M., Fluorescent retrograde double labeling: axonal branching in the ascending raphe and nigral projections, Science, 204 (1979) 873-875. 41 Van der Kooy, D. and Steinbusch, H.W.M., Simultaneous fluorescent retrograde axonal tracing and immunofluorescent characterization of neurons, J. Neurosci. Res., 5 (1980) 479-484.
259
BRE 14104
The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei Vincent A. Pieribone and Gary Aston-Jones Department of Biology, New York University, New York, NY IO003 (U.S.A.)
(Accepted 14 June 1988) Key words: Fluoro-Gold; Retrograde tracing; Fluorescent tracer; Locus coeruleus; Raphe; Substantia nigra; Nucleus basalis; Afferent pathway; Immunofluorescence; Rat
A method is described for identifying the afferents to confined areas within the central nervous system using iontophoretic application of the fluorescent tracer, Fluoro-Gold (FG). Unlike other fluorescent tracers, it is possible to make focal iontophoretic injections through small-tipped micropipettes, and electrophysiological recordings from the injection pipette can be used to define structures prior to injections. Retrograde labeling with FG appears to be as sensitive as wheatgerm agglutinin-conjugated horseradish peroxidase visualized with tetramethylbenzidine. Furthermore, iontophoretically applied FG does not appear to be taken up and trans-. ported retrogradely by fibers of passage. Finally, retrograde transport of FG can be combined with immunofluorescence without: appreciable loss of sensitivity in either label. INTRODUCTION Since their introduction in 1977 by Kuypers and coworkers 4-6'2°-22, fluorescent retrograde tracers have become important tools for determining neuroanatomical connectivity. The use of these tracers offers several advantages over retrograde transport techniques that utilize horseradish peroxidase (HRP) or its conjugates. Fluorescent tracers require minimal tissue processing, they can be combined to demonstrate collateralization of efferents 23'4°, and tissue containing the tracers can be processed for immunocytochemistry to identify neurotransmitters 7"15'16' 24,35.37,41
However, the accuracy of a retrograde tracing study examining the afferents to a small brain region is limited by the ability to retrogradely label only t h o s e neurons which have axons that terminate within the area of interest. It is difficult with most fluorescent tracers to accurately study afferents to small brain nuclei in the rat brain since: (1) iontophoretic injections are generally not possible and it may be im-
possible to make small pressure injections limited to the desired brain site; (2) pressure injections damage tissue; (3) fibers of passage through the injection site transport most fluorescent dyes; and (4) desired injection areas cannot be physiologically localized with electrical recordings from an injection pipette. Schmued and Fallon 36 recently introduced FluoroGold (FG) as a fluorescent retrograde tracer with several novel properties. The initial report did not include details concerning some potential applications of FG. We have now examined this compound in retrograde transport studies as well as in c o m b i n a t i o n with immunocytochemistry, and provide additional information on using this new tracer. In particular, below we present specific details for iontophoretic application of FG combined with electrophysiological recordings from FG-filled pipettes for the study of afferents to small nuclei in the rat brain. MATERIALS AND METHODS Male S p r a g u e - D a w l e y rats (n = 30) were anesthe-
Correspondence: V.A. Pieribone, Department of Biology, New York University, 1009 Main Building, Washington Square, New York, NY 10003, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
260 tized with chloral hydrate (400 mg/kg, intraperitoneal), and placed in a stereotaxic apparatus. A scalp incision was made, a hole was drilled in the skull overlying the injection target, and the dura was reflected. Injections were centered in one of four structures: locus coeruleus (LC), the head of the caudate nucleus (CA), frontal cortex (FCx), or fimbria. Five previously characterized projections were investigated: the nigral-striatal, coeruleocortical, dorsal raphe-cortical, and the paragigantocellularis- and prepositus hypoglossal-to-LC pathways 3. Injections into the CA, FCx, and fimbria were done with the skull horizontal and the following coordinates: 0.2 mm posterior to bregma, 3.5 mm lateral to midline and 5.0 mm from top of skull; 4.0 mm anterior to bregma, 2.0 mm lateral to midline and 3.0 mm from top of skull; and 1.3 mm posterior to bregma, 0.5 mm lateral to midline and 4.0 mm from top of skull, respectively. For LC injections the skull was placed at a 17° angle (nose tilted down) to avoid rupturing the overlying transverse sinus; coordinates were: 3.7 mm caudal to real lambda, 1.2 mm lateral to midline and 6.3 mm from skull. FG (Fluoro-chromes, Englewood, CO) was stored in desiccated light-tight containers at 4 °C until used. Glass capillary tubes (1.5 mm o.d., Omega-Dot) were cleaned by passing deionized water and 95% ethanol through the unpulled pipettes and then airdried. Capillary tubes were then heated, pulled, and the tips broken back to 10-60/~m diameter under microscopic control. These electrodes were filled with a 1% solution of FG in filtered (0.22/~m) 0.1 M sodium acetate buffer (pH 3.3) just prior to use. Injections into LC were made from micropipettes with 10-20/~m tips. Electrophysiological recordings from these pipettes aided in localizing LC by its distinctive discharge characteristics (see Results below) 39. Larger-tipped pipettes (50-60/~m) were used for injections into FCx and CA. Injection parameters for LC were +1.0/~A for 2-10 min, while for FCx and CA currents of +5.0/~A for 15-30 min were used. Iontophoretic injections employed pulsed current (4 s on and 4 s off) to avoid heating or clogging the tip (Finntronics constant-current source). It is also possible to make injections using a duty cycle durations of 7 s on and 7 s off. In some animals, pressure injections (100-200 ni) of the same FG solution were made into CA and FCx via a 50/~m-tipped glass pi-
pette attached to a 1/~1 gas chromatography syringe (Precision Sampling, Baton Rouge, LA) 38. In all cases, electrodes were allowed to remain in place 5 min before and after injections. Animals that received injections into LC were allowed to survive 1-5 days. Animals that received injections into CA and FCx survived 5-40 days. Following the appropriate survival time, animals were deeply anesthetized with chloral hydrate and perfused transcardially with saline (100 ml for 45 s), followed by 450 ml of freshly prepared 4% paraformaldehyde (Merck) in 0.1 M phosphate buffer (pH 7.4) at 80 ml/min and then 450 ml of this same solution at 20 ml/min. All perfusion solutions were at room temperature. Brains were immersed in the same fixative at 4 °C for 90 min then transferred into phosphatebuffered 20% sucrose overnight, both at 4 °C. The following day, 40-/zm-thick frozen sections were collected into ice-cold isotonic 0.1 M phosphate-buffered saline (pH 7.4). Sections not subjected to immunohistochemistry were rinsed twice in buffer and mounted on gelatinized slides. Immunocytochemistry for neurochemical markers was done on free-floating sections. Sections were rinsed twice in Tris-buffered saline (TBS, pH 7.6) and placed in 3% normal goat serum containing 0.25% Triton for 30 min. Following 4-12 h incubation in a dilute solution of primary antisera in TBS with 1% goat serum added, sections were transferred to a solution of fluorescein isothiocyanate (FITC)- or rhodamine isothiocyanate (RITC)-conjugated, affinity purified, goat anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN) in 1% goat serum. Primary antisera were raised in rabbit against dopamine-fl-hydroxylase (DBH; Eugene Tech, N J) or serotonin conjugated to bovine serum albumin (Immuno Nuclear, Indianapolis, IN). Sections were rinsed between each step with 1% goat serum in TBS. Cutting and processing of sections was done under low ambient light conditions to avoid possible quenching of the FG. Slides were allowed to dry overnight in the dark and then quickly dehydrated through graded alcohols (70%, 95%, and 2 x 100%; 20 s each), cleared in xylenes and coverslipped using DPX (BDH, Poole, U.K.). Alternatively some sections were coverslipped in an aqueous media (glycerol/ phosphate buffer, 9:1) with added p-phenylenediamine 19'32 or polyvinyl alcohol (Lehrner, New Ha-
261 ven, CT). Coverslipped sections were kept frozen in the dark at - 2 0 °C until observation. Studies of LC afferents using W G A - H R P have been described 3 previously. Briefly, electrophysiological recordings from W G A - H R P injection pipettes were used to localize LC and iontophoretic injections were made in LC. Animals were allowed to survive one day and then perfused with a glutaraldehyde and paraformaldehyde mixture 27. Forty/~m frozen sections were processed by the technique of Mesulem et al. 27 using tetramethylbenzidine (TMB). A Nikon Microphot microscope equipped with epifluorescence was used for observation and photography of fluorescent material. The Nikon UV-10 (equivalent to the UV-1) fluorescent cube was used, producing an excitation peak at 330 nm and a lowend emission cut-off at 450 nm. On sections processed for FITC double-labeling, a notch filter (Omega Optical) eliminating transmission at 530 + 15 nm was placed in the light path between the emission filter and the ocular/camera to remove FITC fluorescence produced by UV excitation (see Discussion below). Color photographs were made with Kodak Ektachrome 200 or Kodachrome 200 film using an ASA-of 400. Black and white photographs were made on Kodak Panatomic film (ASA 32) or Tmax (ASA 100). RESULTS
Iontophoretic injection experiments Our previous experience indicates that in order to accurately place injections into the relatively small LC nucleus it is very helpful to record cellular activity through the injection pipette and localize LC neurons by their distinctive discharge characteristics 39 (Fig. 1 inset). Towards this end, we tried to dissolve FG (at concentrations of 0.5-4 % ) in the following solutions: 0.1 M phosphate/HC! buffer (pH 7.4), 0.1 M Tris/ HCI buffer (pH 7.6), 0.9% NaCI, Ringer's solution (with and without lactate36), deionized water, and 0.1 M sodium acetate/HCl buffer (pH 6.0 and 3.3). We also varied electrode tip diameters from 4 to 60 /.tm. All concentrations of FG were soluble in all of the above except for the Tris- and phosphate-buffered solutions in which FG precipitated even at low concentrations and after prolonged sonication. Four-
~t.
~
,,J.
Fig. 1. Schematic diagram of the electrophysiologicalrecording and injection paradigm. Single-cell recordings are made through a micropipette filled with FG. Once LC has been identified by its electrical discharge properties, the electrode is connected to a constant current source and iontophoretic current is applied to eject FG. Inset: oscilloscopephotograph of extracellular recording (500 Hz-10 kHz bandpass) from an individual LC neuron made through a FG injection pipette (10/xm tip). Bar = 5 ms, 0.2 mV.
/~m-tipped electrodes filled with the other solutions yielded acceptable single cell recordings. However, we found that only the acetate-buffered solution of FG at pH 3.3 could be used to reliably iontophorese tracer into the brain. Electrodes filled with many of the other solutions extruded FG into a saline solution in vitro but once inserted into the brain, failed to pass current for more than a minute. Although the initial impedance of such electrodes was between 5 and 30 Mr2, the impedance increased to over 200 MQ after applying current for 30-60 s. When such electrodes were subsequently examined, the tips contained air bubbles and solid deposits of FG. Injection sites that resulted from these electrodes were very small (10-50/zm 2) and inconsistent in size. Larger electrode tips and lower concentrations of FG allowed for slightly longer injection times before tips became blocked. Electrodes that were cleaned with water and ethanol prior to use tended to pass current for a longer period of time and produced larger injection sites than uncleaned electrodes, using equal currents. We also found that low current magnitudes were necessary (+ 1.0 # A ) and that current be delivered in pulses rather than continuously. With continuous
262 current the electrodes became clogged and did not p r o d u c e injection sites. Pulses of 4 or 7 s in duration were found to work well and it was concluded that pulse durations on the o r d e r of a few seconds were best to avoid heating or clogging the tip. Overall, we found that a 1% solution of F G in 0.1 M sodium acetate buffer (pH 3.3) iontophoresed through precleaned micropipettes with 10-20 ktm tip diameters using pulsed + 1/~A current gave the most satisfactory and consistent results. Electrodes with 10 lCm tips exhibited impedances between 20 and 25 M Q and yielded clear single-cell recordings from LC neurons (Fig. l inset), allowing identification of the site for accurate and reliable injections. In some areas containing larger neurons (e.g. Purkinje neurons in the cerebellum or sensory neurons in the mesencephalic nucleus of the fifth cra-
nial nerve), excellent single-cell recordings were obtained using 20-~m tipped electrodes, while in other locations excellent multiunit activity was recorded. W e could consistently m a k e injections lasting at least 20 min using the above parameters; longer periods of iontophoresis may be possible but were not explored. Using these procedures all injections were centered in the desired structure and the same injection p a r a m e t e r s p r o d u c e d injection sites of similar volume from animal to animal. A typical injection into LC is depicted in Fig. 2a. This injection lasted 4 min and apparently filled the LC nucleus, producing an injection site covering an a p p r o x i m a t e volume of 0.13 mm 3 (equivalent to a cube 500/~m in each dimension). No necrosis was detected at the injection site from iontophoretic injections of a 1% solution of FG. Neg-
Fig. 2. a: fluorescent photomicrograph of a coronal section containing a typical injection of FG in LC. Photograph is made under episcopic ultraviolet illumination, b: same section as in a except using blue episcopic illumination to show FITC immunofluorescence for dopamine-fl-hydroxylase. Note that the presence of FG does not interfere with FITC immunofluorescence nor does the deposit of FG produce a lesion in LC. Medial (IV ventricle) is to the right and dorsal is at top. Bar = 1001Lm.
263 ative retaining currents were not used since: (1) little or no leakage of tracer was apparent along the injection tract; and (2) negative currents were also found to eject F G appreciably. Pressure injections of this solution (0.1-0.2 /~1) produced similar results but with injection sites of substantially greater volume
FG. Such n o n - a q u e o u s m o u n t i n g did not appear to have any adverse effects on the brightness or persistence of either FITC or R I T C immunofluorescence. P e r m o u n t (Fisher) fluoresces u n d e r ultraviolet illumination and was not used. In some cases mercaptoethanol was added to the D P X as a free radical scavenger 12 in efforts to reduce the quenching rate,
and often with necrotic cores. F G sections coverslipped in aqueous m o u n t a n t s tended to quench rapidly when subjected to highpower microscopic observation. Sections that were dehydrated, cleared with xylenes and coverslipped in D P X quenched at a much slower rate and allowed
fluorescent intensity. W h e n Histoclear (National Diagnostics, N J) was substituted for xylenes, the F G
better optical resolution of the structures containing
cleared material fluoresced brightly and contained
J
~at
~'~
but this was found to increase fading and suppress F G
emission was suppressed and appeared yellowish. Retrogradely labeled F G n e u r o n s in xylene-
W
~
Fig. 3. a: epifluorescent photomicrograph of a coronal section through dorsal raphe containing neurons retrogradely labeled with FG 35 days after an injection in FCx. Note the extensive dendritic fillingand sharp cellular morphology. Midline is in the center and dorsal (third ventricle) is at the top. b: retrogradely labeled neurons in nucleus basalis from the same animal as in a. Medial is to the right and dorsal at the top. c: epifluorescent photomicrograph of a coronal section though the ipsilateral LC from the same animal as in a and b. Note the large number of retrogradely labeled neuronal profiles in LC but the relative paucity of processes extending beyond the bounds of the nucleus proper except ventromedially. Medial (IV ventricle) is to the right and dorsal is at the top. Bar = 100/~m.
264 many white/gold particles. Labeling was particulate in the cytoplasm and the nucleolus often appeared brightly labeled. The particulate nature of FG labeling was very useful in double-labeling studies, providing good contrast with the diffuse immunocytochemical labeling obtained with FITC- or RITC-conjugated antibodies. FITC has an excitation spectra that includes the peak excitation of the UV-10 filter cube (365 nm) resulting in significant excitation or 'bleed-through' with UV excitation. Thus, it was difficult to photograph a neuron with intense FITC labeling and very weak FG labeling. We found that a 530 + 15 nm notch filter removed a substantial portion of the green FITC emission without markedly affecting the FG emission. On the other hand, R I T C immunohistochemistry was also compatible for use with FG transport, and offered the advantage that R I T C did
not fluoresce appreciably during illumination with the UV-10 cube for FG. Additionally, neurons that were heavily labeled with FG were not visible under R I T C illumination, while some bleed-through of FG was apparent under FITC illumination. FG never appeared to 'leak' from retrogradely labeled neurons unless tissue was not well fixed, in which case the retrograde labeling was markedly reduced. In fixed tissue, cellular morphology was sharp and unambiguous. As reported earlier 36, retrograde labeling with FG became more intense as the survival times were extended. With small iontophoretic injections, however, there seemed to be a narrower survival period for optimal labeling than with larger, pressure injections. Staining intensities from iontophoretic deposits seemed to reach a peak after one week and retrograde labeling from animals surviving more than one
Fig. 4. Epifluorescent photomicrograph of a coronal section through the ipsilateral SN in an animal that received a 150 nl injection of FG into CA 35 days previously. Note retrogradely labeled somata in SN pars compacta (upper left) and SN pars reticulata (lower right) whose processes arborize in SN pars reticulata. Medial is to the left and dorsal is at the top. Bar = 100~m.
265 week gradually decreased in intensity. Animals allowed to survive over one month after iontophoretic injections of FG yielded weak or no labeling in areas known to be afferent to the injection site. Conversely, with large, pressure injections the intensity of more distantly labeled neurons became greater as survival times were extended (up to 35 days). However, after survival times of over 40 days the number and intensity of retrogradely labeled cells reached a peak and began to decline. With survival times of one week or greater, the dendritic processes of retrogradely labeled neurons became clearly visible such that secondary and tertiary bifurcations could be seen extending up to 150/tm from retrogradely labeled neurons (Figs. 3, 4).
Pressure injection experiments Pressure injections made into CA (3 rats) pro-
duced robust retrograde neuronal labeling in substantia nigra (SN) (Fig. 4) and injections into FCx (11 rats) produced labeling in nucleus basalis (Fig. 3b), dorsal raphe (Fig. 3a), and LC (Fig. 3c) among other expected sites. In these cases, animals were allowed to survive 35 days, the optimal time for large injections. Note the number and extent of FG-filled dendritic processes of retrogradely labeled neurons in SN pars compacta extending into SN pars reticularis in Fig. 4. For comparison, Fig. 3c shows neurons in LC of the same animal as in Fig. 3a and b that received an injection in FCx. Note that although nucleus basalis and raphe neurons exhibited dendrites extending considerable distances from the somata, the dendritic arborization of labeled LC neurons appears to be predominantly limited to the boundaries of the nucleus proper, with processes extending somewhat further in the ventromedial LC area as
Fig. 5. a: UV illuminated photomicrograph of rostral LC from an animal injected in FCx with FG. b: photomicrograph of the same field as in a but illuminated to reveal FITC immunofluorescence for DBH. Note that all neurons containing FG in LC also contain DBH immunoreactivity. Medial is to the right and dorsal is at the top. Bar = 50/~m.
266
267 previously reported 13. A large fraction (approximately 80%) of the neurons in the LC ipsilateral to the injected FCx were retrogradely labeled. In this same animal, retrogradely labeled neurons in the region of dorsolateral parabrachial nucleus contain processes that extend hundreds of microns from the cell soma into the surrounding parabrachial area (not shown).
Double labeling As documented above, injections of FG into FCx" produced robust retrograde neuronal labeling in LC. When sections were subsequently processed for immunocytochemistry, LC neurons could be readily identified as DBH-positive under blue (FITC) or green (RITC) illumination (Fig. 5b), while neurons containing FG could be unambiguously identified in the same section using U V illumination (Fig. 5a). Even neurons in the injection site containing a dense deposit of FG could be processed for immunohistochemistry without appreciable loss of immunofluorescence (Fig. 2). As expected 13, nearly all of the neurons (approximately 95%) in LC that contained FG also exhibited immunoreactivity for D B H . Similar results were seen for dorsal raphe neurons retrogradely from FCx and subsequently processed for serotonin immunofluorescence. These and other results lead us to conclude that the presence of FG in a neuron does not interfere with the presence or visualization of FITC- or RITC-linked antibodies, and vice-versa.
Sensitivity of the FG method The sensitivity of FG retrograde labeling was compared to that of W G A - H R P revealed by tetramethylbenzidine (TMB), a technique previously shown to be highly sensitive 27-29. We examined afferents to L C 3°'31 as: (i) it is a small subcortical nucleus that requires dense focal deposits of tracer to effectively label afferents; (ii) a sensitive tracer is required to
demonstrate the afferents arising from such small injections; and (iii) our recent retrograde transport study of afferents to LC using W G A - H R P and TMB histochemistry 3 provides a good basis for comparison. Injections of either tracer were judged to be restricted to LC if they did not substantially encroach on neighboring structures (i.e. parabrachial and central grey) and if the injections did not produce retrograde labeling in areas (e.g. nucleus tractus solitarius and vestibular nuclei) afferent to these neighboring structures but that do not project to the LC 3. Injections of FG and W G A - H R P that produced injection sites of apparently similar volume (Fig. 2a and Fig. 6b insert, respectively) resulted in a similar pattern of labeling and similar numbers of retrogradely labeled neurons in the two major sources of afferents to LC, nucleus paragigantoceilularis (Fig. 6) and nuclei prepositus hypoglossi (not shown3).
Fibers of passage Iontophoretic injections placed in the fimbria (+ 1 /~A, 5 min, 2 animals) resulted in no retrograde labeling in septai nuclei and only 1 - 2 labeled neurons per section in hippocampus. Also, an injection into the corpus callosum in one subject resulted in no retrogradely labeled neurons in cerebral cortex. The absence of FG-labeled neurons in areas sending axons through either fimbria or corpus callosum indicates that FG, when applied iontophoretically onto undamaged axons, is not taken up and retrogradely transported to a significant degree. This has been reported previously for pressure injection of FG and SITS (a similar molecule) into various fiber tracts 36. DISCUSSION To study the neurochemical identity of afferents to small deep brain nuclei we sought a double-labeling method that would meet specific criteria: (1) the retrograde tracer must be compatible with immuno-
Fig. 6. a: epifluorescent photomicrograph of a coronal section through PGi showing neurons retrogradely labeled with FG. This animal received an iontophoretic injection of FG in LC (similar to Fig. 2) and survived 5 days. b: a dark-field photomicrograph of PGi containing retrogradely labeled neurons arising from an injection of WGA-HRP in LC. The animal was allowed to survive 24 h and sections of the brain were processed for HRP histochemistry using TMB. Note the similar distribution pattern and numbers of retrogradely labeled neurons. Medial is to the right and dorsal is at the top. Inset: a bright-field photomicrograph of a typical iontophoretic injection of WGA-HRP in LC. Bar = 100/~m (for inset, bar = 130~m).
268 c y t o c h e m i s t r y ; (2) it m u s t a c c u r a t e l y reveal the n e u -
d e c r e a s e d sensitivity for b o t h labels. (ii) T h e r e is n o
r o n s a f f e r e n t to a small i n j e c t i o n site; a n d (3) it can-
sufficiently sensitive s u b s t r a t e f o r H R P t h a t c a n w i t h -
n o t label fibers t h a t pass t h r o u g h but d o not t e r m i -
stand subsequent immunocytochemistry. The sub-
n a t e in t h e i n j e c t i o n site.
s t r a t e T M B o f f e r s t h e g r e a t e s t sensitivity for h i s t o chemical
D o u b l e - l a b e l i n g t e c h n i q u e s e m p l o y i n g H R P histo-
localization o f r e t r o g r a d e l y
HRP 27-29, but
c h e m i s t r y c o m b i n e d w i t h i m m u n o c y t o c h e m i s t r y ~'25"
transported
t h e T M B r e a c t i o n p r o d u c t is n o t s t a b l e
27,33,34,42 h a v e t w o m a j o r d r a w b a c k s . (i) I m m u n o c y t o -
at t h e n e u t r a l p H r e q u i r e d for i m m u n o c y t o c h e m -
c h e m i s t r y f o r d e t e c t i o n o f p r o t e i n s is g e n e r a l l y p e r -
istry. W e h a v e f o u n d , as p r e v i o u s l y r e p o r t e d 3a, t h a t
f o r m e d o n tissue fixed with 4 % p a r a f o r m a l d e h y d e
diaminobenzidine (DAB)-induced
a n d little o r n o a d d e d g l u t a r a l d e h y d e , since t h e l a t t e r
the TMB reaction product decreases the m e t h o d ' s
stabilization
of
can m a r k e d l y r e d u c e t h e a n t i g e n i c i t y o f p r o t e i n s 11'2~.
sensitivity as a r e t r o g r a d e label. In o u r h a n d s , i o n t o -
In c o n t r a s t , the o p t i m a l f i x a t i o n for p r e s e r v a t i o n o f
phoretic injections of WGA-HRP
retrogradely transported
r o b u s t l a b e l i n g in P G i a n d P r H w h e n e x a m i n e d w i t h
contains
substantial
HRP
e n z y m a t i c activity
glutaraldehyde
and
T M B h i s t o c h e m i s t r y a l o n e 3. H o w e v e r , w h e n t h e s e
little p a r a f o r m a l d e h y d e ( 1 % ) , as t h e l a t t e r r e d u c e s
same sections were subsequently subjected to D A B -
H R P e n z y m a t i c activity a n d t h e r e b y d e c r e a s e s t h e
i n d u c e d s t a b i l i z a t i o n t h e r e was a m a r k e d ( a p p r o x i -
m e t h o d ' s sensitivity 27. T h u s ,
(1.25%)
into LC p r o d u c e d
for double
labeling
m a t e l y 4 0 % ) d e c r e a s e in t h e n u m b e r o f r e t r o g r a d e l y
using H R P - b a s e d r e t r o g r a d e t r a n s p o r t , a c o m p r o -
l a b e l e d n e u r o n s . In t h e original p a p e r 34 c o n c e r n i n g
mise fixation p r o c e d u r e is g e n e r a l l y u s e d , r e s u l t i n g in
D A B - i n d u c e d s t a b i l i z a t i o n t h e a u t h o r s r e p o r t an av-
TABLE I
Summary o f retrograde tracer properties Information from chart comes from work in our laboratory and refs. 1,2,4-7,9,10A5-18,20-22,24,40,41. NA = no information available.
Retrograde tracer
Exc. max. (nm)
Era. max. (nm)
No cellular leakage ~
Equivalent sensitivity to HRP
Injection by iontophoresis b
Compatible with immuno.'
Injection site necrosis
Transport by all systems d
No fiber tract uptake e
Fluoro-Gold WGA-HRP Fast blue True blue Diamino yellow Nuclear yellow Prop. iodine Bisbenzimide Granular blue DAPI Primuline Evans blue
330 385 365 385 370 340 360 355 360 400 540
530 460 420 510 520 600 500 425 490 465 735
+ + + + + + + + -
+ + + + - * -
+ + NA NA + NA NA NA NA
+
+ + + + + + + -
+ ** + ** +** + ** NA NA NA NA NA NA -
+ + -
+ + -
+ (-) -
+ + + -
-*** - *** NA - *** - *** NA NA
" Many fluorescent tracers (minus signs) leak from retrogradely labeled neurons resulting in uptake by neighboring cells and producing erroneous retrograde labeling. b Pluses indicate those tracers that can be repeatable iontophoreses (see refs. l, 9). c Acceptable tracers (plus signs) are those that do not yield a substantial reduction in the number of retrogradely labeled neurons when combined with immunohistochemistry and whose labeling does not becomes obscured as a result of the immunohistochemical process• a Certain tracers (minus signs) are not avidly transported transport in some systems when compared to other systems or retrograde tracers 15. c Pluses indicate tracers which do not did result in retrograde labeling when injected into fiber tracts. * Shown not to be as sensitive as HRP in the peripheral nervous system TM. ** These compounds appear to be transported equally in all systems tested. *** Although direct evidence for uptake from undamaged fibers of passage does not exist, these tracers produce considerable necrosis at the injection site-' when administered as recommended and uptake and transport occurs in damaged fibers.
269 erage 25% reduction in the number of retrogradely labeled neurons seen before and after the stabilization of TMB. When this stabilization method is further coupled with the non-optimal fixation (higher than 2% paraformaldehyde) required for the combination of HRP transport and immunocytochemistry, the total number of labeled neurons was further reduced, making this technique unsuitable for our purposes. Additionally, we have found it difficult to unambiguously differentiate all singly and doubly labeled neurons based upon the variable difference in color between the black cobalt/DAB (or cobalt/ DAB/TMB) reaction product and the brown DAB reaction product, as is generally employed with these techniquesS.27,33.34.42. We also examined double-labeling methods employing retrograde transport of various fluorescent retrograde dyes combined with immunocytochemistry. From our experience and after reviewing the literature (see Table I), many of these tracers present problems for determining afferents to small brain nuclei: (i) Many fluorescent tracers are less sensitive than HRP visualized with TMB 2 and therefore presumably much less sensitive than W G A - H R P transport z7. (ii) Most fluorescent tracers are retrogradely transported by fibers of passage either by direct uptake into undamaged fibers or by uptake into fibers damaged as a result of their cytotoxic properties 2' 18,20-22.35. One advantage of conjugated HRP over free HRP stems from the observation that the former is not taken up by undamaged fibers of passage 1°'14. (iii) Many fluorescent tracers 'leak' from retrogradely labeled neurons during the immunocytochemical process 2'4-6-15A8,20,21,35,37, resulting in a loss of retrogradely labeled neurons and possible erroneous labeling of adjacent cells 2. (iv) With the exception of REFERENCES 1 Alheid, G.F. and Carlsen, J., Small injections of fluorescent tracers by iontophoresis or chronic implantation of micropipettes, Brain Research, 235 (1974) 174-178. 2 Aschoff, A. and Hollander, H., Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual systemof the albino rat, J. Neurosci. Methods., 6 (1982) 179-197. 3 Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T., The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network, Science, 234 (1986) 734-737. 4 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berre-
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